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.

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CHAPTER 13 – PHOTOSYNTHESIS IN HIGHER PLANTS

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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.

 

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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.

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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.

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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.

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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.

 

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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.

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

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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.

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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).
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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.

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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.

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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.

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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.

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CHAPTER 11 : BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

CHAPTER 10 – CELL CYCLE AND CELL DIVISION

CELL CYCLE AND CELL DIVISION

  • Growth and reproduction are characteristics of living cells and organisms.

Cell Cycle –

  • The sequence of events by which a cell duplicates its genome, synthesizes the other constituents of the cell and eventually divides into two daughter cells is termed cell cycle.
  • Cell cycle includes three processes cell division, DNA replication and cell growth in coordinated way.
  • Duration of cell cycle can vary from organism to organism and also from cell type to cell type. (e.g., in Yeast cell cycle is of 90 minutes, in human 24 hrs.)

1

Interphase

  • It is divided into 3 further phases G1, S, and G2.

G1 phase (Gap 1 Phase)

  • Corresponds to the interval between mitosis and initiation of DNA replication.
  • During G1 phase the cell is metabolically active and continuously grows but does not replicate its DNA.

S phase (synthesis phase)

  • period during which DNA synthesis or replication takes place.
  • During this time the amount of DNA per cell doubles. (only amount of DNA is doubled, no of chromosomes remain same.)
  • In animal cells, during the S phase, DNA replication begins in the nucleus, and the centriole duplicates in the cytoplasm.

G2 phase (Gap 2 Phase)

  • Proteins are synthesised in preparation for mitosis while cell growth continues.

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  • Some cells do not exhibit division like heart cells, nerve cells etc. these cells enter in an inactive phase called G0 or quiescent phase from G1 phase.
  • Cells in this phase are metabolically active but they do not divide unless they are called on to do so.

Mitosis or M phase

  • In animals, mitotic cell division is only seen in the diploid somatic cells while in the plants mitotic divisions can be seen in both haploid and diploid cells.
  • it is also called as equational division as the number of chromosomes in the parent and progeny cells are the same.
  • Mitosis is divided into the following four stages:
    • Prophase
    • Metaphase
    • Anaphase
    • Telophase

Prophase

  • It follows the S and G2 phases of interphase.
  • The centrioles now begin to move towards opposite poles of the cell.
  • In prophase Chromosomal material condenses to form compact mitotic chromosomes.
  • Initiation of the assembly of mitotic spindle with the help of the microtubules.
  • Cell organelles like Golgi complexes, endoplasmic reticulum, nucleolus and the nuclear envelope disappear.

Metaphase

  • Start of metaphase is marked by the complete disintegration of the nuclear envelope.
  • The chromosomes are spread through the cytoplasm of the cell.
  • condensation of chromosomes is completed and they can be observed clearly under the microscope.
  • This is the stage at which morphology of chromosomes is most easily studied.
  • At this stage, metaphase chromosome is made up of two sister chromatids, which are held together by the centromere.
  • centromere serve as the sites of attachment of spindle fibres to the chromosomes.
  • chromosomes are moved into position at the centre of the cell.
  • the metaphase is characterised by all the chromosomes coming to lie at the equator with one chromatid of each chromosome connected by its kinetochore to spindle fibres from one pole and its sister chromatid connected by its kinetochore to spindle fibres from the opposite pole.
  • The plane of alignment of the chromosomes at metaphase is referred to as the metaphase plate or equatorial plate.

Anaphase

  • At the onset of anaphase, each chromosome arranged at the metaphase plate is split simultaneously and the two daughter chromatids begin to move towards the two opposite poles.
  • As each chromosome moves away from the equatorial plate, the centromere of each chromosome is towards the pole and hence at the leading edge, with the arms of the chromosome trailing behind

Telophase

  • At the beginning of telophase, the chromosomes at their respective poles decondense and form chromatin network.
  • Nuclear envelope assembles around the chromatin network.
  • Nucleolus, Golgi complex and ER etc cell organelles reform.

Cytokinesis

  • After karyokinesis the cell itself is divided into two daughter cells by a separate process called cytokinesis.
  • In an animal cell, this is achieved by the appearance of a furrow in the plasma membrane.
  • The furrow gradually deepens and ultimately joins in the centre dividing the cell cytoplasm into two.
  • Plant cells undergo cytokinesis by cell plate method. In cell plate method wall formation starts in the centre of the cell and grows outward to meet the existing lateral walls.
  • The formation of the new cell wall begins with the formation of a simple precursor, called the cell-plate that represents the middle lamella between the walls of two adjacent cells.
  • At the time of cytoplasmic division, organelles like mitochondria and plastids get distributed between the two daughter cells.
  • In some organisms karyokinesis is not followed by cytokinesis as a result of which multinucleate condition arises leading to the formation of syncytium (e.g., liquid endosperm in coconut). (should be coenocytic)

Significance of mitosis

  • Mitosis results in the production of diploid daughter cells with identical genetic complement usually.
  • The growth of multicellular organisms is due to mitosis.
  • Cell growth results in disturbing the ratio between the nucleus and the cytoplasm. Therefore, cell divide to restore the nucleo-cytoplasmic ratio.
  • mitosis is important in cell repair. The cells of the upper layer of the epidermis, cells of the lining of the gut, and blood cells are being constantly replaced.
  • Mitotic divisions in the meristematic tissues – the apical and the lateral cambium, result in a continuous growth of plants throughout their life.

