PHOTOSYNTHESIS IN HIGHER PLANTS

UNIT The description of structure and variation of living organisms over a period of time, ended up as two, apparently irreconcilable perspectives on biology. The two perspectives essentially rested on two levels of organisation of life forms and phenomena. One described at organismic and above level of organisation while the second described at cellular and molecular level of organisation. The first resulted in ecology and related disciplines.

The second resulted in physiology and biochemistry. Description of physiological processes, in flowering plants as an example, is what is given in the chapters in this unit. The processes of photosynthesis, respiration and ultimately plant growth and development are described in molecular terms but in the context of cellular activities and even at organism level. Wherever appropriate, the relation of the physiological processes to environment is also discussed.

PLANT PHYSIOLOGY Chapter Photosynthesis in Higher Plants Chapter Respiration in Plants Chapter Plant Growth and Development MELVIN CALVIN born in Minnesota in April, , received his Ph.D. in Chemistry from the University of Minnesota. He served as Professor of Chemistry at the University of California, Berkeley. Just after world war II, when the world was under shock after the Hiroshima-Nagasaki bombings, and seeing the ill- effects of radio-activity, Calvin and co-workers put radio- activity to beneficial use.

He along with J.A. Bassham studied reactions in green plants forming sugar and other substances from raw materials like carbon dioxide, water and minerals by labelling the carbon dioxide with C14. Calvin proposed that plants change light energy to chemical energy by transferring an electron in an organised array of pigment molecules and other substances. The mapping of the pathway of carbon assimilation in photosynthesis earned him Nobel Prize in .

The principles of photosynthesis as established by Calvin are, at present, being used in studies on renewable resource for energy and materials and basic studies in solar energy research. Melvin Calvin All animals including human beings depend on plants for their food. Have you ever wondered from where plants get their food? Green plants, in fact, have to make or rather synthesise the food they need and all other organisms depend on them for their needs.

The green plants make or rather synthesise the food they need through photosynthesis and are therefore called autotrophs. You have already learnt that the autotrophic nutrition is found only in plants and all other organisms that depend on the green plants for food are heterotrophs. 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: it is the primary source of all food on earth. It is also responsible for the release of oxygen into the atmosphere by green plants. Have you ever thought what would happen if there were no oxygen to breath?

This chapter focusses on the structure of the photosynthetic machinery and the various reactions that transform light energy into chemical energy. WHAT DO WE KNOW? Let us try to find out what we already know about photosynthesis. Some simple experiments you may have done in the earlier classes have shown that chlorophyll (green pigment of the leaf), light and CO2 are required for photosynthesis to occur.

You may have carried out the experiment to look for starch formation in two leaves – a variegated leaf or a leaf that was partially covered with black paper, and exposed to light. On testing these leaves for the presence of starch it was clear that photosynthesis occurred only in the green parts of the leaves in the presence of light. CHAPTER . What do we Know?

. Early Experiments . Where does Photosynthesis take place? .

How many Pigments are involved in Photosynthesis? . What is Light Reaction? .

The Electron Transport . Where are the ATP and NADPH Used? . The C4 Pathway .

hotorespiration . Factors affecting Photosynthesis Another experiment you may have carried out where 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 the presence of starch later in the two parts of the leaf, you must have found that the exposed part of the leaf tested positive for starch while the portion that was in the tube, tested negative.

This showed that CO2 was required for photosynthesis. Can you explain how this conclusion could be drawn? . EARLY EXPERIMENTS It is interesting to learn about those simple experiments that led to a gradual development in our understanding of photosynthesis.

Joseph Priestley ( - ) in performed a series of experiments that revealed the essential role of air in the growth of green plants. Priestley, you may recall, discovered oxygen in . Priestley observed that a candle burning in a closed space – a bell jar, soon gets extinguished (Figure . a, b, c, d).

Similarly, a mouse would soon suffocate in a closed space. He concluded that a burning candle or an animal that breathe the air, both somehow, damage the air. But when he placed a mint plant in the same bell jar, he found that the mouse stayed alive and the candle continued to burn. Priestley hypothesised as follows: Plants restore to the air whatever breathing animals and burning candles remove.

Can you imagine how Priestley would have conducted the experiment using a candle and a plant? Remember, he would need to rekindle the candle to test whether it burns after a few days. How many different ways can you think of to light the candle without disturbing the set-up? Using a similar setup as the one used by Priestley, but by placing it once in the dark and once in the sunlight, Jan Ingenhousz ( - ) showed that sunlight is essential to the plant process that somehow purifies the air fouled by burning candles or breathing animals.

