Marshall D. Hatch

3 - Enzymes of C4 Photosynthesis

[Extract] The history of the resolution of C4 photosynthesis follows a pattern of demonstrating the operation of unique photosynthetic biochemistry by various means and then identifying the enzymes necessary to support that biochemistry. Critical to the developing understanding of this process was the recognition of two types of photosynthetic cells in C4 plants (mesophyll and bundle sheath) with quite different enzyme complements and distinct biochemical roles (see Fig. 3.1). As currently interpreted (see Edwards and Walker, 1983; Hatch, 1987) the reactions unique to the C4 pathway serve, in association with some remarkable modifications of leaf anatomy and ultrastructure, to concentrate CO2 in bundle sheath cells for utilisatiqn by the photosynthetic carbon reduction cycle carboxylase, ribulose L5-bisphosphate carboxylase-oxygenase (Rubisco). The Rubisco-mediated oxygenase reaction and associated photorespiration are thereby eliminated.

Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants

Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C4-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize PEPC was functioning even though it remained dephosphorylated and less active in the light. 14CO2 labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO2 fixation of transgenic rice plants. Rather, it slightly lowered the CO2 assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower O2 concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized in transgenic rice.

Primary partitioning and storage of photosynthate in sucrose and starch in leaves of C4 plants

A procedure involving pulse labelling of leaves with 14CO2 was developed to measure the primary (initial) partitioning of photosynthate between sucrose and starch. Partitioning of photosynthate into sucrose and starch was determined in leaves of C4 plants and compared with the patterns of storage of carbon in these products during the light period. The ratio of primary partitioning into sucrose and starch varied from about 0.5 in those species that accumulated mostly starch in the leaves (Amaranthus edulis L., Atriplex spongiosa F. Muell. and Flaveria trinervia (Spreng.) C. Mohr) to about 8 in Eleusine indica (L.) Gaertn., which accumulated mostly sucrose. No label was detected in free glucose or fructose. Generally there was a reasonable link between the primary partitioning of photosynthate and the type of carbohydrate stored in the leaf during the day. However, the ratio of carbon initially partitioned into sucrose versus starch was about 3 to 4 times higher in leaves of NADP-malic enzyme-type monocotyledonous species compared with phosphoenolpyruvate carboxykinase-type species, although the ratio of sucrose to starch accumulated in leaves during the day was very similar in the two groups. Sucrose and starch were the principal carbohydrates accumulated in leaves during the day. None of the species examined contained significant amounts of fructan and only one species, Atriplex spongiosa, contained substantial amounts of hexose sugars. In most of the species studied, the proportion of photosynthate partitioned into starch was greater at the end of the day than at the beginning. With the exception of Flaveria trinervia, the rate of CO2 assimilation did not decline during the day, showing that, under our conditions, accumulation of carbohydrate in the leaves did not lead to feedback inhibition of photosynthesis in these C4 species.

Measurement of the Leakage of CO2 from Bundle-Sheath Cells of Leaves during C4 Photosynthesis

During C4 photosynthesis, CO2 is released in bundle-sheath cells by decarboxylation of C4 acids and then refixed via ribulose-1,5-bisphosphate carboxylase. In this study we examined the efficiency of this process by determining the proportion of the released CO2 that diffuses back to mesophyll cells instead of being refixed. This leak of CO2 was assessed by determining the amount of 14CO2 released from leaves during a chase in high [12CO2] following a 70-s pulse in 14CO2. A computer-based analysis of the time-course curve for 14CO2 release indicated a first-order process and provided an estimate of the initial velocity of 14CO2 release from leaves. From this value and the net rate of photosynthesis determined from the 14CO2 fixed in the pulse, the CO2 leak rate from bundle-sheath cells (expressed as a percentage of the rate of CO2 production from C4 acids) could be deduced. For nine species of Gramineae representing the different subgroups of C4 plants and two NAD-malic enzyme-type dicotyledonous species, the CO2 leak ranged between 8 and 14%. However, very high CO2 leak rates (averaging about 27%) were recorded for two NADP-malic enzyme-type dicotyledonous species of Flaveria. The results are discussed in terms of the efficiency of C4 photosynthesis and observed quantum yields.

Photosynthetic metabolism in bundle sheath cells of the C4 species Zea mays: Sources of ATP and NADPH and the contribution of photosystem II

Abstract Rates of ATP and NADPH consumption during photosynthesis by isolated strands of Zea mays bundle sheath cells were calculated from an analysis of intermediates labeled from assimilated 14 C0 2 . The NADPH generated during decarboxylation of added malate (via malic enzyme) was also calculated so that the component of total NADPH produced by photoreduction could be deduced. Adding ribose 5-phosphate, malate, and aspartate to cells provided with HCO 3 − substantially increased the rates of ATP and NADPH utilization by the reactions of the photosynthetic carbon reduction cycle. The rate of NADP + photoreduction was reduced by adding malate and aspartate. The ratio of ATP produced by photophosphorylation to NADP + photoreduced was highest (5 to 11) in systems provided with malate and aspartate. Only low rates of oxygen evolution were observed in the presence of HCO 3 − and ribose 5-phosphate; these rates were further reduced when malate and aspartate were added. Observed rates of oxygen evolution were in close agreement with the rates of NADP + photoreduction estimated by analysis of labeled intermediates. Simultaneous measurements of oxygen evolution and oxygen uptake, using isotopic oxygen, indicated that the rate of pseudocyclic electron transport was low. Oxygen evolution was light dependent and inhibited by NH 4 C1 and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Cells provided with malate and sufficient 3-(3,4-dichlorophenyl)-l,1-dimethylurea to completely inhibit NADP + photoreduction retained a substantial capacity for ATP synthesis. Reducing the oxygen concentration partially reversed 3-(3,4-dichlorophenyl)-l,l-dimethylurea inhibition. We propose that these treatments modify the poising of the cyclic electron transport system and that cyclic photophosphorylation is responsible for up to 80% of the ATP produced in these cells.

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C4 grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C4 acid metabolized during C4 photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C4 dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO2 to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O2 at rates between 1.5 and 2 μmol · min−1 · mg−1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO3−or aspartate plus 2-oxoglutarate. Rates of more than 2 μmol O2 · min−1 · mg−1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO2 fixation and malate decarboxylation to pyruvate recorded were about 1.7 μmol · min−1 · mg−1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C4 acids from 14CO2 indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.

C4 photosynthesis: discovery and resolution

This Minireview provides a brief account of the scene and interesting turn of events surrounding the discovery and resolution of the mechanism of C4 photosynthesis, as well as the recognition of the process by the wider plant science community.

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: Pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH4)2SO4, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; Km values at pH 8.5 are oxaloacetate, 18 microM; malate, 24 mM; NADPH, 50 microM; and NADP, 45 microM. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, Ki = 50 microM). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of Mr 38,000. Active enzyme is much more sensitive (greater than 50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10(2) from the binding affinities for active NADP-malate dehydrogenase and 10(4) for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.

Light-dark modulation of leaf pyruvate,Pi dikinase

Abstract Pyruvate,P i dikinase catalyses a unique type of reaction in which two separate substrates are phosphorylated. In leaves this enzyme has a key role in the photosynthetic process known as the C 4 pathway. This article discusses the complex mechanism by which pyruvate,P i dikinase activity is modulated in leaves in response to changing light intensity. Regulation involves interconversion between an active non-phosphorylated form and an inactive phosphorylated form of the enzyme but both reactions proceed by unique mechanisms. The single enzyme catalysing these reactions apparently has two separate active sites.