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Meiosis

  • The specialised kind of cell division that reduces the chromosome number by half results in the production of haploid daughter cells called
  • It is responsible for formation of haploid gametes, which during sexual reproduction form diploid zygote by fusion.
  • Meiosis involves two sequential cycles of nuclear and cell division called meiosis I and meiosis II but only a single cycle of DNA replication.
  • Interphase of meiosis is similar to interphase of mitosis.

 

Meiosis I

Prophase I

  • Prophase of the meiosis I division is typically longer and more complex than prophase of mitosis.
  • It has been further subdivided into the following five phases based on chromosomal behavior.

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 Metaphase I:

  • The bivalent chromosomes align on the equatorial plate.
  • The microtubules from the opposite poles of the spindle attach to the pair of homologous chromosomes.

Anaphase I:

  • The homologous chromosomes separate, while sister chromatids remain associated at their centromeres.

Telophase I

  • The nuclear membrane and nucleolus reappear.
  • cytokinesis follows telophase I.
  • Although in many cases the chromosomes do undergo some dispersion, they do not reach the extremely extended state of the interphase nucleus. The stage between the two meiotic divisions is called interkinesis and is generally short lived.
  • Interkinesis is followed by prophase II, a much simpler prophase than prophase I.

 

Meiosis II

Meiosis II resembles a normal mitosis.

Prophase II:

  • Meiosis II is initiated immediately after cytokinesis.
  • The nuclear membrane disappears by the end of prophase II.
  • The chromosomes again become compact.

Metaphase II:

  • At this stage the chromosomes align at the equator and the microtubules from opposite poles of the spindle get attached to the kinetochores of sister chromatids.

Anaphase II:

  • splitting of the centromere of each chromosome.
  • Chromosomes move toward opposite poles of the cell.

Telophase II:

  • the two groups of chromosomes once again get enclosed by a nuclear envelope.
  • cytokinesis follows resulting in the formation of four haploid daughter cells).

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SIGNIFICANCE OF MEIOSIS

  • by meiosis conservation of specific chromosome number of each species is achieved across generations in sexually reproducing organisms.
  • It also increases the genetic variability in the population of organisms from one generation to the next. Variations are very important for the process of evolution.

 

 

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CHAPTER 10 – CELL CYCLE AND CELL DIVISION

 

CHAPTER 9 – BIOMOLECULES

BIOMOLECULES

  • All living organisms are made up of similar elements
  • In living organisms Carbon and Hydrogen are in abundance with respect to other elements.           

            How to Analyse Chemical Composition?

  • To analyze the chemical composition, We can take any living tissue and grind it in trichloroacetic acid (Cl3CCOOH) using a mortar and a pestle. We obtain a thick slurry. If we were to strain this through a cheesecloth or cotton we would obtain two fractions
  1. filtrate or the acid-soluble pool,
  2. retentate or the acid-insoluble fraction.
  • Scientists have found thousands of organic compounds in the acid-soluble pool.
  • All the carbon compounds that we get from living tissues can be called ‘biomolecules’.

Living organisms have also got inorganic elements and compounds in them.

  • Wet weight – weight of living tissue/structure.
  • Dry Weight – weight of structure after drying it. (Wet weight – water).
  • Ash – ifthe tissue is fully burnt, all the carbon compounds are oxidised to gaseous form (CO2, water vapour) and are removed. What is remaining is called ‘ash’. This ash contains inorganic elements (like calcium, magnesium etc). (Dry weight – carbon compound)

Table : A Comparison of Elements Present in Non-living and Living Matter

Element % Weight of
Earth’s crust Human body
Hydrogen (H) 0.14 0.5
Carbon   (C) 0.03 18.5
Oxygen (O) 46.6 65.0
Nitrogen (N) very little 3.3
Sulphur (S) 0.03 0.3
Sodium (Na) 2.8 0.2
Calcium (Ca) 3.6 1.5
Magnesium (Mg) 2.1 0.1
Silicon (Si) 27.7 negligible

 

Table : A List of Representative Inorganic Constituents of Living Tissues

Component Formula
Sodium Na+
Potassium K+
Calcium Ca+2
Magnesium Mg+2
Water H2O
Compounds NaCl, CaCO3, PO4–3, SO4–2

Amino acids

  • Amino acids are organic compounds containing an amino group and an acidic group as substituents on the same carbon i.e., the α-carbon. Hence, they are called α-amino acids. They are substituted methanes.
  • There are four substituent groups occupying the four valency positions. These are hydrogen, carboxyl group, amino group and a variable group designated as R group.
  • Based on the nature of R group there are many amino acids. However, those which occur in proteins are only of twentyone types.
    • R group = hydrogen e.g., glycine
    • R group = methyl group e.g., alanine
    • R group = hydroxy methyl e.g., serine.

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  • The chemical and physical properties of amino acids are essentially of the amino, carboxyl and the R functional groups.
    • Acidic amino acid – glutamic acid etc.
    • Basic amino acid – lysine
    • Neutral amino acid – valine.
    • aromatic amino acids – tyrosine, phenylalanine, tryptophan.
  • A particular property of amino acids is the ionizable nature of-NH2 and -COOH groups. Hence in solutions of different pHs, the structure of amino acids changes.

