Serine hydroxymethyltransferase6 is involved in growth and resistance against pathogens via ethylene and lignin production in Arabidopsis.

Photorespiratory serine hydroxymethyltransferases (SHMTs) are important enzymes of cellular one-carbon metabolism. In this study, we investigated the potential role of SHMT6 in Arabidopsis thaliana. We found that SHMT6 is localized in the nucleus and expressed in different tissues during development. Interestingly SHMT6 is inducible in response to avirulent, virulent Pseudomonas syringae and to Fusarium oxysporum infection. Overexpression of SHMT6 leads to larger flowers, siliques, seeds, roots, and consequently an enhanced overall biomass. This enhanced growth was accompanied by increased stomatal conductance and photosynthetic capacity as well as ATP, protein, and chlorophyll levels. By contrast, a shmt6 knockout mutant displayed reduced growth. When challenged with Pseudomonas syringae pv tomato (Pst) DC3000 expressing AvrRpm1, SHMT6 overexpression lines displayed a clear hypersensitive response which was characterized by enhanced electrolyte leakage and reduced bacterial growth. In response to virulent Pst DC3000, the shmt6 mutant developed severe disease symptoms and becomes very susceptible, whereas SHMT6 overexpression lines showed enhanced resistance with increased expression of defense pathway associated genes. In response to Fusarium oxysporum, overexpression lines showed a reduction in symptoms. Moreover, SHMT6 overexpression lead to enhanced production of ethylene and lignin, which are important components of the defense response. Collectively, our data revealed that SHMT6 plays an important role in development and defense against pathogens.

High Mitochondrial Activity but Incomplete Engagement of the Cyanide-Resistant Alternative Pathway in Guard Cell Protoplasts of Pea.

The respiratory properties of guard cell protoplasts (GCP) were examined in comparison with those of mesophyll protoplasts (MCP) from the same leaves of pea (Pisum sativum L. cv Arkel). The rates of respiratory O2 uptake by GCP were extremely high (280 [mu]mol mg-1 Chl h-1) and were several times greater than those of MCP. On the other hand, the rates of photosynthetic O2 evolution by GCP were similar to those of MCP. Also on the basis of protoplast volume, the respiratory rates of GCP were higher: more than three times those of MCP. The enzymes of the tricarboxylic acid cycle, per unit protein or unit protoplast volume, had a 2- to 5-fold higher activity in GCP than in MCP, indicating an enrichment of mitochondrial activity in GCP relative to that in MCP. Respiratory inhibitors were used to assess the activity of the cytochrome (cyanide-sensitive) and alternative (cyanide-resistant) pathways in GCP and MCP. The inhibition of respiration by KCN or antimycin A was more in GCP than that in MCP. The marked inhibition of respiratory O2 uptake by salicylhydroxamic acid in the presence of KCN showed the presence of the cyanide-resistant pathway in GCP. The activity of the cyanide-resistant electron transport path constituted only one-third of total respiration in GCP but accounted for two-thirds of respiration in MCP. The alternative pathway was not completely engaged in GCP but reached its full capacity in MCP.

Importance of the cytochrome pathway of mitochondrial electron transport over the alternative pathway during the Kok effect in leaf discs of pea (Pisum sativum).

The Kok effect refers to the progressive light-induced inhibition of dark respiration at low light intensities, which saturates around the light compensation point. This appears as a sudden break around the light compensation point in the plot of photosynthesis versus light intensity. The magnitude of the break can be considered as a measure of the Kok effect. In the present work, the importance of different components of dark respiration during the Kok effect was investigated by using low concentrations of mitochondrial inhibitors in leaf discs of pea (Pisum sativum L. cv. Azad P1). The effects of glucose (stimulates respiration) and 0.8 M sorbitol (imposes osmotic stress and inhibits photosynthesis) were also studied for comparison. The magnitude of the break decreased significantly in the presence of antimycin A or oligomycin (inhibitors of cytochrome pathway of mitochondrial electron transport and ATP synthase, respectively). In contrast, there was no significant change with salicylhydroxamic acid (SHAM; an inhibitor of alternative pathway of mitochondrial electron transport). The magnitude of the break increased significantly with glucose, and decreased on exposure to osmotic stress. Our results suggest that the Kok effect (inhibition of dark respiration in light) is modulated by inhibitors of cytochrome pathway and ATP synthesis, but not that of the alternative pathway.

Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes.

The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea (Pisum sativum) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO2 (0.1 mM NaHCO3) than that at optimal CO2 (1.0 mM NaHCO3). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.

