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

Sodium nitroprusside affects the level of anthocyanin and flavonol glycosides in pea (Pisum sativum L. cv. Arkel) leaves

The effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor were investigated on the levels of anthocyanin and flavonol glycoside in pea (Pisum sativum L.) cv. Arkel leaves. The study was conducted in leaf discs (ca. 20 mm2) prepared from the youngest leaves. The anthocyanin and flavonol glycosides content diminish significantly (~ 21% of each) in leaf discs following 1 mM SNP (1 mM) treatment for 3 h under light (600 μmol M-2.s-1). However, a huge increase both in the levels of anthocyanin and flavonol glycosides, 72 and 53% respectively was recorded after 2 h of 1 mM SNP treatment. 0.5 mM SNP treatment of the leaf discs did not change the anthocyanin level but considerable declined (~13%) was observed in the level of flavonol glycosides as compared to the control. Surprisingly, the anthocyanin content in no SNP treated leaf discs after 3 h of incubation under light (600 μmol M-2.s-1) increased rapidly by 72% while flavonol glycosides content by 15% only. The photosynthetic capacities of SNP treated leaf discs were drastically inhibited. The study prelude that NO in combination of light influence the accumulation of anthocyanin and flavonol glycosides in pea leaves

Marked modulation by phosphate of phosphoenolpyruvate carboxylase in leaves of Amaranthus hypochondriacus, a NAD-ME type C<SUB>4</SUB> plant: decrease in malate sensitivity but no change in the phosphorylation status

The effect of Pi on the properties of phosphoenolpyruvate carboxylase (PEPC) from Amaranthus hypochondriacus, a NAD-ME type C4 plant, was studied in leaf extracts as well as with purified protein. Efforts were also made to modulate the Pi status of the leaf by feeding leaves with either Pi or mannose. Inclusion of 30 mM Pi during the assay enhanced the enzyme activity in leaf extracts or of purified protein by &gt;2-fold. The effect of Pi on the enzyme purified from dark-adapted leaves was more pronounced than that from light-adapted ones. The Ki for malate increased &gt;2.3-fold and &gt;1.9-fold by Pi in the enzyme purified from dark-adapted leaves and light-adapted leaves, respectively. Pi also induced an almost 50-60% increase in Km for PEP or Ka for glucose-6-phosphate. Feeding the leaves with Pi also increased the activity of PEPC in leaf extracts, while decreasing the malate sensitivity of the enzyme. On the other hand, Pi sequestering by mannose marginally decreased the activity, while markedly suppressing the light activation, of PEPC. There was no change in phosphorylation of PEPC in leaves of A. hypochondriacus due to the feeding of 30 mM Pi. However, feeding with mannose decreased the light-enhanced phosphorylation of PEPC. The marked decrease in malate sensitivity of PEPC with no change in phosphorylation state indicates that the changes induced by Pi are independent of the phosphorylation of PEPC. It is suggested here that Pi is an important factor in regulating PEPC in vivo and could also be used as a tool to analyse the properties of PEPC

Photosynthesis research in India: transition from yield physiology into molecular biology

Photosynthesis research in India can be traced back several thousand years, with the mention of the Sun energizing the plants, which form food for all living creatures on the earth (from the Mahabharata, the great epic, ca. 2600 B.C.) and the report of Sage Parasara (ca. 100 B.C.) on the ability of plants to make their own food, due to their pigments. With the pioneering studies by Sir Jagdish Chandra Bose, work on photosynthesis proceeded steadily during the first half of the 20th century. Some of the classic reports during this period are: malate metabolism in Hydrilla, spectrophotometric estimation of chlorophylls, importance of spectral quality for photosynthesis-an indication of two photosystems, photoinactivation of photosynthesis, and importance of flag leaf photosynthesis to grain yield. After the 1960s, there was a burst of research in the areas of physiology and biochemistry of carbon assimilation and photochemistry. A significant transition occurred, before the beginning of new millennium, into the area of molecular biology of chloroplasts, regulation of photosynthesis and stress tolerance. Future research work in India is geared to focus on the following aspects of photosynthesis: elucidation/analysis of genes, molecular biology/evolution of enzymes, development/use of transgenics and modeling

Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C<SUB>3</SUB> and C<SUB>4</SUB> plants

