Reversible changes in structure and function of photosynthetic apparatus of pea (Pisum sativum) leaves under drought stress.

The effects of drought on photosynthesis have been extensively studied, whereas those on thylakoid organization are limited. We observed a significant decline in gas exchange parameters of pea (Pisum sativum) leaves under progressive drought stress. Chl a fluorescence kinetics revealed the reduction of photochemical efficiency of photosystem (PS)II and PSI. The non-photochemical quenching (NPQ) and the levels of PSII subunit PSBS increased. Furthermore, the light-harvesting complexes (LHCs) and some of the PSI and PSII core proteins were disassembled in drought conditions, whereas these complexes were reassociated during recovery. By contrast, the abundance of supercomplexes of PSII-LHCII and PSII dimer were reduced, whereas LHCII monomers increased following the change in the macro-organization of thylakoids. The stacks of thylakoids were loosely arranged in drought-affected plants, which could be attributed to changes in the supercomplexes of thylakoids. Severe drought stress caused a reduction of both LHCI and LHCII and a few reaction center proteins of PSI and PSII, indicating significant disorganization of the photosynthetic machinery. After 7 days of rewatering, plants recovered well, with restored chloroplast thylakoid structure and photosynthetic efficiency. The correlation of structural changes with leaf reactive oxygen species levels indicated that these changes were associated with the production of reactive oxygen species.

Suppression of photorespiratory metabolism by low O2 and presence of aminooxyacetic acid induces oxidative stress in Arabidopsis thaliana leaves.

Photorespiration, an essential component of plant metabolism, was upregulated under abiotic stress conditions, such as high light or drought. One of the signals for such upregulation was the rise in reactive oxygen species (ROS). Photorespiration was expected to mitigate oxidative stress by reducing ROS levels. However, it was unclear if ROS levels would increase when photorespiration was lowered. Our goal was to examine the redox status in leaves when photorespiratory metabolism was restricted under low O2 (medium flushed with N2 gas) or by adding aminooxyacetic acid (AOA), a photorespiratory inhibitor. We examined the impact of low O2 and AOA in leaves of Arabidopsis thaliana under dark, moderate, or high light. Downregulation of typical photorespiratory enzymes, including catalase (CAT), glycolate oxidase (GO), and phosphoglycolate phosphatase (PGLP) under low O2 or with AOA confirmed the lowering of photorespiratory metabolism. A marked increase in ROS levels (superoxide and H2O2) indicated the induction of oxidative stress. Thus, our results demonstrated for the first time that restricted photorespiratory conditions increased the extent of oxidative stress. We propose that photorespiration is essential to sustain normal ROS levels and optimize metabolism in cellular compartments of Arabidopsis leaves. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01388-4.

Photorespiration is complemented by cyclic electron flow and the alternative oxidase pathway to optimize photosynthesis and protect against abiotic stress.

Optimization of photosynthetic performance and protection against abiotic stress are essential to sustain plant growth. Photorespiratory metabolism can help plants to adapt to abiotic stress. The beneficial role of photorespiration under abiotic stress is further strengthened by cyclic electron flow (CEF) and alternative oxidase (AOX) pathways. We have attempted to critically assess the literature on the responses of these three phenomena-photorespiration, CEF and AOX, to different stress situations. We emphasize that photorespiration is the key player to protect photosynthesis and upregulates CEF as well as AOX. Then these three processes work in coordination to protect the plants against photoinhibition and maintain an optimal redox state in the cell, while providing ATP for metabolism and protein repair. H2O2 generated during photorespiratory metabolism seems to be an important signal to upregulate CEF or AOX. Further experiments are necessary to identify the signals originating from CEF or AOX to modulate photorespiration. The mutants deficient in CEF or AOX or both could be useful in this regard. The mutual interactions between CEF and AOX, so as to keep their complementarity, are also to be examined further.

Mechanism of Stomatal Closure in Plants Exposed to Drought and Cold Stress.