Ingenhousz in an elegant experiment with an aquatic plant showed that in bright sunlight, small bubbles were formed around the green parts while in the dark they did not. Later he identified these bubbles to be of oxygen. Hence he showed that it is only the green part of the plants that could release oxygen. (a) (c) (b) (d) Figure .

Priestley’s experiment It was not until about that Julius von Sachs provided evidence for production of glucose when plants grow. Glucose is usually stored as starch. His later studies showed that the green substance in plants (chlorophyll as we know it now) is located in special bodies (later called chloroplasts) within plant cells. He found that the green parts in plants is where glucose is made, and that the glucose is usually stored as starch.

Now consider the interesting experiments done by T.W Engelmann ( – ). Using a prism he 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. He observed that the bacteria accumulated mainly in the region of blue and red light of the split spectrum.

A first action spectrum of photosynthesis was thus described. It resembles roughly the absorption spectra of chlorophyll a and b (discussed in section . ). By the middle of the nineteenth century the key features of plant photosynthesis were known, namely, that plants could use light energy to make carbohydrates from CO2 and water.

The empirical equation representing the total process of photosynthesis for oxygen evolving organisms was then understood as: CO H O CH O O Light  →  [ ] where [CH2O] represented a carbohydrate (e.g., glucose, a six-carbon sugar). A milestone contribution to the understanding of photosynthesis was that made by a microbiologist, Cornelius van Niel ( - ), who, based on his 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. This can be expressed by: H A CO A CH O H O Light  →  In green plants H2O is the hydrogen donor and is oxidised to O2. Some organisms do not release O2 during photosynthesis.

When H2S, instead is the hydrogen donor for purple and green sulphur bacteria, the ‘oxidation’ product is sulphur or sulphate depending on the organism 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: CO H O C H O H O O Light  →  where C6 H12 O6 represents glucose.

The O2 released is from water; this was proved using radio isotope techniques. Note that this is not a single reaction but description of a multistep process called photosynthesis. Can you explain why twelve molecules of water as substrate are used in the equation given above? WHERE DOES PHOTOSYNTHESIS TAKE PLACE?

You would of course answer: in ‘the green leaf’ or ‘in the chloroplasts’, based on what you earlier read in Chapter . You are definitely right. Photosynthesis does take place in the green leaves of plants but it does so also in other green parts of the plants. Can you name some other parts where you think photosynthesis may occur?

You would recollect from previous unit that the 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. When do you think the chloroplasts will be aligned with their flat surfaces parallel to the walls? When would they be perpendicular to the incident light?

You have studied the structure of chloroplast in Chapter . Within the chloroplast there is membranous system consisting of grana, the stroma lamellae, and the matrix stroma (Figure . ). 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 synthesise sugar, which in turn forms starch. The former set of reactions, since they are directly light driven are called light reactions (photochemical 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 (carbon reactions). However, this should not be construed to mean that they occur in darkness or that they are not light-dependent. Figure . Diagrammatic representation of an electron micrograph of a section of chloroplast Outer membrane Inner membrane Stromal lamella Grana Stroma Ribosomes Starch granule Lipid droplet INVOLVED IN PHOTOSYNTHESIS?

Looking at plants have you ever wondered why and how there are so many shades of green in their leaves – even in the same plant? We can look for an answer to this question by trying to separate the leaf pigments of any green plant through paper chromatography. 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 aaaaa (bright or blue green in the chromatogram), chlorophyll bbbbb (yellow green), xanthophylls (yellow) and carotenoids (yellow to yellow-orange). Let us now see what roles various pigments play in photosynthesis.

Pigments are substances that have an ability to absorb light, at specific wavelengths. Can you guess which is the most abundant plant pigment in the world? Let us study the graph showing the ability of chlorophyll a pigment to absorb lights of different wavelengths (Figure . a).

Of course, you are familiar with the wavelength of the visible spectrum of light as well as the VIBGYOR. From Figure .3a can you determine the wavelength (colour of light) at which chlorophyll a shows the maximum absorption? Does it show another absorption peak at any other wavelengths too? If yes, which one?

Now look at Figure .3b showing the wavelengths at which maximum photosynthesis occurs in a plant. Can you see that the wavelengths at which there is maximum absorption by chlorophyll a, i.e., 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. But by looking at Figure .3c can you say that there is a complete one- to-one overlap between the absorption spectrum of chlorophyll a and the action spectrum of photosynthesis?