Modulation of osmotic stress effects on photosynthesis and respiration by temperature in mesophyll protoplast of pea.

Exposure of mesophyll protoplast of pea to osmotic stress decreases the rate of photosynthesis while stimulating marginally the respiratory rate of mesophyll protoplasts. The interaction of osmotic and temperature stress during the modulation of photosynthetic and respiratory rates of pea (Pisum sativum var Azad P1) mesophyll protoplasts was investigated. The protoplasts were exposed to either iso-osmotic (0.4 M) or hyper-osmotic (1.0 M) concentration of sorbitol at 15 degrees and 25 degrees C. The rates of photosynthesis and respiration were studied. At optimum temperature of 25 degrees C, there was a decrease in photosynthesis (< 10%) at hyper-osmoticum (osmotic effect), whereas respiration increased marginally (by about 15%). Low temperature (15 degrees C) aggravated the sensitivity of both respiration and photosynthesis to osmotic stress. At 15 degrees C, the decrease in photosynthesis due to osmotic stress was > 35%, while the respiratory rate was stimulated by 30%. The relative proportion of cytochrome pathway decreased by about 50% at both 15 degrees C and 25 degrees C while that of alternative pathway increased, more so, at 15 degrees C, when the mesophyll protoplasts were subjected to hyper-osmoticum stress. The titration experiments showed that extent of engagement of alternative pathway was higher, the slope value was slightly higher for 15 degrees C compared to 25 degrees C. Low temperature modulates the effect of hyper-osmoticum stress on photosynthesis and respiration, and results in increased participation of alternative pathway.

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants.

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.

Emerging concept for the role of photorespiration as an important part of abiotic stress response.

When plants are exposed to stress, generation of reactive oxygen species (ROS) is often one of the first responses. In order to survive, cells attempt to down-regulate the production of ROS, while at the same time scavenging ROS. Photorespiration is now appreciated as an important part of stress responses in green tissues for preventing ROS accumulation. Photorespiratory reactions can dissipate excess reducing equivalents and energy either directly (using ATP, NAD(P)H and reduced ferredoxin) or indirectly (e.g., via alternative oxidase (AOX) and providing an internal CO2 pool). Photorespiration, however, is also a source of H2 O2 that is possibly involved in signal transduction, resulting in modulation of gene expression. We propose that photorespiration can assume a major role in the readjustment of redox homeostasis. Protection of photosynthesis from photoinhibition through photorespiration is well known. Photorespiration can mitigate oxidative stress under conditions of drought/water stress, salinity, low CO2 and chilling. Adjustments to even mild disturbances in redox status, caused by a deficiency in ascorbate, AOX or chloroplastic NADP-malate dehydrogenase, comprise increases in photorespiratory components such as catalase, P-protein of glycine decarboxylase complex (GDC) and glycine content. The accumulation of excess reducing equivalents or ROS in plant cells also affects mitochondria. Therefore, a strong interaction between the chloroplast redox status and photorespiration is not surprising, but highlights interesting properties evident in plant cells. We draw attention to the fact that a complex network of multiple and dynamic systems, including photorespiration, prevents oxidative damage while optimising photosynthesis. Further experiments are necessary to identify and validate the direct targets of redox signals among photorespiratory components.

Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of Arabidopsis.

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H(2)O(2) in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H(2)DCF-DA). Bicarbonate-induced stomatal closure as well as H(2)O(2) production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H(2)O(2) production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H(2)O(2) was quicker and greater with ABA than that with bicarbonate. Such pattern of H(2)O(2) production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H(2)O(2) is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.

Evolution of C(4) phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C(4) isozyme and its orthologues in C(3) and C(3)/C(4) Alternantheras.

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C(4) photosynthesis. It has evolved from ancestral non-photosynthetic (C(3)) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C(4) PEPCases were adapted to their new function in C(4) photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C(4) plant Alternanthera pungens H.B.K., the C(3)/C(4) intermediate plant A. tenella Colla, and the C(3) plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K (0.5) PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C(4)) as a C(4) PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C(3)) and A. tenella (C(3)/C(4)) were found to be typical C(3) PEPCase isozymes. The C(4) characteristics of the PEPCase of A. pungens were accompanied by the presence of the C(4)-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C(4) PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3' untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis.

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O(2) and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and alpha-ketoglutarate) in CO(2)-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO(2)-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO(2)-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.

