Redox basis of photosynthesis inhibition at supra-optimal bicarbonate in mesophyll protoplasts of Arabidopsis thaliana.

We examined the patterns of photosynthetic O2 evolution at 1 mM (optimal) and 10 mM (supra-optimal) bicarbonate in mesophyll protoplasts of Arabidopsis thaliana. The photosynthetic rate of protoplasts reached the maximum at an optimal concentration of 1 mM bicarbonate and got suppressed at supra-optimal levels of bicarbonate. We examined the basis of such photosynthesis inhibition by mesophyll protoplasts at supra-optimal bicarbonate. The wild-type protoplasts exposed to supra-optimal bicarbonate showed up signs of oxidative stress. Besides the wild-type, two mutants were used: nadp-mdh (deficient in chloroplastic NADP-MDH) and vtc1 (deficient in mitochondrial ascorbate biosynthesis). The protoplasts of the nadp-mdh mutant exhibited a higher photosynthetic rate and greater sensitivity to supra-optimal bicarbonate than the wild-type. The ascorbate-deficient vtc1 mutant had a low photosynthetic rate and no significant inhibition at high bicarbonate. The nadp-mdh mutants had elevated activities, protein, and transcript levels of key antioxidant enzymes. On the other hand, the antioxidant enzyme systems in vtc1 mutants were not much affected at supra-optimal bicarbonate. We propose that the inhibition of photosynthesis at supra-optimal bicarbonate depends on the redox state of mesophyll protoplasts. The robust antioxidant enzyme systems in protoplasts of nadp-mdh mutant might be priming the plants to sustain high photosynthesis at supra-optimal bicarbonate.

Modulation of Photorespiratory Enzymes by Oxidative and Photo-Oxidative Stress Induced by Menadione in Leaves of Pea (Pisum sativum).

Photorespiration, an essential component of plant metabolism, is concerted across four subcellular compartments, namely, chloroplast, peroxisome, mitochondrion, and the cytoplasm. It is unclear how the pathway located in different subcellular compartments respond to stress occurring exclusively in one of those. We attempted to assess the inter-organelle interaction during the photorespiratory pathway. For that purpose, we induced oxidative stress by menadione (MD) in mitochondria and photo-oxidative stress (high light) in chloroplasts. Subsequently, we examined the changes in selected photorespiratory enzymes, known to be located in other subcellular compartments. The presence of MD upregulated the transcript and protein levels of five chosen photorespiratory enzymes in both normal and high light. Peroxisomal glycolate oxidase and catalase activities increased by 50% and 25%, respectively, while chloroplastic glycerate kinase and phosphoglycolate phosphatase increased by ~30%. The effect of MD was maximum in high light, indicating photo-oxidative stress was an influential factor to regulate photorespiration. Oxidative stress created in mitochondria caused a coordinative upregulation of photorespiration in other organelles. We provided evidence that reactive oxygen species are important signals for inter-organelle communication during photorespiration. Thus, MD can be a valuable tool to modulate the redox state in plant cells to study the metabolic consequences across membranes.

Photosynthesis is sensitive to nitric oxide and respiration sensitive to hydrogen peroxide: Studies with pea mesophyll protoplasts.

Reports on the effect of nitric oxide (NO) or reactive oxygen species (ROS) on photosynthesis and respiration in leaf tissues are intriguing; therefore, the effects of exogenous addition of sodium nitroprusside (SNP, releases NO) or H2O2 on the photosynthetic O2 evolution and respiratory O2 uptake by mesophyll protoplasts in pea (Pisum sativum) were evaluated in the present study. Low concentrations of SNP or H2O2 were used to minimize nonspecific effects. The effects of NO or H2O2 on respiration and photosynthesis were different. The presence of NO decreased the rate of photosynthesis but caused a marginal stimulation of dark respiration. Conversely, externally administered H2O2 drastically decreased the rate of respiration but only slightly decreased photosynthesis. The PS I activity was more sensitive to NO than PS II. On the other hand, 100 µM H2O2 had no effect on the photochemical reactions of either PS I or PS II. The sensitivity of photosynthesis to antimycin A or SHAM (reflecting the interplay between chloroplasts and mitochondria) was not affected by NO. By contrast, H2O2 markedly decreased the sensitivity of photosynthesis to antimycin A and SHAM. It can be concluded that chloroplasts are the primary targets of NO, while mitochondria are the primary targets of ROS in plant cells. We propose that H2O2 can be an important signal to modulate the crosstalk between chloroplasts and mitochondria.

Oxidative stress induced in chloroplasts or mitochondria promotes proline accumulation in leaves of pea (Pisum sativum): another example of chloroplast-mitochondria interactions.

Oxidative stress can occur in different parts of plant cells. We employed two oxidants that induce reactive oxygen species (ROS) in different intracellular compartments: methyl viologen (MV, in chloroplasts) and menadione (MD, in mitochondria). The responses of pea (Pisum sativum) leaf discs to MV or MD after 4-h incubation in dark or moderate (300 µE m-2 s-1) or high light (1200 µE m-2 s-1) were examined. Marked increase in ROS levels was observed, irrespective of compartment targeted. The levels of proline, a compatible solute, increased markedly much more than that of ascorbate or glutathione during oxidative/photo-oxidative stress, emphasizing the importance of proline. Further, the activities and transcripts of enzymes involved in biosynthesis or oxidation of proline were studied. An upregulation of biosynthesis and downregulation of oxidation was the basis of proline accumulation. Pyrroline-5-carboxylate synthetase (P5CS, involved in biosynthesis) and proline dehydrogenase (PDH, involved in oxidation) were the key enzymes regulated under oxidative stress. Since these two enzymes-P5CS and PDH-are located in chloroplasts and mitochondria, respectively, we suggest that proline metabolism can help to mediate inter-organelle interactions and achieve redox homeostasis under photo-oxidative stress.

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in Pisum sativum.

MAIN CONCLUSION: Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N',N'-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

Ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosynthesis and protect against photoinhibition.

The possible role of L-ascorbate (AsA) as a biochemical signal during the interactions between photosynthesis and respiration was examined in leaf discs of Arabidopsis thaliana. AsA content was either decreased as in AsA-deficient vtc1 mutants or increased by treatment with L-galactono-1, 4-lactone (L-GalL, a precursor of AsA; EC 1.3.2.3). In mutants, photosynthesis was extremely sensitive to both antimycin A (inhibitor of the cytochrome c oxidase pathway [COX pathway]) and salicylhydroxamic acid (SHAM, inhibitor of the alternative pathway [AOX pathway]), particularly at high light conditions. Mitochondrial inhibitors lowered the ratio of reduced AsA to total AsA, at high light, indicating oxidative stress in leaf discs. Elevation of AsA by L-GalL decreased the sensitivity of photosynthesis at high light to antimycin A or SHAM, sustained photosynthesis at supraoptimal light and relieved the extent of photoinhibition. High ratios of reduced AsA to total AsA in L-GalL-treated leaf discs suggests that L-GalL lowers oxidative stress. The protection by L-GalL of photosynthesis against the mitochondrial inhibitors and photoinhibition was quite pronounced in vtc1 mutants. Our results suggest that the levels and redox state of AsA modify the pattern of modulation of photosynthesis by mitochondrial metabolism. The extent of the AOX pathway as a percentage of the total respiration in Arabidopsis mesophyll protoplasts was much higher in vtc1 than in wild type. We suggest that the role of AsA becomes pronounced at high light and/or when the AOX pathway is inhibited. While acknowledging the importance of the COX pathway, we hypothesize that AsA and the AOX pathway may complement each other to protect photosynthesis against photoinhibition.