Drought is one of the abiotic stresses which impairs the plant growth/development and restricts the yield of many crops throughout the world. Stomatal closure is a common adaptation response of plants to the onset of drought condition. Stomata are microscopic pores on the leaf epidermis, which regulate the transpiration/CO2 uptake by leaves. Stomatal guard cells can sense various abiotic and biotic stress stimuli from the internal and external environment and respond quickly to initiate closure under unfavorable conditions. Stomata also limit the entry of pathogens into leaves, restricting their invasion. Drought is accompanied by the production and/or mobilization of the phytohormone, abscisic acid (ABA), which is well-known for its ability to induce stomatal closure. Apart from the ABA, various other factors that accumulate during drought and affect the stomatal function are plant hormones (auxins, MJ, ethylene, brassinosteroids, and cytokinins), microbial elicitors (salicylic acid, harpin, Flg 22, and chitosan), and polyamines . The role of various signaling components/secondary messengers during stomatal opening or closure has been a matter of intense investigation. Reactive oxygen species (ROS) , nitric oxide (NO) , cytosolic pH, and calcium are some of the well-documented signaling components during stomatal closure. The interrelationship and interactions of these signaling components such as ROS, NO, cytosolic pH, and free Ca2+ are quite complex and need further detailed examination.Low temperatures can have deleterious effects on plants. However, plants evolved protection mechanisms to overcome the impact of this stress. Cold temperature inhibits stomatal opening and causes stomatal closure. Cold-acclimated plants often exhibit marked changes in their lipid composition, particularly of the membranes. Cold stress often leads to the accumulation of ABA, besides osmolytes such as glycine betaine and proline. The role of signaling components such as ROS, NO, and Ca2+ during cold acclimation is yet to be established, though the effects of cold stress on plant growth and development are studied extensively. The information on the mitigation processes is quite limited. We have attempted to describe consequences of drought and cold stress in plants, emphasizing stomatal closure. Several of these factors trigger signaling components in roots, shoots, and atmosphere, all leading to stomatal closure. A scheme is presented to show the possible signaling events and their convergence and divergence of action during stomatal closure. The possible directions for future research are discussed.

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.

Perspectives for a better understanding of the metabolic integration of photorespiration within a complex plant primary metabolism network.

Photorespiration is an essential high flux metabolic pathway that is found in all oxygen-producing photosynthetic organisms. It is often viewed as a closed metabolic repair pathway that serves to detoxify 2-phosphoglycolic acid and to recycle carbon to fuel the Calvin-Benson cycle. However, this view is too simplistic since the photorespiratory cycle is known to interact with several primary metabolic pathways, including photosynthesis, nitrate assimilation, amino acid metabolism, C1 metabolism and the Krebs (TCA) cycle. Here we will review recent advances in photorespiration research and discuss future priorities to better understand (i) the metabolic integration of the photorespiratory cycle within the complex network of plant primary metabolism and (ii) the importance of photorespiration in response to abiotic and biotic stresses.

Nitric oxide as a secondary messenger during stomatal closure as a part of plant immunity response against pathogens.

Stomata facilitate the loss of water, as well as CO2 uptake for photosynthesis. In addition, stomatal closure restricts the entry of pathogens into leaves and forms a part of plant defense response. Plants have evolved ways to modulate stomata by plant hormones as well as microbial elicitors, including pathogen/microbe associated molecular patterns. Stomatal closure initiated by signals of either abiotic or biotic factors results from the loss of guard cell turgor due mainly to K(+)/anion efflux. Nitric oxide (NO) is a key element among the signaling elements leading to stomatal closure, hypersensitive response and programmed cell death. Due to the growing importance of NO as signaling molecule in plants, and the strong relation between stomata and pathogen resistance, we attempted to present a critical overview of plant innate immunity, in relation to stomatal closure. The parallel role of NO during plant innate immunity and stomatal closure is highlighted. The cross-talk between NO and other signaling components, such as reactive oxygen species (ROS) is discussed. The possible sources of NO and mechanisms of NO action, through post-translational modification of proteins are discussed. The mini-review is concluded with remarks on the existing gaps in our knowledge and suggestions for future research.

Role of mitochondria and chloroplasts during stomatal closure: Subcellular location of superoxide and H2O2 production in guard cells of Arabidopsis thaliana.

