Science

Acids, bases and pH

Atomic structure

Biochemical nomenclature

Biotransformation

Carbohydrates

Carnivorous plants

Cell communication

Cell cycle

Chromatography and electrophoresis

Cladistics

Core metabolism

DNA replication

Electron microscopy

Enzymes

Enzyme mechanisms

Essays

Extracellular matrix

Genetics

Genomes

Kinetics

Lipids and membranes

Molecular biology methods

Moles

Natural selection

Nervous system

Nitrogen metabolism

Nuclear structure

Nucleic acids

Oxidative phosphorylation

Phloem

Photomorphogenesis

Photorespiration

Photosynthesis

Plant action potential

Plant circadian rhythms

Plant growth

Plant mineral nutrition

Plant secondary metabolites

Prokaryotes and eukaryotes

Proteins

Protein targeting

Reaction mechanisms

Seeds

Spectroscopy

Statistics

Thermodynamics

Transcription

Translation

Water potential

You are what you eat

Reference

Domains

Genetic code

Geological timeline

Model organisms

Molecules

Plants

Vegetable empire

Steve's Place

Search

Guestbook

Me

Perl

Science

Rants

Photorespiration

Contents

• Photorespiration
• C4 and CAM metabolism
• Diagrams

Photorespiration

Many land plants take up oxygen and release CO₂ in the light. There is a superficial resemblance to true respiration, but the process is much faster. However, it is normally masked by photosynthesis, which is even faster. Photorespiration differs from true respiration. Although plants do respire normally (with mitochondria, etc.) this is useful (produces ATP and NADH), and occurs mostly in the dark. In contrast, photorespiration is wasteful and occurs mostly in the light. Photorespiration appears to serve no useful purpose. Its main effect is to reduce the apparent rate of photosynthesis. Most of our important crops photorespire about half of their potential yield away!

Photorespiration is not easy to see because it is normally masked by photosynthesis. It was discovered accidentally by Decker (1955), who measured the CO₂ exchange of a leaf just after a light was switched off. In the light, there was a steady rate of CO₂ uptake, but when the light was switched off, there was a sudden burst of CO₂ release before settling down to true respiration. The burst peaked at about 3 times the rate of true respiration.

Decker interpreted this as being due to a very rapid rate of respiration, which occurred in light alongside photosynthesis, but did not fall off as rapidly as photosynthesis, when the light went off.

We can measure the height of the CO₂ burst, but this isn't hugely accurate, as photorespiration is already declining (it's at its greatest in bright light). Alternatively we can measure the rate of CO₂ release into a CO₂-free air in the light - photorespiration shows as a steady CO₂ output with no photosynthesis to mask it - but some of the CO₂ released will be refixed before it can be measured. Both techniques underestimate photorespiration, but both give high figures: half of the photosynthetic rate.

In the dark, respiration rises from 0 to 3% O₂, then levels off. This is typical of true respiration: provided the O₂ concentration is above a critical level, the rate of respiration is controlled by ATP requirement, not oxygen availability.

In the light, respiration (defined as the consumption of oxygen) rises all the way from 0 to 100% O₂. Most of the oxygen uptake in the light is not due to true respiration: the rate is controlled by oxygen availability, not ATP requirement.

Ogren and Bowes (1971) noticed that RuBP carboxylase was competitively inhibited by oxygen. Further work by this group and their rivals, Andrews, Lorimer and Tolbert showed that oxygen was acting as an alternative substrate for this enzyme, and renamed the enzyme Rubisco: ribulose-1,5-bis-phosphate carboxylase/oxygenase.

Instead of producing 2 molecules of PGA, oxygenase activity produces 1 molecule of PGA, and one of a C₂ compound, phosphoglycolate (below). Rubisco malfunctions because it cannot completely distinguish between CO₂ and O₂.

Under normal atmospheric conditions, there is one oxygenase reaction for every two carboxylase reactions, i.e. a third of RuBP is destroyed without fixing CO₂. Consequently, 2 carbon atoms would be lost by the oxygenase for every 2 fixed by the carboxylase if the phosphoglycolate were simply discarded. This would cause the Calvin cycle to rapidly grind to a halt. This indicates that phosphoglycolate must be re-metabolised and returned to the Calvin cycle, and it is this metabolic pathways that causes the 'symptom' (CO₂ production) of photorespiration.

