CHAPTER 18 – BODY FLUIDS AND CIRCULATION

CHAPTER 18

BODY FLUIDS AND CIRCULATION

  • Simple organisms like sponges and coelenterates circulate water from their surroundings through their body cavities to facilitate the cells to exchange these substances.
  • More complex organisms use special fluids within their bodies to transport such materials like Blood and lymph (tissue fluid).

BLOOD

Blood is a special connective tissue consisting of a fluid matrix, plasma, and formed elements.

Plasma

  • Plasma is a straw coloured, viscous fluid constituting nearly 55 per cent of the blood.
  • 90-92 per cent of plasma is water and proteins contribute 6-8 per cent of it.
  • Fibrinogen, globulins and albumins are the major proteins
  • Fibrinogens are needed for clotting or coagulation of blood.
  • Globulins involved in defense mechanisms of the body and the albumins help in osmotic balance.
  • Plasma also contains small amounts of minerals like Na+, Ca++, Mg++, HCO3, Cl, etc.
  • Glucose, amino acids, lipids, etc., are also present in the plasma as they are always in transit in the body.
  • Factors for coagulation or clotting of blood are also present in the plasma in an inactive form.
  • Plasma without the clotting factors is called serum.

Formed Elements

  • Erythrocytes, leucocytes and platelets are collectively called formed elements and they constitute nearly 45 per cent of the blood.

Erythrocytes or red blood cells (RBC)

  • Most abundant of all the cells in blood.
  • Count – (In a healthy adult man) 5 – 5.5 millions of RBCs mm-3 of blood.
  • Formed in – Red bone marrow in the adults.
  • Nucleus – absent in most of the mammals
  • Shape – biconcave
  • Haemoglobin – red coloured, iron containing complex protein. hence the colour and name of cells.
  • Haemoglobin count – 12-16 gms of haemoglobin in every 100 ml of blood. These molecules play a significant role in transport of respiratory gases.
  • Average life span – 120 days.
  • Destroyed in – spleen (graveyard of RBCs).

Leucocytes or white blood cells (WBC)

  • Colourless due to the lack of haemoglobin.
  • Nucleus – present
  • Count (TLC – total leucocyte count) – 6000-8000 mm-3 of blood.
  • Leucocytes are generally short lived.
  • We have two main categories of WBCs – granulocytes and agranulocytes.
  • Neutrophils, eosinophils and basophils are different types of granulocytes, while lymphocytes and monocytes are the agranulocytes.
  • Neutrophils – most abundant cells (60-65 per cent), phagocytic.
  • Basophils – least abundant (0.5-1 per cent), secrete histamine, serotonin, heparin, etc., and are involved in inflammatory reactions.
  • Monocytes – (6-8 per cent), phagocytic cells.
  • Eosinophils – (2-3 per cent), resist infections and are also associated with allergic reactions.
  • Lymphocytes – (20-25 per cent) are of two major types – ‘B’ and ‘T’ forms. Both B and T lymphocytes are responsible for immune responses of the body.

Platelets or thrombocytes

  • Cell fragments produced from megakaryocytes (special cells in the bone marrow).
  • Count – 1,500,00-3,500,00 platelets mm-3 of blood.
  • Role – release a variety of substances most of which are involved in the coagulation or clotting of blood.
  • A reduction in their number can lead to clotting disorders which will lead to excessive loss of blood from the body.

Screenshot (91)

 BLOOD GROUPS

  • Various types of grouping of blood has been done.
  • Two such groupings – the ABO and Rh – are widely used all over the world.

ABO grouping

  • ABO grouping is based on the presence or absence of two surface antigens (chemicals that can induce immune response) on the RBCs namely A and B.
  • Similarly, the plasma of different individuals contain two natural antibodies (proteins produced in response to antigens).
  • During blood transfusion, any blood cannot be used; the blood of a donor has to be carefully matched with the blood of a recipient before any blood transfusion to avoid severe problems of clumping (destruction of RBC).

Blood Groups and Donor Compatibility

Blood Group Antigens on RBCs Antibodies in Plasma Donor’s Group
A A anti-B A, O
B B anti-A B, O
AB A, B nil AB, A, B, O
O nil anti-A, B O
  • Group ‘O’ blood can be donated to persons with any other blood group and hence ‘O’ group individuals are called ‘universal donors’.
  • ‘AB’ group can accept blood from persons with AB as well as the other groups of blood. Therefore, such persons are called ‘universal recipients’.

Rh grouping

  • Another antigen, the Rh antigen similar to one present in Rhesus monkeys (hence Rh), is also observed on the surface of RBCs of majority (nearly 80 per cent) of humans. Such individuals are called Rh positive (Rh+ve) and those in whom this antigen is absent are called Rh negative (Rh-ve).
  • An Rh-ve person, if exposed to Rh+ve blood, will form specific antibodies against the Rh antigens. Therefore, Rh group should also be matched before transfusions.

Erythroblastosis foetalis –

  • A special case of Rh incompatibility (mismatching) observed between the Rh-ve blood of a pregnant mother with Rh+ve blood of the foetus.
  • Rh antigens of the foetus do not get exposed to the Rh-ve blood of the mother in the first pregnancy as the two bloods are well separated by the placenta.
  • However, during the delivery of the first child, there is a possibility of exposure of the maternal blood to small amounts of the Rh+ve blood from the foetus.
  • In such cases, the mother starts preparing antibodies against Rh in her blood.
  • in case of her subsequent pregnancies, the Rh antibodies from the mother (Rh-ve) can leak into the blood of the foetus (Rh+ve) and destroy the foetal RBCs.
  • This could be fatal to the foetus or could cause severe anaemia and jaundice to the baby.
  • This condition is called erythroblastosis foetalis.
  • This can be avoided by administering anti-Rh antibodies to the mother immediately after the delivery of the first child.

Coagulation of Blood

  • Blood exhibits coagulation or clotting in response to an injury or trauma. This is a mechanism to prevent excessive loss of blood from the body.
  • a dark reddish brown scum formed at the site of a cut or an injury over a period of time is a clot or coagulam, formed mainly of a network of threads called fibrins in which dead and damaged formed elements of blood are trapped.
  • Fibrins are formed by the conversion of inactive fibrinogens in the plasma by the enzyme thrombin.
  • Thrombins, in turn are formed from another inactive substance present in the plasma called prothrombin.
  • An enzyme complex, thrombokinase, is required for the above reaction.
  • This complex is formed by a series of linked enzymic reactions (cascade process) involving a number of factors present in the plasma in an inactive state.
  • An injury or a trauma stimulates the platelets in the blood to release certain factors which activate the mechanism of coagulation.
  • Certain factors released by the tissues at the site of injury also can initiate coagulation.
  • Calcium ions play a very important role in clotting.

LYMPH (TISSUE FLUID)

  • As the blood passes through the capillaries in tissues, some water along with many small water soluble substances move out into the spaces between the cells of tissues leaving the larger proteins and most of the formed elements in the blood vessels.
  • This fluid released out is called the interstitial fluid or tissue fluid.
  • It has the same mineral distribution as that in plasma.
  • Exchange of nutrients, gases, etc., between the blood and the cells always occur through this fluid.
  • An elaborate network of vessels called the lymphatic system collects this fluid and drains it back to the major veins. The fluid present in the lymphatic system is called the lymph.
  • Lymph is a colourless fluid containing specialised lymphocytes which are responsible for the immune responses of the body.
  • Lymph is also an important carrier for nutrients, hormones, etc. Fats are absorbed through lymph in the lacteals present in the intestinal villi.

 CIRCULATORY PATHWAYS

  • The circulatory patterns are of two types – open or closed.
  • Open circulatory system is present in arthropods and molluscs in which blood pumped by the heart passes through large vessels into open spaces or body cavities called sinuses.
  • Annelids and chordates have a closed circulatory system in which the blood pumped by the heart is always circulated through a closed network of blood vessels. This pattern is considered to be more advantageous as the flow of fluid can be more precisely regulated.
  • All vertebrates possess a muscular chambered heart.
  • Fishes have a 2-chambered heart with an atrium and a ventricle. Amphibians and the reptiles (except crocodiles) have a 3-chambered heart with two atria and a single ventricle, whereas crocodiles, birds and mammals possess a 4-chambered heart with two atria and two ventricles.
  • In fishes the heart pumps out deoxygenated blood which is oxygenated by the gills and supplied to the body parts from where deoxygenated blood is returned to the heart (single circulation).
  • In amphibians and reptiles, the left atrium receives oxygenated blood from the gills/lungs/skin and the right atrium gets the deoxygenated blood from other body parts. However, they get mixed up in the single ventricle which pumps out mixed blood (incomplete double circulation).
  • In birds and mammals, oxygenated and deoxygenated blood received by the left and right atria respectively passes on to the ventricles of the same sides. The ventricles pump it out without any mixing up, i.e., two separate circulatory pathways are present in these organisms, hence, these animals have double circulation.

 HUMAN CIRCULATORY SYSTEM

  • Human circulatory system, also called the blood vascular system consists of a muscular chambered heart, a network of closed branching blood vessels and blood, the fluid which is circulated.

HEART

  • Heart, the mesodermally derived organ, is situated in the thoracic cavity, in between the two lungs, slightly tilted to the left.
  • It has the size of a clenched fist.
  • It is protected by a double walled membranous bag, pericardium, enclosing the pericardial fluid.
  • Our heart has four chambers, two relatively small upper chambers called atria and two larger lower chambers called
  • A thin, muscular wall called the inter­atrial septum separates the right and the left atria, whereas a thick-walled, the inter-ventricular septum, separates the left and the right ventricles.
  • The atrium and the ventricle of the same side are also separated by a thick fibrous tissue called the atrio-ventricular septum.
  • However, each of these septa are provided with an opening through which the two chambers of the same side are connected.
  • The opening between the right atrium and the right ventricle is guarded by a valve formed of three muscular flaps or cusps, the tricuspid valve, whereas a bicuspid or mitral valve guards the opening between the left atrium and the left ventricle.
  • The openings of the right and the left ventricles into the pulmonary artery and the aorta respectively are provided with the semilunar valves. The valves in the heart allows the flow of blood only in one direction, i.e., from the atria to the ventricles and from the ventricles to the pulmonary artery or aorta. These valves prevent any backward flow.
  • The entire heart is made of cardiac muscles.
  • The walls of ventricles are much thicker than that of the atria.

Screenshot (92)

  • A specialised cardiac musculature called the nodal tissue is also distributed in the heart.
  • A patch of this tissue is present in the right upper corner of the right atrium called the sino-atrial node (SAN).
  • Another mass of this tissue is seen in the lower left corner of the right atrium close to the atrio-ventricular septum called the atrio-ventricular node (AVN).
  • A bundle of nodal fibres, atrio­ventricular bundle (AV bundle) continues from the AVN which passes through the atrio-ventricular septa to emerge on the top of the inter­ventricular septum and immediately divides into a right and left bundle.
  • These branches give rise to minute fibres throughout the ventricular musculature of the respective sides and are called purkinje fibres.
  • These fibres alongwith right and left bundles are known as bundle of HIS.
  • The nodal musculature has the ability to generate action potentials without any external stimuli, i.e., it is autoexcitable.
  • However, the number of action potentials that could be generated in a minute vary at different parts of the nodal system.
  • The SAN can generate the maximum number of action potentials, i.e., 70-75 min-1, and is responsible for initiating and maintaining the rhythmic contractile activity of the heart. Therefore, it is called the pacemaker.
  • Our heart normally beats 70-75 times in a minute (average 72 beats min-1).

CARDIAC CYCLE

  • To begin with, all the four chambers of heart are in a relaxed state, i.e., they are in joint diastole.
  • As the tricuspid and bicuspid valves are open, blood from the pulmonary veins and vena cava flows into the left and the right ventricle respectively through the left and right atria. The semilunar valves are closed at this stage.
  • The SAN now generates an action potential which stimulates both the atria to undergo a simultaneous contraction – the atrial systole. This increases the flow of blood into the ventricles by about 30 per cent.
  • The action potential is conducted to the ventricular side by the AVN and AV bundle from where the bundle of HIS transmits it through the entire ventricular musculature.
  • This causes the ventricular muscles to contract, (ventricular systole), the atria undergo relaxation (diastole), coinciding with the ventricular systole.
  • Ventricular systole increases the ventricular pressure causing the closure of tricuspid and bicuspid valves due to attempted backflow of blood into the atria.
  • As the ventricular pressure increases further, the semilunar valves guarding the pulmonary artery (right side) and the aorta (left side) are forced open, allowing the blood in the ventricles to flow through these vessels into the circulatory pathways.
  • The ventricles now relax (ventricular diastole) and the ventricular pressure falls causing the closure of semilunar valves which prevents the backflow of blood into the ventricles.
  • As the ventricular pressure declines further, the tricuspid and bicuspid valves are pushed open by the pressure in the atria exerted by the blood which was being emptied into them by the veins. The blood now once again moves freely to the ventricles.
  • The ventricles and atria are now again in a relaxed (joint diastole) state, as earlier. Soon the SAN generates a new action potential and the events described above are repeated in that sequence and the process continues.
  • This sequential event in the heart which is cyclically repeated is called the cardiac cycle and it consists of systole and diastole of both the atria and ventricles.
  • The heart beats 72 times per minute, i.e., that many cardiac cycles are performed per minute.
  • From this it could be deduced that the duration of a cardiac cycle is 0.8 seconds.
  • During a cardiac cycle, each ventricle pumps out approximately 70 mL of blood which is called the stroke volume.
  • The stroke volume multiplied by the heart rate (no. of beats per min.) gives the cardiac output.
  • Therefore, the cardiac output can be defined as the volume of blood pumped out by each ventricle per minute and averages 5000 mL or 5 litres in a healthy individual.
  • The body has the ability to alter the stroke volume as well as the heart rate and thereby the cardiac output. For example, the cardiac output of an athlete will be much higher than that of an ordinary man.
  • During each cardiac cycle two prominent sounds are produced which can be easily heard through a stethoscope.
  • The first heart sound (lub) is associated with the closure of the tricuspid and bicuspid valves whereas the second heart sound (dub) is associated with the closure of the semilunar valves. These sounds are of clinical diagnostic significance.

 ELECTROCARDIOGRAPH (ECG)

  • ECG is a graphical representation of the electrical activity of the heart during a cardiac cycle.
  • To obtain a standard ECG, a patient is connected to the machine with three electrical leads (one to each wrist and to the left ankle) that continuously monitor the heart activity.
  • For a detailed evaluation of the heart’s function, multiple leads are attached to the chest region.
  • Each peak in the ECG is identified with a letter from P to T that corresponds to a specific electrical activity of the heart.
  • The P-wave represents the electrical excitation (or depolarisation) of the atria, which leads to the contraction of both the atria.
  • The QRS complex represents the depolarisation of the ventricles, which initiates the ventricular contraction. The contraction starts shortly after Q and marks the beginning of the systole.
  • The T-wave represents the return of the ventricles from excited to normal state (repolarisation). The end of the T-wave marks the end of systole.
  • By counting the number of QRS complexes that occur in a given time period, one can determine the heart beat rate of an individual.
  • Since the ECGs obtained from different individuals have roughly the same shape for a given lead configuration, any deviation from this shape indicates a possible abnormality or disease. Hence, it is of a great clinical significance.

 Screenshot (93)

 DOUBLE CIRCULATION

  • The blood pumped by the right ventricle enters the pulmonary artery, whereas the left ventricle pumps blood into the aorta.
  • The deoxygenated blood pumped into the pulmonary artery is passed on to the lungs from where the oxygenated blood is carried by the pulmonary veins into the left atrium. This pathway constitutes the pulmonary circulation.
  • The oxygenated blood entering the aorta is carried by a network of arteries, arterioles and capillaries to the tissues from where the deoxygenated blood is collected by a system of venules, veins and vena cava and emptied into the right atrium. This is the systemic circulation.
  • The systemic circulation provides nutrients, O2 and other essential substances to the tissues and takes CO2 and other harmful substances away for elimination.
  • A unique vascular connection exists between the digestive tract and liver called hepatic portal system. The hepatic portal vein carries blood from intestine to the liver before it is delivered to the systemic circulation.
  • A special coronary system of blood vessels is present in our body exclusively for the circulation of blood to and from the cardiac musculature.

Screenshot (94)

REGULATION OF CARDIAC ACTIVITY

  • Normal activities of the heart are regulated intrinsically, i.e., auto regulated by specialised muscles (nodal tissue), hence the heart is called myogenic.
  • A special neural centre in the medulla oblangata can moderate the cardiac function through autonomic nervous system (ANS).
  • Neural signals through the sympathetic nerves (part of ANS) can increase the rate of heart beat, the strength of ventricular contraction and thereby the cardiac output.
  • On the other hand, parasympathetic neural signals (another component of ANS) decrease the rate of heart beat, speed of conduction of action potential and thereby the cardiac output.
  • Adrenal medullary hormones can also increase the cardiac output.

DISORDERS OF CIRCULATORY SYSTEM

High Blood Pressure (Hypertension):

  • Hypertension is the term for blood pressure that is higher than normal (120/80).
  • In this measurement 120 mm Hg (millimetres of mercury pressure) is the systolic, or pumping, pressure and 80 mm Hg is the diastolic, or resting, pressure.
  • If repeated checks of blood pressure of an individual is 140/90 (140 over 90) or higher, it shows hypertension.
  • High blood pressure leads to heart diseases and also affects vital organs like brain and kidney.

Coronary Artery Disease (CAD):

  • Coronary Artery Disease, often referred to as atherosclerosis, affects the vessels that supply blood to the heart muscle.
  • It is caused by deposits of calcium, fat, cholesterol and fibrous tissues, which makes the lumen of arteries narrower.

Angina:

  • It is also called ‘angina pectoris’.
  • A symptom of acute chest pain appears when no enough oxygen is reaching the heart muscle.
  • Angina can occur in men and women of any age but it is more common among the middle-aged and elderly.
  • It occurs due to conditions that affect the blood flow.

Heart Failure:

  • Heart failure means the state of heart when it is not pumping blood effectively enough to meet the needs of the body.
  • It is sometimes called congestive heart failure because congestion of the lungs is one of the main symptoms of this disease.
  • Heart failure is not the same as cardiac arrest (when the heart stops beating) or a heart attack (when the heart muscle is suddenly damaged by an inadequate blood supply).

 

PRINTABLE Pdf file of chapter notes are available…please click on the link…

CHAPTER 18 – BODY FLUIDS AND CIRCULATION

CHAPTER 17 – BREATHING AND EXCHANGE OF GASES

CHAPTER 17

BREATHING AND EXCHANGE OF GASES

  • Oxygen (O2) is utilised by the organisms to indirectly break down nutrient molecules like glucose and to derive energy for performing various activities.
  • Carbon dioxide (CO2) which is harmful is also released during the above catabolic reactions.
  • This process of exchange of O2 from the atmosphere with CO2 produced by the cells is called breathing, commonly known as  respiration.

Respiratory Organs

  • Lower invertebrates like sponges, coelenterates, flatworms, etc., exchange O2 with CO2 by simple diffusion over their entire body surface.
  • Earthworms use their moist cuticle and insects have a network of tubes (tracheal tubes) to transport atmospheric air within the body.
  • Special vascularised structures called gills are used by most of the aquatic arthropods and molluscs whereas vascularised bags called lungs are used by the terrestrial forms for the exchange of gases.
  • Among vertebrates, fishes use gills whereas reptiles, birds and mammals respire through lungs.
  • Amphibians like frogs can respire through their moist skin also.
  • Mammals have a well developed respiratory system.

Human Respiratory System

  • We have a pair of external nostrils opening out above the upper lips. It leads to a nasal chamber through the nasal passage.
  • The nasal chamber opens into nasopharynx, which is a portion of pharynx, the common passage for food and air.
  • Nasopharynx opens through glottis of the larynx region into the trachea.
  • Larynx is a cartilaginous box which helps in sound production and hence called the sound box.
  • During swallowing glottis can be covered by a thin elastic cartilaginous flap called epiglottis to prevent the entry of food into the larynx.
  • Trachea is a straight tube extending up to the mid-thoracic cavity, which divides at the level of 5th thoracic vertebra into a right and left primary bronchi.
  • Each bronchi undergoes repeated divisions to form the secondary and tertiary bronchi and bronchioles ending up in very thin terminal bronchioles.
  • The tracheae, primary, secondary and tertiary bronchi, and initial bronchioles are supported by incomplete cartilaginous rings.
  • Each terminal bronchiole gives rise to a number of very thin, irregular- walled and vascularised bag-like structures called alveoli.