Stomatal guard cells are unique in that they have more mitochondria than chloroplasts. Several reports emphasized the importance of mitochondria as the major energy source during stomatal opening. We re-examined their role during stomatal closure. The marked sensitivity of stomata to both menadione (MD) and methyl viologen (MV) demonstrated that both mitochondria and chloroplasts helped to promote stomatal closure in Arabidopsis. As in the case of abscisic acid (ABA), a plant stress hormone, MD and MV induced stomatal closure at micromolar concentration. All three compounds generated superoxide and H2O2, as indicated by fluorescence probes, BES-So-AM and CM-H2DCFDA, respectively. Results from tiron (a superoxide scavenger) and catalase (an H2O2 scavenger) confirmed that both the superoxide and H2O2 were requisites for stomatal closure. Co-localization of the superoxide and H2O2 in mitochondria and chloroplasts using fluorescent probes revealed that exposure to MV initially triggered higher superoxide and H2O2 generation in mitochondria. In contrast, MD elevated superoxide/H2O2 levels in chloroplasts. However, with prolonged exposure, MD and MV induced ROS production in other organelles. We conclude that ROS production in mitochondria and chloroplasts leads to stomatal closure. We propose that stomatal guard cells can be good models for examining inter-organellar interactions.

Measurement of Photorespiratory Cycle Enzyme Activities in Leaves Exposed to Abiotic Stress.

Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress. This chapter describes the procedures for assaying several individual enzyme activities of key photorespiratory enzymes and evaluating their response to oxidative/photooxidative stress. It is essential to ascertain the presence of stress in the experimental material. Therefore, procedures for typical abiotic stress induction in leaves by highlighting without or with menadione (an oxidant that targets mitochondria) are also included.

Moderate modulation by S-nitrosoglutathione of photorespiratory enzymes in pea (Pisum sativum) leaves, compared to the strong effects of high light.

When plants are exposed to water stress, photosynthesis is downregulated due to enhanced reactive oxygen species (ROS) and nitric oxide (NO). In contrast, photorespiratory metabolism protected photosynthesis and sustained yield. Modulation of photorespiration by ROS was established, but the effect of NO on photorespiratory metabolism was unclear. We, therefore, examined the impact of externally added NO by using S-nitrosoglutathione (GSNO), a natural NO donor, in leaf discs of pea (Pisum sativum) under dark or light: moderate or high light (HL). Maximum NO accumulation with GSNO was under high light. The presence of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, prevented the increase in NO, confirming the release of NO in leaves. The increase in S-nitrosothiols and tyrosine-nitrated proteins on exposure to GSNO confirmed the nitrosative stress in leaves. However, the changes by GSNO in the activities and transcripts of five photorespiratory enzymes: glycolate oxidase, hydroxypyruvate reductase, catalase, glycerate kinase, and phosphoglycolate phosphatase activities were marginal. The changes in photorespiratory enzymes caused by GSNO were much less than those with HL. Since GSNO caused only mild oxidative stress, we felt that the key modulator of photorespiration might be ROS, but not NO.

Optimization of photosynthesis by multiple metabolic pathways involving interorganelle interactions: resource sharing and ROS maintenance as the bases.

The bioenergetic processes of photosynthesis and respiration are mutually beneficial. Their interaction extends to photorespiration, which is linked to optimize photosynthesis. The interplay of these three pathways is facilitated by two major phenomena: sharing of energy/metabolite resources and maintenance of optimal levels of reactive oxygen species (ROS). The resource sharing among different compartments of plant cells is based on the production/utilization of reducing equivalents (NADPH, NADH) and ATP as well as on the metabolite exchange. The responsibility of generating the cellular requirements of ATP and NAD(P)H is mostly by the chloroplasts and mitochondria. In turn, besides the chloroplasts, the mitochondria, cytosol and peroxisomes are common sinks for reduced equivalents. Transporters located in membranes ensure the coordinated movement of metabolites across the cellular compartments. The present review emphasizes the beneficial interactions among photosynthesis, dark respiration and photorespiration, in relation to metabolism of C, N and S. Since the bioenergetic reactions tend to generate ROS, the cells modulate chloroplast and mitochondrial reactions, so as to ensure that the ROS levels do not rise to toxic levels. The patterns of minimization of ROS production and scavenging of excess ROS in intracellular compartments are highlighted. Some of the emerging developments are pointed out, such as model plants, orientation/movement of organelles and metabolomics.

