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 : BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Chapter 11

Biotechnology : Principles and Processes

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

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

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

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

PRINCIPLES OF BIOTECHNOLOGY

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

TOOLS OF RECOMBINANT DNA TECHNOLOGY

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

  1. Restriction Enzymes

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

Naming of enzymes –

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

Action of enzyme –

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

5—— GAATTC —— 3

3—— CTTAAG —— 5

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

1.jpg

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

1.jpg

Fig: Diagrammatic representation of recombinant DNA technology

 

Separation and isolation of DNA fragments :

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

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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.

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

CHAPTER 11 : BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

NCERT class 11th (ENGLISH)

NCERT TEXT BOOK

Biology

class 11th (ENGLISH)

CONTENT PAGE

UNIT I – DIVERSITY IN THE LIVING WORLD

Chapter 1 : The Living World

Chapter 2 : Biological Classification

Chapter 3 : Plant Kingdom

Chapter 4 : Animal Kingdom

UNIT II – STRUCTURAL ORGANISATION IN PLANTS AND ANIMALS

Chapter 5 : Morphology of Flowering Plants

Chapter 6 : Anatomy of Flowering Plants

Chapter 7 : Structural Organisation in Animals

UNIT III – CELL : STRUCTURE AND FUNCTIONS

Chapter 8 : Cell : The Unit of Life

Chapter 9 : Biomolecules

Chapter 10 : Cell Cycle and Cell Division

UNIT IV – PLANT PHYSIOLOGY

Chapter 11 : Transport in Plants

Chapter 12 : Mineral Nutrition

Chapter 13 : Photosynthesis in Higher Plants

Chapter 14 : Respiration in Plants

Chapter 15 : Plant Growth and Development

UNIT V – HUMAN PHYSIOLOGY

Chapter 16 : Digestion and Absorption

Chapter 17 : Breathing and Exchange of Gases

Chapter 18 : Body Fluids and Circulation

Chapter 19 : Excretory Products and their Elimination

Chapter 20 : Locomotion and Movement

Chapter 21 : Neural Control and Coordination

Chapter 22 : Chemical Coordination and Integration