Figure .3a Graph showing the absorption spectrum of chlorophyll a, b and the carotenoids Figure .3b Graph showing action spectrum of photosynthesis Figure .3c Graph showing action spectrum of photosynthesis superimposed on absorption spectrum of chlorophyll a 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. Let us see how this happens. 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.

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 protein 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 (Figure . ).

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 nm, hence is called P700, while in PS II it has absorption maxima at nm, and is called P680.

THE ELECTRON TRANSPORT In photosystem II the reaction centre chlorophyll a absorbs 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 Photon Reaction centre Pigment molecules Primary acceptor Figure . The light harvesting complex system consisting of cytochromes (Figure . ).

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 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 acceptor, down the electron transport chain to PS I, excitation of electrons, transfer to another acceptor, and finally down hill to NADP+ reducing it to NADPH + H+ is called the Z scheme, due to its characterstic shape (Figure . ). This shape is formed when all the carriers are placed in a sequence on a redox potential scale.

. . Splitting of Water You would then ask, How does PS II supply electrons continuously? 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 2H+, [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.

H O H O e  →  − We need to emphasise here that the water splitting complex is associated with the PS II, which itself is physically located on the inner side of the membrane of the thylakoid. Then, where are the protons and O2 formed likely to be released – in the lumen? or on the outer side of the membrane? .

. 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 through which Electron transport system - - e acceptor e acceptor Light Photosystem II Photosystem I NADPH NADP+ LHC LHC H O 2e + 2H + [O] - ADP+iP ATP Figure .

Z scheme of light reaction ATP 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 (Figure . ). When only PS I is functional, the electron is circulated within the photosystem and the phosphorylation occurs due to cyclic flow of electrons (Figure . ).

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 (Figure . ).

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 nm are available for excitation. . .

Chemiosmotic Hypothesis Let us now try and understand how actually ATP is synthesised in the chloroplast. The chemiosmotic hypothesis has been put forward to explain the mechanism. Like in respiration, in photosynthesis too, ATP synthesis is linked to development of a proton gradient across a membrane. This time these are the membranes of 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 (Chapter ). Let us understand what causes the proton gradient across the membrane. We need to consider again the processes that take place during the activation of electrons and their transport to determine the steps that cause a proton gradient to develop (Figure .

). (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. Figure . Cyclic photophosphorylation Photosystem I Light e- acceptor Electron transport system Chlorophyll P ADP+iP ATP (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 acceptor 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.

Why are we so interested in the proton gradient? This gradient is important because it is the breakdown of this gradient that leads to the synthesis of ATP. The gradient is broken down due to the movement of protons across the membrane to the stroma through the transmembrane Figure . ATP synthesis through chemiosmosis channel of the CF0 of the ATP synthase.

The ATP synthase enzyme consists of two parts: one called the CF0 is embedded in the thylakoid membrane and forms a transmembrane channel that carries out facilitated diffusion of protons across the membrane. The other portion is called CF1 and protrudes on the outer surface of the thylakoid membrane on the side that faces the stroma. The break down of the gradient provides enough energy to cause a conformational change in the CF1 particle of the ATP synthase, which makes the enzyme synthesise several molecules of energy- packed ATP. Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATP synthase.

Energy is used to pump protons across a membrane, to create a gradient or a high concentration of protons within the thylakoid lumen. ATP synthase has a channel that allows diffusion of protons back across the membrane; this releases enough energy to activate ATP synthase 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. WHERE ARE THE ATP AND NADPH USED?

We learnt that 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 H2O.

You may wonder how this could be verified; it is simple: 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. Can we, hence, say that calling the biosynthetic phase as the dark dark dark dark dark reaction reaction reaction reaction reaction is a misnomer? Discuss this amongst yourselves.

Let us now see how the ATP and NADPH are used in the biosynthetic phase. We saw earlier that CO2 is combined with H2O to produce (CH2O)n or sugars. It was of interest to scientists to find out how this reaction proceeded, or rather what was the first product formed when CO2 is taken into a reaction or fixed. Just after world war II, among the several efforts to put radioisotopes to beneficial use, the work of Melvin Calvin is exemplary.

The use of radioactive 14C by him in algal photosynthesis studies led to the discovery that the first CO2 fixation product was a -carbon organic acid. He also contributed to working out the complete biosynthetic pathway; hence it was called Calvin cycle after him. The first product identified was -phosphoglyceric acid or in short PGA. How many carbon atoms does it have?