Screenshot (84)

  • The branching network of bronchi, bronchioles and alveoli comprise the lungs.
  • We have two lungs which are covered by a double layered pleura, with pleural fluid between them. It reduces friction on the lung- surface.
  • The outer pleural membrane is in close contact with the thoracic lining whereas the inner pleural membrane is in contact with the lung surface.
  • The part starting with the external nostrils up to the terminal bronchioles constitute the conducting part whereas the alveoli and their ducts form the respiratory or exchange part of the respiratory system.
  • The conducting part transports the atmospheric air to the alveoli, clears it from foreign particles, humidifies and also brings the air to body temperature.
  • Exchange part is the site of actual diffusion of O2 and CO2 between blood and atmospheric air.
  • The lungs are situated in the thoracic chamber which is anatomically an air-tight chamber.
  • The thoracic chamber is formed dorsally by the vertebral column, ventrally by the sternum, laterally by the ribs and on the lower side by the dome-shaped diaphragm.
  • The anatomical setup of lungs in thorax is such that any change in the volume of the thoracic cavity will be reflected in the lung (pulmonary) cavity. Such an arrangement is essential for breathing, as we cannot directly alter the pulmonary volume.
  • Respiration involves the following steps:
    • Breathing or pulmonary ventilation by which atmospheric air is drawn in and CO2 rich alveolar air is released out.
    • Diffusion of gases (O2 and CO2) across alveolar membrane.
    • Transport of gases by the blood.
    • Diffusion of O2 and CO2 between blood and tissues.
    • Utilisation of O2 by the cells for catabolic reactions and resultant release of CO2 (cellular respiration)

Mechanism of Breathing

  • Breathing involves two stages: inspiration during which atmospheric air is drawn in and expiration by which the alveolar air is released out.
  • The movement of air into and out of the lungs is carried out by creating a pressure gradient between the lungs and the atmosphere.
  • Inspiration can occur if the pressure within the lungs (intra-pulmonary pressure) is less than the atmospheric pressure, i.e., there is a negative pressure in the lungs with respect to atmospheric pressure.
  • Similarly, expiration takes place when the intra-pulmonary pressure is higher than the atmospheric pressure.
  • The diaphragm and a specialised set of muscles – external and internal intercostals between the ribs, help in generation of such gradients.
  • Inspiration is initiated by the contraction of diaphragm which increases the volume of thoracic chamber in the antero-posterior axis. The contraction of external inter-costal muscles lifts up the ribs and the sternum causing an increase in the volume of the thoracic chamber in the dorso-ventral axis.
  • The overall increase in the thoracic volume causes a similar increase in pulmonary volume.
  • An increase in pulmonary volume decreases the intra-pulmonary pressure to less than the atmospheric pressure which forces the air from outside to move into the lungs, i.e., inspiration.
  • Relaxation of the diaphragm and the inter-costal muscles returns the diaphragm and sternum to their normal positions and reduce the thoracic volume and thereby the pulmonary volume. This leads to an increase in intra-pulmonary pressure to slightly above the atmospheric pressure causing the expulsion of air from the lungs, i.e., expiration.
  • We have the ability to increase the strength of inspiration and expiration with the help of additional muscles in the abdomen.
  • On an average, a healthy human breathes 12-16 times/minute.
  • The volume of air involved in breathing movements can be estimated by using a spirometer which helps in clinical assessment of pulmonary functions.

Screenshot (85)

Respiratory Volumes and Capacities

Tidal Volume (TV):

Volume of air inspired or expired during a normal respiration. It is approx. 500 mL., i.e., a healthy man can inspire or expire approximately 6000 to 8000 mL of air per minute.

Inspiratory Reserve Volume (IRV):

Additional volume of air, a person can inspire by a forcible inspiration. This averages 2500 mL to 3000 mL.

Expiratory Reserve Volume (ERV):

Additional volume of air, a person can expire by a forcible expiration. This averages 1000 mL to 1100 mL.

Residual Volume (RV):

Volume of air remaining in the lungs even after a forcible expiration. This averages 1100 mL to 1200 mL.

By adding up a few respiratory volumes described above, one can derive various pulmonary capacities, which can be used in clinical diagnosis.

Inspiratory Capacity (IC):

Total volume of air a person can inspire after a normal expiration. This includes tidal volume and inspiratory reserve volume (TV+IRV).

Expiratory Capacity (EC):

Total volume of air a person can expire after a normal inspiration. This includes tidal volume and expiratory reserve volume (TV+ERV).

Functional Residual Capacity (FRC):

Volume of air that will remain in the lungs after a normal expiration. This includes ERV+RV.

Vital Capacity (VC):

The maximum volume of air a person can breathe in after a forced expiration. This includes ERV, TV and IRV or the maximum volume of air a person can breathe out after a forced inspiration.

Total Lung Capacity:

Total volume of air accommodated in the lungs at the end of a forced inspiration. This includes RV, ERV, TV and IRV or vital capacity + residual volume.

Exchange of Gases

  • Alveoli are the primary sites of exchange of gases. Exchange of gases also occur between blood and tissues.
  • O2 and CO2 are exchanged in these sites by simple diffusion mainly based on pressure/concentration gradient.
  • Solubility of the gases as well as the thickness of the membranes involved in diffusion are also some important factors that can affect the rate of diffusion.
  • Pressure contributed by an individual gas in a mixture of gases is called partial pressure and is represented as pO2 for oxygen and pCO2 for carbon dioxide.

 

Respiratory Gas Atmospheric Air Alveoli Blood (Deoxygenated) Blood (Oxygenated) Tissues
O2 159 104 40 95 40
CO2 0.3 40 45 40 45

The data given in the table clearly indicates a concentration gradient for oxygen from alveoli to blood and blood to tissues. Similarly, a gradient is present for CO2 in the opposite direction, i.e., from tissues to blood and blood to alveoli.

  • As the solubility of CO2 is 20-25 times higher than that of O2, the amount of CO2 that can diffuse through the diffusion membrane per unit difference in partial pressure is much higher compared to that of O2.
  • The diffusion membrane is made up of three major layers namely, the thin squamous epithelium of alveoli, the endothelium of alveolar capillaries and the basement substance in between them. However, its total thickness is much less than a millimetre.

Screenshot (87)

  • Therefore, all the factors in our body are favourable for diffusion of O2 from alveoli to tissues and that of CO2 from tissues to alveoli.

Screenshot (89)

Transport of Gases

  • Blood is the medium of transport for O2 and CO2. About 97 per cent of O2 is transported by RBCs in the blood.
  • The remaining 3 per cent of O2 is carried in a dissolved state through the plasma.
  • Nearly 20-25 per cent of CO2 is transported by RBCs whereas 70 per cent of it is carried as bicarbonate. About 7 per cent of CO2 is carried in a dissolved state through plasma.

Transport of Oxygen

  • Haemoglobin is a red coloured iron containing pigment present in the RBCs. O2 can bind with haemoglobin in a reversible manner to form
  • Each haemoglobin molecule can carry a maximum of four molecules of O2.
  • Binding of oxygen with haemoglobin is primarily related to partial pressure of O2.
  • Partial pressure of CO2, hydrogen ion concentration and temperature are the other factors which can interfere with this binding.
  • A sigmoid curve is obtained when percentage saturation of haemoglobin with O2 is plotted against the pO2. This curve is called the Oxygen dissociation curve and is highly useful in studying the effect of factors like pCO2, H+ concentration, etc., on binding of O2 with haemoglobin.
  • In the alveoli, where there is high pO2, low pCO2, lesser H+ concentration and lower temperature, the factors are all favourable for the formation of oxyhaemoglobin, whereas in the tissues, where low pO2, high pCO2, high H+ concentration and higher temperature exist, the conditions are favourable for dissociation of oxygen from the oxyhaemoglobin.
  • This clearly indicates that O2 gets bound to haemoglobin in the lung surface and gets dissociated at the tissues.
  • Every 100 ml of oxygenated blood can deliver around 5 ml of O2 to the tissues under normal physiological conditions.

 Screenshot (88)

Transport of Carbon dioxide

  • CO2 is carried by haemoglobin as carbamino-haemoglobin (about 20-25 per cent).
  • This binding is related to the partial pressure of CO2. pO2 is a major factor which could affect this binding.
  • When pCO2 is high and pO2 is low as in the tissues, more binding of carbon dioxide occurs whereas, when the pCO2 is low and pO2 is high as in the alveoli, dissociation of CO2 from carbamino-haemoglobin takes place, i.e., CO2 which is bound to haemoglobin from the tissues is delivered at the alveoli.
  • RBCs contain a very high concentration of the enzyme, carbonic anhydrase and minute quantities of the same is present in the plasma too.
  • This enzyme facilitates the following reaction in both directions.

Screenshot (90)

  • At the tissue site where partial pressure of CO2 is high due to catabolism, CO2 diffuses into blood (RBCs and plasma) and forms HCO3 and H+.
  • At the alveolar site where pCO2 is low, the reaction proceeds in the opposite direction leading to the formation of CO2 and H2
  • Thus, CO2 trapped as bicarbonate at the tissue level and transported to the alveoli is released out as CO2.
  • Every 100 ml of deoxygenated blood delivers approximately 4 ml of CO2 to the alveoli.

 Regulation of Respiration

  • Human beings have a significant ability to maintain and moderate the respiratory rhythm to suit the demands of the body tissues. This is done by the neural system.
  • A specialised centre present in the medulla region of the brain called respiratory rhythm centre is primarily responsible for this regulation.
  • Another centre present in the pons region of the brain called pneumotaxic centre can moderate the functions of the respiratory rhythm centre.
  • Neural signal from this centre can reduce the duration of inspiration and thereby alter the respiratory rate.
  • A chemosensitive area is situated adjacent to the rhythm centre which is highly sensitive to CO2 and hydrogen ions.
  • Increase in these substances can activate this centre, which in turn can signal the rhythm centre to make necessary adjustments in the respiratory process by which these substances can be eliminated.
  • Receptors associated with aortic arch and carotid artery also can recognise changes in CO2 and H+ concentration and send necessary signals to the rhythm centre for remedial actions.
  • The role of oxygen in the regulation of respiratory rhythm is quite insignificant.

 Disorders of Respiratory System

  • Asthma is a difficulty in breathing causing wheezing due to inflammation of bronchi and bronchioles.
  • Emphysema is a chronic disorder in which alveolar walls are damaged due to which respiratory surface is decreased. One of the major causes of this is cigarette smoking.
  • Occupational Respiratory Disorders: In certain industries, especially those involving grinding or stone-breaking, so much dust is produced that the defense mechanism of the body cannot fully cope with the situation. Long exposure can give rise to inflammation leading to fibrosis (proliferation of fibrous tissues) and thus causing serious lung damage. Workers in such industries should wear protective masks.

 

PRINTABLE Pdf file of chapter notes are available…please click on the link…

CHAPTER 17 – BREATHING AND EXCHANGE OF GASES

CHAPTER 16 – DIGESTION AND ABSORPTION

Chapter 16

Digestion and Absorption

  • The major components of our food are carbohydrates, proteins and fats. Vitamins and minerals are also required in small quantities.
  • Food provides energy and organic materials for growth and repair of tissues.
  • The water we take in, plays an important role in metabolic processes and also prevents dehydration of the body.
  • Biomacromolecules in food cannot be utilised by our body in their original form. They have to be broken down and converted into simple substances in the digestive system. This process of conversion of complex food substances to simple absorbable forms is called digestion and is carried out by our digestive system by mechanical and biochemical methods.

 DIGESTIVE SYSTEM

The human digestive system consists of the alimentary canal and the associated glands.

Screenshot (74)

 Alimentary Canal

  • The alimentary canal begins with an anterior opening – the mouth, and it opens out posteriorly through the anus.
  • The mouth leads to the buccal cavity or oral cavity.
  • The oral cavity has a number of teeth and a muscular tongue.
  • Each tooth is embedded in a socket of jaw bone. This type of attachment is called
  • Majority of mammals including human being forms two sets of teeth during their life, a set of temporary milk or deciduous teeth replaced by a set of permanent or adult teeth. This type of dentition is called
  • An adult human has 32 permanent teeth which are of four different types (Heterodont dentition), namely, incisors (I), canine (C), premolars (PM) and molars (M).
  • Arrangement of teeth in each half of the upper and lower jaw in the order I, C, PM, M is represented by a dental formula which in human is 2123/2123.

Screenshot (75)

  • The hard chewing surface of the teeth, made up of enamel, helpsin the mastication of food.
  • The tongue is a freely movable muscular organ attached to the floor of the oral cavity by the frenulum.
  • The upper surface of the tongue has small projections called papillae, some of which bear taste buds.
  • The oral cavity leads into a short pharynx which serves as a common passage for food and air. The oesophagus and the trachea (wind pipe) open into the pharynx.
  • A cartilaginous flap called epiglottis prevents the entry of food into the glottis – opening of the wind pipe – during swallowing.
  • The oesophagus is a thin, long tube which extends posteriorly passing through the neck, thorax and diaphragm and leads to a ‘J’ shaped bag like structure called stomach.
  • A muscular sphincter (gastro-oesophageal) regulates the opening of oesophagus into the stomach.
  • The stomach, located in the upper left portion of the abdominal cavity, has three major parts – a cardiac portion into which the oesophagus opens, a fundic region and a pyloric portion which opens into the first part of small intestine.

Screenshot (76)

  • Small intestine is distinguishable into three regions, a ‘U’ shaped duodenum, a long coiled middle portion jejunum and a highly coiled ileum.
  • The opening of the stomach into the duodenum is guarded by the pyloric sphincter.
  • Ileum opens into the large intestine. It consists of caecum, colon and rectum.
  • Caecum is a small blind sac which hosts some symbiotic micro-organisms.
  • A narrow finger-like tubular projection, the vermiform appendix which is a vestigial organ, arises from the caecum.
  • The caecum opens into the colon. The colon is divided into three parts – an ascending, a transverse and a descending part.
  • The descending part opens into the rectum which opens out through the anus.
  • The wall of alimentary canal from oesophagus to rectum possesses four layers namely serosa, muscularis, sub-mucosa and mucosa.
  • Serosa is the outermost layer and is made up of a thin mesothelium (epithelium of visceral organs) with some connective tissues.
  • Muscularis is formed by smooth muscles usually arranged into an inner circular and an outer longitudinal layer. An oblique muscle layer may be present in some regions(Stomach).

Screenshot (77)

  • The sub­mucosal layer is formed of loose connective tissues containing nerves, blood and lymph vessels. In duodenum, glands are also present in sub-mucosa.
  • The innermost layer lining the lumen of the alimentary canal is the mucosa. This layer forms irregular folds (rugae) in the stomach and small finger-like foldings called villi in the small intestine.
  • The cells lining the villi produce numerous microscopic projections called microvilli giving a brush border appearance. These modifications increase the surface area enormously.
  • Villi are supplied with a network of capillaries and a large lymph vessel called the lacteal.

Screenshot (78)

  • Mucosal epithelium has goblet cells which secrete mucus that help in lubrication.
  • Mucosa also forms glands in the stomach (gastric glands) and crypts in between the bases of villi in the intestine (crypts of Lieberkuhn).
  • All the four layers show modifications in different parts of the alimentary canal.

 Digestive Glands

  • The digestive glands associated with the alimentary canal include the salivary glands, the liver and the pancreas.
  • Saliva is mainly produced by three pairs of salivary glands, the parotids (cheek), the sub-maxillary/sub-mandibular (lower jaw) and the sub-lingual (below the tongue).
  • These glands situated just outside the buccal cavity secrete salivary juice into the buccal cavity.
  • Liver is the largest gland of the body weighing about 1.2 to 1.5 kg in an adult human.
  • It is situated in the abdominal cavity, just below the diaphragm and has two lobes.
  • The hepatic lobules are the structural and functional units of liver containing hepatic cells arranged in the form of cords.
  • Each lobule is covered by a thin connective tissue sheath called the Glisson’s capsule.
  • The bile secreted by the hepatic cells passes through the hepatic ducts and is stored and concentrated in a thin muscular sac called the gall bladder.
  • The duct of gall bladder (cystic duct) along with the hepatic duct from the liver forms the common bile duct.
  • The bile duct and the pancreatic duct open together into the duodenum as the common hepato-pancreatic duct which is guarded by a sphincter called the sphincter of Oddi.
  • The pancreas is a compound (both exocrine and endocrine) elongated organ situated between the limbs of the ‘U’ shaped duodenum.
  • The exocrine portion secretes an alkaline pancreatic juice containing enzymes and the endocrine portion secretes hormones, insulin and glucagon.

Screenshot (79)

DIGESTION OF FOOD

  • The process of digestion is accomplished by mechanical and chemical processes.
  • The buccal cavity performs two major functions, mastication of food and facilitation of swallowing. The teeth and the tongue with the help ofsaliva masticate and mix up the food thoroughly.
  • Mucus in saliva helps in lubricating and adhering the masticated food particles into a BOLUS.
  • The bolus is then conveyed into the pharynx and then into the oesophagus by swallowing or DEGLUTITION.
  • The bolus further passes down through the oesophagus by successive waves of muscular contractions called peristalsis.
  • The gastro-oesophageal sphincter controls the passage of food into the stomach.
  • The saliva secreted into the oral cavity contains electrolytes (Na+, K+, Cl, HCO) and enzymes, salivary amylase and lysozyme.
  • The chemical process of digestion is initiated in the oral cavity by the hydrolytic action of the carbohydrate splitting enzyme, the salivary amylase.

Screenshot (80)

  • About 30 per cent of starch is hydrolysed here by this enzyme (optimum pH 6.8) into a disaccharide – maltose.
  • Lysozyme present in saliva acts as an antibacterial agent that prevents infections.
  • The mucosa of stomach has gastric glands. Gastric glands have three major types of cells namely –
    • mucus neck cells which secrete mucus;
    • peptic or chief cells which secrete the proenzyme pepsinogen; and
    • parietal or oxyntic cells which secrete HCl and intrinsic factor (factor essential for absorption of vitamin B12).
  • The stomach stores the food for 4-5 hours.
  • The food mixes thoroughly with the acidic gastric juice of the stomach by the churning movements of its muscular wall and is called the CHYME.
  • The proenzyme pepsinogen, on exposure to hydrochloric acid gets converted into the active enzyme pepsin, the proteolytic enzyme of the stomach.
  • Pepsin converts proteins into proteoses and peptones (peptides).
  • The mucus and bicarbonates present in the gastric juice play an important role in lubrication and protection of the mucosal epithelium from excoriation by the highly concentrated hydrochloric acid. HCl provides the acidic pH (pH 1.8) optimal for pepsins.
  • Rennin is a proteolytic enzyme found in gastric juice of infants which helps in the digestion of milk proteins. Small amounts of lipases are also secreted by gastric glands.
  • Various types of movements are generated by the muscularis layer of the small intestine.
  • These movements help in a thorough mixing up of the food with various secretions in the intestine and thereby facilitate digestion.
  • The bile, pancreatic juice and the intestinal juice are the secretions released into the small intestine.
  • Pancreatic juice and bile are released through the hepato-pancreatic duct.
  • The pancreatic juice contains inactive enzymes – trypsinogen, chymotrypsinogen, procarboxypeptidases, amylases, lipases and nucleases.
  • Trypsinogen is activated by an enzyme, enterokinase, secreted by the intestinal mucosa into active trypsin, which in turn activates the other enzymes in the pancreatic juice.
  • The bile released into the duodenum contains bile pigments (bilirubin and bili-verdin), bile salts, cholesterol and phospholipids but no enzymes.
  • Bile helps in emulsification of fats, i.e., breaking down of the fats into very small micelles. Bile also activates lipases.
  • The intestinal mucosal epithelium has goblet cells which secrete mucus.
  • The secretions of the brush border cells of the mucosa alongwith the secretions of the goblet cells constitute the intestinal juice or succus entericus.
  • This juice contains a variety of enzymes like disaccharidases (e.g., maltase), dipeptidases, lipases, nucleosidases, etc.
  • The mucus alongwith the bicarbonates from the pancreas protects the intestinal mucosa from acid as well as provide an alkaline medium (pH 7.8) for enzymatic activities.
  • Sub-mucosal glands (Brunner’s glands) also help in this.
  • Proteins, proteoses and peptones (partially hydrolysed proteins) in the chyme reaching the intestine are acted upon by the proteolytic enzymes of pancreatic juice as given below:

Screenshot (81)

  • Carbohydrates in the chyme are hydrolysed by pancreatic amylase into disaccharides.

Screenshot (81) - Copy

  • Fats are broken down by lipases with the help of bile into di-and monoglycerides.

Screenshot (83) - Copy

  • Nucleases in the pancreatic juice acts on nucleic acids to form nucleotides and nucleosides

Screenshot (82)

  • The enzymes in the succus entericus act on the end products of the above reactions to form the respective simple absorbable forms. These final steps in digestion occur very close to the mucosal epithelial cells of the intestine.

