Twenty-five years ago research had already established a firm biochemical and physiological understanding of the CO2-concentrating mechanism that creates a high CO2 environment (1,000–3,000 μbar) in bundle-sheath cells in leaves of C4 plants and accounts for most of their distinctive

In this lecture the author reminisced about three critical phases of research on C-4 photosynthesis. These case histories had their origins in the very early studies of the process but then spanned most of the intervening period.

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.

A simple four-step procedure for the purification of thioredoxin-m from Zea mays leaves is described. The procedure provides pure protein with recoveries of 20-25%. This thioredoxin mediates in the regulation of NADP-malate dehydrogenase involved in photosynthesis in C4 plants.

Journal Article C4 Photosynthesis: An Unlikely Process Full of Surprises Get access Marshall D. Hatch Marshall D. Hatch Division of Plant IndustryCSIRO, GPO Box 1600, Canberra 2601, Australia Search for other works by this author on: Oxford Academic Google Scholar Plant and Cell Physiology, Volume 33, Issue 4, June 1992, Pages 333–342, https://doi.org/10.1093/oxfordjournals.pcp.a078260 Published: 01 June 1992 Article history Received: 02 March 1992 Published: 01 June 1992

The time course of thioredoxin-mediated reductive activation of isolated Zea mays nicotinamide adenine dinucleotide phosphatemalate dehydrogenase is highly sigmoidal in nature. We examined the factors affecting these kinetics, including the thiol-disulfide status of unactivated and activated forms of the enzyme. The maximum steady rate of activation was increased, and the length of the lag in activation decreased, as the concentrations of thioredoxin-m, dithiothreitol, and KCl were increased. The lag in activation (sigmoidicity) was eliminated by preincubating the unactivated enzyme with 100 mm 2-mercaptoethanol; this pretreatment did not activate the enzyme. Unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase was found to contain approximately two SH groups per subunit, increasing to about four SH per subunit after pretreatment with 2-mercaptoethanol and six SH per subunit after activation by incubating the enzyme with dithiothreitol. We suggest that reduction of one particular higher redox potential disulfide group in unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase facilitates the subsequent reduction of the critical S-S group (regulatory S-S) necessary to generate the active form of the enzyme.

Effects of adenylates on the activity of mitochondrial NAD-malic enzyme from NAD-malic-enzyme (NAD-ME)-type and phosphoenolpyruvate-carboxykinase-(PCK)-type C4 plants are examined. At physiological concentrations, ATP, ADP, and AMP all inhibit the enzyme from Atriplex spongiosa and Panicum miliaceum (NAD-ME-type plants), with ATP the most inhibitory species. The degree of inhibition is greater with subsaturating levels of activator, malate, and Mn2+. NAD-malic enzyme from Urochloa panicoides (PCK-type) is activated by ATP (up to 10-fold) and inhibited by ADP and AMP. These effects are discussed in relation to regulation of C4 photosynthesis.

The relationship between overcycling of the C4 acid cycle in C4 photosynthesis (due to CO2 leakage) and the quantum yield of photosynthesis is considered. From a comparison of theoretical and measured quantum yields we suggest that the high efficiency of light utilisation by most C4 plants can only be explained by the mandatory involvement of both the Q-cycle and cyclic or pseudocyclic electron transport in the proton partitioning process. The existence of the Q-cycle mechanism may have been a prerequisite for the evolution of the C4 pathway.

The mechanism and possible regulation of C4 acid decarboxylation in NAD-malic enzyme-type C4 plants was studied using isolated bundle sheath cells and mitochondria from Panicum miliaceum. Rates of C4 acid-dependent photosynthetic O2 evolution equalled those observed with saturating NaHCO3; the rates ranged from 3 to 5 mumol min-1 (mg chlorophyll)-1. C4 acid-dependent O2 evolution required the addition of aspartate and 2-oxoglutarate (as a source of oxaloacetate) and also malate and orthophosphate. C4 acid decarboxylation by both isolated cells and mitochondria, measured as pyruvate production, also required all four of these components. The scheme previously proposed to account for aspartate decarboxylation in NAD-malic enzyme-type C4 plants does not envisage a role for externally derived malate. However, the mandatory requirement for malate (with orthophosphate), together with the observation that C4 acid decarboxylation is blocked by an inhibitor of the mitochondrial dicarboxylate transporter, suggests that a net flux of malate from outside the mitochondria is required to sustain this process. Arsenate was found to substitute for orthophosphate favoring a role for orthophosphate in malate transport rather than a metabolic one. The results are discussed in terms of likely mitochondrial metabolite transport mechanisms and regulation of the C4 acid decarboxylation process.