 

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Lipids

  • Lipids are generally water insoluble. They could be simple fatty acids.
  • A fatty acid has a carboxyl group attached to an R group. The R group could be a methyl (-CH3), or ethyl (-C2H5) or higher number of-CH2 groups (1 carbon to 19 carbons).
    • Palmitic acid has 16 carbons including carboxyl carbon.
    • Arachidonic acid has 20 carbon atoms including the carboxyl carbon.
  • Fatty acids could be saturated (without double bond) or unsaturated (with one or more C=C double bonds).
  • Another simple lipid is glycerol which is trihydroxy propane.
  • Many lipids have both glycerol and fatty acids. Here the fatty acids are found esterified with glycerol. They can be then monoglycerides, diglycerides and triglycerides.
  • These are also called fats and oils based on melting point. Oils have lower melting point (e.g., gingely oil) and hence remain as oil in winters.
  • Some lipids have phosphorous and a phosphorylated organic compound in them. These are phospholipids. They are found in cell membrane. Lecithin is one example.
  • Some tissues especially the neural tissues have lipids with more complex structures.

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Nucleotides

  • Many carbon compounds have heterocyclic rings like nitrogen bases -adenine, guanine, cytosine, uracil, and thymine.
  • When found attached to a sugar, they are called nucleosides.(nucleoside = sugar + nitrogen base). Adenosine, guanosine, thymidine, uridine and cytidine are nucleosides.
  • If a phosphate group is also found esterified to the sugar they are called nucleotides. (Nucleotides = nucleosides + phosphate). Adenylic acid, thymidylic acid, guanylic acid, uridylic acid and cytidylic acid are nucleotides.
  • Nucleic acids like DNA and RNA consist of nucleotides only. DNA and RNA function as genetic material.

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Primary and Secondary Metabolites

  • Primary metabolites – Biomolecules which are present in all organisms and have identifiable functions and play known roles in normal physiologial processes.
  • Secondary metabolites – In plants, fungus and microbes many compounds other than primary metabolites are present. e.g,alkaloides, flavonoides, rubber, essential oils, antibiotics,coloured pigments, scents, gums, spices. The role or functions of all the secondary metabolitesare not known yet. many of them are useful to ‘human welfare’ (e.g., rubber, drugs, spices, scents and pigments). Some secondary metabolites have ecological importance.

 

Table : Some Secondary Metabolites
Pigments Carotenoids, Anthocyanins, etc.
Alkaloids Morphine, Codeine, etc.
Terpenoides Monoterpenes, Diterpenes etc.
Essential oils Lemon grass oil, etc.
Toxins Abrin, Ricin
Lectins Concanavalin A
Drugs Vinblastin, curcumin, etc.
Polymeric substances Rubber, gums, cellulose

BIOMACROMOLECULES

  • There is one feature common to all those compounds found in the acid soluble pool. They have molecular weights ranging from 18 to around 800 daltons (Da) approximately. (Micromolecules) (Mw= <1000 daltons)
  • The acid insoluble fraction, has only four types of organic compounds i.e., proteins, nucleic acids, polysaccharides and lipids. These classes of compounds with the exception of lipids, have molecular weights in the range of ten thousand daltons and above. (Macromolecules) (Mw= >1000 daltons)
  • The molecules in the insoluble fraction with the exception of lipids are polymeric substances.
  • Lipids are small molecular weightcompounds and are present not only as such but also arranged into structures like cell membrane and other membranes. When we grind a tissue, we are disrupting the cell structure. Cell membrane and other membranes are broken into pieces, and form vesicles which are not water soluble. Therefore, these membrane fragments in the form of vesicles get separated along with the acid insoluble pool and hence in the macromolecular fraction. Lipids are not strictly macromolecules.
  • The acid soluble pool represents roughly the cytoplasmic composition. The macromolecules from cytoplasm and organelles become the acid insoluble fraction. Together they represent the entire chemical composition of living tissues or organisms.

Table : Average Composition of Cells

Component % of the total cellular mass
Water 70-90
Proteins 10-15
Carbohydrates 3
Lipids 2
Nucleic acids 5-7
Ions 1

 

PROTEINS

  • Proteins are polypeptides. They are linear chains of amino acids linked by peptide bonds.
  • Each protein is a polymer of amino acids. As there are 21 types of amino acids (e.g., alanine, cysteine, proline, tryptophan, lysine, etc.), a protein is a heteropolymer and not a homopolymer.
  • A homopolymer has only one type of monomer repeating ‘n’ number of times.
  • Amino acids can be essential or non-essential. Essential amino acids are supplied in diet while our body prepares non essential amino acids.
  • Proteins carry out many functions in living organisms, some transport nutrients across cell membrane, some fight infectious organisms, some are hormones, some are enzymes,etc.
  • Collagen is the most abundant protein in animal world.
  • Ribulose bisphosphate Carboxylase-Oxygenase (RUBISCO) is the most abundant protein in the whole of the biosphere.

Table : Some Proteins and their Functions

Protein Functions
Collagen Intercellular ground substance
Trypsin Enzyme
Insulin Hormone
Antibody Fights infectious agents
Receptor Sensory reception (smell, taste, hormone, etc.)
GLUT-4 Enables glucose transport into cells

 

POLYSACCHARIDES

  • Polysaccharides are long chains of sugars. They are threads (literally a cotton thread) containing different monosaccharides as building blocks.