Screenshot (82) - Copy

  • The breakdown of biomacromolecules mentioned above occurs in the duodenum region of the small intestine.
  • The simple substances thus formed are absorbed in the jejunum and ileum regions of the small intestine.
  • The undigested and unabsorbed substances are passed on to the large intestine.
  • No significant digestive activity occurs in the large intestine. The functions of large intestine are:
    • absorption of some water, minerals and certain drugs;
    • secretion of mucus which helps in adhering the waste (undigested) particles together and lubricating it for an easy passage.
  • The undigested, unabsorbed substances called faeces enters into the caecum of the large intestine through ileo-caecal valve, which prevents the back flow of the faecal matter.
  • It is temporarily stored in the rectum till defaecation.
  • The activities of the gastro-intestinal tract are under neural and hormonal control for proper coordination of different parts.
  • The sight, smell and/or the presence of food in the oral cavity can stimulate the secretion of saliva.
  • Gastric and intestinal secretions are also, similarly, stimulated by neural signals.
  • The muscular activities of different parts of the alimentary canal can also be moderated by neural mechanisms, both local and through CNS.
  • Hormonal control of the secretion of digestive juices is carried out by the local hormones produced by the gastric and intestinal mucosa.

ABSORPTION OF DIGESTED PRODUCTS

  • Absorption is the process by which the end products of digestion pass through the intestinal mucosa into the blood or lymph.
  • It is carried out by passive, active or facilitated transport mechanisms.
  • Small amounts of monosacharides like glucose, amino acids and some of electrolytes like chloride ions are generally absorbed by simple diffusion.
  • The passage of these substances into the blood depends upon the concentration gradients. However, some of the substances like fructose and some amino acids are absorbed with the help of the carrier ions like Na+. This mechanism is called the facilitated transport.
  • Transport of water depends upon the osmotic gradient.
  • Active transport occurs against the concentration gradient and hence requires energy.
  • Various nutrients like amino acids, monosacharides like glucose, electrolytes like Na+ are absorbed into the blood by this mechanism.
  • Fatty acids and glycerol being insoluble, cannot be absorbed into the blood.
  • They are first incorporated into small droplets called micelles which move into the intestinal mucosa.
  • They are re-formed into very small protein coated fat globules called the chylomicrons which are transported into the lymph vessels (lacteals) in the villi.
  • These lymph vessels ultimately release the absorbed substances into the blood stream.
  • Absorption of substances takes place in different parts of the alimentary canal, like mouth, stomach, small intestine and large intestine.
  • However, maximum absorption occurs in the small intestine.

Table : The Summary of Absorption in Different Parts of Digestive System

Mouth Stomach Small Intestine Large Intestine
Certain drugs coming in contact with the mucosa of mouth and lower side of the tongue are absorbed into the blood capillaries lining them. Absorption of water, simple sugars, and alcohol etc. takes place. Principal organ for absorption of nutrients. The digestion is completed here and the final products of digestion such as glucose, fructose, fatty acids, glycerol and amino acids are absorbed through the mucosa into the blood stream and lymph. Absorption of water, some minerals and drugs takes place.
  • The absorbed substances finally reach the tissues which utilise them for their activities. This process is called assimilation.
  • The digestive wastes, solidified into coherent faeces in the rectum initiate a neural reflex causing an urge or desire for its removal. The egestion of faeces to the outside through the anal opening (defaecation) is a voluntary process and is carried out by a mass peristaltic movement.

DISORDERS OF DIGESTIVE SYSTEM

  • The inflammation of the intestinal tract is the most common ailment due to bacterial or viral infections. The infections are also caused by the parasites of the intestine like tape worm, round worm, thread worm, hook worm, pin worm, etc.
  • Jaundice: The liver is affected, skin and eyes turn yellow due to the deposit of bile pigments.
  • Vomiting: It is the ejection of stomach contents through the mouth. This reflex action is controlled by the vomit centre in the medulla. A feeling of nausea precedes vomiting.
  • Diarrhoea: The abnormal frequency of bowel movement and increased liquidity of the faecal discharge is known as diarrhoea. It reduces the absorption of food.
  • Constipation: In constipation, the faeces are retained within the rectum as the bowel movements occur irregularly.
  • Indigestion: In this condition, the food is not properly digested leading to a feeling of fullness. The causes of indigestion are inadequate enzyme secretion, anxiety, food poisoning, over eating, and spicy food.

 

PRINTABLE Pdf file of chapter notes are available…please click on the link…

CHAPTER 16 – DIGESTION AND ABSORPTION

CHAPTER 15 – PLANT GROWTH AND DEVELOPMENT

CHAPTER 15

PLANT GROWTH AND DEVELOPMENT

  • Trees continue to increase in height or girth over a period of time.
  • However, the leaves, flowers and fruits of the same tree not only have limited dimensions but also appear and fall periodically and sometime repeatedly.
  • Development is the sum of two processes: growth and differentiation.

Screenshot (63)

GROWTH

  • Growth is regarded as one of the most fundamental and conspicuous characteristics of a living being.
  • Growth can be defined as an irreversible permanent increase in size of an organ or its parts or even of an individual cell.
  • Generally, growth is accompanied by metabolic processes (both anabolic and catabolic), that occur at the expense of energy. Therefore, expansion of a leaf is growth while swelling of piece of wood when placed in water is not.

Plant Growth Generally is Indeterminate

  • Plant growth is unique because plants retain the capacity for unlimited growth throughout their life.
  • This ability of the plants is due to the presence of meristems at certain locations in their body.
  • The cells of such meristems have the capacity to divide and self-perpetuate.
  • The product, however, soon loses the capacity to divide and such cells make up the plant body.
  • This form of growth wherein new cells are always being added to the plant body by the activity of the meristem is called the open form of growth.
  • The root apical meristem and the shoot apical meristem are responsible for the primary growth of the plants and principally contribute to the elongation of the plants along their axis.
  • In dicotyledonous plants and gymnosperms, the lateral meristems, vascular cambium and cork-cambium appear later in life and cause the increase in the girth of the organs in which they are active. This is known as secondary growth of the plant.

Screenshot (64)

Growth is Measurable

  • Growth, at a cellular level, is principally a consequence of increase in the amount of protoplasm. Since increase in protoplasm is difficult to measure directly, one generally measures some quantity which is more or less proportional to it.
  • Growth is, therefore, measured by a variety of parameters some of which are: increase in fresh weight, dry weight, length, area, volume and cell number.

Phases of Growth

  • The period of growth is generally divided into three phases, namely, meristematic, elongation and maturation.
  • The constantly dividing cells, both at the root apex and the shoot apex, represent the meristematic phase of growth. The cells in this region are rich in protoplasm, possess large conspicuous nuclei. Their cell walls are primary in nature, thin and cellulosic with abundant plasmodesmatal connections.
  • The cells proximal (just next, away from the tip) to the meristematic zone represent the phase of elongation. Increased vacuolation, cell enlargement and new cell wall deposition are the characteristics of the cells in this phase.
  • Further away from the apex, i.e., more proximal to the phase of elongation, lies the portion of axis which is undergoing the phase of maturation. The cells of this zone, attain their maximal size in terms of wall thickening and protoplasmic modifications.

Screenshot (65)

Growth Rates

  • The increased growth per unit time is termed as growth rate.
  • Thus, rate of growth can be expressed mathematically.
  • The growth rate shows an increase that may be arithmetic or geometrical.
  • In arithmetic growth, following mitotic cell division, only one daughter cell continues to divide while the other differentiates and matures. The simplest expression of arithmetic growth is exemplified by a root elongating at a constant rate.
  • On plotting the length of the organ against time, a linear curve is obtained. Mathematically, it is expressed as  Lt = Lo + rt

Lt = length at time ‘t’

L0 = length at time ‘zero’

r = growth rate / elongation per unit time.

  • In geometrical growth, the initial growth is slow (lag phase), and it increases rapidly thereafter – at an exponential rate (log or exponential phase). Here, both the progeny cells following mitotic cell division retain the ability to divide and continue to do so. However, with limited nutrient supply, the growth slows down leading to a stationary phase.
  • If we plot the parameter of growth against time, we get a typical sigmoid or S-curve. A sigmoid curve is a characteristic of living organism growing in a natural environment. It is typical for all cells, tissues and organs of a plant.
  • The exponential growth can be expressed as

Wt = Woert

Wt = final size (weight, height, number etc.)

Wo = initial size at the beginning of the period

r = growth rate

t = time of growth

e = base of natural logarithms.

Here, r is the relative growth rate and is also the measure of the ability of the plant to produce new plant material, referred to as efficiency index. Hence, the final size of W1 depends on the initial size, Wo.

Screenshot (66)

Screenshot (67)

  • Quantitative comparisons between the growth of living system can also be made in two ways :

(i) measurement and the comparison of total growth per unit time is called the absolute growth rate.

(ii) The growth of the given system per unit time expressed on a common basis, e.g., per unit initial parameter is called the relative growth rate.

Screenshot (68)

Conditions for Growth

  • water, oxygen and nutrients are very essential elements for growth.
  • The plant cells grow in size by cell enlargement which in turn requires water. Turgidity of cells helps in extension growth. Thus, plant growth and further development is intimately linked to the water status of the plant. Water also provides the medium for enzymatic activities needed for growth.
  • oxygen helps in releasing metabolic energy essential for growth activities.
  • Nutrients (macro and micro essential elements) are required by plants for the synthesis of protoplasm and act as source of energy.
  • In addition, every plant organism has an optimum temperature range best suited for its growth. Any deviation from this range could be detrimental to its survival.
  • Environmental signals such as light and gravity also affect certain phases/stages of growth.

Differentiation, Dedifferentiation and Redifferentiation

  • The cells derived from root apical and shoot-apical meristems and cambium differentiate and mature to perform specific functions. This act leading to maturation is termed as
  • During differentiation, cells undergo few to major structural changes both in their cell walls and protoplasm. For example, to form a tracheary element, the cells would lose their protoplasm. They also develops a very strong, elastic, lignocellulosic secondary cell walls, to carry water to long distances even under extreme tension.
  • differentiated cells, that have lost the capacity to divide can regain the capacity of division under certain conditions. This phenomenon is termed as For example, formation of meristems – interfascicular cambium and cork cambium from fully differentiated parenchyma cells.

While doing so, such meristems/tissues are able to divide and produce cells that once again lose the capacity to divide but mature to perform specific functions, i.e., get redifferentiated.

  • Growth in plants is open, i.e., it can be indeterminate or determinate. Now, we may say that even differentiation in plants is open, because cells/tissues arising out of the same meristem have different structures at maturity.
  • The final structure at maturity of a cell/tissue is also determined by the location of the cell within. For example, cells positioned away from root apical meristems differentiate as root-cap cells, while those pushed to the periphery mature as epidermis.

DEVELOPMENT

  • Development is a term that includes all changes that an organism goes through during its life cycle from germination of the seed to senescence.

Screenshot (69)

  • Plants follow different pathways in response to environment or phases of life to form different kinds of structures. This ability is called plasticity, g., heterophylly in cotton, coriander and larkspur.

In such plants, the leaves of the juvenile plant are different in shape from those in mature plants.

  • On the other hand, difference in shapes of leaves produced in air and those produced in water in buttercup also represent the heterophyllous development due to environment. This phenomenon of heterophylly is an example of plasticity.

Screenshot (70)

  • Thus, growth, differentiation and development are very closely related events in the life of a plant. Broadly, development is considered as the sum of growth and differentiation.
  • Development in plants (i.e., both growth and differentiation) is under the control of intrinsic and extrinsic factors. The former includes both intracellular (genetic) or intercellular factors (chemicals such as plant growth regulators) while the latter includes light, temperature, water, oxygen, nutrition, etc.

PLANT GROWTH REGULATORS

Characteristics

  • The plant growth regulators (PGRs) are small, simple molecules of diverse chemical composition.
  • They could be
    • indole compounds (indole-3-acetic acid, IAA);
    • adenine derivatives (N6-furfurylamino purine, kinetin),
    • derivatives of carotenoids (abscisic acid, ABA);
    • terpenes (gibberellic acid, GA3) or
    • gases (ethylene, C2H4).
  • Plant growth regulators are variously described as plant growth substances, plant hormones or phytohormones in literature.
  • The PGRs can be broadly divided into two groups based on their functions in a living plant body.
  • One group of PGRs are involved in growth promoting activities, such as cell division, cell enlargement, pattern formation, tropic growth, flowering, fruiting and seed formation. These are also called plant growth promoters, e.g., auxins, gibberellins and cytokinins.
  • The PGRs of the other group play an important role in plant responses to wounds and stresses of biotic and abiotic origin. They are also involved in various growth inhibiting activities such as dormancy and abscission. The PGR abscisic acid belongs to this group.
  • The gaseous PGR, ethylene, could fit either of the groups, but it is largely an inhibitor of growth activities.

The Discovery of Plant Growth Regulators

  • Interestingly, the discovery of each of the five major groups of PGRs have been accidental.
  • Charles Darwin and his son Francis Darwin – observed phototropism in Canary grass.
  • W. Went – isolated Auxin from tips of coleoptiles of oat seedlings.
  • Kurosawa – reported the appearance of symptoms of the ‘Bakane disease’ in uninfected rice seedlings when they were treated with sterile filtrates of the fungus Gibberalla fujikuroi. The active substances were later identified as gibberellic acid.
  • Skoog and co-workers – observed callus proliferation in tobacco callus in presence of extracts of vascular tissues/yeast extract/coconut milk or DNA in addition to Auxins.
  • Skoog and Miller – identified and crystallised the cytokinesis promoting active substance and termed it kinetin.
  • Cousins – confirmed the release of a volatile substance from ripened oranges that hastened the ripening of stored unripened bananas. Later this volatile substance was identified as ethylene, a gaseous PGR
  • During mid-1960s, three independent researches reported the purification and chemical characterisation of three different kinds of inhibitors: inhibitor-B, abscission II and dormin. Later all the three were proved to be chemically identical. It was named abscisic acid (ABA).

Screenshot (71)

PHYSIOLOGICAL EFFECTS OF PLANT GROWTH REGULATORS

AUXINS

  • Auxins (from Greek ‘auxein’ : to grow) was first isolated from human urine.
  • The term ‘auxin’ is applied to the indole-3-acetic acid (IAA), and to other natural and synthetic compounds having certain growth regulating properties.
  • They are generally produced by the growing apices of the stems and roots, from where they migrate to the regions of their action.
  • Auxins like IAA and indole butyric acid (IBA) have been isolated from plants.
  • NAA (naphthalene acetic acid) and 2, 4-D (2, 4-dichlorophenoxyacetic) are synthetic auxins.
  • All these auxins have been used extensively in agricultural and horticultural practices.
  • They help to initiate rooting in stem cuttings, an application widely used for plant propagation.
  • Auxins promote flowering e.g. in pineapples.
  • They help to prevent fruit and leaf drop at early stages but promote the abscission of older mature leaves and fruits.
  • In most higher plants, the growing apical bud inhibits the growth of the lateral (axillary) buds, a phenomenon called apical dominance. Removal of shoot tips (decapitation) usually results in the growth of lateral buds. It is widely applied in tea plantations, hedge-making.
  • Auxins also induce parthenocarpy, e.g., in tomatoes.
  • They are widely used as herbicides. 2, 4-D, widely used to kill dicotyledonous weeds, does not affect mature monocotyledonous plants.
  • It is used to prepare weed-free lawns by gardeners.
  • Auxin also controls xylem differentiation and helps in cell division.

GIBBERELLINS

  • There are more than 100 gibberellins reported from widely different organisms such as fungi and higher plants. They are denoted as GA1, GA2, GA3 and so on.
  • However, Gibberellic acid (GA3) was one of the first gibberellins to be discovered and remains the most intensively studied form.
  • All GAs are acidic.
  • They have ability to cause an increase in length of axis. It is used to increase the length of grapes stalks.
  • Gibberellins, cause fruits like apple to elongate and improve its shape.
  • They also delay senescence. Thus, the fruits can be left on the tree longer so as to extend the market period.
  • GA3 is used to speed up the malting process in brewing industry.
  • Sugarcane stores carbohydrate as sugar in their stems. Spraying sugarcane crop with gibberellins increases the length of the stem, thus increasing the yield by as much as 20 tonnes per acre.
  • Spraying juvenile conifers with GAs hastens the maturity period, thus leading to early seed production.
  • Gibberellins also promotes bolting (internode elongation just prior to flowering) in beet, cabbages and many plants with rosette habit.

CYTOKININS

  • Cytokinins have specific effects on cytokinesis, and were discovered as kinetin (a modified form of adenine, a purine) from the autoclaved herring sperm DNA.
  • Kinetin does not occur naturally in plants.
  • Search for natural substances with cytokinin-like activities led to the isolation of zeatin from corn-kernels and coconut milk.
  • Since the discovery of zeatin, several naturally occurring cytokinins, and some synthetic compounds with cell division promoting activity, have been identified.
  • Natural cytokinins are synthesised in regions where rapid cell division occurs, for example, root apices, developing shoot buds, young fruits etc.
  • It helps to produce new leaves, chloroplasts in leaves, lateral shoot growth and adventitious shoot formation.
  • Cytokinins help overcome the apical dominance.
  • They promote nutrient mobilisation which helps in the delay of leaf senescence.

ETHYLENE

  • Ethylene is a simple gaseous PGR.
  • It is synthesised in large amounts by tissues undergoing senescence and ripening fruits.
  • Influences of ethylene on plants include horizontal growth of seedlings, swelling of the axis and apical hook formation in dicot seedlings.
  • Ethylene promotes senescence and abscission of plant organs especially of leaves and flowers.
  • Ethylene is highly effective in fruit ripening.
  • It enhances the respiration rate during ripening of the fruits. This rise in rate of respiration is called respiratory climactic.
  • Ethylene breaks seed and bud dormancy, initiates germination in peanut seeds, sprouting of potato tubers.
  • Ethylene promotes rapid internode/petiole elongation in deep water rice plants.
  • It helps leaves/ upper parts of the shoot to remain above water.
  • Ethylene also promotes root growth and root hair formation, thus helping the plants to increase their absorption surface.
  • Ethylene is used to initiate flowering and for synchronising fruit-set in pineapples.
  • It also induces flowering in mango.
  • Since ethylene regulates so many physiological processes, it is one of the most widely used PGR in agriculture.
  • The most widely used compound as source of ethylene is ethephon.
  • Ethephon in an aqueous solution is readily absorbed and transported within the plant and releases ethylene slowly.
  • Ethephon hastens fruit ripening in tomatoes and apples and accelerates abscission in flowers and fruits (thinning of cotton, cherry, walnut).
  • It promotes female flowers in cucumbers thereby increasing the yield.

ABSCISIC ACID

  • Abscisic acid (ABA) was discovered for its role in regulating abscission and dormancy.
  • It acts as a general plant growth inhibitor and an inhibitor of plant metabolism.
  • ABA inhibits seed germination.
  • ABA stimulates the closure of stomata in the epidermis and increases the tolerance of plants to various kinds of stresses. Therefore, it is also called the stress hormone.
  • ABA plays an important role in seed development, maturation and dormancy.
  • By inducing dormancy, ABA helps seeds to withstand desiccation and other factors unfavourable for growth.
  • In most situations, ABA acts as an antagonist to GAs.

 

  • There are a number of events in the life of a plant where more than one PGR interact to affect that event, e.g., dormancy in seeds/ buds, abscission, senescence, apical dominance, etc.
  • The role of PGR is of only one kind of intrinsic control. Along with genomic control and extrinsic factors, they play an important role in plant growth and development.
  • Many of the extrinsic factors such as temperature and light, control plant growth and development via PGR.
  • Some of such events could be: vernalisation, flowering, dormancy, seed germination, plant movements, etc.

PHOTOPERIODISM

  • Some plants require a periodic exposure to light to induce flowering. Such plants are able to measure the duration of exposure to light.
  • For example, some plants require the exposure to light for a period exceding a well defined critical duration, while others must be exposed to light for a period less than this critical duration before the flowering is initiated in them. The former group of plants are called long day plants while the latter ones are termed short day plants.
  • The critical duration is different for different plants.
  • There are many plants, however, where there is no such correlation between exposure to light duration and inducation of flowering response; such plants are called day-neutral plants.
  • It is now also known that not only the duration of light period but that the duration of dark period is also of equal importance.
  • Hence, it can be said that flowering in certain plants depends not only on a combination of light and dark exposures but also their relative durations. This response of plants to periods of day/night is termed
  • It is also interesting to note that while shoot apices modify themselves into flowering apices prior to flowering, they (i.e., shoot apices of plants) by themselves cannot percieve photoperiods.
  • The site of perception of light/dark duration are the leaves.
  • It has been hypothesised that there is a hormonal substance(s) that is responsible for flowering.
  • This hormonal substance migrates from leaves to shoot apices for inducing flowering only when the plants are exposed to the necessary inductive photoperiod.