Sensitivity of photosynthesis by spinach chloroplast membranes to osmotic stress in vitro: Rapid inhibition of O2 evolution in presence of magnesium.

Thylakoids prepared from spinach (Spinacea oleracea L.) chloroplasts were exposed to osmotic stress in vitro in the presence or absence of different inorganic salts. By an hour after incubation in 1.0 M sorbitol and 10 mM (or more) MgCl2, the thylakoids lost approximately 80% of their photosystem (PS) II activity, but not PS I. The inhibition occurred only in presence of magnesium as indicated by the combinations of several cations/anions. The PS II activity was relatively insensitive to osmotic stress in the presence of diphenyl carbazide. We therefore conclude that under conditions of water stress in the presence of 10 mM or higher Mg(2+), the oxygen evolving system in chloroplasts is rapidly inactivated.

Ammonium ions stimulate in vitro the activity of phosphoenolpyruvate carboxylase from leaves of Amaranthus hypochondriacus, a C4 plant: evidence for allosteric activation.

Ammonium ions stimulated in vitro the activity of PEP carboxylase (PEPC) extracted from dark-adapted leaves of Amaranthus hypochondriacus. Maximum stimulation of 80 to 85% occurred at 50 microM ammonium chloride. There was a marginal inhibition of PEPC at 5 mM ammonium chloride. Among several ions tested, potassium ions stimulated PEPC to a limited extent of about 30%. In presence of ammonium, there was no change either in the sensitivity of enzyme to malate or in the affinity for substrate, PEP. On the other hand, glucose-6-phosphate, an allosteric activator, which stimulated the enzyme by two-fold, could enhance PEPC activity by < 20% in the presence of ammonium. The light-activated form of PEPC from leaves of Amaranthus hypochondriacus was not stimulated, but was inhibited in the presence of ammonium. Our results demonstrate that ammonium ions stimulate PEPC by acting at the allosteric site. Ammonium ion being a component of plant metabolism could be an important regulator of PEPC, particularly in C4 plants.

Photorespiration in C3-C 4 intermediate species of Alternanthera and Parthenium: Reduced ammonia production and increased capacity of CO2 refixation in the light.

The pattern of photorespiratory ammonia (PR-NH3) formation and its modulation by exogenous bicarbonate or glycine were investigated in C3-C4 intermediates of Alternanthera (A. ficoides and A. tenella) and Parthenium hysterophorus in comparison to those of C3 or C4 species. The average rates of PR-NH3 accumulation in leaves of the intermediates were slightly less than (about 25% reduced) those in C3 species, and were further low in C4 plants (40% of that in C3). The levels of PR-NH3 in leaf discs decreased markedly when exogenous bicarbonate was present in the incubation medium. The inhibitory effect of bicarbonate on PR-NH3 accumulation was pronounced in C3 plants, very low in C4 species and was moderate in the C3-C4 intermediates. Glycine, an intermediate of photorespiratory metabolism, raised the levels of PR-NH3 in leaves of not only C4 but also C3-C4 intermediates, bringing the rates close to those of C3 species. The rate of mitochondrial glycine decarboxylation in darkness in C3-C4 intermediates was partially reduced (about 80% of that in C3 species), corresponding to the activity-levels of glycine decarboxylase and serine hydroxymethyltransferase in leaves. The intermediates had a remarkable capacity of reassimilating photorespiratory CO2 in vivo, as indicated by the apparent refixation of about 85% of the CO2 released from exogenous glycine in the light. We suggest that the reduced photorespiration in the C3-C4 intermediate species of Alternanthera and Parthenium is due to both a limitation in the extent of glycine production/decarboxylation and an efficient refixation/recycling of internal CO2.

Tetrazolium Reduction by Guard Cells in Abaxial Epidermis of Vicia faba: Blue Light Stimulation of a Plasmalemma Redox System.

The stomata in the abaxial epidermis of Vicia faba were examined for the location of redox systems using tetrazolium salts. Three distinct redox systems could be demonstrated: chloroplast, mitochondrial, and plasmalemma. The chloroplast activity required light and NADP. Mitochondrial activity required added NADH and was suppressed by preincubation with KCN. The plasmalemma redox system in guard cells also required NADH, but was insensitive to KCN and was stimulated by blue light. The involvement of an NADH dehydrogenase in the blue light stimulated redox system in guard cells was suggested by the sensitivity to plantanetin, an inhibitor of NADH dehydrogenase. The redox system of mitochondria was the most active followed by that of plasmalemma. The activity of chloroplasts was the least among the three redox systems. The plasmalemma mediated tetrazolium reduction was stimulated by exogenous flavins and suppressed by Kl or phenylacetate, inhibitors of flavin excitation. We therefore conclude that an NADH-dependent, flavin mediated electron transport system, sensitive to blue light, operates in the plasmalemma of guard cells.