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  • Celluloseis a polymeric polysaccharide consisting of only one type of monosaccharide i.e., glucose. Cellulose is a homopolymer.
  • Starch is a variant of this but present as a store house of energy in plant tissues. Animals have another variant called glycogen.
  • Inulin is a polymer of fructose.
  • In a polysaccharide chain (say glycogen), the right end is called the reducing end and the left end is called the non-reducing end. It has branches.
  • Starch forms helical secondary structures. In fact, starch can hold I2 molecules in the helical portion. The starch-I2 is blue in colour. Cellulose does not contain complex helices and hence cannot hold I2.
  • Plant cell walls are made of cellulose. Paper made from plant pulp is cellulose. Cotton fibre is cellulose.
  • There are more complex polysaccharides in nature. They act as building blocks, amino-sugars and chemically modified sugars (e.g., glucosamine, N-acetyl galactosamine, etc.).
  • Exoskeletons of arthropods, for example, have a complex polysaccharide called chitin. These complex polysaccharides are heteropolymers.

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NUCLEIC ACIDS

  • Present in acid insoluble fraction of all living tissues.
  • These are polynucleotides. For nucleic acids, the building block is a nucleotide. A nucleotide has three chemically distinct components. One is a heterocyclic compound (N2 bases, the second is a monosaccharide and the third a phosphoric acid or phosphate.)
  • Adenine, Guanine, Uracil, Cytosine, and Thymine are N2 containing bases. Adenine and Guanine are substituted purines while the rest are substituted pyrimidines.
  • The sugar found in polynucleotides is either ribose (a monosaccharide pentose) or 2′ deoxyribose.
  • A nucleic acid containing deoxyribose is called deoxyribonucleic acid (DNA) while that which contains ribose is called ribonucleic acid (RNA).

STRUCTURE OF PROTEINS

  • Proteins are heteropolymers containing strings of amino acids. \
  • Biologists describe the protein structure at four levels.
    • Primary structure –

It is linear structure of protein.

the left end represented by the first amino acid and the right end represented by the last amino acid.

The first aminoacid is also called as N-terminal amino acid. The last amino acid is called the C-terminal amino acid.

Secondary structure –

The linear protein thread is folded in the form of a helix (similar to a revolving staircase).In proteins, only right handed helices are observed.

Tertiary structure –

The long protein chain is also folded upon itself like a hollow wollen ball, giving rise to the tertiary structure. This gives us a 3-dimensional view of a protein. Tertiary structure is absolutely necessary for the many biological activities of proteins.

Quaternary structure –

Some proteins are an assembly of more than one polypeptide or subunits. The manner in which these individual folded polypeptides or subunits are arranged with respect to each other (e.g. linear string of spheres, spheres arranged one upon each other in the form of a cube or plate etc.) is the architecture of a protein otherwise called the quaternary structure of a protein.

e.g., Adulthuman haemoglobin consists of 4 subunits. Two of these are identical to each other. Hence, two subunits of α type and two subunits of β type together constitute the human haemoglobin (Hb).

 

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NATURE OF BOND LINKING MONOMERS IN A POLYMER

  • In a polypeptide or a protein, amino acids are linked by a peptide bond which is formed when the carboxyl (-COOH) group of one amino acid reacts with the amino (-NH2) group of the next amino acid with the elimination of a water moiety (the process is called dehydration).
  • In a polysaccharide the individual monosaccharides are linked by a glycosidic bond. This bond is also formed by dehydration. This bond is formed between two carbon atoms of two adjacent monosaccharides.
  • In a nucleicacid a phosphate moiety links the 3′-carbon of one sugar of one nucleotide to the 5′-carbon of the sugar of the succeeding nucleotide. The bond between the phosphate and hydroxyl group of sugar is an ester As there is one such ester bond on either side, it is called phosphodiester bond.

Nucleic acids exhibit a wide variety of secondary structures.

one of the secondary structures exhibited by DNA is the famous Watson-Crick model.

Watson-Crick Model

  • According to this model DNA exists as a double helix. The two strands of polynucleotides are antiparallel i.e., run in the opposite direction.
  • The backbone is formed by the sugar-phosphate-sugar chain.
  • The nitrogen bases are projected more or less perpendicular to this backbone but face inside.
  • A and G of one strand compulsorily base pairswith T and C, respectively, on the other strand. There are two hydrogen bonds between A and T. There are three hydrogen bonds between G and C.
  • Each strand appears like a helical staircase.
  • Each step of ascent is represented by a pair of bases. At each step of ascent, the strand turns 36°.
  • One full turn of the helical strand would involve ten steps or ten base pairs.
  • On drawing a line diagram, the pitch would be 34Å. The rise per base pair would be 3.4Å. This form of DNA with the above mentioned salient features is called B-DNA.
  • There are more than a dozen forms of DNA named after English alphabets with unique structural features.

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DYNAMIC STATE OF BODY CONSTITUENTS – CONCEPT OF METABOLISM

  • living organisms contain thousands of organic compounds. These compounds or biomolecules are present in certain concentrations (expressed as mols/cell or mols/litre etc.).
  • allthese biomolecules have a turn over. This means that they are constantly being changed into some other biomolecules and also made from some other biomoleculesthrough chemical reactions. Together all these chemical reactions are called
  • These metabolic reactions result in the transformation of biomolecules like removal of CO2 from amino acids making an amino acid into an amine, removal of amino group in a nucleotide base; hydrolysis of a glycosidic bond in a disaccharide, etc.
  • Majority of these metabolic reactions are always linked to some other reactions or the metabolites are converted into each other in a series of linked reactions called metabolic pathways.
  • Flow of metabolites through metabolic pathway has a definite rate and direction. This metabolite flow is called the dynamic state of body constituents.
  • Another feature of these metabolic reactions is that every chemical reaction is a catalysed reaction. There is no uncatalysed metabolic conversion in living systems.
  • The catalysts which hasten the rate of a given metabolic conversation are also proteins. These proteins with catalytic power are named

METABOLIC BASIS FOR LIVING

  • Metabolic pathways can lead to a more complex structure from a simpler structure (for example, acetic acid becomes cholesterol) =anabolic pathways,or lead to a simpler structure from a complex structure (for example, glucose becomes lactic acid in our skeletal muscle)=catabolic pathways.
  • Anabolic pathways, as expected, consume energy. While, catabolic pathways lead to the release of energy, which is stored in the form of chemical bonds in ATP(adenosine triphosphate).