 

VERNALISATION

  • There are plants for which flowering is either quantitatively or qualitatively dependent on exposure to low temperature. This phenomenon is termed
  • It prevents precocious reproductive development late in the growing season, and enables the plant to have sufficient time to reach maturity.
  • Vernalisation refers specially to the promotion of flowering by a period of low temperature.
  • Some important food plants, wheat, barley, rye have two kinds of varieties: winter and spring varieties.
  • The ‘spring’ variety are normally planted in the spring and come to flower and produce grain before the end of the growing season.
  • Winter varieties, however, if planted in spring would normally fail to flower or produce mature grain within a span of a flowering season. Hence, they are planted in autumn.
  • They germinate, and over winter come out as small seedlings, resume growth in the spring, and are harvested usually around mid-summer.
  • Another example of vernalisation is seen in biennial plants.
  • Biennials are monocarpic plants that normally flower and die in the second season.
  • Sugerbeet, cabbages, carrots are some of the common biennials.
  • Subjecting the growing of a biennial plant to a cold treatment stimulates a subsequent photoperiodic flowering response.

 

TO DOWNLOAD PRINTABLE PDF FILE, CLICK ON FOLLOWING LINK…

CHAPTER 15 – PLANT GROWTH AND DEVELOPMENT

CHAPTER 14 – RESPIRATION IN PLANTS

CHAPTER 14

RESPIRATION IN PLANTS

  • All the energy required for ‘life’ processes is obtained by oxidation of some macromolecules that we call ‘food’.
  • Only green plants and cyanobacteria can prepare their own food; by the process of photosynthesis they trap light energy and convert it into chemical energy that is stored in the bonds of carbohydrates like glucose, sucrose and starch.
  • In green plants too, not all cells, tissues and organs photosynthesise; only cells containing chloroplasts, that are most often located in the superficial layers, carry out photosynthesis.
  • Hence, even in green plants all other organs, tissues and cells that are non-green, need food for oxidation. Hence, food has to be translocated to all non- green parts.
  • Animals are heterotrophic, i.e., they obtain food from plants directly (herbivores) or indirectly (carnivores). Saprophytes like fungi are dependent on dead and decaying matter.
  • Ultimately all the food that is respired for life processes comes from photosynthesis.
  • Photosynthesis, of course, takes place within the chloroplasts (in the eukaryotes), whereas the breakdown of complex molecules to yield energy takes place in the cytoplasm and in the mitochondria (also only in eukaryotes).
  • The breaking of the C-C bonds of complex compounds through oxidation within the cells, leading to release of considerable amount of energy is called
  • The compounds that are oxidised during this process are known as respiratory substrates. Usually carbohydrates are oxidised to release energy, but proteins, fats and even organic acids can be used as respiratory substances in some plants, under certain conditions.
  • During oxidation within a cell, all the energy contained in respiratory substrates is not released free into the cell, or in a single step. It is released in a series of slow step-wise reactions controlled by enzymes, and it is trapped as chemical energy in the form of ATP.
  • The energy released by oxidation in respiration is not used directly but is used to synthesise ATP, which is broken down whenever and wherever energy needs to be utilised. Hence, ATP acts as the energy currency of the cell.
  • This energy trapped in ATP is utilised in various energy-requiring processes of the organisms, and the carbon skeleton produced during respiration is used as precursors for biosynthesis of other molecules in the cell.
  • Plants require O2 for respiration to occur and they also give out CO2. Hence, plants have systems in place that ensure the availability of O2. Plants, unlike animals, have no specialised organs for gaseous exchange but they have stomata and lenticels for this purpose.
  • Reasons why plants can get along without respiratory organs.
    • First, each plant part takes care of its own gas-exchange needs. There is very little transport of gases from one plant part to another.
    • Second, plants do not present great demands for gas exchange. Roots, stems and leaves respire at rates far lower than animals do. Only during photosynthesis are large volumes of gases exchanged and, each leaf is well adapted to take care of its own needs during these periods. When cells photosynthesise, availability of O2 is not a problem in these cells since O2 is released within the cell.
    • Third, the distance that gases must diffuse even in large, bulky plants is not great. Each living cell in a plant is located quite close to the surface of the plant. This is true for leaves, in stems, the ‘living’ cells are organised in thin layers inside and beneath the bark. They also have openings called lenticels. The cells in the interior are dead and provide only mechanical support. Thus, most cells of a plant have at least a part of their surface in contact with air. This is also facilitated by the loose packing of parenchyma cells in leaves, stems and roots, which provide an interconnected network of air spaces.
  • The complete combustion of glucose, which produces CO2 and H2O as end products, yields energy most of which is given out as heat.

  C6H12O6 + 6O2 ————– > 6CO2 + 6H2O + Energy

  • If this energy is to be useful to the cell, it should be able to utilise it to synthesise other molecules that the cell requires.
  • Plant cell uses is to catabolise the glucose molecule in such a way that not all the liberated energy goes out as heat. The key is to oxidise glucose not in one step but in several small steps enabling some steps to be just large enough such that the energy released can be coupled to ATP synthesis.
  • During the process of respiration, oxygen is utilised, and carbon dioxide, water and energy are released as products. The combustion reaction requires oxygen.
  • But some cells live where oxygen may or may not be available.
  • All living organisms retain the enzymatic machinery to partially oxidise glucose without the help of oxygen. This breakdown of glucose to pyruvic acid is called

GLYCOLYSIS

  • The term glycolysis has originated from the Greek words, glycos for sugar, and lysis for splitting.
  • The scheme of glycolysis was given by Gustav Embden, Otto Meyerhof, and J. Parnas, and is often referred to as the EMP pathway.
  • In anaerobic organisms, it is the only process in respiration.
  • Glycolysis occurs in the cytoplasm of the cell and is present in all living organisms.
  • In this process, glucose undergoes partial oxidation to form two molecules of pyruvic acid.
  • In plants, this glucose is derived from sucrose, which is the end product of photosynthesis, or from storage
  • Sucrose is converted into glucose and fructose by the enzyme, invertase, and these two monosaccharides readily enter the glycolytic pathway.
  • Glucose and fructose are phosphorylated to give rise to glucose-6- phosphate by the activity of the enzyme hexokinase.
  • This phosphorylated form of glucose then isomerises to produce fructose-6-phosphate. subsequent steps of metabolism of glucose and fructose are same.
  • In glycolysis, a chain of ten reactions, under the control of different enzymes, takes place to produce pyruvate from glucose.
  • ATP is utilised at two steps: first in the conversion of glucose into glucose 6-phosphate and second in the conversion of fructose 6-phosphate to fructose 1, 6-diphosphate.
  • The fructose 1, 6-diphosphate is split into dihydroxyacetone phosphate and 3-phosphoglyceraldehyde (PGAL).
  • We find that there is one step where NADH + H+ is formed from NAD+; this is when 3-phosphoglyceraldehyde (PGAL) is converted to 1, 3-bisphosphoglycerate (DPGA). Two redox-equivalents are removed (in the form of two hydrogen atoms) from PGAL and transferred to a molecule of NAD+.
  • PGAL is oxidised and with inorganic phosphate to get converted into DPGA. The conversion of DPGA to 3-phosphoglyceric acid (PGA), is also an energy yielding process; this energy is trapped by the formation of ATP.
  • Another ATP is synthesised during the conversion of PEP to pyruvic acid.
  • Total 4 ATP molecules are directly synthesised in this pathway from one glucose molecule.
  • Pyruvic acid is then the key product of glycolysis.
  • The metabolic fate of pyruvate depends on the cellular need. There are three major ways in which different cells handle pyruvic acid produced by glycolysis. These are lactic acid fermentation, alcoholic fermentation and aerobic respiration.
  • Fermentation takes place under anaerobic conditions in many prokaryotes and unicellular eukaryotes.
  • For the complete oxidation of glucose to CO2 and H2O, however, organisms adopt Krebs’ cycle which is also called as aerobic respiration. This requires O2

Screenshot (61)

FERMENTATION

  • In fermentation, the incomplete oxidation of glucose is achieved under anaerobic conditions by sets of reactions where pyruvic acid is converted to CO2 and ethanol.
  • The enzymes, pyruvic acid decarboxylase and alcohol dehydrogenase catalyse these reactions.
  • Other organisms like some bacteria produce lactic acid from pyruvic acid.
  • In animal cells also, like muscles during exercise, when oxygen is inadequate for cellular respiration pyruvic acid is reduced to lactic acid by lactate dehydrogenase.
  • The reducing agent is NADH+H+ which is reoxidised to NAD+ in both the processes.
  • In both lactic acid and alcohol fermentation not much energy is released; less than seven per cent of the energy in glucose is released and not all of it is trapped as high energy bonds of ATP. Also, the processes are hazardous – either acid or alcohol is produced.
  • Yeasts poison themselves to death when the concentration of alcohol reaches about 13 per cent.
  • The maximum concentration of alcohol in beverages that are naturally fermented is 13%.
  • Alcoholic beverages of alcohol content greater than this concentration are obtained by distillation.

Screenshot (52)

AEROBIC RESPIRATION

  • For aerobic respiration to take place within the mitochondria, the final product of glycolysis, pyruvate is transported from the cytoplasm into the mitochondria.
  • The crucial events in aerobic respiration are:
    • The complete oxidation of pyruvate by the stepwise removal of all the hydrogen atoms, leaving three molecules of CO2
    • The passing on of the electrons removed as part of the hydrogen atoms to molecular O2 with simultaneous synthesis of ATP.
  • The first process takes place in the matrix of the mitochondria while the second process is located on the inner membrane of the mitochondria.
  • Pyruvate, which is formed by the glycolytic catabolism of carbohydrates in the cytosol, after it enters mitochondrial matrix undergoes oxidative decarboxylation by a complex set of reactions catalysed by pyruvic dehydrogenase.
  • The reactions catalysed by pyruvic dehydrogenase require the participation of several coenzymes, including NAD+ and Coenzyme A.

1

  • During this process, two molecules of NADH are produced from the metabolism of two molecules of pyruvic acid (produced from one glucose molecule during glycolysis).
  • The acetyl CoA then enters a cyclic pathway, tricarboxylic acid cycle, more commonly called as Krebs’ cycle after the scientist Hans Krebs who first elucidated it.

TRICARBOXYLIC ACID CYCLE

  • The TCA cycle starts with the condensation of acetyl group with oxaloacetic acid (OAA) and water to yield citric acid. The reaction is catalysed by the enzyme citrate synthase and a molecule of CoA is released.
  • Citrate is then isomerised to isocitrate.
  • It is followed by two successive steps of decarboxylation, leading to the formation of a-ketoglutaric acid and then succinyl-CoA.
  • In the remaining steps of citric acid cycle, succinyl-CoA is oxidised to OAA allowing the cycle to continue.
  • During the conversion of succinyl-CoA to succinic acid a molecule of GTP is synthesised. This is a substrate level phosphorylation.
  • In a coupled reaction GTP is converted to GDP with the simultaneous synthesis of ATP from ADP.
  • Also there are three points in the cycle where NAD+ is reduced to NADH + H+ and one point where FAD+ is reduced to FADH2.
  • The continued oxidation of acetic acid via the TCA cycle requires the continued replenishment of oxaloacetic acid, the first member of the cycle.
  • In addition it also requires regeneration of NAD+ and FAD+ from NADH and FADH2 The summary equation for this phase of respiration may be written as follows:

Screenshot (54)

Screenshot (53)

ELECTRON TRANSPORT SYSTEM (ETS) AND OXIDATIVE PHOSPHORYLATION

  • The following steps in the respiratory process are to release and utilise the energy stored in NADH+H+ and FADH2.
  • This is accomplished when they are oxidised through the electron transport system and the electrons are passed on to O2 resulting in the formation of H2
  • The metabolic pathway through which the electron passes from one carrier to another, is called the electron transport system (ETS) and it is present in the inner mitochondrial membrane.
  • Electrons from NADH produced in the mitochondrial matrix during citric acid cycle are oxidised by an NADH dehydrogenase (complex I), and electrons are then transferred to ubiquinone located within the inner membrane.
  • Ubiquinone also receives reducing equivalents via FADH2 (complex II) that is generated during oxidation of succinate in the citric acid cycle.
  • The reduced ubiquinone (ubiquinol) is then oxidised with the transfer of electrons to cytochrome c via cytochrome bc1 complex (complex III).
  • Cytochrome c is a small protein attached to the outer surface of the inner membrane and acts as a mobile carrier for transfer of electrons between complex III and IV.
  • Complex IV refers to cytochrome c oxidase complex containing cytochromes a and a3, and two copper centres.

Screenshot (55)

  • When the electrons pass from one carrier to another via complex I to IV in the electron transport chain, they are coupled to ATP synthase (complex V) for the production of ATP from ADP and inorganic phosphate.
  • The number of ATP molecules synthesised depends on the nature of the electron donor. Oxidation of one molecule of NADH gives rise to 3 molecules of ATP, while that of one molecule of FADH2 produces 2 molecules of ATP.
  • Although the aerobic process of respiration takes place only in the presence of oxygen, the role of oxygen is limited to the terminal stage of the process. Yet, the presence of oxygen is vital, since it drives thewhole process by removing hydrogen from the system. Oxygen acts as the final hydrogen acceptor.
  • Unlike photophosphorylation where it is the light energy that is utilised for the production of proton gradient required for phosphorylation, in respiration it is the energy of oxidation-reduction utilised for the same process. It is for this reason that the process is called oxidative phosphorylation.
  • The energy released during the electron transport system is utilised in synthesising ATP with the help of ATP synthase (complex V).
  • This complex consists of two major components, F1 and F0. The F1 headpiece is a peripheral membrane protein complex and contains the site for synthesis of ATP from ADP and inorganic phosphate. F0 is an integral membrane protein complex that forms the channel through which protons cross the inner membrane. The passage of protons through the channel is coupled to the catalytic site of the F: component for the production of ATP. For each ATP produced, 2H+ passes through F0 from the intermembrane space to the matrix down the electrochemical proton gradient.

Screenshot (56)

THE RESPIRATORY BALANCE SHEET

  • The calculations of the net gain of ATP for every glucose molecule oxidised; are in reality only a theoretical exercise as these calculations can be made only on certain assumptions that:
    • There is a sequential, orderly pathway functioning, with one substrate forming the next and with glycolysis, TCA cycle and ETS pathway following one after another.
    • The NADH synthesised in glycolysis is transferred into the mitochondria and undergoes oxidative phosphorylation.
    • None of the intermediates in the pathway are utilised to synthesise any other compound.
    • Only glucose is being respired – no other alternative substrates are entering in the pathway at any of the intermediary stages.

But this kind of assumptions are not really valid in a living system; all pathways work simultaneously and do not take place one after another; substrates enter the pathways and are withdrawn from it as and when necessary; ATP is utilised as and when needed; enzymatic rates are controlled by multiple means.

  • There can be a net gain of 36 ATP molecules during aerobic respiration of one molecule of glucose.
  • Comparisons of fermentation and aerobic respiration:
    • Fermentation accounts for only a partial breakdown of glucose whereas in aerobic respiration it is completely degraded to CO2 and H2
    • In fermentation there is a net gain of only two molecules of ATP for each molecule of glucose degraded to pyruvic acid whereas many more molecules of ATP are generated under aerobic conditions.
    • NADH is oxidised to NAD+ rather slowly in fermentation, however the reaction is very vigorous in case of aerobic respiration.

AMPHIBOLIC PATHWAY

  • Glucose is the favoured substrate for respiration. All carbohydrates are usually first converted into glucose before they are used for respiration.
  • Other substrates can also be respired, as has been mentioned earlier, but then they do not enter the respiratory pathway at the first step.
  • Fats would need to be broken down into glycerol and fatty acids first. If fatty acids were to be respired they would first be degraded to acetyl CoA and enter the pathway. Glycerol would enter the pathway after being converted to PGAL.
  • The proteins would be degraded by proteases and the individual amino acids (after deamination) depending on their structure would enter the pathway at some stage within the Krebs’ cycle or even as pyruvate or acetyl CoA.
  • Since respiration involves breakdown of substrates, the respiratory process has traditionally been considered a catabolic process and the respiratory pathway as a catabolic pathway.

Screenshot (57)

  • The respiratory pathway comes into the picture both during breakdown and synthesis of fatty acids. similarly, during breakdown and synthesis of protein too, respiratory intermediates form the link. Breaking down processes within the living organism is catabolism, and synthesis is anabolism. Because the respiratory pathway is involved in both anabolism and catabolism, it would hence be better to consider the respiratory pathwayas an amphibolic pathway rather than as a catabolic one.

 

RESPIRATORY QUOTIENT

  • The ratio of the volume of CO2 evolved to the volume of O2 consumed in respiration is called the respiratory quotient (RQ) or respiratory ratio.

Screenshot (58)

  • The respiratory quotient depends upon the type of respiratory substrate used during respiration.
  • When carbohydrates are used as substrate and are completelyoxidised, the RQ will be 1, because equal amounts of CO2 and O2 are evolved and consumed, respectively, as shown in the equation below :

Screenshot (59)

  • When fats are used in respiration, the RQ is less than 1. Calculations for a fatty acid, tripalmitin, if used as a substrate is shown:

Screenshot (60)

  • When proteins are respiratory substrates the ratio would be about 0.9.
  • In living organisms respiratory substances are often more than one; pure proteins or fats are never used as respiratory substrates.

 

For Printable PDF file please click on the following link

CHAPTER 14 – RESPIRATION IN PLANTS

CHAPTER 13 – PHOTOSYNTHESIS IN HIGHER PLANTS

CHAPTER 13

PHOTOSYNTHESIS IN HIGHER PLANTS

  • Green plants carry out ‘photosynthesis’, a physico-chemical process by which they use light energy to drive the synthesis of organic compounds.
  • Ultimately, all living forms on earth depend on sunlight for energy.
  • The use of energy from sunlight by plants doing photosynthesis is the basis of life on earth. Photosynthesis is important due to two reasons:

(a) it is the primary source of all food on earth.

(b) It is also responsible for the release of oxygen into the atmosphere by green plants.

  • chlorophyll (green pigment of the leaf), light and CO2 are required for photosynthesis to occur.

 

  • Two leaves experiment for importance of chlorophyll for starch formation – a variegated leaf or a leaf that was partially covered with black paper, and one that was exposed to light. On testing these leaves for starch it was clear that photosynthesis occurred only in the green parts of the leaves in the presence of light.
  • Half-leaf experiment for importance of CO2 for starch formation – a part of a leaf is enclosed in a test tube containing some KOH soaked cotton (which absorbs CO2), while the other half is exposed to air. The setup is then placed in light for some time. On testing for starch later in the two halves of the leaf the exposed part of the leaf tested positive for starch while the portion that was in the tube, tested negative.

 

Early Experiments

Joseph Priestley (1770)               – revealed the essential role of air in the growth of green plants

– discovered oxygen in 1774.

– Bell jar experiment

– hypothesised that Plants restore to the air whatever breathing animals and burning candles remove.

1

 

Jan Ingenhousz (1730-1799)      – showed that sunlight is essential to the photosynthesis.

Julius von Sachs (1854)              – provided evidence for production of glucose when plants grow.

                                                      – Glucose is usually stored as starch.

– showed that the green substance in plants is located in special bodies (later called chloroplasts) within plant cells.

T.W Engelmann (1843 – 1909)   – Used a prism to split light into its spectral components and then illuminated a green alga, Cladophora, placed in a suspension of aerobic bacteria. The bacteria were used to detect the sites of O2 evolution.

observed that the bacteria accumulated mainly in the region of blue and red light of the split spectrum.

described first action spectrum of photosynthesis, which resembles roughly the absorption spectra of chlorophyll a and b.

The empirical equation representing the total process of photosynthesis for oxygen evolving organisms was then understood as:

CO2 + H2O —Light –> [CH2O] + O2

where [CH2O] represented a carbohydrate (e.g., glucose, a six-carbon sugar).

Cornelius van Niel (1897-1985)  – a microbiologist

– studies of purple and green bacteria,

– demonstrated that photosynthesis is essentially a light-dependent   reaction in which hydrogen from a suitable oxidisable compound reduces carbon dioxide to carbohydrates.

2H2A + CO2Light –> 2A + CH2O + H2O

– In green plants H2O is the hydrogen donor and is oxidised to O2.

– When H2S is the hydrogen donor for purple and green sulphur bacteria, the ‘oxidation’ product is sulphur or sulphate and not O2.

– Hence, he inferred that the O2 evolved by the green plant comes from H2O, not from carbon dioxide.

– This was later proved by using radioisotopic techniques.