Temperature caused phenomenal modulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaf discs of Amaranthus hypochondriacus (NAD-ME type C4 species), compared to the pattern in Pisum sativum (a C3 plant). The optimal incubation temperature for PEPC in A. hypochondriacus (C4) was 45&#176;C compared to 30&#176;C in P. sativum (C3). A. hypochondriacus (C4) lost nearly 70% of PEPC activity on exposure to a low temperature of 15&#176;C, compared to only about a 35% loss in the case of P. sativum (C3). Thus, the C4 enzyme was less sensitive to supra- optimal temperature and more sensitive to sub- optimal temperature than that of the C3 species. As the temperature was raised from 15&#176;C to 50&#176;C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C4 plants (45%) was more than that in C3 species (30%). The maintenance of high enzyme activity at warm temperatures, together with a sharp decrease in the malate sensitivity of PEPC was also noticed in other C4 plants. The temperature-induced changes in PEPC of both A. hypochondriacus (C4) and P. sativum (C3) were reversible to a large extent. There was no difference in the extent of phosphorylation of PEPC in leaves of A. hypochondriacus on exposure to varying temperatures, unlike the marked increase in the phosphorylation of enzyme on illumination of the leaves. These results demonstrate that (i) there are marked differences in the temperature sensitivity of PEPC in C3 and C4 plants, (ii) the temperature induced changes are reversible, and (iii) these changes are not related to the phosphorylation state of the enzyme. The inclusion of PEG-6000, during the assay, dampened the modulation by temperature of malate sensitivity of PEPC in A. hypochondriacus. It is suggested that the variation in temperature may cause significant conformational changes in C4-PEPC

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO2. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components-ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate-glutathione (Asc-GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways

Modulation by S-nitrosoglutathione (a natural nitric oxide donor) of photosystem in Pisum sativum leaves, as revealed by chlorophyll fluorescence: Light-dependent aggravation of nitric oxide effects

The reported effects of nitric oxide (NO), a signaling molecule, on the photochemical components of leaves are ambiguous. We examined the changes by a natural NO donor, S-nitrosoglutathione (GSNO). The effect of GSNO on Pisum sativum leaves was studied after a 3-hour exposure in dark, moderate (ML), or high light (HL). The NO levels in GSNO-treated samples were at their maximum under HL, compared to those under ML or dark. Most of the elevated NO was decreased by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, confirming the NO increase. Treatment with GSNO caused inhibition of photosynthesis/respiration and restricted electron transport mediated by both photosystem (PS)II and PSI. However, the inhibition by NO-donor of PSII components was stronger than those of PSI. A marked increase in the PSI acceptor side limitation [Y(NA)] and a decrease in PSI donor side limitation [Y(ND)] indicated an upregulation of cyclic electron transport, possibly to balance the damage to PSII by GSNO. We suggest that NO aggravated the HL-induced inhibition of photosynthesis and dark respiration

In vitro stability of various enzymes by proline from H2O2 mediated oxidative damage

111-125Plants under stress need to favour certain pathways so as to survive the stress period. Protection of specific enzymes by proline and other osmolytes could be one such mechanism to favour some pathways/processes. Therefore, the influence of osmolyte proline on conformational changes of various proteins caused by hydrogen peroxide (H2O2) was studied by intrinsic and extrinsic fluorescence emissions. H2O2 caused conformational change in proteins. Results indicated that for Alcohol dehydrogenase (AD) and Glutamate dehydrogenase (GD) enzymes, H2O2 induced conformational change was high and that for Glucose 6-phosphate dehydrogenase (G6PDH) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was low. Fluorescence and far-UV, CD measurements of catalase demonstrated that the H2O2 stabilized the protein secondary structure at low concentrations but destabilized it at higher concentrations. Intrinsic and ANS fluorescence results showed that proline at a concentration of 1.0 M prompted a reduction in the H2O2-induced exposed hydrophobic surfaces of studied enzymes, to different degrees which suggests its differential protective effect. Furthermore, SDS-PAGE studies revealed that proline was not able to reduce or inhibit the H2O2 mediated aggregation of GAPDH

Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels

The interactive effects of light and temperature on C4 phosphoenolpyruvate carboxylase (PEPC) were examined both in vivo and in situ using the leaves of Amaranthus hypochondriacus collected at different times during a day and in each month during the year. The maximum activity of PEPC, least inhibition by malate, and highest activation by glucose-6-phosphate were at 15.00 h during a typical day, in all the months. This peak was preceded by maximum ambient light but coincided with high temperature in the field. The highest magnitude in such responses was in the summer (e.g. May) and least in the winter (e.g. December). Light appeared to dominate in modulating the PEPC catalytic activity, whereas temperature had a strong influence on the regulatory properties, suggesting interesting molecular interactions. The molecular mechanisms involved in such interactive effects were determined by examining the PEPC protein/phosphorylation/mRNA levels. A marked diurnal rhythm could be seen in the PEPC protein levels and phosphorylation status during May (summer month). In contrast, only the phosphorylation status increased during the day in December (winter month). The mRNA peaks were not as strong as those of phosphorylation. Thus, the phosphorylation status and the protein levels of PEPC were crucial in modulating the daily and seasonal patterns in C4 leaves in situ. This is the first detailed study on the diurnal as well as seasonal patterns in PEPC activity, its regulatory properties, protein levels, phosphorylation status, and mRNA levels, in relation to light and temperature intensities in the field