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.

Pyrabactin, an ABA agonist, induced stomatal closure and changes in signalling components of guard cells in abaxial epidermis of Pisum sativum.

Pyrabactin, a synthetic agonist of abscisic acid (ABA), inhibits seed germination and hypocotyl growth and stimulates gene expression in a very similar way to ABA, implying the possible modulation of stomatal function by pyrabactin as well. The effect of pyrabactin on stomatal closure and secondary messengers was therefore studied in guard cells of Pisum sativum abaxial epidermis. Pyrabactin caused marked stomatal closure in a pattern similar to ABA. In addition, pyrabactin elevated the levels of reactive oxygen species (ROS), nitric oxide (NO), and cytoplasmic pH levels in guard cells, as indicated by the respective fluorophores. However, apyrabactin, an inactive analogue of ABA, did not affect either stomatal closure or the signalling components of guard cells. The effects of pyrabactin-induced changes were reversed by pharmalogical compounds that modulate ROS, NO or cytoplasmic pH levels, quite similar to ABA effects. Fusicoccin, a fungal toxin, could reverse the stomatal closure caused by pyrabactin, as well as that caused by ABA. Experiments on stomatal closure by varying concentrations of ABA, in the presence of fixed concentration of pyrabactin, and vice versa, revealed that the actions of ABA and pyrabactin were additive. Further kinetic analysis of data revealed that the apparent K(D) of ABA was increased almost 4-fold in the presence of ABA, suggesting that pyrabactin and ABA were competing with each other either at the same site or close to the active site. It is proposed that pyrabactin could be used to examine the ABA-related signal-transduction components in stomatal guard cells as well as in other plant tissues. It is also suggested that pyrabactin can be used as an antitranspirant or as a priming agent for improving the drought tolerance of crop plants.

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 C(3) 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.

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 C(4) 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 C(4) 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.

Abscisic Acid-Induced Stomatal Closure: An Important Component of Plant Defense Against Abiotic and Biotic Stress.

Abscisic acid (ABA) is a stress hormone that accumulates under different abiotic and biotic stresses. A typical effect of ABA on leaves is to reduce transpirational water loss by closing stomata and parallelly defend against microbes by restricting their entry through stomatal pores. ABA can also promote the accumulation of polyamines, sphingolipids, and even proline. Stomatal closure by compounds other than ABA also helps plant defense against both abiotic and biotic stress factors. Further, ABA can interact with other hormones, such as methyl jasmonate (MJ) and salicylic acid (SA). Such cross-talk can be an additional factor in plant adaptations against environmental stresses and microbial pathogens. The present review highlights the recent progress in understanding ABA's multifaceted role under stress conditions, particularly stomatal closure. We point out the importance of reactive oxygen species (ROS), reactive carbonyl species (RCS), nitric oxide (NO), and Ca2+ in guard cells as key signaling components during the ABA-mediated short-term plant defense reactions. The rise in ROS, RCS, NO, and intracellular Ca2+ triggered by ABA can promote additional events involved in long-term adaptive measures, including gene expression, accumulation of compatible solutes to protect the cell, hypersensitive response (HR), and programmed cell death (PCD). Several pathogens can counteract and try to reopen stomata. Similarly, pathogens attempt to trigger PCD of host tissue to their benefit. Yet, ABA-induced effects independent of stomatal closure can delay the pathogen spread and infection within leaves. Stomatal closure and other ABA influences can be among the early steps of defense and a crucial component of plants' innate immunity response. Stomatal guard cells are quite sensitive to environmental stress and are considered good model systems for signal transduction studies. Further research on the ABA-induced stomatal closure mechanism can help us design strategies for plant/crop adaptations to stress.