The correct equation, that would represent the overall process of photosynthesis is therefore:

6CO2 +12H2O —Light –> C6H12O6 + 6H2O + 6O2

 

SITE OF PHOTOSYNTHESIS

  • Photosynthesis occurs in green leaf in the chloroplasts.
  • mesophyll cells in the leaves, have a large number of chloroplasts. Usually the chloroplasts align themselves along the walls of the mesophyll cells, such that they get the optimum quantity of the incident light.
  • Within the chloroplast there is the membranous system consisting of grana, the stroma lamellae, and the fluid stroma. There is a clear division of labour within the chloroplast. The membrane system is responsible for trapping the light energy and also for the synthesis of ATP and NADPH. In stroma, enzymatic reactions incorporate CO2 into the plant leading to the synthesis of sugar, which in turn forms starch.
  • The former set of reactions, since they are directly light driven are called light reactions. The latter are not directly light driven but are dependent on the products of light reactions (ATP and NADPH). Hence, to distinguish the latter they are called, by convention, as dark reactions. However, this should not be construed to mean that they occur in darkness or that they are not light- dependent.

Screenshot (42)  HOW MANY PIGMENTS ARE INVOLVED IN PHOTOSYNTHESIS?

  • A chromatographic separation of the leaf pigments shows that the colour that we see in leaves is not due to a single pigment but due to four pigments:

Chlorophyll a (bright or blue green in the chromatogram),

chlorophyll b (yellow green),

xanthophylls (yellow) and

carotenoids (yellow to yellow-orange).

  • Pigments are substances that have an ability to absorb light, at specific wavelengths.
  • wavelengths at which there is maximum absorption by chlorophyll a, is in the blue and the red regions, also shows higher rate of photosynthesis. Hence, we can conclude that chlorophyll a is the chief pigment associated with photosynthesis.
  • These graphs, together, show that most of the photosynthesis takes place in the blue and red regions of the spectrum; some photosynthesis does take place at the other wavelengths of the visible spectrum.
  • Though chlorophyll is the major pigment responsible for trapping light, other thylakoid pigments like chlorophyll b, xanthophylls and carotenoids, which are called accessory pigments, also absorb light and transfer the energy to chlorophyll a. Indeed, they not only enable a wider range of wavelength of incoming light to be utilised for photosyntesis but also protect chlorophyll a from photo-oxidation.

Screenshot (43)

WHAT IS LIGHT REACTION?

  • Light reactions or the ‘Photochemical’ phase include light absorption, water splitting, oxygen release, and the formation of high-energy chemical intermediates, ATP and NADPH.
  • several complexes are involved in the process.
  • The pigments are organised into two discrete photochemical light harvesting complexes (LHC) within the Photosystem I (PS I) and Photosystem II (PS II). These are named in the sequence of their discovery, and not in the sequence in which they function during the light reaction.
  • The LHC are made up of hundreds of pigment molecules bound to proteins.
  • Each photosystem has all the pigments (except one molecule of chlorophyll a) forming a light harvesting system also called antennae.
  • These pigments help to make photosynthesis more efficient by absorbing different wavelengths of light. The single chlorophyll a molecule forms the reaction centre.
  • The reaction centre is different in both the photosystems.
  • In PS I the reaction centre chlorophyll a has an absorption peak at 700 nm, hence is called P700, while in PS II it has absorption maxima at 680 nm, and is called P680

Screenshot (44) THE ELECTRON TRANSPORT

  • In photosystem II the reaction centre chlorophyll a absorbs 680 nm wavelength of red light causing electrons to become excited and jump into an orbit farther from the atomic nucleus. These electrons are picked up by an electron acceptor which passes them to an electrons transport system consisting of cytochromes.
  • This movement of electrons is downhill, in terms of an oxidation-reduction or redox potential scale.
  • The electrons are not used up as they pass through the electron transport chain, but are passed on to the pigments of photosystem PS I.
  • Simultaneously, electrons in the reaction centre of PS I are also excited when they receive red light of wavelength 700 nm and are transferred to another accepter molecule that has a greater redox potential.
  • These electrons then are moved downhill again, this time to a molecule of energy-rich NADP+.
  • The addition of these electrons reduces NADP+ to NADPH + H+.
  • This whole scheme of transfer of electrons, starting from the PS II, uphill to the accepter, down the electron transport chain to PS I, excitation of electrons,transfer to another accepter, and finally down hill to NADP+ causing it to be reduced to NADPH + H+ is called the Z scheme, due to its characterstic shape. This shape is formed when all the carriers are placed in a sequence on a redox potential scale.

Screenshot (45)  Splitting of Water

  • The electrons that were moved from photosystem II must be replaced.
  • This is achieved by electrons available due to splitting of water.
  • The splitting of water is associated with the PS II; water is split into H+, [O] and electrons.
  • This creates oxygen, one of the net products of photosynthesis.
  • The electrons needed to replace those removed from photosystem I are provided by photosystem II.
  • 2H2O ——-> 4H+ + O2 + 4e
  • water splitting complex is associated with the PS II, which itself is physically located on the inner side of the membrane of the thylakoid.

Cyclic and Non-cyclic Photo-phosphorylation

  • Living organisms have the capability of extracting energy from oxidisable substances and store this in the form of bond energy.
  • Special substances like ATP, carry this energy in their chemical bonds.
  • The process of whichATP is synthesised by cells (in mitochondria and chloroplasts) is named phosphorylation.
  • Photo- phosphorylation is the synthesis of ATP from ADP and inorganic phosphate in the presence of light.
  • When the two photosystems work in a series, first PS II and then the PS I, a process called non-cyclic photo-phosphorylation occurs.
  • The two photosystems are connected through an electron transport chain, as seen earlier – in the Z scheme.
  • Both ATP and NADPH + H+ are synthesised by this kind of electron flow.
  • When only PS I is functional, the electron is circulated within the photosystem and the phosphorylation occurs due to cyclic flow of electrons.
  • A possible location where this could be happening is in the stroma lamellae.
  • While the membrane or lamellae of the grana have both PS I and PS II the stroma lamellae membranes lack PS II as well as NADP reductase enzyme.
  • The excited electron does not pass on to NADP+ but is cycled back to the PS I complex through the electron transport chain. The cyclic flow hence, results only in the synthesis of ATP, but not of NADPH + H+.
  • Cyclic photophosphorylation also occurs when only light of wavelengths beyond 680 nm are available for excitation.

Chemiosmotic Hypothesis

  • The chemiosmotic hypothesis has been put forward to explain the mechanism of synthesis of ATP.
  • Like in respiration, in photosynthesis too, ATP synthesis is linked to development of a proton gradient across a membrane.
  • This time these are membranes of the thylakoid.
  • There is one difference though, here the proton accumulation is towards the inside of the membrane, i.e., in the lumen.
  • In respiration, protons accumulate in the intermembrane space of the mitochondria when electrons move through the ETS.
  • Processes that take place during the activation of electrons and their transport to determine the steps that cause a proton gradient to develop are –

(a) Since splitting of the water molecule takes place on the inner side of the membrane, the protons or hydrogen ions that are produced by the splitting of water accumulate within the lumen of the thylakoids.

(b) As electrons move through the photosystems, protons are transported across the membrane. This happens because the primary accepter of electron which is located towards the outer side of the membrane transfers its electron not to an electron carrier but to an H carrier. Hence, this molecule removes a proton from the stroma while transporting an electron. When this molecule passes on its electron to the electron carrier on the inner side of the membrane, the proton is released into the inner side or the lumen side of the membrane.

(c) The NADP reductase enzyme is located on the stroma side of the membrane. Along with electrons that come from the accepter of electrons of PS I, protons are necessary for the reduction of NADP+ to NADPH+ H+. These protons are also removed from the stroma.

  • Hence, within the chloroplast, protons in the stroma decrease in number, while in the lumen there is accumulation of protons.
  • This creates a proton gradient across the thylakoid membrane as well as a measurable decrease in pH in the lumen.
  • This proton gradient is important because it is the breakdown of this gradient that leads to release of energy.
  • The gradient is broken down due to the movement of protons across the membrane to the stroma through the transmembrane channel of the F0 of the ATPase.
  • The ATPase enzyme consists of two parts:

one called the F0 is embedded in the membrane and forms a transmembrane channel that carries out facilitated diffusion of protons across the membrane.

The other portion is called F1 and protrudes on the outer surface of the thylakoid membrane on the side that faces the stroma. The breakdown of the gradient provides enough energy to cause a conformational change in the F1 particle of the ATPase, which makes the enzyme synthesise several molecules of energy-packed ATP.

  • Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATPase.
  • Energy is used to pump protons across a membrane, to create a gradient or a high concentration of protons within the thylakoid lumen.
  • ATPase has a channel that allows diffusion of protons back across the membrane; this releases enough energy to activate ATPase enzyme that catalyses the formation of ATP.
  • Along with the NADPH produced by the movement of electrons, the ATP will be used immediately in the biosynthetic reaction taking place in the stroma, responsible for fixing CO2, and synthesis of sugars.

Screenshot (46)

WHERE ARE THE ATP AND NADPH USED?

  • The products of light reaction are ATP, NADPH and O2.
  • Of these O2 diffuses out of the chloroplast while ATP and NADPH are used to drive the processes leading to the synthesis of food, more accurately, sugars.
  • This is the biosynthetic phase of photosynthesis.
  • This process does not directly depend on the presence of light but is dependent on the products of the light reaction, i.e., ATP and NADPH, besides CO2 and H2
  • Immediately after light becomes unavailable, the biosynthetic process continues for some time, and then stops. If then, light is made available, the synthesis starts again.
  • CO2 is combined with H2O to produce (CH2O)n or sugars.
  • Melvin Calvin used radioactive 14C in algal photosynthesis studies which led to the discovery that the first CO2 fixation product was a 3-carbon organic acid or 3-phosphoglyceric acid (PGA).
  • He also contributed to working out the complete biosynthetic pathway; hence it was called Calvin cycle after him.
  • In another group of plants, first stable product of CO2 fixation is 4 carbon organic acid oxaloacetic acid or OAA.
  • The Primary Acceptor of CO2 is a 5-carbon ketose sugar – ribulose bisphosphate (RuBP).

THE CALVIN CYCLE

  • Calvin and his co-workers then worked out the whole pathway and showed that the pathway operated in a cyclic manner; the RuBP was regenerated.
  • The Calvin pathway occurs in all photosynthetic plants; it does not matter whether they have C3 or C4 (or any other) pathways.
  • The Calvin cycle can be described under three stages: carboxylation, reduction and regeneration.
  1. Carboxylation –

Carboxylation is the fixation of CO2 into a stable organic intermediate.

Carboxylation is the most crucial step of the Calvin cycle where CO2 is utilised for the carboxylation of RuBP.

This reaction is catalysed by the enzyme RuBP carboxylase which results in the formation of two molecules of 3-PGA.

Since this enzyme also has an oxygenation activity it would be more correct to call it RuBP carboxylase-oxygenase or RuBisCO.

  1. Reduction –

These are a series of reactions that lead to the formation of glucose.

The steps involve utilisation of 2 molecules of ATP for phosphorylation and two of NADPH for reduction per CO2 molecule fixed.

The fixation of six molecules of CO2 and 6 turns of the cycle are required for the removal of one molecule of glucose from the pathway.

  1. Regeneration –

Regeneration of the CO2 acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted.

The regeneration steps require one ATP for phosphorylation to form RuBP.

  • Hence for every CO2 molecule entering the Calvin cycle, 3 molecules of ATP and 2 of NADPH are required.
  • It is probably to meet this difference in number of ATP and NADPH used in the dark reaction that the cyclic phosphorylation takes place.
  • To make one molecule of glucose 6 turns of the cycle are required.
  • Total 18 ATP, 12 NADPH, 12 CO2 are used synthesis of for 1 molecule of glucose.

 Screenshot (47)

THE C4 PATHWAY

  • Plants that are adapted to dry tropical regions have the C4 pathway
  • Though these plants have the C4 oxaloacetic acid as the first CO2 fixation product they use the C3 pathway or the Calvin cycle as the main biosynthetic pathway.
  • C4 plants are special: They have a special type of leaf anatomy, they tolerate higher temperatures, they show a response to highlight intensities, they lack a process called photorespiration and have greater productivity of biomass.
  • The particularly large cells around the vascular bundles of the C4 pathway plants are called bundle sheath cells, and the leaves which have such anatomy are said to have ‘Kranz’ anatomy.
  • ‘Kranz’ means ‘wreath’ and is a reflection of the arrangement of cells.
  • The bundle sheath cells may form several layers around the vascular bundles; they are characterised by having a large number of chloroplasts, thick walls impervious to gaseous exchange and no intercellular spaces.
  • This pathway also known as Hatch and Slack Pathway, is a cyclic process.
  • The primary CO2 acceptor is a 3-carbon molecule phosphoenol pyruvate (PEP) and is present in the mesophyll cells. The enzyme responsible for this fixation is PEP carboxylase or PEPcase.
  • The mesophyll cells lack RuBisCO enzyme.
  • The C4 acid OAA is formed in the mesophyll cells.
  • It then forms other 4-carbon compounds like malic acid or aspartic acid in the mesophyll cells itself, which are transported to the bundle sheath cells.
  • In the bundle sheath cells these C4 acids are broken down to release CO2 and a 3-carbon molecule.
  • The 3-carbon molecule is transported back to the mesophyll where it is converted to PEP again, thus, completing the cycle.
  • The CO2 released in the bundle sheath cells enters the C3 or the Calvin pathway, a pathway common to all plants. The bundle sheath cells are rich in an enzyme Ribulose bisphosphate carboxylase-oxygenase (RuBisCO), but lack PEPcase.
  • Thus, the basic pathway that results in the formation of the sugars, the Calvin pathway, is common to the C3 and C4
  • the Calvin pathway occurs in all the mesophyll cells of the C3 plants while in the C4 plants it does not take place in the mesophyll cells but does so only in the bundle sheath cells.

Screenshot (48)

PHOTORESPIRATION

  • RuBisCO is the most abundant enzyme in the world.
  • It is characterised by the fact that its active site can bind to both CO2 and O2 – hence the name.
  • RuBisCO has a much greater affinity for CO2 than for O2.
  • This binding is competitive. It is the relative concentration of O2 and CO2 that determines which of the two will bind to the enzyme.
  • In C3 plants some O2 does bind to RuBisCO, and hence CO2 fixation is decreased. Here the RuBP instead of being converted to 2 molecules of PGA binds with O2 to form one molecule and phosphoglycolate in a pathway called photorespiration.
  • In the photorespiratory pathway, there is neither synthesis of sugars, nor of ATP. Rather it results in the release of CO2 with the utilisation of ATP.
  • In the photorespiratory pathway there is no synthesis of ATP or NADPH. Therefore, photorespiration is a wasteful process.
  • In C4 plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of CO2 at the enzyme site.
  • This takes place when the C4 acid from the mesophyll is broken down in the bundle cells to release CO2 – this results in increasing the intracellular concentration of CO2.
  • In turn, this ensures that the RuBisCO functions as a carboxylase minimising the oxygenase activity.
  • Because of absence of photorespiration in C4 plants, productivity and yields are better in these plants.
  • These plants show tolerance to higher temperatures.
Characteristics C3 Plants C4 Plants
Cell type in which the Calvin cycle takes place Both Bundle sheath
Cell type in which the initial carboxylation reaction occurs Both Mesophyll
How many cell types does the leaf have that fix Co2. Three: Bundle sheath, palisade, spongy mesophyll Two: Bundle sheath and

Mesophyll

Which is the primary Co2 acceptor RuBP PEP
Number of carbons in the primary Co2 acceptor 5 3
Which is the primary Co2 fixation product PGA OAA
No. of carbons in the primary Co2 fixation product 3 4
Does the plant have RuBisCo? Yes Yes
Does the plant have PEP Case? No Yes
Which cells in the plant have Rubisco? Mesophyll/Bundle sheath/none Mesophyll/Bundle sheath/none
Co2 fixation rate under high light conditions Medium High
Whether photorespiration is present at low light intensities Sometimes Negligible
Whether photorespiration is present at high light intensities Sometimes Negligible
Whether photorespiration would be present at low CO2 concentrations High Negligible
Whether photorespiration would be present at high CO2 concentrations Sometimes Negligible
Temperature optimum 20-25C 30-40 C

FACTORS AFFECTING PHOTOSYNTHESIS

  • The rate of photosynthesis is very important in determining the yield of plants including crop plants.
  • Photosynthesis is under the influence of several factors, both internal (plant) and external.
  • The plant factors include the number, size, age and orientation of leaves, mesophyll cells and chloroplasts, internal CO2 concentration and the amount of chlorophyll. The plant or internal factors are dependent on the genetic predisposition and the growth of the plant.
  • The external factors include the availability of sunlight, temperature, CO2 concentration and water.
  • As a plant photosynthesises, all these factors will simultaneously affect its rate. Hence, though several factors interact and simultaneously affect photosynthesis or CO2 fixation, usually one factor is the major cause or is the one that limits the rate. Hence, at any point the rate will be determined by the factor available at sub-optimal levels.
  • Blackman’s (1905) Law of Limiting Factors – If a chemical process is affected by more than one factor, then its rate will be determined by the factor which is nearest to its minimal value: it is the factor which directly affects the process if its quantity is changed
  • For example, despite the presence of a green leaf and optimal light and CO2 conditions, the plant may not photosynthesise if the temperature is very low. This leaf, if given the optimal temperature, will start photosynthesising.

Light

  • It includes light quality, light intensity and the duration of exposure to light.
  • There is a linear relationship between incident light and CO2 fixation rates at low light intensities.
  • At higher light intensities, gradually the rate does not show further increase as other factors become limiting.
  • Light saturation occurs at 10 per cent of the full sunlight.
  • Hence, except for plants in shade or in dense forests, light is rarely a limiting factor in nature.
  • Increase in incident light beyond a point causes the breakdown of chlorophyll and a decrease in photosynthesis.

Screenshot (50)

Carbon dioxide Concentration

  • Carbon dioxide is the major limiting factor for photosynthesis.
  • The concentration of CO2 is very low in the atmosphere (between 0.03 and 0.04 per cent).
  • Increase in concentration upto 0.05 per cent can cause an increase in CO2 fixation rates; beyond this the levels can become damaging over longer periods.
  • The C3 and C4 plants respond differently to CO2 At low light conditions neither group responds to high Co2 conditions. At high light intensities, both C3 and C4 plants show increase in the rates of photosynthesis.
  • C4 plants show saturation at about 360 µlL-1 while in C3 plants saturation is seen only beyond 450 µlL-1. Thus, current availability of CO2 levels is limiting to the C3
  • The fact that C3 plants respond to higher CO2 concentration by showing increased rates of photosynthesis leading to higher productivity has been used for some greenhouse crops such as tomatoes and bell pepper. They are allowed to grow in carbon dioxide enriched atmosphere that leads to higher yields.

Temperature

  • The dark reactions being enzymatic are temperature controlled.
  • Though the light reactions are also temperature sensitive they are affected to a much lesser extent. The C4 plants respond to higher temperatures and show higher rate of photosynthesis while C3 plants have a much lower temperature optimum.
  • The temperature optimum for photosynthesis of different plants also depends on the habitat that they are adapted to.
  • Tropical plants have a higher temperature optimum than the plants adapted to temperate climates.

Water

  • Even though water is one of the reactants in the light reaction, the effect of water as a factor is more through its effect on the plant, rather than directly on photosynthesis.
  • Water stress causes the stomata to close hence reducing the CO2 Besides, water stress also makes leaves wilt, reducing the surface area of the leaves and their metabolic activity as well.

Printable PDF file is given in below link…..

CHAPTER 13 – PHOTOSYNTHESIS IN HIGHER PLANTS

CHAPTER 12 – MINERAL NUTRITION

CHAPTER 12

MINERAL NUTRITION

  • All living organisms require macromolecules, such as carbohydrates, proteins and fats, and water and minerals for their growth and development.

Methods to Study the Mineral Requirements of Plants

  • In 1860, Julius von Sachs, a prominent German botanist, demonstrated, for the first time, that plants could be grown to maturity in a defined nutrient solution in complete absence of soil. This technique of growing plants in a nutrient solution is known as hydroponics.
  • Culture of plants in a soil-free mineral solution require purified water and mineral nutrient salts.
  • After a series of experiments in which the roots of the plants were immersed in nutrient solutions and wherein an element was added / removed or given in varied concentration, a mineral solution suitable for the plant growth was obtained. By this method, essential elements were identified and their deficiency symptoms discovered.
  • Hydroponics has been successfully employed as a technique for the commercial production of vegetables such as tomato, seedless cucumber and lettuce.
  • It must be emphasised that the nutrient solutions must be adequately aerated to obtain the optimum growth.