Phosphoglycolate and PGA must be returned to the Calvin cycle to regenerate RuBP. PGA is a normal Calvin cycle intermediate and can feed straight back in; however, it feeds in before the reductive stage in the cycle so ATP and NADPH must be spent, so this is wasteful.

Phosphoglycolate is not a Calvin cycle intermediate. Two molecules of phosphoglycolate are converted to one of PGA and one of CO₂, which enter the Calvin cycle. The overall reaction (keep this in mind!) is approximately:

Phosphoglycolate + O₂ + ATP → PGA + CO₂ + ADP + 2P_i

The pathway is surprisingly complex, uses a lot of energy, and takes place in no less than three organelles: chloroplasts, peroxisomes, and mitochondria.

1. Chloroplasts

The first stage occurs in the chloroplast. Phosphoglycolate is hydrolysed by a phosphatase to glycolate. This effectively wastes the ATP that was originally used to phosphorylate RuBP.

2. Peroxisomes

Glycolate leaves the chloroplasts and enters the peroxisomes. These are small organelles surrounded by a single membrane, of which there are many different kinds. Some contain catalase to destroy hydrogen peroxide (in fact, there is so much enzyme in them, it crystallises out, giving a characteristic shape under EM).

Leaf peroxisomes contain glycolic oxidase which consumes glycolate and O₂ and produces H₂O₂. The H₂O₂ is degraded by catalase to O₂. There is a net uptake of oxygen at this point. There is then a short pathway which aminates glycolate to glycine by adding an amino group.

3. Mitochondria

The glycine leaves the peroxisomes and enters the mitochondria. It is converted to serine, via a complicated pair of reaction involving the C₁ metabolism (folic acid cofactor).

2 Glycine (C₂) + NAD → Serine (C₃) + CO₂ + NH₃

This reaction is the origin of the CO₂ released in photorespiration.

4. Peroxisomes again

Serine leaves the mitochondria and returns to the peroxisomes. It loses its amino group and is reduced to glycerate by NADH. The last two reactions are coupled to the amination of glycine at stage 2, so this pathway doesn't actually use up any nitrogen or NADH.

5. Back to the chloroplasts

Glycerate then returns to the chloroplasts, where it is phosphorylated to PGA using ATP. It then has to go around the Calvin cycle, using even more ATP and NADPH, before arriving back at RuBP.

The whole pathway to recover RuBP from PGA/phosphoglycolate is very expensive. In round figures, a plant uses as much energy in the photorespiratory pathway as it does in the fixation of CO₂.

How could such an inefficient process evolve? Rubisco evolved in photosynthetic sulfur bacteria before there was any oxygen in the earth's atmosphere. Later, when oxygenic photosynthesis evolved in the cyanobacteria, atmospheric oxygen concentrations increased, and oxygenase reaction began to occur. Initially, there was selection to increase the specificity of Rubisco for CO₂ - photorespiratory rates are much higher in modern green plants than in anaerobic bacteria - but this increase in specificity was at the expense of the rate of the carboxylase reaction.

Unfortunately, if the active site is modified to reduce access to oxygen, it may also reduce the access to CO₂. Plants compensate for this by increasing the concentration of Rubisco. Rubisco comprises half of the protein in the chloroplast, which makes it the most abundant protein on earth. For all practical purposes, Rubisco has reached the limit of its specificity.

The evolution of phosphoglycolate recovery is instructive: The glycine/serine conversion to PGA had already evolved to allow catabolism of protein for respiration. Glyoxylate feeds in via a shortcut through the Krebs cycle. In fact, all that was needed to regenerate PGA from phosphoglycolate was one new enzyme, phosphoglycolate phosphatase. This explains the curious pathway, and the seemingly unnecessary involvement of amino acids. Other less cumbersome pathways might be possible, but these would have to evolve from scratch, and evolution doesn't like running up a hill in the adaptive landscape. Natural selection adopts the most expedient solutions, not the most elegant.

There are other ways to reduce photorespiration. Algae inhibit the oxygenase reaction by concentrating CO₂. Ion pumps in the plasmalemma actively pump in HCO₃⁻, and carbonic anhydrase converts this to CO₂. Consequently, most algae show only low levels of photorespiration. When plants became terrestrial, they were no longer surrounded by a sea of bicarbonate, so they had to evolve other mechanisms.