C4 plants suppress photorespiration by concentrating CO2 at the site of ribulose-l,5-bisphosphate carboxylase (Rubisco). During photosynthesis in C4 leaves an inorganic carbon pool develops which is up to 10 x that expected by simple equilibration with external CO2 (1). The effective concentration of CO2 in bundle sheath cells requires that the mesophyll-bundle sheath cell interface be resistant to CO2 diffusion. The more “leaky” this interface is to CO2 the more energy must be expended by “overcycling” of the C4 cycle relative to net CO2 assimilation to maintain a high bundle sheath CO2 concentration. The work presented here quantitatively examines the permeability of the bundle sheath-mesophyll interface to CO2 and its implications for C4 photosynthesis.

In C(4) plants carbonic anhydrase catalyzes the critical first step of C(4) photosynthesis, the hydration of CO(2) to bicarbonate. The maximum activity of this enzyme in C(4) leaf extracts, measured by H(+) production with saturating CO(2) and extrapolated to 25 degrees C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C(3) leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO(2) concentrations was apparently just sufficient to prevent the rate of conversion of CO(2) to HCO(3) (-) from limiting C(4) photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.

Exogenous Mg2+ inhibited PGA- and OAA-dependent photosynthetic O2 evolution by isolated mesophyll chloroplasts from Zea mays and also HCO3--dependent O2 evolution by chloroplasts from Panicum miliaceum bundle sheath cells. Inhibition of 50-75% was observed with 3-5 mM Mg2+; this varied to some extent with pH but was not reversed by K+ . Inhibition of HCO3-- and PGA-dependent O2 evolution by the divalent cation ionophore A23187 was reversed by adding Mg2+ . Notably, HCO3-- dependent O2 evolution by isolated bundle sheath cells from P. miliaceum was not inhibited by [Mg2+] up to 20 mM. Addition of Mg2+ in the light decreased the stromal pH of mesophyll chloroplasts by less than 0.2 units but reduced apparent stromal volume by as much as 25%. At least for bundle sheath chloroplasts, stromal pH varied with external pH over the range from 7 to 8 but remained about 0.3 units higher throughout this range. Oxygen evolution by isolated mesophyll chloroplasts, and bundle sheath cells and chloroplasts, was relatively insensitive to external pH in the range from 7 to 8. The results are considered in terms of likely mechanisms for the effects of exogenous Mg2+ and pH on photosynthesis by isolated chloroplasts and the physiological significance of Mg2+ effects.

Diffusion of inorganic carbon into isolated bundle sheath cells from a variety of C(4) species was characterized by coupling inward diffusion of CO(2) to photosynthetic carbon assimilation. The average permeability coefficient for CO(2) (P(CO(2) )) for five representatives from the three decarboxylation types was approximately 20 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. The average value for the NAD-ME species Panicum miliaceum (10 determinations) was 26 with a standard deviation of 6 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. A P(CO(2) ) of at least 500 micromoles per minute per milligram chlorophyll per millimolar was determined for cells isolated from the C(3) plant Xanthium strumarium. It is concluded that bundle sheath cells are one to two orders of magnitude less permeable to CO(2) than C(3) photosynthetic cells. These data also suggest that CO(2) diffusion in bundle sheath cells may be made up of two components, one involving an apoplastic path and the other a symplastic (plasmodesmatal) path, each contributing approximately equally.