Essential Mineral Elements

  • Most of the minerals present in soil can enter plants through roots.
  • In fact, more than sixty elements of the 105 discovered so far are found in different plants.
  • Some plant species accumulate selenium, some others gold, while some plants growing near nuclear test sites take up radioactive strontium.
  • There are techniques that are able to detect the minerals even at a very low concentration (10-8 g/ mL).

Criteria for Essentiality

The criteria for essentiality of an element are given below:

  1. The element must be absolutely necessary for supporting normal growth and reproduction. In the absence of the element the plants do not complete their life cycle or set the seeds.
  2. The requirement of the element must be specific and not replaceable by another element. In other words, deficiency of any one element cannot be met by supplying some other element.
  3. The element must be directly involved in the metabolism of the plant.

Based upon the above criteria only a few elements have been found to be absolutely essential for plant growth and metabolism.

These elements are further divided into two broad categories based on their quantitative requirements.

Macronutrients, and
Micronutrients

  • Macronutrients are generally present in plant tissues in large amounts (in excess of 10 mmole Kg -1 of dry matter).
    • The macronutrients include carbon, hydrogen, oxygen, nitrogen, phosphorous, sulphur, potassium, calcium and magnesium.
    • Of these, carbon, hydrogen and oxygen are mainly obtained from CO2 and H2O, while the others are absorbed from the soil as mineral nutrition.
  • Micronutrients or trace elements, are needed in very small amounts (less than 10 mmole Kg 1 of dry matter).
    • These include iron, manganese, copper, molybdenum, zinc, boron, chlorine and nickel.
    • In addition to the 17 essential elements named above, there are some beneficial elements such as sodium, silicon, cobalt and selenium. They are required by higher plants.

Essential elements can also be grouped into four broad categories on the basis of their diverse functions. These categories are:

  1. Essential elements as components of biomolecules and hence structural elements of cells (e.g., carbon, hydrogen, oxygen and nitrogen).
  2. Essential elements that are components of energy-related chemical compounds in plants (e.g., magnesium in chlorophyll and phosphorous in ATP).
  3. Essential elements that activate or inhibit enzymes, for example Mg2+ is an activator for both ribulose bisphosphate carboxylase- oxygenase and phosphoenol pyruvate carboxylase, both of which are critical enzymes in photosynthetic carbon fixation; Zn2+ is an activator of alcohol dehydrogenase and Mo of nitrogenase during nitrogen metabolism. For this, you will need to recollect some of the biochemical pathways you have studied earlier.
  4. Some essential elements can alter the osmotic potential of a cell. Potassium plays an important role in the opening and closing of stomata. You may recall the role of minerals as solutes in determining the water potential of a cell.

Role of Macro- and Micro-nutrients

Nitrogen :

  • This is the mineral element required by plants in the greatest amount.
  • absorbed mainly as – NO3 though some are also taken up as NO2 or NH4+.
  • Nitrogen is required by all parts of a plant, particularly the meristematic tissues and the metabolically active cells.
  • Nitrogen is one of the major constituents of proteins, nucleic acids, vitamins and hormones.

Phosphorus:

  • Phosphorus is absorbed by the plants from soil in the form of phosphate ions (either as H2PO4 or HPO2-).
  • Phosphorus is a constituent of cell membranes, certain proteins, all nucleic acids and nucleotides, and is required for all phosphorylation reactions.

Potassium:

  • It is absorbed as potassium ion (K+).
  • In plants, this is required in more abundant quantities in the meristematic tissues, buds, leaves and root tips.
  • Potassium helps to maintain an anion-cation balance in cells and is involved in protein synthesis, opening and closing of stomata, activation of enzymes and in the maintenance of the turgidity of cells.

Calcium:

  • Plant absorbs calcium from the soil in the form of calcium ions (Ca2+).
  • Calcium is required by meristematic and differentiating tissues.
  • During cell division it is used in the synthesis of cell wall, particularly as calcium pectate in the middle lamella.
  • It is also needed during the formation of mitotic spindle.
  • It accumulates in older leaves.
  • It is involved in the normal functioning of the cell membranes. It activates certain enzymes and plays an important role in regulating metabolic activities.

Magnesium:

  • It is absorbed by plants in the form of divalent Mg2+.
  • It activates the enzymes of respiration, photosynthesis and are involved in the synthesis of DNA and RNA.
  • Magnesium is a constituent of the ring structure of chlorophyll and helps to maintain the ribosome structure.

Sulphur:

  • Plants obtain sulphur in the form of sulphate (SO2-).
  • Sulphur is present in two amino acids – cysteine and methionine and is the main constituent of several coenzymes, vitamins (thiamine, biotin, Coenzyme A) and ferredoxin.

Iron:

  • Plants obtain iron in the form of ferric ions (Fe3+).
  • It is required in larger amounts in comparison to other micronutrients.
  • It is an important constituent of proteins involved in the transfer of electrons like ferredoxin and cytochromes.
  • It is reversibly oxidised from Fe2+ to Fe3+ during electron transfer.
  • It activates catalase enzyme, and is essential for the formation of chlorophyll.

Manganese:

  • It is absorbed in the form of manganous ions (Mn2+).
  • It activates many enzymes involved in photosynthesis, respiration and nitrogen metabolism.
  • The best defined function of manganese is in the splitting of water to liberate oxygen during photosynthesis.

Zinc:

  • Plants obtain zinc as Zn2+ ions.
  • It activates various enzymes, especially carboxylases.
  • It is also needed in the synthesis of auxin.

Copper:

  • It is absorbed as cupric ions (Cu2+).
  • It is essential for the overall metabolism in plants.
  • Like iron, it is associated with certain enzymes involved in redox reactions and is reversibly oxidised from Cu+ to Cu2+.

Boron:

  • It is absorbed as BO|- or B4O2-.
  • Boron is required for uptake and utilisation of Ca2+, membrane functioning, pollen germination, cell elongation, cell differentiation and carbohydrate translocation.

Molybdenum:

  • Plants obtain it in the form of molybdate ions (MoO2+).
  • It is a component of several enzymes, including nitrogenase and nitrate reductase both of which participate in nitrogen metabolism.

Chlorine:

  • It is absorbed in the form of chloride anion (Cl).
  • Along with Na+ and K+, it helps in determining the solute concentration and the anion- cation balance in cells.
  • It is essential for the water-splitting reaction in photosynthesis, a reaction that leads to oxygen evolution.

Deficiency Symptoms of Essential Elements

  • Whenever the supply of an essential element becomes limited, plant growth is retarded. The concentration of the essential element below which plant growth is retarded is termed as Critical concentration. The element is said to be deficient when present below the critical concentration.
  • Since each element has one or more specific structural or functional role in plants, in the absence of any particular element, plants show certain morphological changes. These morphological changes are indicative of certain element deficiencies and are called deficiency symptoms.
  • The deficiency symptoms vary from element to element and they disappear when the deficient mineral nutrient is provided to the plant. However, if deprivation continues, it may eventually lead to the death of the plant.
  • The parts of the plants that show the deficiency symptoms also depend on the mobility of the element in the plant. For elements that are actively mobilised within the plants and exported to young developing tissues, the deficiency symptoms tend to appear first in the older tissues. For example, the deficiency symptoms of nitrogen, potassium and magnesium are visible first in the senescent leaves. In the older leaves, biomolecules containing these elements are broken down, making these elements available for mobilising to younger leaves.
  • The deficiency symptoms tend to appear first in the young tissues whenever the elements are relatively immobile and are not transported out of the mature organs, for example, elements like sulphur and calcium are a part of the structural component of the cell and hence are not easily released.
  • The kind of deficiency symptoms shown in plants include chlorosis, necrosis, stunted plant growth, premature fall of leaves and buds, and inhibition of cell division.
  • Chlorosis – loss of chlorophyll leading to yellowing in leaves.
  • caused by the deficiency of elements N, K, Mg, S, Fe, Mn, Zn and Mo.
  • Necrosis – death of tissue, particularly leaf tissue.
  • Caused by the deficiency of Ca, Mg, Cu, K.
  • Inhibition of cell division – Lack or low level of N, K, S, Mo.
  • Delayed flowering – N, S, Mo.

Toxicity of Micronutrients

  • The requirement of micronutrients is always in low amounts while their moderate decrease causes the deficiency symptoms and a moderate increase causes toxicity.
  • There is a narrow range of concentration at which the elements are optimum.
  • Any mineral ion concentration in tissues that reduces the dry weight of tissues by about 10 per cent is considered toxic.
  • Such critical concentrations vary widely among different micronutrients.
  • The toxicity symptoms are difficult to identify.
  • Toxicity levels for any element also vary for different plants.
  • Many a times, excess of an element may inhibit the uptake of another element.
  • Manganese toxicity – the appearance of brown spots surrounded by chlorotic veins.
  • Manganese competes with iron and magnesium for uptake
  • Manganese competes with magnesium for binding with enzymes.
  • Manganese also inhibit calcium translocation in shoot apex.
  • Therefore, excess of manganese may, in fact, induce deficiencies of iron, magnesium and calcium.
  • Thus, what appears as symptoms of manganese toxicity may actually be the deficiency symptoms of iron, magnesium and calcium.

Mechanism of Absorption of Elements

  • The process of absorption can be demarcated into two main phases.
  • In the first phase, an initial rapid uptake of ions into the ‘free space’ or ‘outer space’ of cells – the apoplast, is passive.
  • In the second phase of uptake, the ions are taken in slowly into the ‘inner space’ – the symplast of the cells is active.
  • The passive movement of ions into the apoplast usually occurs through ion-channels, the trans-membrane proteins that function as selective pores. On the other hand, the entry or exit of ions to and from the symplast requires the expenditure of metabolic energy, which is an active process.
  • The movement of ions is usually called the flux.
  • inward movement into the cells is influx and the outward movement, efflux.

Translocation of Solutes

  • Mineral salts are translocated through xylem along with the ascending stream of water, which is pulled up through the plant by transpirational pull.
  • Analysis of xylem sap shows the presence of mineral salts in it.
  • Use of radioisotopes of mineral elements also substantiate the view that they are transported through the xylem.

Soil as Reservoir of Essential Elements

  • Majority of the nutrients that are essential for the growth and development of plants become available to the roots due to weathering and breakdown of rocks.
  • These processes enrich the soil with dissolved ions and inorganic salts.
  • Since they are derived from the rock minerals, their role in plant nutrition is referred to as mineral nutrition.
  • Soil consists of a wide variety of substances. Soil not only supplies minerals but also harbours nitrogen-fixing bacteria, other microbes, holds water, supplies air to the roots and acts as a matrix that stabilises the plant.
  • Since deficiency of essential minerals affect the crop-yield, there is often a need for supplying them through fertilisers.
  • Both macro-nutrients (N, P, K, S, etc.) and micro-nutrients (Cu, Zn, Fe, Mn, etc.) form components of fertilisers and are applied as per need.

Metabolism of Nitrogen

Nitrogen Cycle

  • Apart from carbon, hydrogen and oxygen, nitrogen is the most prevalent element in living organisms.
  • Nitrogen is a constituent of amino acids, proteins, hormones, chlorophylls and many of the vitamins.
  • Plants compete with microbes for the limited nitrogen that is available in soil. Thus, nitrogen is a limiting nutrient for both natural and agricultural eco-systems.
  • Nitrogen exists as two nitrogen atoms joined by a very strong triple covalent bond (N = N). The process of conversion of nitrogen (N2) to ammonia is termed as Nitrogen Fixation.
  • in nature, lightning and ultraviolet radiation provide enough energy to convert nitrogen to nitrogen oxides (NO, NO2, N2O).
  • Industrial combustions, forest fires, automobile exhausts and power-generating stations are also sources of atmospheric nitrogen oxides.
  • Decomposition of organic nitrogen of dead plants and animals into ammonia is called ammonification.

2NH3 + 3O2 ——–>2NO2 + 2H+ + 2H2O
…. (i)

2NO2 + O2 ———>2NO3
…. (ii)

  • Some of this ammonia volatilises and re-enters the atmosphere but most of it is converted into nitrate by soil bacteria in the following steps:
  • Ammonia is first oxidised to nitrite by the bacteria Nitrosomonas and/or Nitrococcus. The nitrite is further oxidised to nitrate with the help of the bacterium Nitrobacter. These steps are called Nitrification.
  • The nitrifying bacteria are Chemoautotrophs.
  • The nitrate thus formed is absorbed by plants and is transported to the leaves.
  • In leaves, it is reduced to form ammonia that finally forms the amine group of amino acids.
  • Nitrate present in the soil is also reduced to nitrogen is by the process of denitrification.
  • Denitrification is carried by bacteria Pseuodomonas and Thiobacillus.

Biological Nitrogen Fixation

  • Reduction of nitrogen to ammonia by living organism is called biological nitrogen fixation.
  • The enzyme Nitrogenase which is capable of nitrogen reduction is present exclusively in prokaryotes.
  • Such microbes are called nitrogen fixers.
  • The nitrogen-fixing microbes could be free-living or symbiotic.
  • Free-living nitrogen-fixing aerobic microbes – Azotobacter and Beijerinckia.
  • Free-living nitrogen-fixing aerobic microbes – Rhodospirilhim and Bacillus.
  • A number of cyanobacteria such as Anabaena and Nostoc are also free living nitrogen-fixers.

Symbiotic biological nitrogen fixation

  • Rod-shaped Rhizobium – Symbiosis with the root nodules of several legumes such as alfalfa, sweet clover, sweet pea, lentils, garden pea, broad bean, clover beans, etc.
  • The microbe. Frankia, also produces nitrogen-fixing nodules on the roots of non-leguminous plants like Alnus.
  • Both Rhizobium and Frankia are free-living in soil but as symbionts, can fix atmospheric nitrogen.
  • Red or pink colour of the nodules is due to the presence of leguminous haemoglobin or leg-haemoglobin.

Nodule formation

  • Nodule formation involves a sequence of multiple interactions between Rhizobium and roots of the host plant.
  • Principal stages in the nodule formation are summarised as follows:
  • Rhizobia multiply and colonise the surroundings of roots and get attached to epidermal and root hair cells.
  • The root-hairs curl and the bacteria invade the root-hair.
  • An infection thread is produced carrying the bacteria into the cortex of the root, where they initiate the nodule formation in the cortex of the root.
  • Then the bacteria are released from the thread into the cells which leads to the differentiation of specialised nitrogen fixing cells.
  • The nodule thus formed, establishes a direct vascular connection with the host for exchange of nutrients.

  • The nodule contains all the necessary biochemical components, such as the enzyme nitrogenase and leghaemoglobin.
  • The enzyme nitrogenase is a Mo-Fe protein and catalyses the conversion of atmospheric nitrogen to ammonia, the first stable product of nitrogen fixation.

N2 + 8H+ + 16ATP ———–> 2NH3 + H2 + 16ADP + 16Pi

  • The enzyme nitrogenase is highly sensitive to the molecular oxygen; it requires anaerobic conditions.
  • The nodules have adaptations that ensure that the enzyme is protected from oxygen.
  • To protect these enzymes, the nodule contains an oxygen scavenger called leg-haemoglobin.
  • It is interesting to note that these microbes live as aerobes under free-living conditions (where nitrogenase is not operational), but during nitrogen-fixing events, they become anaerobic (thus protecting the nitrogenase enzyme).
  • Ammonia synthesis by nitrogenease requires a very high input of energy (8 ATP for each NH3 produced). The energy required, thus, is obtained from the respiration of the host cells.

Fate of ammonia

  • At physiological pH, the ammonia is protonated to form NH4+ (ammonium) ion.
  • While most of the plants can assimilate nitrate as well as ammonium ions, the latter is quite toxic to plants and hence cannot accumulate in them.
  • NH4+ is used to synthesise amino acids in plants.
  • There are two main ways in which this can take place:
  • Reductive amination: In these processes, ammonia reacts with α- ketoglutaric acid and forms glutamic acid.

α-ketogluatric acid + NH4+ + NADPH ——–> glutamate + H2O + NADP

  • Transamination: It involves the transfer of amino group from one amino acid to the keto group of a keto acid.
  • Glutamic acid is the main amino acid from which the transfer of NH2, the amino group takes place and other amino acids are formed through transamination.
  • The enzyme transaminase catalyses all such reactions. For example,

  • The two most important amides – asparagine and glutamine, found in plants are a structural part of proteins.
  • They are formed from two amino acids, namely aspartic acid and glutamic acid, respectively, by addition of another amino group to each.
  • The hydroxyl part of the acid is replaced by another NH2- radicle.
  • Since amides contain more nitrogen than the amino acids, they are transported to other parts of the plant via xylem vessels.
  • In addition, along with the transpiration stream the nodules of some plants (e.g., soyabean) export the fixed nitrogen as ureides.
  • These compounds also have a particularly high nitrogen to carbon ratio.

 

PRINTABLE Pdf file of chapter notes are available…please click on the link…

CHAPTER 12 – MINERAL NUTRITION

CHAPTER 11 – TRANSPORT IN PLANTS

Chapter 11

Transport in Plants

  • Plants need to move molecules over very long distances, much more than animals do; they also do not have a circulatory system in place.
  • Water taken up by the roots has to reach all parts of the plant, up to the very tip of the growing stem. The photosynthates or food synthesised by the leaves have also to be moved to all parts including the root tips embedded deep inside the soil.
  • Movement across short distances, say within the cell, across the membranes and from cell to cell within the tissue has also to take place.
  • In a flowering plant the substances that would need to be transported are water, mineral nutrients, organic nutrients and plant growth regulators.
  • Small distance transport – by diffusion and by cytoplasmic streaming supplemented by active transport.
  • Long distance Transport – through the vascular system (the xylem and the phloem) and is called translocation.
  • In rooted plants, transport in xylem (of water and minerals) is essentially unidirectional, from roots to the stems.
  • Organic and mineral nutrients however, undergo multidirectional transport.
  • Organic compounds synthesised in the photosynthetic leaves are exported to all other parts of the plant Including storage organs. From the storage organs they are later re-exported.
  • The mineral nutrients are taken up by the roots and transported upwards into the stem, leaves and the growing regions. When any plant part undergoes senescence, nutrients may be withdrawn from such regions and moved to the growing parts.
  • Hormones or plant growth regulators and other chemical stimuli are also transported, though in very small amounts, sometimes in a strictly polarised or unidirectional manner from where they are synthesised to other parts.

Means of Transport

Diffusion

  • Passive movement and may be from one part of the cell to the other, or from cell to cell, or over short distances, say, from the intercellular spaces of the leaf to the outside.
  • No energy expenditure takes place.
  • Molecules move in a random fashion, the net result being substances moving from regions of higher concentration to regions of lower concentration.
  • Diffusion is a slow process and is not dependent on a ‘living system’.
  • Diffusion is obvious in gases and liquids, but diffusion in solids rather than of solids is more likely.
  • Diffusion is very important to plants since it the only means for gaseous movement within the plant body.
  • Diffusion rates are affected by the gradient of concentration, the permeability of the membrane separating them, size of the substances, temperature and pressure.

Facilitated Diffusion

  • The diffusion of any substance across a membrane also depends on its solubility in lipids, the major constituent of the membrane.
  • Substances soluble in lipids diffuse through the membrane faster.
  • Substances that have a hydrophilic moiety, find it difficult to pass through the membrane; their movement has to be facilitated. Membrane proteins provide sites at which such molecules cross the membrane.
  • They do not set up a concentration gradient: a concentration gradient must already be present for molecules to diffuse even if facilitated by the proteins. This process is called facilitated diffusion.
  • In facilitated diffusion special proteins help move substances across membranes without expenditure of ATP energy.
  • Facilitated diffusion cannot cause net transport of molecules from a low to a high concentration – this would require input of energy.
  • Transport rate reaches a maximum when all of the protein transporters are being used (saturation).
  • Facilitated diffusion is very specific: it allows cell to select substances for uptake.
  • It is sensitive to inhibitors which react with protein side chains.
  • The proteins form channels in the membrane for molecules to pass through. Some channels are always open; others can be controlled. Some are large, allowing a variety of molecules to cross.
  • The porins are proteins that form huge pores in the outer membranes of the plastids, mitochondria and some bacteria allowing molecules up to the size of small proteins to pass through.
  • When an extracellular molecule bound to the transport protein; the transport protein then rotates and releases the molecule inside the cell, e.g., water channels (made up of eight different types of aquaporins.)

Passive symports and antiports

  • Some carrier or transport proteins allow diffusion only if two types of molecules move together.
  • In a symport, both molecules cross the membrane in the same direction; in an antiport, they move in opposite directions.
  • When a molecule moves across a membrane independent of other molecules, the process is called uniport.