As a side note, if the oxygenase reaction could be prevented, a crop plant would have twice as much energy to spend on CO₂ fixation. Crop yields could be doubled. Can we do this? Yes: we can grow plants in 4% oxygen (just enough for dark respiration) instead of 21%, but this is not used commercially, because the cost of the atmosphere greatly exceeds the value of the extra yield (and someone, somewhere would inevitably end up suffocating in a greenhouse). If a reduction in O₂ has this effect, so should an increase in CO₂, and this is routinely done for many greenhouse crops, by enriching the CO₂ in the atmosphere from 0.04% to 0.12%. This requires very little extra CO₂, but it is a threefold increase in CO₂'s ability to compete with O₂. The cost is often much less than the value of increase in yield.

The final way of reducing photorepiration is to use a different receptor for CO₂ instead of Rubisco, and if possible, to do it in the dark. This biochemical pumping mechanism is used before CO₂ is presented to Rubisco. The first-formed product of C4 photosynthesis is a C4 organic acid. C4 plants do not photorespire significantly, and this makes them produce double the yield of C3 (PGA producing) plants. Unfortunately they are nearly all tropical.

C4 and CAM metabolism

C4 photosynthesis is an adaptation to growth in hot climates. The first products of photosynthesis are C4 acids: 'normal' plants produce PGA, a 3 carbon acid, and hence are termed C3 plants. C4 photosynthesis has evolved independently in at least 16 families of flowering plants. C4 metabolism is a way of getting round the problem of Rubisco's unfortunate oxygenase activity. C4 plants concentrate CO₂ biochemically in a variety of ways, so Rubisco is exposed to a very high CO₂/O₂ ratio, which inhibits photorespiration.

Some of the most productive crops are C4, including maize and sugarcane; as are some of the worst weeds, such as Amaranthus (love-lies-bleeding) and Tribulus terrestris. Provided with a high light intensity, their maximum rates of photosynthesis may be double those of C3 plants.

C4 also metabolism reduces transpiration by lowering the CO₂ compensation point. Photosynthesis is slower when there is less CO₂ about, and the compensation point is defined as the [CO₂] needed to give net photosynthesis.

At the compensation point, photosynthesis is just too slow to outrun (mostly photo)respiration. C3 plants photorespire so much, that their compensation point is c. 0.005%. C4 plants do not photorespire, so they have near zero compensation points.

Stomata close under dry conditions, and when this happens, the levels of CO₂ in the mesophyll air spaces fall towards the compensation point. Since this is lower in C4 plants, they can photosynthesise with narrower stomatal apertures, so only lose half as much water per CO₂ fixed as C3 plants. So although we may regard the C4 syndrome as an adaptation that reduces photorespiration, as far as plants are 'concerned', it is an adaptation to reducing water loss in hot climates.

All C4 plants show kranz anatomy. The bulk of the photosynthetic tissue is concentrated in two layers around the vascular bundles: the inner bundle-sheath layer and the outer mesophyll layer. C4 leaf veins show up as a darker green than the rest of the leaf: you can usually spot this just by holding a leaf up to the light.

C4 photosynthesis is found in both monocotyledons and dicotyledons. Most of the economically important ones are found in the Poaceae (grasses), and there are some interesting intermediates between C3 and C4, such as Panicum milioides. The genus Atriplex contains both C3 and C4 types which are still closely enough related to interbreed.

The C4 syndrome was discovered when radioactive ¹⁴CO₂ was fed to sugarcane, and the first products seen were C4 acids, not C3 PGA.

The receptor for CO₂ in C4 plants is not RuBP, but phosphoenolpyruvate (PEP), which is generally carboxylated by PEP carboxylase.

PEP + CO₂ + H₂O → Oxaloacetate + P_i

Oxaloacetate is then either reduced to malate; or transaminated to aspartate.

CO₂ fixation into C4 acids occurs in the mesophyll. C4 acids are translocated into the bundle-sheath, through plasmodesmata, where they are decarboxylated to give CO₂, and this CO₂ enters the Calvin cycle as normal. The C₃ fragment is returned to the mesophyll and is metabolised back to PEP.