THE LIVING STATE

  • Many chemical compounds or metabolites, or biomolecules, are present at concentrations characteristic of each of them.

e.g., the blood concentration of glucose in a normal healthy individual is 4.5-5.0 mM, while that of hormones would be nanograms/ mL.

  • all living organisms exist in a steady-state characterised by concentrations of each of these biomolecules. These biomolecules are in a metabolic flux. Any chemical or physical process moves spontaneously to equilibrium.
  • The steady state is a non-equilibirium state. Because systems at equilibrium cannot perform work.
  • the living state is a non-equilibrium steady-state to be able to perform work; living process is a constant effort to prevent falling into equilibrium. This is achieved by energy input. Metabolism provides a mechanism for the production of energy. Hence the living state and metabolism are synonymous. Without metabolism there cannot be a living state.

 

ENZYMES

  • Almost all enzymes are proteins. There are some nucleic acids that behave like enzymes. These are called ribozymes.
  • An enzyme like any protein has a primary structure,secondary and the tertiary structure.
  • In tertiary structure, the backbone of the protein chain folds upon itself, the chain criss-crosses itself and hence, many crevices or pockets are made. One such pocket is the ‘active site’.
  • An active site of an enzyme is a crevice or pocket into which the substrate fits. Thus enzymes, through their active site, catalyse reactions at a high rate.
  • Enzyme catalysts differ from inorganic catalysts in many ways. Inorganic catalysts work efficiently at high temperatures and high pressures, while enzymes get damaged at high temperatures (above 40°C).

However, enzymes isolated from organisms who normally live under extremely high temperatures (e.g., hot vents and sulphur springs), are stable and retain their catalytic power even at high temperatures (upto 80°-90°C). Thermal stability is thus an important quality of such enzymes isolated from thermophilic organisms.

Chemical Reactions

  • Chemical compounds undergo two types of changes.

A physical change simply refers to a change in shape without breaking of bonds. This is a physical process. Another physical process is a change in state of matter: when ice melts into water, or when water becomes a vapour.

when bonds are broken and new bonds are formed during transformation, this will be called a chemical reaction. For example:

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hydrolysis of starch into glucose is an organic chemical reaction.

  • Rate of a physical or chemical process refers to the amount of product formed per unit time. It can be expressed as:

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Rate can also be called velocity if the direction is specified.

  • Rates of physical and chemical processes are influenced by temperature among other factors.
  • A general rule is that rate doubles or decreases by half for every 10°C change in either direction. Catalysed reactions proceed at rates vastly higher than that of uncatalysed ones. e.g.,

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In the absence of any enzyme this reaction is very slow, with about 200 molecules of H2CO3 being formed in an hour. However, by using the enzyme carbonic anhydrase, the reaction speeds about 600,000 molecules being formed every second.

  • A multistep chemical reaction, when each of the steps is catalysed by the same enzyme complex or different enzymes, is called a metabolic pathway. For example,

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This reaction is actually a metabolic pathway in which glucose becomes pyruvic acid through ten different enzyme catalysed metabolic reactions.

  • This pathway provides different products in different conditions –

In our skeletal muscle, under anaerobic conditions, lactic acid is formed.

Under normal aerobic conditions, pyruvic acid is formed.

In yeast, during fermentation, the same pathway leads to the production of ethanol (alcohol).

 How do Enzymes bring about such High Rates of Chemical Conversions?

  • Enzymes, i.e. proteins with three dimensional structures including an ‘active site’, convert a substrate (S) into a product (P). Symbolically, this can be depicted as:

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  • Substrate ‘S’ has to bind the enzyme at its ‘active site’ within a given cleft or pocket. The substrate has to diffusetowards the ‘active site’.
  • There is thus, an obligatory formation of an ‘ES’ complex. E stands for enzyme. This complex formation is a transient phenomenon.
  • During the state where substrate is bound to the enzyme active site, a new structure of the substrate called transition state structure is formed.
  • Very soon, after the expected bond breaking/making is completed, the product is released from the active site. In other words, the structure of substrate gets transformed into the structure of product(s).
  • There could be many more ‘altered structural states’ between the stable substrate and the product. all otherintermediate structural states are unstable. Stability is something related to energy status of the molecule or the structure.
  • If ‘P’ is at a lower level than’S’, the reaction is an exothermic reaction. One need not supply energy (by heating) in order to form the product.
  • However, whether it is an exothermic or spontaneous reaction or an endothermic or energy requiring reaction, the ‘S’ has to go through a much higher energy state or transition state.
  • The difference in average energy content of’S’ from that of this transition state is called ‘activation energy’.
  • Enzymes eventually bring down this energy barrier making the transition of’S’ to ‘P’ more easy.