Metabolomics of Withania somnifera (L.) Dunal: Advances and applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera L. (Solanaceae), commonly known as Ashwagandha or Indian ginseng, is used in Ayurveda (Indian system of traditional medicine) for vitality, cardio-protection and treating other ailments, such as neurological disorders, gout, and skin diseases. AIM OF THE REVIEW: We present a critical overview of the information on the metabolomics of W. somnifera and highlight the significance of the technique for use in quality control of medicinal products. We have also pointed out the use of metabolomics to distinguish varieties and to identify best methods of cultivation, collection, as well as extraction. MATERIAL AND METHODS: The relevant information on medicinal value, phytochemical studies, metabolomics of W. somnifera, and their applications were collected from a rigorous electronic search through scientific databases, including Scopus, PubMed, Web of Science and Google Scholar. Structures of selected metabolites were from the PubChem. RESULTS: The pharmacological activities of W. somnifera were well documented. Roots are the most important parts of the plant used in Ayurvedic preparations. Stem and leaves also have a rich content of bioactive phytochemicals like steroidal lactones, alkaloids, and phenolic acids. Metabolomic studies revealed that metabolite profiles of W. somnifera depended on plant parts collected and the developmental stage of the plant, besides the season of sample collection and geographical location. The levels of withanolides were variable, depending on the morpho/chemotypes within the species of W. somnifera. Although studies on W. somnifera were initiated several years ago, the complexity of secondary metabolites was not realized due to the lack of adequate and fool-proof technology for phytochemical fingerprinting. Sophistications in chromatography coupled to mass spectrometry facilitated the discovery of several new metabolites. Mutually complementary techniques like LC-MS, GC-MS, HPTLC, and NMR were employed to obtain a comprehensive metabolomic profile. Subsequent data analyses and searches against spectral databases enabled the annotation of signals and dereplication of metabolites in several numbers without isolating them individually. CONCLUSIONS: The present review provides a critical update of metabolomic data and the diverse application of the technique. The identification of parameters for standardization and quality control of herbal products is essential to facilitate mandatory checks for the purity of formulation. Such studies would enable us to identify the best geographical location of plants and the time of collection. We recommend the use of metabolomic analysis of herbal products based on W. somnifera for quality control as well as the discovery of novel bioactive compounds.

Variable Secondary Metabolite Profiles Across Cultivars of Curcuma longa L. and C. aromatica Salisb.

Background: Curcuma spp. (Zingiberaceae) are used as a spice and coloring agent. Their rhizomes and essential oils are known for medicinal properties, besides their use in the flavoring and cosmetic industry. Most of these biological activities were attributed to volatile and nonvolatile secondary metabolites present in the rhizomes of Curcuma spp. The metabolite variations among the species and even cultivars need to be established for optimized use of Curcuma spp. Objectives: We compared the phytochemical profiles of rhizomes and their essential oils to establish the variability among seven cultivars: five of Curcuma longa L. (Alleppey Supreme, Duggirala Red, Prathibha, Salem, Suguna) and two of C. aromatica Salisb. (Kasturi Araku, Kasturi Avidi). The GC-MS and LC-MS-based analyses were employed to profile secondary metabolites of these selected cultivars. Methods: Rhizomes of Curcuma spp. were subjected to hydro-distillation to collect essential oil and analyzed by GC-MS. The methanol extracts of fresh rhizomes were subjected to LC-MS analyses. The compounds were identified by using the relevant MS library databases as many compounds as possible. Results: The essential oil content of the cultivars was in the range of 0.74-1.62%. Several compounds were detected from the essential oils and rhizome extracts by GC-MS and LC-MS, respectively. Of these, 28 compounds (13 from GCMS and 15 from LCMS) were common in all seven cultivars, e.g., α-thujene, and diarylheptanoids like curcumin. Furthermore, a total of 39 new compounds were identified from C. longa L. and/or C. aromatica Salisb., most of them being cultivar-specific. Of these compounds, 35 were detected by GC-MS analyses of essential oils, 1,2-cyclohexanediol, 1-methyl-4-(1-methylethyl)-, and santolina alcohol, to name a few. The other four compounds were detected by LC-MS of the methanolic extracts of the rhizomes, e.g., kaempferol-3,7-O-dimethyl ether and 5,7,8-trihydroxy-2',5'-dimethoxy-3',4'-methylene dioxyisoflavanone. Conclusions: We identified and recorded the variability in the metabolite profiles of essential oils and whole rhizome extracts from the seven cultivars of Curcuma longa L. and C. aromatica Salisb. As many as 39 new metabolites were detected in these seven Indian cultivars of Curcuma spp. Many of these compounds have health benefits.

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.

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.