There are several advantages to C4 metabolism. Firstly CO₂ is released at a much higher concentration (about 10 times atmospheric) in the bundle sheath, where is competes better with oxygen for Rubisco (Rubisco is only found in the bundle sheath). Secondly, it allows net photosynthesis with less carbon dioxide, so the stomatal aperture may be held more nearly closed, and less water is lost. However, there are also disadvantages (or else almost all plants would run C4). The C₃ acids produced by the decarboxylation stage must be converted back to PEP. There are three ways of doing this, and they all use up ATP. This means C4 plants need higher light levels to generate the extra ATP. This helps to explain why they are found only in hot, sunny climates.

1. NADP-ME in maize

This is the first of the three C4 metabolic routes. PEP is carboxylated to oxaloacetate, reduced to malate and shunted into the bundle sheath. The malate is then decarboxylated to pyruvate by NADP-linked malic enzyme in the bundle-sheath chloroplasts. This generates NADPH, so the chloroplasts lack PSII, and make no oxygen in situ either, which is an unexpected bonus. Pyruvate is then translocated back to the mesophyll, and phosphorylated back to PEP, using 2 ATP.

2. NAD-ME in millet

PEP is carboxylated to oxaloacetate, transaminated to aspartate and shunted into the bundle sheath. Aspartate then is transaminated back to oxaloacetate in the mesophyll, reduced to malate by NADH; and decarboxylated to pyruvate by NAD-linked malic enzyme in the bundle-sheath mitochondria. There is no net production of NAD(P)H so the chloroplasts have PSII and may be less effective at inhibiting photorespiration. Pyruvate is phosphorylated in the mesophyll back to PEP, using 2 ATP.

3. PEP-carboxykinase in Guinea grass

PEP is carboxylated to oxaloacetate and shunted into the mesophyll. The oxaloacetate is decarboxylated and phosphorylated to PEP in one step using PEP carboxykinase in the cytosol. This only uses 1 ATP and may be more energy-efficient than the other types; however, the reaction is freely reversible, so may not generate such a high level of CO₂ in the bundle-sheath.

There is a second way of inhibiting photorespiration and transpiration, and that is using a process termed CAM. Whereas C4 metabolism physically separates CO₂ fixation and RuBP carboxylation - CO₂ is fixed in the mesophyll, and RuBP is carboxylated in the bundle-sheath - CAM (crassulacean acid metabolism) plants separate these processes in time. CAM is even better than C4 at inhibiting photorespiration, and is found in plants from really arid regions.

• Cacti.
• Crassula.
• Pineapple.

The biochemistry of CAM is very similar to C4. PEP carboxylase generates oxaloacetate; this is reduced to malate; and malate is split by NADP-malic enzyme to make pyruvate and CO₂. Hence photorespiration is inhibited. However, the anatomy is nothing special: there is no kranz anatomy; vacuoles in the mesophyll are often large, but not always. So how do they manage to inhibit photorespiration? The clue is in the fact that the pH of the vacuolar sap increases during the day and decreases at night. CAM plants synthesise acids (malate) during the night, and these are used up during the day. This is a temporal separation of fixation and RuBP carboxylation.

CO₂ is absorbed by PEP carboxylase during the night when the stomata are open. Malate is stored in the vacuole until morning and used to generate CO₂ during the day. The stomata can stay closed all day, increasing the internal [CO₂], and this reduces photorespiration and water loss hugely.

That concludes this little foray into the joys of photosynthesis and respiration. Although these processes may seem very complex at first glance, I hope this essay has helped to show their underlying similarities, and the fantastically ad hoc nature of evolved systems.

Diagrams

You may find these diagrams useful:

• Photorespiration.
• Calvin cycle.

Test yourself

1. How does photorespiration differ from true respiration?
2. Why is photorespiration wasteful?
3. Why is barley a C₃ plant, whilst maize is a C4 plant?
4. Why don't you find many CAM plants in the UK?

Answers

Bibliography

• Berg, J. M., Tymoczko, J. L. and Stryer, L. (2006). Biochemistry. 6th edition. W. H. Freeman and Company, New York. 574-577. "The activity of the Calvin cycle depends on environmental conditions"
• Taiz, L. and Zeiger, E. (2002). Plant Physiology. 3rd edition. Sinauer Associates Incorporated, Sunderland, Massachusetts. 145-170. "Photosynthesis: carbon reactions"