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     Nature of Enzyme Action

  • Each enzyme (E) has a substrate (S) binding site in its molecule so that a highly reactive enzyme-substrate complex (ES) is produced. This complex is short-lived and dissociates into its product(s) P and the unchanged enzyme with an intermediate formation of the enzyme-product complex (EP).

The formation of the ES complex is essential for catalysis.

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  • The catalytic cycle of an enzyme action can be described in the following steps:
  1. First, the substrate binds to the active site of the enzyme, fitting into the active site.
  2. The binding of the substrate induces the enzyme to alter its shape, fitting more tightly around the substrate.
  3. The active site of the enzyme, now in close proximity of the substrate breaks the chemical bonds of the substrate and the new enzyme- product complex is formed.
  4. The enzyme releases the products of the reaction and the free enzyme is ready to bind to another molecule of the substrate and run through the catalytic cycle once again.

Factors Affecting Enzyme Activity

Factors affecting Enzyme activity are temperature, pH, change in substrate concentration or binding of specific chemicals that regulate its activity.

  1. Temperature and pH

Enzymes generally function in a narrow range of temperature and pH.

Each enzyme shows its highest activity at a particular temperature and pH called the optimum temperature and optimum pH.

Low temperature preserves the enzyme in a temporarily inactive state whereas high temperature destroys enzymatic activity because proteins are denatured by heat.

  1. Concentration of Substrate

With the increase in substrate concentration, the velocity of the enzymatic reaction rises at first. The reaction ultimately reaches a maximum velocity (Vmax) which is not exceeded by any further rise in concentration of the substrate. This is because the enzyme molecules are fewer than the substrate molecules and after saturation of these molecules, there are no free enzyme molecules to bind with the additional substrate molecules.

The activity of an enzyme is also sensitive to the presence of specific chemicals that bind to the enzyme. When the binding of the chemical shuts off enzyme activity, the process is called inhibition and the chemical is called an inhibitor.

Competitive inhibition –

When the inhibitor closely resembles the substrate in its molecular structure and inhibits the activity of the enzyme, it is known as competitive inhibitor. Due to its close structural similarity with the substrate, the inhibitor competes with the substrate for the substrate-binding site of the enzyme. Consequently, the substrate cannot bind and as a result, the enzyme action declines,

e.g., inhibition of succinic dehydrogenase by malonate which closely resembles the substrate succinate in structure.

Such competitive inhibitors are often used in the control of bacterial pathogens.

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Classification and Nomenclature of Enzymes

Enzymes are divided into 6 classes each with 4-13 subclasses and named accordingly by a four-digit number.

  1. Oxidoreductases/dehydrogenases: Enzymes which catalyse oxidoreduction between two substrates S and S’ e.g.,
  2. Transferases: Enzymes catalysing a transfer of a group, G (other than hydrogen) between a pair of substrate S and S’ e.g.,
  3. Hydrolases: Enzymes catalysing hydrolysis of ester, ether, peptide, glycosidic, C-C, C-halide or P-N bonds.
  4. Lyases: Enzymes that catalyse removal of groups from substrates by mechanisms other than hydrolysis leaving double bonds.
  5. Isomerases: Includes all enzymes catalysing inter-conversion of optical, geometric or positional isomers.
  6. Ligases: Enzymes catalysing the linking together of 2 compounds, e.g., enzymes which catalyse joining of C-O, C-S, C-N, P-O etc. bonds.

   Co-factors        

  • Enzymes are composed of one or several polypeptide chains. However, there are a number of cases in which non-protein constituents called co-factors are bound to the enzyme to make the enzyme catalytically active.
  • In these instances, the protein portion of the enzymes is called the apoenzyme.
  • Three kinds of cofactors may be identified: prosthetic groups, co-enzymes and metal ions.
  • Prosthetic groups are organic compounds and are distinguished from other cofactors in that they are tightly bound to the apoenzyme.

For example, in peroxidase and catalase, which catalyze the breakdown of hydrogen peroxide to water and oxygen, haem is the prosthetic group and it is a part of the active site of the enzyme.

  • Co-enzymes are also organic compounds but their association with the apoenzyme is only transient, usually occurring during the course of catalysis. Furthermore, co-enzymes serve as co-factors in a number of different enzyme catalyzed reactions. The essential chemical components of many coenzymes are vitamins, e.g., coenzyme nicotinamide adenine dinucleotide (NAD) and NADP contain the vitamin niacin.
  • Metal ions – A number of enzymes require metal ions for their activity which form coordination bonds with side chains at the active site and at the same time form one or more cordination bonds with the substrate, e.g., zinc is a cofactor for the proteolytic enzyme carboxypeptidase.

Catalytic activity is lost when the co-factor is removed from the enzyme which testifies that they play a crucial role in the catalytic activity of the enzyme.

 

 

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

CHAPTER 9 – BIOMOLECULES

 

CH 8 – CELL: THE UNIT OF LIFE

CELL: THE UNIT OF LIFE

  • Cell   is —  Basic unit of life

—  Fundamental structural and functional unit of all living organisms.

  • Cytology – study of cell and cellular structures.
  • Types of organisms –

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  • All unicellular organisms are capable of
    • Independent existence.
    • Performing the essential functions of life.

Anything less than a complete structure of a cell does not ensure independent living. Hence, cell is the fundamental structural and functional unit of all living organisms.