Active Transport

  • Active transport uses energy to pump molecules against a concentration gradient.
  • Active transport is carried out by membrane-proteins. Hence different proteins in the membrane play a major role in both active as well as passive transport.
  • Pumps are proteins that use energy to carry substances across the cell membrane. These pumps can transport substances from a low concentration to a high concentration (‘uphill’ transport).
  • Transport rate reaches a maximum when all the protein transporters are being used or are saturated.
  • Like enzymes the carrier protein is very specific in what it carries across the membrane.
  • These proteins are sensitive to inhibitors that react with protein side chains.

Comparison of Different Transport Processes

  • Proteins in the membrane are responsible for facilitated diffusion and active transport and hence show common characterstics of being highly selective; they are liable to saturate, respond to inhibitors and are under hormonal regulation.
  • But diffusion whether facilitated or not – take place only along a gradient and do not use energy.

Plant-Water Relations

  • Water is essential for all physiological activities of the plant and plays a very important role in all living organisms.
  • It provides the medium in which most substances are dissolved. The protoplasm of the cells is nothing but water in which different molecules are dissolved and (several particles) suspended. A watermelon has over 92 per cent water; most herbaceous plants have only about 10 to 15 per cent of its fresh weight as dry matter.
  • Of course, distribution of water within a plant varies – woody parts have relatively very little water, while soft parts mostly contain water. A seed may appear dry but it still has water – otherwise it would not be alive and respiring!
  • Terrestrial plants take up huge amount water daily but most of it is lost to the air through evaporation from the leaves, i.e., transpiration. A mature corn plant absorbs almost three litres of water in a day, while a mustard plant absorbs water equal to its own weight in about 5 hours. Because of this high demand for water, it is not surprising that water is often the limiting factor for plant growth and productivity in both agricultural and natural environments.

Water Potential

  • Water potential (Psi w) is a concept fundamental to understanding water movement. Solute potential (Psi s) and pressure potential (Psi p) are the two main components that determine water potential.
  • Water molecules possess kinetic energy. In liquid and gaseous form they are in random motion that is both rapid and constant. The greater the concentration of water in a system, the greater is its kinetic energy or ‘water potential’. Hence, it is obvious that pure water will have the greatest water potential.
  • If two systems containing water are in contact, random movement of water molecules will result in net movement of water molecules from the system with higher energy to the one with lower energy. Thus water will move from the system containing water at higher water potential to the one having low water potential.
  • This process of movement of substances down a gradient of free energy is called diffusion.
  • Water potential is denoted by the Greek symbol Psi and is expressed in pressure units such as pascals (Pa).
  • By convention, the water potential of pure water at standard temperatures, which is not under any pressure, is taken to be zero.
  • If some solute is dissolved in pure water, the solution has fewer free water and the concentration of water decreases, reducing its water potential. Hence, all solutions have a lower water potential than pure water; the magnitude of this lowering due to dissolution of a solute is called solute potential or Psi s. Psi s is always negative. The more the solute molecules, the lower (more negative) is the Psi s.
  • For a solution at atmospheric pressure (water potential) Psi w = (solute potential) Psi s.
  • If a pressure greater than atmospheric pressure is applied to pure water or a solution, its water potential increases. It is equivalent to pumping water from one place to another.
  • Pressure can build up in a plant system when water enters a plant cell due to diffusion causing a pressure built up against the cell wall, it makes the cell turgid, this increases the pressure potential. Pressure potential is usually positive.
  • Though in plants negative potential or tension in the water column in the xylem plays a major role in water transport up a stem. Pressure potential is denoted as (p.
  • Water potential of a cell is affected by both solute and pressure potential. The relationship between them is as follows:

Osmosis

  • The plant cell is surrounded by a cell membrane and a cell wall. The cell wall is freely permeable to water and substances in solution hence is not a barrier to movement.
  • In plants the cells usually contain a large central vacuole, whose contents, the vacuolar sap, contribute to the solute potential of the cell.
  • In plant cells, the cell membrane and the membrane of the vacuole, the tonoplast together are important determinants of movement of molecules in or out of the cell.
  • Osmosis is the term used to refer specifically to the diffusion of water across a differentially- or semi-permeable membrane. Osmosis occurs spontaneously in response to a driving force.
  • The net direction and rate of osmosis depends on both the pressure gradient and concentration gradient.
  • Water will move from its region of higher chemical potential (or concentration) to its region of lower chemical potential until equilibrium is reached. At equilibrium the two chambers should have the same water potential.


In above Fig two chambers, A and B, containing solutions are separated by a semi-permeable membrane.

  • Solution of which chamber has a lower water potential? – B
  • Solution of which chamber has a lower solute potential? – B
  • In which direction will osmosis occur? – A(B
  • Which solution has a higher solute potential?- A
  • At equilibrium which chamber will have lower water potential?- Equal
  • If one chamber has a psi of -2000 kPa, and the other -1000 kPa, which is the chamber that has the higher psi ?- second with -1000kP.

  • Experiment – a solution of sucrose in water taken in a funnel is separated from pure water in a beaker through a semi-permeable membrane (Egg membrane – Can be obtained by removing the yolk and albumin through a small hole at one end of the egg, and placing the shell in dilute solution of hydrochloric acid for a few hours. The egg shell dissolves leaving the membrane intact). Water will move into the funnel, resulting in rise in the level of the solution in the funnel. This will continue till the equilibrium is reached. External pressure can be applied from the upper part of the funnel such that no water diffuses into the funnel through the membrane. This pressure required to prevent water from diffusing is the osmotic pressure and this is the function of the solute concentration; more the solute concentration, greater will be the pressure required to prevent water from diffusing in. Numerically osmotic pressure is equivalent to the osmotic potential, but the sign is opposite. Osmotic pressure is the positive pressure applied, while osmotic potential is negative.

Plasmolysis

    • The behaviour of the plant cells (or tissues) with regard to water movement depends on the surrounding solution.
    • If the external solution balances the osmotic pressure of the cytoplasm, it is said to be isotonic.
    • If the external solution is more dilute than the cytoplasm, it is hypotonic and if the external solution is more concentrated, it is hypertonic.
    • Cells swell in hypotonic solutions and shrink in hypertonic ones.
    • Plasmolysis occurs when water moves out of the cell and the cell membrane of a plant cell shrinks away from its cell wall. This occurs when the cell (or tissue) is placed in a solution that is hypertonic (has more solutes) to the protoplasm. Water moves out; it is first lost from the cytoplasm and then from the vacuole.
    • The water when drawn out of the cell through diffusion into the extracellular (outside cell) fluid causes the protoplast to shrink away from the walls. The cell is said to be plasmolysed.
    • The movement of water occurred across the membrane moving from an area of high water potential (i.e., the cell) to an area of lower water potential outside the cell.
    • The process of plamolysis is usually reversible.
    • When the cells are placed in a hypotonic solution (higher water potential or dilute solution as compared to the cytoplasm), water diffuses into the cell causing the cytoplasm to build up a pressure against the wall, that is called turgor pressure. The pressure exerted by the protoplasts due to entry of water against the rigid walls is called pressure potential. Because of the rigidity of the cell wall, the cell does not rupture.
    • This turgor pressure is ultimately responsible for enlargement and extension growth of cells.

Imbibition

  • Imbibition is a special type of diffusion when water is absorbed by solids – colloids – causing them to enormously increase in volume. e.g., absorption of water by seeds and dry wood.
  • The pressure that is produced by the swelling of wood had been used by prehistoric man to split rocks and boulders.
  • If it were not for the pressure due to imbibition, seedlings would not have been able to emerge out of the soil into the open.
  • Imbibition is also diffusion since water movement is along a concentration gradient; the seeds and other such materials have almost no water hence they absorb water easily.
  • Water potential gradient between the absorbent and the liquid imbibed is essential for imbibition.
  • In addition, for any substance to imbibe any liquid, affinity between the adsorbant and the liquid is also a pre-requisite.

Long Distance Transport of Water

  • Long distance transport of substances within a plant cannot be by diffusion alone. Diffusion is a slow process. It can account for only short distance movement of molecules. For example, the movement of a molecule across a typical plant cell (about 50µm) takes approximately 2.5 s.
  • In large and complex organisms, often substances have to be moved across very large distances. sometimes the sites of production or absorption and sites of storage are too far from each other; diffusion or active transport would not suffice. Special long distance transport systems become necessary so as to move substances across long distances and at a much faster rate.
  • Water and minerals, and food are generally moved by a mass or bulk flow system.
  • Mass flow is the movement of substances in bulk or en masse from one point to another as a result of pressure differences between the two points. It is a characteristic of mass flow that substances, whether in solution or in suspension, are swept along at the same pace, as in a flowing river.
  • This is unlike diffusion where different substances move independently depending on their concentration gradients.
  • Bulk flow can be achieved either through a positive hydrostatic pressure gradient (e.g., a garden hose) or a negative hydrostatic pressure gradient (e.g., suction through a straw).
  • The bulk movement of substances through the conducting or vascular tissues of plants is called translocation.
  • The higher plants have highly specialised vascular tissues – xylem and phloem.
  • Xylem is associated with translocation of mainly water, mineral salts, some organic nitrogen and hormones, from roots to the aerial parts of the plants.
  • The phloem translocates a variety of organic and inorganic solutes, mainly from the leaves to other parts of the plants.

How do Plants Absorb Water?

  • The responsibility of absorption of water and minerals is of the root hairs.
  • Root hairs are thin-walled slender extensions of root epidermal cells that greatly increase the surface area for absorption.
  • Water is absorbed along with mineral solutes, by the root hairs, purely by diffusion.
  • once water is absorbed by the root hairs, it can move deeper into root layers by two distinct pathways: Apoplast pathway, Symplast pathway.
  • Apoplast is the system of adjacent cell walls that is continuous throughout the plant, except at the casparian strips of the endodermis in the roots.
  • The apoplastic movement of water occurs exclusively through the intercellular spaces and the walls of the cells.
  • Movement through the apoplast does not involve crossing the cell membrane.
  • This movement is dependent on the gradient.
  • The apoplast does not provide any barrier to water movement and water movement is through mass flow.
  • As water evaporates into the intercellular spaces or the atmosphere, tension develop in the continuous stream of water in the apoplast, hence mass flow of water occurs due to the adhesive and cohesive properties of water.
  • The symplastic system is the system of interconnected protoplasts. Neighbouring cells are connected through cytoplasmic strands that extend through plasmodesmata.
  • During symplastic movement, the water travels through the cells – their cytoplasm; intercellular movement is through the plasmodesmata.
  • Water has to enter the cells through the cell membrane, hence the movement is relatively slower.
  • Movement is again down a potential gradient.
  • symplastic movement may be aided by cytoplasmic streaming.

  • Most of the water flow in the roots occurs via the apoplast since the cortical cells are loosely packed, and hence offer no resistance to water movement. However, the inner boundary of the cortex, the endodermis, is impervious to water because of a band of suberised matrix called the casparian strip.
  • Water molecules are unable to penetrate the layer, so they are directed to wall regions that are not suberised, into the cells proper through the membranes.
  • The water then moves through the symplast and again crosses a membrane to reach the cells of the xylem.
  • The movement of water through the root layers is ultimately symplastic in the endodermis. This is the only way water and other solutes can enter the vascular cylinder.
  • Once inside the xylem, water is again free to move between cells as well as through them. In young roots, water enters directly into the xylem vessels and/or tracheids. These are non-living conduits and so are parts of the apoplast.

  • Some plants have additional structures associated with them that help in water (and mineral) absorption. A mycorrhiza is a symbiotic association of a fungus with a root system.
  • The fungal filaments form a network around the young root or they penetrate the root cells. The hyphae have a very large surface area that absorb mineral ions and water from the soil from a much larger volume of soil that perhaps a root cannot do.
  • The fungus provides minerals and water to the roots, in turn the roots provide sugars and N-containing compounds to the mycorrhizae.
  • Some plants have an obligate association with the mycorrhizae. e.g., Pinus seeds cannot germinate and establish without the presence of mycorrhizae.

Water Movement up a Plant

Root Pressure

  • As various ions from the soil are actively transported into the vascular tissues of the roots, water follows (its potential gradient) and increases the pressure inside the xylem. This positive pressure is called root pressure, and can be responsible for pushing up water to small heights in the stem.
  • Effects of root pressure is also observable at night and early morning when evaporation is low, and excess water collects in the form of droplets around special openings of veins near the tip of grass blades, and leaves of many herbaceous parts. Such water loss in its liquid phase is known as guttation.
  • Root pressure can, at best, only provide a modest push in the overall process of water transport. They obviously do not play a major role in water movement up tall trees.
  • The greatest contribution of root pressure may be to re-establish the continuous chains of water molecules in the xylem which often break under the enormous tensions created by transpiration.
  • Root pressure does not account for the majority of water transport; most plants meet their need by transpiratory pull.

Transpiration pull

  • The flow of water upward through the xylem in plants can achieve fairly high rates, up to 15 metres per hour.
  • Most researchers agree that water is mainly ‘pulled’ through the plant, and that the driving force for this process is transpiration from the leaves. This is referred to as the cohesion-tension-transpiration pull model of water transport. But, what generates this transpirational pull?
  • Water is transient in plants. Less than 1 per cent of the water reaching the leaves is used in photosynthesis and plant growth. Most of it is lost through the stomata in the leaves. This water loss is known as transpiration.

TRANSPIRATION

  • Transpiration is the evaporative loss of water by plants. It occurs mainly through the stomata in the leaves.
  • Besides the loss of water vapour in transpiration, exchange of oxygen and carbon dioxide in the leaf also occurs through pores called stomata (sing.: stoma).
  • Normally stomata are open in the day time and close during the night.
  • The immediate cause of the opening or closing of the stomata is a change in the turgidity of the guard cells.
  • The inner wall of each guard cell, towards the pore or stomatal aperture, is thick and elastic. When turgidity increases within the two guard cells flanking each stomatal aperture or pore, the thin outer walls bulge out and force the inner walls into a crescent shape.
  • The opening of the stoma is also aided due to the orientation of the microfibrils in the cell walls of the guard cells. Cellulose microfibrils are oriented radially rather than longitudinally making it easier for the stoma to open.
  • When the guard cells lose turgor, due to water loss (or water stress) the elastic inner walls regain their original shape, the guard cells become flaccid and the stoma closes.

  • Usually the lower surface of a dorsiventral (often dicotyledonous) leaf has a greater number of stomata while in an isobilateral (often monocotyledonous) leaf they are about equal on both surfaces.
  • Transpiration is affected by several external factors: temperature, light, humidity, wind speed. Plant factors that affect transpiration include number and distribution of stomata, number of stomata open, per cent, water status of the plant, canopy structure etc.
  • The transpiration driven ascent of xylem sap depends mainly on the following physical properties of water:
  • Cohesion – mutual attraction between water molecules.
  • Adhesion – attraction of water molecules to polar surfaces (such as the surface of tracheary elements).
  • Surface Tension – water molecules are attracted to each other in the liquid phase more than to water in the gas phase.
  • These properties give water high tensile strength, i.e., an ability to resist a pulling force, and high capillarity, i.e., the ability to rise in thin tubes. In plants capillarity is aided by the small diameter of the tracheary elements – the tracheids and vessel elements.
  • The process of photosynthesis requires water. The system of xylem vessels from the root to the leaf vein can supply the needed water.
  • As water evaporates through the stomata, since the thin film of water over the cells is continuous, it results in pulling of water, molecule by molecule, into the leaf from the xylem.
  • Also, because of lower concentration of water vapour in the atmosphere as compared to the substomatal cavity and intercellular spaces, water diffuses into the surrounding air. This creates a ‘pull’.
  • Measurements reveal that the forces generated by transpiration can create pressures sufficient to lift a xylem sized column of water over 130 metres high.

Transpiration and Photosynthesis – a Compromise

  • Transpiration has more than one purpose; it
    • createstranspiration pull for absorption and transport of plants
    • supplieswater for photosynthesis
    • transportsminerals from the soil to all parts of the plant
    • cools leaf surfaces, sometimes 10 to 15 degrees, by evaporative cooling
    • maintainsthe shape and structure of the plants by keeping cells turgid
  • An actively photosynthesising plant has an insatiable need for water. Photosynthesis is limited by available water which can be swiftly depleted by transpiration.
  • The humidity of rainforests is largely due to this vast cycling of water from root to leaf to atmosphere and back to the soil.
  • The evolution of the C4 photosynthetic system is probably one of the strategies for maximising the availability of CO2 while minimising water loss.
  • C4 plants are twice as efficient as C3 plants in terms of fixing carbon (making sugar). However, a C4 plant loses only half as much water as a C3 plant for the same amount of CO2 fixed.

Uptake and Transport of Mineral Nutrients

  • Plants obtain their carbon and most of their oxygen from CO2 in the atmosphere. However, their remaining nutritional requirements are obtained from minerals and water for hydrogen in the soil.

Uptake of Mineral Ions

  • Unlike water, all minerals cannot be passively absorbed by the roots.
  • Two factors account for this:

(i) minerals are present in the soil as charged particles (ions) which cannot move across cell membranes and
(ii) the concentration of minerals in the soil is usually lower than the concentration of minerals in the root.

  • Therefore, most minerals must enter the root by active absorption into the cytoplasm of epidermal cells.
  • This needs energy in the form of ATP.
  • The active uptake of ions is partly responsible for the water potential gradient in roots, and therefore for the uptake of water by osmosis. Some ions also move into the epidermal cells passively.
  • Ions are absorbed from the soil by both passive and active transport.
  • Specific proteins in the membranes of root hair cells actively pump ions from the soil into the cytoplasms of the epidermal cells.
  • Like all cells, the endodermal cells have many transport proteins embedded in their plasma membrane; they let some solutes cross the membrane, but not others.
  • Transport proteins of endodermal cells are control points, where a plant adjusts the quantity and types of solutes that reach the xylem.
  • the root endodermis because of the layer of suberin has the ability to actively transport ions in one direction only.

Translocation of Mineral Ions

  • After the ions have reached xylem through active or passive uptake, or a combination of the two, their further transport up the stem to all parts of the plant is through the transpiration stream.
  • The chief sinks for the mineral elements are the growing regions of the plant, such as the apical and lateral meristems, young leaves, developing flowers, fruits and seeds, and the storage organs.
  • Unloading of mineral ions occurs at the fine vein endings through diffusion and active uptake by these cells.
  • Mineral ions are frequently remobilised, particularly from older, senescing parts.
  • Older dying leaves export much of their mineral content to younger leaves.
  • Similarly, before leaf fall in decidous plants, minerals are removed to other parts.
  • Elements most readily mobilised are phosphorus, sulphur, nitrogen and potassium.
  • Some elements that are structural components like calcium are not remobilised.
  • An analysis of the xylem exudates shows that though some of the nitrogen travels as inorganic ions, much of it is carried in the organic form as amino acids and related compounds.
  • Similarly, small amounts of P and S are carried as organic compounds.
  • In addition, small amount of exchange of materials does take place between xylem and phloem.
  • Hence, it is not that we can clearly make a distinction and say categorically that xylem transports only inorganic nutrients while phloem transports only organic materials.

Phloem Transport: Flow from Source to Sink

  • Food, primarily sucrose, is transported by the vascular tissue phloem from a source to a sink.
  • Usually the source is understood to be that part of the plant which synthesises the food, i.e., the leaf, and sink, the part that needs or stores the food.
  • But, the source and sink may be reversed depending on the season, or the plant’s needs.
  • Sugar stored in roots may be mobilised to become a source of food in the early spring when the buds of trees, act as sink; they need energy for growth and development of the photosynthetic apparatus.
  • Since the source-sink relationship is variable, the direction of movement in the phloem can be upwards or downwards, i.e., bi-directional. This contrasts with that of the xylem where the movement is always unidirectional, i.e., upwards.
  • Hence, unlike one-way flow of water in transpiration, food in phloem sap can be transported in any required direction so long as there is a source of sugar and a sink able to use, store or remove the sugar.
  • Phloem sap is mainly water and sucrose, but other sugars, hormones and amino acids are also transported or translocated through phloem.