Bundle sheath cells prepared from C4 leaves have a variety of experimental applications because of their high permeability to metabolites. We determined the factors affecting the physical and metabolic integrity of Panicum miliaceum bundle sheath cell strands immediately following isolation and during subsequent storage. Cell integrity was monitored by determining chloroplast intactness (as ferricyanidedependent O2 evolution) and photosynthetic activity (usually as HCO3-, 3-phosphoglycerate- or C4 acid-dependent O2 evolution). In freshly isolated cell preparations, at least 85-90% of the chloroplasts were apparently intact and there was no significant decline in this value during storage for up to 5 h. With the best extraction conditions, light-dependent O2 evolution in response to adding HCO3- or C4 acids ranged between 3 and 6 mol min-1 mg-1 chlorophyll. Preillumination of leaves was critical for good isolated cell photosynthetic activity and the activity due to C4 acids in particular was increased substantially by including EDTA. During storage for up to 5 h at 0°C as much as 80% of the capacity for HCO3-- or C4 acid-dependent O2 evolution was lost; this loss was very largely prevented by storing the cells with EDTA. Large kinetic lags (induction phase) also developed during storage of cells. Both the loss of photosynthetic activity and the kinetic lags were reduced or eliminated by including photosynthetic intermediates such as dihydroxyacetone phosphate or ribose 5-phosphate in the storage medium. Under the best conditions, bundle sheath cell preparations could be stored for at least 3 h with little apparent change in cell integrity or metabolic capacity.

Bundle sheath cells from leaves of C 4 plants can be isolated as strands surrounding vascular tissue. In this form these cells are highly permeable to metabolites and, as a consequence, they have a variety of experimental uses. The present paper reports on anatomical and ultrastructural features of isolated bundle sheath cell strands in relation to their integrity and permeability. This analysis shows that the cells retain a high degree of structural integrity during isolation. The plasmodesmata that originally connected the bundle sheath cytosol with mesophyll cells are apparently also retained in their entirety. However, at the external surface (mesophyll side) a membranous sac was commonly observed protruding from the end of plasmodesmata. The functional integrity of cells and the molecular weight exclusion limit for entry of compounds was assessed by following plasmolysis and cytorrhysis induced by polyethylene glycol solutions of varying molecular weights. Other evidence for the retention of cell compartment semipermeability is also provided.

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO(2) assimilation or oxygen evolution, over a wide range of CO(2) concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO(2) near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO(2) concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO(3) (-), indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of (14)C-incorporation from (14)CO(2) into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO(2) occurs predominantly by direct CO(2) fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO(2) concentrations, permeability coefficients for CO(2) flux into bundle sheath cells were determined for a range of C(4) species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C(3) plants. These results support the concept that a CO(2) permeability barrier exists to allow the development of high CO(2) concentrations in bundle sheath cells during C(4) photosynthesis.

A theoretical model of the composition of the inorganic carbon pool generated in C(4) leaves during steady-state photosynthesis was derived. This model gives the concentrations of CO(2) and O(2) in the bundle sheath cells for any given net photosynthesis rate and inorganic carbon pool size. The model predicts a bundle sheath CO(2) concentration of 70 micromolar during steady state photosynthesis in a typical C(4) plant, and that about 13% of the inorganic carbon generated in bundle sheath cells would leak back to the mesophyll cells, predominantly as CO(2). Under these circumstances the flux of carbon through the C(4) acid cycle would have to exceed the net rate of CO(2) assimilation by 15.5%. With the calculated O(2) concentration of 0.44 millimolar, the potential photorespiratory CO(2) loss in bundle sheath cells would be about 3% of CO(2) assimilation. Among the factors having a critical influence on the above values are the permeability of bundle sheath chloroplasts to HCO(3) (-), the activity of carbonic anhydrase within these chloroplasts, the assumed stromal volume, and the permeability coefficients for CO(2) and O(2) diffusion across the interface between bundle sheath and mesophyll cells. The model suggests that as the net photosynthesis rate changes in C(4) plants, the level and distribution of the components of the inorganic carbon pool change in such a way that C(4) acid overcycling is maintained in an approximately constant ratio with respect to the net photosynthesis rate.

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.