  • Some important scientists –
Name of scientist Their work
Robert hooke Discovered cell
Anton von Leeuwenhoek first saw and described a live cell
Robert Brown Discovered nucleus
Schleiden (German botanist), Schwann (British Zoologist) Formulated Cell Theory
  • Robert hooke first time describe about cell in his book ‘Micrographia’. He actually saw cell wall of dead cells not cell itself.

 

  • CELL THEORY

    • Formulated by Schleiden and Schwann.
    • Modified by Rudolf Virchow – he explained that new cells develop from pre existing cells by cell division (Omnis cellula-e cellula).
    • Exception of cell theory – virus, viriods,
  1. All living organisms are composed of cells and products of cells.
  2. Cell is structural unit of life.
  • All cells arise from pre-existing cells.

 

  • CELL SIZE AND SHAPE

    • Smallest cell – mycoplasmas (PPLO – Pleuro Pneumonia Like Organisms)
    • Largest cell – egg of an ostrich.
    • Smallest cell in human body – Red Blood Cell.
    • Largest cell in human body – Ovum.
    • Longest cell in human body – Nerve Cell.

Even shape of cells may vary with the functions they perform.

 

  • TYPES OF CELL

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PROKARYOTIC CELL

  • Represented by Blue Green Algae, mycoplasmas, bacteria etc.

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  • Cell wall
    • Determine shape of cell.
    • Provide strong, structural support
    • Prevent bacteria from bursting or collapsing

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  • Plasma membrane
    • Semipermeable
    • Structurally similar to that of eukaryotes.
  • Mesosomes
    • Formed by extension of plasma membrane into cell.
    • In the form of vesicles, tubules and lamella.
    • Help in cell wall formation, DNA replication and distribution to daughter cells.
    • Also help in respiration, secretion processes, to increase the surface area of the plasma membrane and enzymatic content.
  • Chromatophores
    • Membranous extensions into cytoplasm.
    • Contain pigments.
    • In cyanobacteria.
  • Flagella
    • Present in motile cells.
    • Thin filamentous extensions from their cell wall.
    • Composed of three parts – filament, hook and basal body.
  • Pili and Fimbriae
    • Pili are elongated tubular structure while fimbriae are small bristle like fibres.
    • Help in attachment of bacteria.
  • Ribosomes
    • Associated with the plasma membrane of the cell.
    • Made of two subunits – 50S and 30S units which when present together form 70S.

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  • Site of protein synthesis.
  • Ribosome of a polysome translate the mRNA into protein.

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  • Inclusion bodies
    • For storage of reserve material in prokaryotic cells.
    • These are not bounded by any membrane system and lie free in the cytoplasm.
    • g., phosphate granules, cyanophycean granules and glycogen granules.
    • Gas vacuoles are found in blue green and purple and green photosynthetic bacteria.

 

EUKARYOTIC CELLS

  • Include all the protists, plants, animals and fungi.
  • Extensive compartmentalisation of cytoplasm through the presence of membrane bound organelles present.
  • possess an organised nucleus with a nuclear envelope.
  • genetic material is organised into chromosomes.

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  • Cell wall
    • non-living, rigid structure
    • forms an outer covering for the plasma membrane of fungi and plants.
    • gives shape to the cell and protects the cell from mechanical damage and infection.
    • it also helps in cell-to-cell interaction and provides barrier to undesirable macromolecules.

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  • Layers of cell wall
  1. Middle lamella
  • Outermost
  • Made up of mainly calcium pectate.
  • Holds or glues the different neighbouring cell together.
  1. Primary wall
    • Capable of growth.
    • Present in young cell.
    • Gradually diminishes as cell matures.
    • Madeup of cellulose, hemicelluloses.
    • Present in meristem, pith, cortex etc.
  2. Secondary wall
    • Innermost layer.
    • Lignified (in sclerenchyma, vesels, tracheids), suberinised (casparian strips, endodermis)
    • Suberin, lignin make cell wall impermeable.
    • Present in sclerenchyma, collenchyma, and vessels, tracheids.

 

  • Cell wall and middle lamella maybe traversed by plasmodesmata which connects the cytoplasm of neighbouring cells.

 

  • Cell membrane
    • Mainly composed of bilayer phospholipids, also possess protein and carbohydrate.
    • lipids are arranged within the membrane with the polar head (hydrophilic) towards the outer sides and the nonpolar tails (hydrophobic) towards the inner part.

This ensures that the nonpolar tail of saturated hydrocarbons is protected from the aqueous environment.

  • The ratio of protein and lipid varies in different cell types.

( In human RBC membrane has 52% protein and 40% lipids.)

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  • Structure of cell membrane is explained by Fluid Mosaic Model which was given by Singer and Nicolsan.
  • According to this model the quasi-fluid nature of lipid enables lateral movement of proteins within the overall bilayer.
  • The fluid nature of the membrane is important for functions like cell growth, formation of intercellular junctions, secretion, endocytosis, cell division etc.

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Fluid Mosaic Model

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  • Mitochondria
    • Double membrane bound cell organelle.

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  • Mitochondria are site of aerobic respiration. They produce ATP, hence called ‘Power House Of Cell’.
  • The matrix also possesses single circular DNA molecule, a few RNA molecules, ribosomes (70S) and the components required for the synthesis of proteins. So, mitochondria also known as ‘semi autonomous organelle’.
  • The mitochondria divide by fission and produce new mitochondria.

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  • Plastids
    • Found in all plant cells and in euglenoides.
    • They bear some specific pigments, thus imparting specific colours to the plants.

