Agepati S. Raghavendra

Cytoplasmic Alkalization Precedes Reactive Oxygen Species Production during Methyl Jasmonate- and Abscisic Acid-Induced Stomatal Closure

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H2O2 production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H2O2 production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H2O2 production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.

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.

Interdependence of photosynthesis and respiration in plant cells: interactions between chloroplasts and mitochondria

Abstract Photosynthesis and respiration in an illuminated plant cell are not only interdependent but also mutually beneficial. Respiratory rates increase after hours of illumination due to carbohydrate (substrate) accumulation. Besides such long-term effects, photosynthesis and respiration interact even during short illumination periods of a few minutes. The rate of respiration in isolated leaf protoplasts increases severalfold after 10–15 min of illumination. Such light-enhanced dark respiration (LEDR) has been demonstrated in protoplasts as well as in leaves. The stimulation of LEDR by bicarbonate and its sensitivity to inhibitors of photosynthesis (DCMU) or the Calvin cycle ( d,l -glyceraldehyde point out the importance of photosynthetic carbon metabolism for respiration. From metabolite analyses of protoplasts, the majority of LEDR is due to mitochondrial oxidation of malate produced by chloroplasts. Simultaneous measurements of photosynthesis and respiration, using mass spectrometry, demonstrate that mitochondrial TCA cycle-based CO2 evolution is inhibited by illumination while O2 uptake is either unaffected or stimulated. The marked sensitivity of photosynthesis in leaves or protoplasts to classic mitochondrial inhibitors such as oligomycin, sodium azide or antimycin A implies that mitochondrial metabolism is essential for photosynthesis. Respiration not only benefits photosynthesis but also protects illuminated leaf protoplasts against photoinhibition. Oxidative electron transport and phosphorylation play a much more important role than the reactions of glycolysis and the TCA cycle in this beneficial interaction. The metabolite shuttles involving PGA-DHAP and/or OAA-malate across the chloroplast and mitochondrial membranes could form the biochemical basis of the interaction between photosynthesis and respiration. Alternatively, cytosolic NAD(P)H, derived from photosynthetic products, can be directly acted upon by the mitochondrial external NAD(P)H dehydrogenase and oxidised through the mitochondrial electron transport system. Mitochondrial oxidation of NAD(P)H (even if indirect) helps to prevent the over-reduction of the cytosol and, consequently, the chloroplast in illuminated leaf cells. Besides the direct interaction with chloroplasts, mitochondria can supply reducing equivalents through malate to peroxisomes during photorespiration and provide citrate as the precursor of oxoglutarate, necessary for glutamine and glutamate formation. These two phenomena further complement the strong interdependence of photosynthesis and mitochondrial metabolism.

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

Referee: Christine Foyer, Dept. of Biochemistry and Physiology, IACR - Rothamsted, Harpemden, Herts AL5 2JQ, England, United Kingdom 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 interesting topic.

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A.

Plant respiration is characterized by two pathways for electron transfer to O(2), namely the cytochrome pathway (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O(2) via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOX1A. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosynthesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize photosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3 plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

Nitric oxide production occurs after cytosolic alkalinization during stomatal closure induced by abscisic acid.

Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum. Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min. These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production. We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.

Different signaling pathways involved during the suppression of stomatal opening by methyl jasmonate or abscisic acid

Abstract The stomatal opening in abaxial epidermal strips of Nicotiana glauca is suppressed by the presence of abscisic acid (ABA) or methyl jasmonate (MJ). The role of calcium and related secondary messengers/signaling compounds during the restriction of stomatal opening by ABA or MJ was assessed. External calcium promoted, while EGTA prevented the process of stomatal closure, in case of both ABA and MJ. However, the effect of EGTA was more pronounced in the case of MJ than that of ABA. These observations established that the intracellular calcium is an important factor during stomatal closure induced not only by ABA but also by MJ. Lanthanum, verapamil, ruthenium red (Ca 2+ channel blockers), trifluoperazine and W7 (calmodulin (CaM) antagonists) prevented MJ-induced stomatal closure but had only a partial effect on ABA-mediated stomatal closure. In contrast, U73122, 1-butanol (phospholipase C/D inhibitors) and phorbol myristate acetate (PMA, protein kinase C activator) reversed the stomatal closure caused by ABA but not that by MJ. Thus, the suppression of stomatal opening by ABA appears to be more dependent on phospholipases and protein kinase C than that by MJ. Similarly, the action of MJ on stomata seems to be crucially dependent on CaM and calcium channels of the guard cells. We suggest that MJ could be a useful tool, besides ABA, to analyze the sequence and pattern of signal transduction in stomatal guard cells.

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 C4ppc 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 His138, His579 and Arg587 in catalysis and/or substrate-binding by the E. coli enzyme, Ser8 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.

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.