The Pressure Flow or Mass Flow Hypothesis

  • The accepted mechanism used for the translocation of sugars from source to sink is called the pressure flow hypothesis.
  • As glucose is prepared at the source (by photosynthesis) it is converted to sucrose (a dissacharide). The sugar is then moved in the form of sucrose into the companion cells and then into the living phloem sieve tube cells by active transport.
  • This process of loading at the source produces a hypertonic condition in the phloem.
  • Water in the adjacent xylem moves into the phloem by osmosis. As osmotic pressure builds up the phloem sap will move to areas of lower pressure.
  • At the sink osmotic pressure must be reduced. Again active transport is necessary to move the sucrose out of the phloem sap and into the cells which will use the sugar – converting it into energy, starch, or cellulose.
  • As sugars are removed, the osmotic pressure decreases and water moves out of the phloem.
  • the movement of sugars in the phloem begins at the source, where sugars are loaded (actively transported) into a sieve tube. Loading of the phloem sets up a water potential gradient that facilitates the mass movement in the phloem.
  • Phloem tissue is composed of sieve tube cells, which form long columns with holes in their end walls called sieve plates.
  • Cytoplasmic strands pass through the holes in the sieve plates, so forming continuous filaments.
  • As hydrostatic pressure in the phloem sieve tube increases, pressure flow begins, and the sap moves through the phloem.
  • Meanwhile, at the sink, incoming sugars are actively transported out of the phloem and removed as complex carbohydrates.
  • The loss of solute produces a high water potential in the phloem, and water passes out, returning eventually to xylem.
  • A simple experiment, called girdling, was used to identify the tissues through which food is transported.
  • On the trunk of a tree a ring of bark up to a depth of the phloem layer, can be carefully removed.
  • In the absence of downward movement of food, the portion of the bark above the ring on the stem becomes swollen after a few weeks.
  • This simple experiment shows that phloem is the tissue responsible for translocation of food; and that transport takes place in one direction, i.e., towards the roots.

 

PRINTABLE Pdf file of chapter notes are available…please click on the link…

CHAPTER 11- TRANSPORT IN PLANTS

CHAPTER 12 : BIOTECHNOLOGY AND ITS APPLICATIONS

CHAPTER 12

BIOTECHNOLOGY AND ITS APPLICATIONS

  • Biotechnology essentially deals with industrial scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals.
  • The applications of biotechnology include therapeutics, diagnostics, and genetically
    modified crops for agriculture, processed food, bioremediation, waste treatment, and
    energy production.
  • Three critical research areas of biotechnology are:
    (i) Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme.
    (ii) Creating optimal conditions through engineering for a catalyst to act, and
    (iii) Downstream processing technologies to purify the protein/organic compound.

BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE

There are three options that can be thought for increasing food production
(i) agro-chemical based agriculture;
(ii) organic agriculture; and
(iii) Genetically engineered crop-based agriculture.

  •  We have succeeded in tripling the food supply by Green Revolution but yet it was not
    enough to feed the growing human population.
  • Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of better management practices and use of agrochemicals (fertilisers and pesticides).
  • However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with existing varieties are not possible using conventional breeding.
  • So there is a need to find alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields and to minimise the use of fertilisers and chemicals so that their harmful effects on the environment can be reduced. Use of genetically modified crops is a possible solution.
  • Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).
  • Genetic modification has:
    (i) Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
    (ii) Reduced reliance on chemical pesticides (pest-resistant crops).
    (iii) Helped to reduce post-harvest losses.
    (iv) Increased efficiency of mineral usage by plants (this prevents early exhaustion of
    fertility of soil).
    (v) Enhanced nutritional value of food, e.g., Vitamin ‘A’ enriched rice.
    In addition to these uses, GM has been used to create tailor-made plants to supply
    alternative resources to industries, in the form of starches, fuels and pharmaceuticals.
  • By applications of biotechnology in agriculture, pest resistant plants are produced,
    which could decrease the amount of pesticide used.
  • Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short).
  • Bt toxin gene has been cloned from the bacteria and been expressed in plants to
    provide resistance to insects without the need for insecticides; in effect created a
    bio-pesticide. Examples are Bt cotton, Bt corn, rice, tomato, potato and soyabean etc.

Bt Cotton:

  • Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes).
  • B. thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein.
  • This toxin does not kill the Bacillus because this protein exists as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.
  • Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into the several crop plants such as cotton. The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific.
  • The toxin is coded by a gene named cry. There are a number of them, for example, the proteins encoded by the genes crylAc and cryllAb control the cotton bollworms, that of crylAb controls corn borer.

1

Pest Resistant Plants:

  • Several nematodes parasitise a wide variety of plants and animals including human beings.
  • A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a
    great reduction in yield.
  • A novel strategy was adopted to prevent this infestation which was based on the
    process of RNA interference (RNAi).
  • RNAi takes place in all eukaryotic organisms as a method of cellular defense.
  • This method involves silencing of a specific mRNA due to a complementary dsRNA
    molecule that binds to and prevents translation of the mRNA (silencing).
  • The source of this complementary RNA could be from an infection by viruses having
    RNA genomes or mobile genetic elements (transposons) that replicate via an RNA
    intermediate.
  • Using Agrobacterium vectors, nematode-specific genes were introduced into the host
    plant.
  • The introduction of DNA was such that it produced both sense and anti-sense RNA in
    the host cells. These two RNA’s being complementary to each other formed a double
    stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the
    nematode.
  • The consequence was that the parasite could not survive in a transgenic host
    expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

1

BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

  • By enabling mass production of safe and more effective therapeutic drugs.
  • Further, the recombinant therapeutics do not induce unwanted immunological
    responses as is common in case of similar products isolated from non-human sources.
  • At present, about 30 recombinant therapeutics have been approved for human-use the world over. In India, 12 of these are presently being marketed.

    Genetically Engineered Insulin

  • Management of adult-onset diabetes is possible by taking insulin at regular time
    intervals.
  • if enough human-insulin was not available, that one would have to isolate and use
    insulin from other animals.
  • Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and
    pigs.
  • Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein.
  • Insulin consists of two short polypeptide chains: chain A and chain B, which are linked together by disulphide bridges.
  • In mammals, including humans, insulin is synthesised as a prohormone (like a
    pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully
    mature and functional hormone) which contains an extra stretch called the C peptide.
  • This C peptide is not present in the mature insulin and is removed during maturation
    into insulin.
  • The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form.
  • In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

    1.JPG

Gene Therapy

  • Gene therapy is the corrective therapy for hereditary disease.
    Gene therapy is a collection of methods that allows correction of a gene defect that has
    been diagnosed in a child/embryo. Here genes are inserted into a person’s cells and
    tissues to treat a disease.
  • Correction of a genetic defect involves delivery of a normal gene into the individual or
    embryo to take over the function of and compensate for the non-functional gene.
  • The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine
    deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function.
  • The disorder is caused due to the deletion of the gene for adenosine deaminase.
  • ADA deficiency can be cured by bone marrow transplantation or by enzyme
    replacement therapy, in which functional ADA is given to the patient by injection.
    But the problem with both of these approaches that they are not completely curative.
  • In gene therapy, lymphocytes from the blood of the patient are grown in a culture
    outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced
    into these lymphocytes, which are subsequently returned to the patient. However, as
    these cells are not immortal, the patient requires periodic infusion of such genetically
    engineered lymphocytes. However, if the gene isolate from marrow cells producing
    ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Molecular Diagnosis

  • For effective treatment of a disease, early diagnosis and understanding its
    pathophysiology is very important but using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible.
  • Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked
    Immuno-sorbent Assay (ELISA) are some of the techniques that serve the purpose of
    early diagnosis.
  • Presence of a pathogen (bacteria, viruses, etc.) is normally suspected only when the
    pathogen has produced a disease symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus (at a time when the symptoms of the disease are not yet visible) can be detected by amplification of their nucleic acid by PCR.
  • PCR is now routinely used to detect HIV in suspected AIDS patients. It is being used to
    detect mutations in genes in suspected cancer patients too. It is a powerful techqnique
    to identify many other genetic disorders.
  • PCR –
    A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using
    autoradiography. The clone having the mutated gene will hence not appear on the
    photographic film, because the probe will not have complimentarity with the mutated
    gene.
  • ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesised against the pathogen.

TRANSGENIC ANIMALS

  • Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals.
  • Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 per cent of all existing transgenic animals are mice.
  • common reasons to produce transgenic animals:
    (i) Normal physiology and development:
    Transgenic animals can be specifically designed to allow the study of how genes are
    regulated, and how they affect the normal functions of the body and its
    development, e.g., study of complex factors involved in growth such as insulin-like
    growth factor.
    By introducing genes from other species that alter the formation of this factor and
    studying the biological effects that result, information is obtained about the
    biological role of the factor in the body.
    (ii) Study of disease:
    Many transgenic animals are designed to increase our understanding of how genes
    contribute to the development of disease. These are specially made to serve as
    models for human diseases so that Investigation of new treatments for diseases is
    made possible.
    Today transgenic models exist for many human diseases such as cancer, cystic
    fibrosis, rheumatoid arthritis and Alzheimer’s.
    (iii) Biological products:
    Medicines required to treat certain human diseases can contain biological products,
    but such products are often expensive to make.
    Transgenic animals that produce useful biological products can be created by the
    introduction of the portion of DNA (or genes) which codes for a particular product
    such as human protein (α-1-antitrypsin) used to treat emphysema.
    Similar attempts are being made for treatment of phenylketonuria (PKU) and cystic
    fibrosis.
    In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4
    grams per litre). The milk contained the human alpha-lactalbumin and was
    nutritionally a more balanced product for human babies than natural cow-milk.
    (iv) Vaccine safety:
    Transgenic mice are being developed for use in testing the safety of vaccines before
    they are used on humans.
    Transgenic mice are being used to test the safety of the polio vaccine. If successful
    and found to be reliable, they could replace the use of monkeys to test the safety of
    batches of the vaccine.
    (v) Chemical safety testing:
    This is known as toxicity/safety testing. The procedure is the same as that used for
    testing toxicity of drugs.
    Transgenic animals are made that carry genes which make them more sensitive to
    toxic substances than non-transgenic animals. They are then exposed to the toxic
    substances and the effects studied. Toxicity testing in such animals will allow us to
    obtain results in less time.

    ETHICAL ISSUES

    The manipulation of living organisms by the human race cannot go on any further, without
    regulation. Some ethical standards are required to evaluate the morality of all human
    activities that might help or harm living organisms.
    Going beyond the morality of such issues, the biological significance of such things is also
    important. Genetic modification of organisms can have unpredicatable results when such
    organisms are introduced into the ecosystem.
    Therefore, the Indian Government has set up organisations such as GEAC (Genetic
    Engineering Approval Committee), which will make decisions regarding the validity of GM
    research and the safety of introducing GM-organisms for public services.

Bio-patent:

  • The modification/usage of living organisms for public services (as food and medicine
    sources, for example) has also created problems with patents granted for the same.
  • There is growing public anger that certain companies are being granted patents for
    products and technologies that make use of the genetic materials, plants and other
    biological resources that have long been identified, developed and used by farmers and
    indigenous people of a specific region/country.
  • Rice is an important food grain, the presence of which goes back thousands of years in
    Asia’s agricultural history. There are an estimated 200,000 varieties of rice in India
    alone. The diversity of rice in India is one of the richest in the world.
  • Basmati rice is distinct for its unique aroma and flavour and 27 documented varieties of Basmati are grown in India. There is reference to Basmati in ancient texts, folklore and poetry, as it has been grown for centuries.
  • In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed the company to sell a ‘new’ variety of Basmati, in the US and abroad.
  • This ‘new’ variety of Basmati had actually been derived from Indian farmer’s varieties.
    Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or a novelty.
  • The patent extends to functional equivalents, implying that other people selling
    Basmati rice could be restricted by the patent.
  • Several attempts have also been made to patent uses, products and processes based
    on Indian traditional herbal medicines, e.g., turmeric neem.
  • If we are not vigilant and we do not immediately counter these patent applications,
    other countries/individuals may encash on our rich legacy and we may not be able to
    do anything about it.

Biopiracy

  • It is the term used to refer to the use of bio-resources by multinational companies and
    other organisations without proper authorisation from the countries and people
    concerned without compensatory payment.
  • Most of the industrialised nations are rich financially but poor in biodiversity and
    traditional knowledge. In contrast the developing and the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources. Traditional
    knowledge related to bio-resources can be exploited to develop modern applications
    and can also be used to save time, effort and expenditure during their
    commercialisation.
  • There has been growing realisation of the injustice, inadequate compensation and
    benefit sharing between developed and developing countries. Therefore, some nations
    are developing laws to prevent such unauthorised exploitation of their bio-resources
    and traditional knowledge.
  • The Indian Parliament has recently cleared the second amendment of the Indian
    Patents Bill, that takes such issues into consideration, including patent terms
    emergency provisions and research and development initiative.

 

To download notes in pdf format please click on the following link.

CHAPTER 12 : BIOTECHNOLOGY AND ITS APPLICATIONS

CHAPTER 11 : BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Chapter 11

Biotechnology : Principles and Processes

[you can download the notes from the link given at the end of theory]

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

  • Traditional form – based on natural capabilities of microorganisms. making curd, bread or wine, which are all microbe-mediated processes, could also be thought as a form of biotechnology. However, it is used in a restricted sense today,
  • Modern form – it uses genetically modified organisms to achieve the same on a larger scale. Further, many other processes/techniques are also included under biotechnology. For example, in vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it, developing a DNA vaccine or correcting a defective gene, are all part of biotechnology.
  • The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modern molecular biotechnology. The definition given by EFB is as follows:

‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

PRINCIPLES OF BIOTECHNOLOGY

  • Among many, the two core techniques that enabled birth of modern biotechnology are :
    • Genetic engineering: Techniques to alter the chemistry of genetic material (DNA and RNA),to introduce these into host organisms and thus change the phenotype of the host organism.
    • Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc.
  • Sexual reproduction has many advantages over asexual reproduction. The former provides opportunities for variations and formulation of unique combinations of genetic setup, some of which may be beneficial to the organism as well as the population. Asexual reproduction preserves the genetic information, while sexual reproduction permits variation.
  • Traditional hybridisation procedures used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with the desired genes. The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer, overcome this limitation and allow us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.
  • A piece of DNA, which is somehow transferred into an alien organism, most likely would not be able to multiply itself in the progeny cells of the organism. But, when it gets integrated into the genome of the recipient, it may multiply and be inherited along with the host DNA. This is because the alien piece of DNA has become part of a chromosome, which has the ability to replicate.
  • In a chromosome there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication. Therefore, for the multiplication of any alien piece of DNA in an organism it needs to be a part of a chromosome(s) which has a specific sequence known as ‘origin of replication’. Thus, an alien DNA is linked with the origin of replication, so that, this alien piece of DNA can replicate and multiply itself in the host organism. This can also be called as cloning or making multiple identical copies of any template DNA.
  • The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of  Salmonella typhimurium.
  • Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance.
  • The cutting of DNA at specific locations became possible with the discovery of the so-called ‘molecular scissors’- restriction enzymes.
  • The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it. A plasmid can be used as vector to deliver an alien piece of DNA into the host organism.
  • The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA.
  • When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance gene in coli was called cloning of antibiotic resistance gene in E. coli.
  • there are three basic steps in genetically modifying an organism
    • identification of DNA with desirable genes;
    • introduction of the identified DNA into the host;
    • maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

Key tools of Recombinant DNA technology are – restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.

  1. Restriction Enzymes

  • In 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. One of these added methyl groups to DNA, while the other cut DNA. The later was called restriction endonuclease.
  • The first restriction endonuclease isolated – Hind II.
  • Restriction endonuclease cut DNA molecules at a particular point by recognising a specific sequence of base pairs. This specific base sequence is known as the recognition sequence.(For Hind II – sequence of 6 base pairs).
  • Today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Naming of enzymes –

  • First letter of the name comes from the genes
  • The second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13.
  • Next letter derived from the name of strain.
  • Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Action of enzyme –

  • Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds; exonucleasesand endonucleases.
  • Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
  • Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar -phosphate backbones.
  • Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA.
  • The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same. For example, the following sequences reads the same on the two strands in 5→3 This is also true if read in the 3→5direction.

5—— GAATTC —— 3

3—— CTTAAG —— 5

  • Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. There are overhanging stretches called sticky ends on each strand.
  • These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.
  • Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different sources/genomes.
  • When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases .
  • Normally, unless one cuts the vector and the source DNA with the same restriction enzyme, the recombinant vector molecule cannot be created.

1.jpg

Fig: Steps in formation of recombinant DNA by action of restriction endonuclease enzyme – EcoRI

1.jpg

Fig: Diagrammatic representation of recombinant DNA technology

 

Separation and isolation of DNA fragments :

  • The cutting of DNA by restriction endonucleases results in the fragmentes of DNA. These fragments can be separated by a technique known as gel electrophoresis.
  • Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds.
  • The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.
  • The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
  • We can see bright orange coloured bands of DNA in aethidium bromide stained gel exposed to UV light.
  • The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

1.jpg

Fig: A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested set of DNA fragments (lane 2 to 4)
  1. Cloning Vectors

  • Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
  • Bacteriophages because of their high number per cell, have very high copy numbers of their genome within the bacterial cells.
  • If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.
  • Vectors used at present, are engineered in such way that they help easy linking of foreign DNA and selection of recombinants from non-recombinants.

Features required to facilitate cloning into a vector.

Origin of replication (ori):

  • This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells.
  • This sequence is also responsible for controlling the copy number of the linked DNA.
  • So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

    Selectable marker :

  • In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating nontransformants and selectively permitting the growth of the transformants.
  • Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.
  • Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for coli. The normal E. coli cells do not carry resistance against any of these antibiotics.

    Cloning sites:

  • In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.
  • Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning.
  • The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance
  • For example, you can ligate a foreign DNA at the Bam H I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, nonrecombinants will grow on the medium containing both the antibiotics. In this case, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in selection of recombinants.
  • Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
  • In this, a recombinant DNA is inserted within the coding sequence of an enzyme, a-galactosidase. This results into inactivation of the enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the a-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

    Vectors for cloning genes in plants and animals :

  • Viruses and bacteria are used to transfer genes into plants and animals which transform eukaryotic cells and force them to do what the bacteria or viruses want.
  • For example, Agrobacterioumtumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
  • Similarly, retroviruses in animals have the ability to transform normal cells into cancerous
  • A better understanding of the art of delivering genes by pathogens in their eukaryotic hosts has generated knowledge to transform these tools of pathogens into useful vectors for delivering genes of interest to humans.
  • The tumor inducing (Ti) plasmid of Agrobacterium tumifacienshas now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants. Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells. So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).
    1.jpg
Fig: E. coli cloning vector pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I, PvuII, PstI, ClaI), ori and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid.
  1. Competent Host (For Transformation with Recombinant DNA)

  • Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA.
  • This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
  • Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 420oC (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
  • In micro-injection method, recombinant DNA is directly injected into the nucleus of an animal cell.
  • In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA in a method known as biolisticsor gene gun.
  • And the last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology involves several steps in specific sequence such as –

  • isolation of DNA,
  • fragmentation of DNA by restriction endonucleases,
  • isolation of a desired DNA fragment,
  • ligation of the DNA fragment into a vector,
  • transferring the recombinant DNA into the host,
  • culturing the host cells in a medium at large scale and
  • extraction of the desired product.
  1. Isolation of the Genetic Material (DNA)

  • Nucleic acid is the genetic material of all organisms without exception. In majority of organisms this is deoxyribonucleic acid or DNA.
  • In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules. Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase(plant cells), chitinase(fungus).
  • genes are located on long molecules of DNA interwined with proteins such as histones.
  • RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension.

1.jpg

Fig: DNA thatseparates out can beremoved by spooling
  1. Cutting of DNA at Specific Locations

  • Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.
  • Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).
  • The process is repeated with the vector DNA also.
  • The joining of DNA involves several processes. After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added. This results in the preparation of recombinant DNA.
  1. Amplification of Gene of Interest using PCR (Polymerase Chain Reaction)

  • In this reaction, multiple copies of the gene (or DNA) of interest is synthesisedin vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase.
  • The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
  • If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times.
  • Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermusaquaticus), which remain active during the high temperature induced denaturation of double stranded DNA.
  • The amplified fragment if desired can now be used to ligate with a vector for further cloning.

1.jpg

Fig: Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation;

(ii) Primer annealing; and (iii) Extension of primers

  1. Insertion of Recombinant DNA into the Host Cell/Organism

  • There are several methods of introducing the ligated DNA into recipient cells. Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
  • So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into coli cells, the host cells become transformed into ampicillin-resistant cells. If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow, untransformed recipient cells will die. Since, due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin. The ampicillin resistance gene in this case is called a selectable marker.
  1. Obtaining the Foreign Gene Product

  • When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial, plant or animal cell, the alien DNA gets multiplied.
  • In almost all recominant technologies, the ultimate aim is to produce a desirable protein. Hence, there is a need for the recombinant DNA to be expressed.
  • The foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involve understanding many technical details.
  • After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale.
  • If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein.
  • The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.
  • The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.
  • Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
  • A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor.
  • The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

1.jpg

Fig: (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through whichsterile air bubbles are sparged

 

  1. Downstream Processing
  • After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it is ready for marketing as a finished The processes include separation and purification, which are collectively referred to as downstream processing.
  • The product has to be formulated with suitable preservatives. Such formulation has to undergo thorough clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing and quality control testing vary from product to product.

To download notes in pdf format please click on the following link.

CHAPTER 11 : BIOTECHNOLOGY: PRINCIPLES AND PROCESSES