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  • Chloroplasts are mainly found in the mesophyll cells of the leaves.
  • These are various shaped like lens, oval, spherical, discoid, ribbon.
  • Double membrane bound Cell organelle. Inner is less permeable than outer.

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  • There are also stroma lamellae connecting the thylakoids of the different grana.
  • Stroma also contains small, double-stranded circular DNA molecules and ribosomes (70S). so, it is also known ‘semi autonomous organelle’.

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  • Endoplasmic Reticulum
    • a network or reticulum of tiny tubular structures scattered in the cytoplasm that is called the endoplasmic reticulum (ER)
    • Hence, ER divides the intracellular space into two distinct compartments, i.e., luminal(inside ER) and extra luminal(cytoplasm).

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  • Golgi apparatus
    • Discovered by Camillo Golgi.
    • They consist of many flat, disc-shaped sacs or cisternae stacked parallely.
    • The Golgi cisternae are concentrically arranged near the nucleus with distinct convex cis or the forming face and concave trans or the maturing face, which are interconnected.
    • The golgi apparatus principally performs the function of packaging materials.
    • golgi apparatus remains in close association with the endoplasmic reticulum as materials to be packaged in the form of vesicles from the ER fuse with the cis face of the golgi apparatus and move towards the maturing face.
    • A number of proteins synthesised by ribosomes on the endoplasmic reticulum are modified in the cisternae of the golgi apparatus before they are released from its trans
    • Golgi apparatus is the important site of formation of glycoproteins and glycolipids

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  • Lysosomes
    • These are membrane bound vesicular structures formed by the process of packaging in the golgi apparatus.
    • The isolated lysosomal vesicles have been found to be very rich in almost all types of hydrolytic enzymes (hydrolases – lipases, proteases, carbohydrases) optimally active at the acidic pH.
    • These enzymes are capable of digesting carbohydrates, proteins, lipids and nucleic acids.
  • Vacuoles
    • Membrane-bound space found in the cytoplasm. Membrane known as tonoplast.
    • It contains water, sap, excretory product and other materials not useful for the cell.
    • In plant cells the vacuoles are very large.
    • In plants, the tonoplast facilitates the transport of a number of ions and other materials against concentration gradients into the vacuole.
    • In Amoeba the contractile vacuole is important for excretion.
    • In many cells food vacuoles are formed by engulfing the food particles.

 

  • Ribosome
    • first observed under the electron microscope by George Palade.
    • They are composed of ribonucleic acid (RNA) and proteins.
    • Not Bounded by any membrane.
    • The eukaryotic ribosomes are 80S while the prokaryotic ribosomes are 70S.

(‘S’ stands for the sedimentation coefficient).

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  • Cytoskeleton
    • An elaborate network of filamentous proteinaceous structures present in the cytoplasm
    • Functions are mechanical support, motility, maintenance of the shape of the cell.
  • Cilia and Flagella
    • They are hair like outgrowths of cell membrane responsible for locomotion and movement of cell.
    • Cilia are small structures which work like oars, causing the movement of either the cell or the surrounding fluid. Flagella are comparatively longer.
    • Eukaryotic cilium and flagellum are covered with plasma membrane.
    • Their core called the axoneme, possesses a number of microtubules running parallel to the long axis. The axoneme usually has nine pairs of doublets of radially arranged peripheral microtubules, and a pair of centrally located microtubules. (9+2)
    • Both the cilium and flagellum emerge from centriole-like structure called the basal bodies.

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  • Centrosome and centriole
    • Centrosome is an organelle usually containing two perpendicularly lying centrioles surrounded by amorphous pericentriolar materials.
    • Centriole has an organisation like the cartwheel. They are made up of nine evenly spaced triplet peripheral fibrils of tubulin.
    • The central part of the centriole is also proteinaceous and called the hub, connected with peripheral tubules by radial
    • The centrioles form the basal body of cilia or flagella, and spindle fibres that give rise to spindle apparatus during cell division in animal cells.

 

  • Microbodies
    • Many membrane bound minute vesicles called microbodies that contain various enzymes.
    • They are present in both plant and animal cells.

 

  • Nucleus
    • first described by Robert Brown.
    • the material of the nucleus stained by the basic dyes was given the name chromatin by Flemming.
    • The interphase nucleus has nucleoprotein fibres called chromatin, nuclear matrix and one or more spherical bodies called
    • the nuclear envelope is consists of two parallel membranes with a space inbetween called perinuclear space.
    • The outer membrane usually remains continuous with the endoplasmic reticulum and also bears ribosomes on it.
    • At a number of places the nuclear envelope is interrupted by minute pores. These nuclear pores provide passages for movement of RNA and protein molecules.
    • Normally, there is only one nucleus per cell.Some mature cells even lack nucleus, e.g., erythrocytes of many mammals and sieve tube cells of vascular plants.
    • The nuclear matrix or the nucleoplasm contains nucleolus and chromatin.
    • The nucleoli are spherical structures present in the nucleoplasm. It is non-membrane bound. It is a site for active ribosomal RNA synthesis.
    • During cell division, chromatin network condenses into c
    • Chromatin contains DNA and some basic proteins called histones, some non-histone proteins and also RNA.
    • Every chromosome essentially has a primary constriction or the centromere on the sides of which disc shaped structures called kinetochores are present.

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  • Sometimes a few chromosomes have non-staining secondary constrictions at a constant location. This gives the appearance of a small fragment called the satellite.

 

 

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CHAPTER 8 – CELL: THE UNIT OF LIFE