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum).

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.

Action of proline on stomata differs from that of abscisic Acid, g-substances, or methyl jasmonate.

Methyl jasmonate (MJ) and a mixture of G(1), G(2), and G(3) (G-substances) inhibited stomatal opening in abaxial epidermis of Commelina benghalensis and complete closure occurred at 10(-6) molar MJ, or 10(-3) molar G-substances compared to 10(-5) molar abscisic acid (ABA). Proline, even at 10(-3) molar caused only a partial stomatal closure. Apart from ABA, other endogenous plant growth regulators do regulate stomata. Reduction in the stimulation by fusicoccin and complete stomatal closure, at 30 millimolar KCl or less, were affected by ABA, MJ, or G-substances, but not by proline. The action of MJ or G-substances was similar to ABA in decreasing proton efflux and the levels of potassium, malate, or reducing sugars. Proline, however, interfered with starch-sugar interconversion but had no effect on proton efflux or potassium content of epidermis.

Light-enhanced dark respiration in mesophyll protoplasts from leaves of pea.

The respiratory oxygen uptake by mesophyll protoplasts of pea (Pisum sativum cv Arkel) was stimulated up to threefold after 15 minutes of illumination at an intensity of 1250 microeinsteins per square meter per second in the presence of 5 millimolar bicarbonate at 30 degrees C. The extent of light-enhanced dark respiration (LEDR) increased progressively with duration of preillumination. The LEDR exhibited two phases. The initial high rate of respiration decreased in about 10 minutes to a lower steady value similar to that before illumination. The promotion of LEDR by the presence of bicarbonate and inhibition by glyceraldehyde or 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that LEDR was dependent on products of photosynthetic carbon assimilation/electron transport. Thus, the photosynthetic products exert a markedly quick influence on dark respiration in mesophyll protoplasts.

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure, regulation and genetic engineering.

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C4-specific, C3 or etiolated, CAM and root forms. C4 leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C4-dicot Flaveria trinervia. Selective expression of ppc in only C4-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C4-PEPC pinpoint the phosphorylatable serine near the N-terminus, C4-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO2, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO3 (-)-dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C4 ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C4 PEPC and the transgenic tobacco plants expressed both C3 and C4 isoforms. Site-directed mutagenesis of ppc indicates the importance of His(138), His(579) and Arg(587) in catalysis and/or substrate-binding by the E. coli enzyme, Ser(8) in the regulation of sorghum PEPC. Important areas for further research on C4 PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.

Essentiality of mitochondrial oxidative metabolism for photosynthesis: optimization of carbon assimilation and protection against photoinhibition.

The review emphasizes the essentiality of mitochondrial oxidative metabolism for photosynthetic carbon assimilation. Photosynthetic activity in chloroplasts and oxidative metabolism in mitochondria interact with each other and stimulate their activities. During light, the partially modified TCA cycle supplies oxoglutarate to cytosol and chloroplasts. The marked stimulation of O2 uptake after few minutes of photosynthetic activity, termed as light enhanced dark respiration (LEDR), is now a well-known phenomenon. Both the cytochrome and alternative pathways of mitochondrial electron transport are important in such interactions. The function of chloroplast is optimized by the complementary nature of mitochondrial metabolism in multiple ways: facilitation of export of excess reduced equivalents from chloroplasts, shortening of photosynthetic induction, maintenance of photorespiratory activity, and supply of ATP for sucrose biosynthesis as well as other cytosolic needs. Further, the mitochondrial oxidative electron transport and phosphorylation also protects chloroplasts against photoinhibition. Besides mitochondrial respiration, reducing equivalents (and ATP) are used for other metabolic phenomena, such as sulfur or nitrogen metabolism and photorespiration. These reactions often involve peroxisomes and cytosol. The beneficial interaction between chloroplasts and mitochondria therefore extends invariably to also peroxisomes and cytosol. While the interorganelle exchange of metabolites is the known basis of such interaction, further experiments are warranted to identify other biochemical signals between them. The uses of techniques such as on-line mass spectrometric measurement, novel mutants/transgenics, and variability in metabolism by growth conditions hold a high promise to help the plant biologist to understand this