Debashree Sengupta

Plant aldo-keto reductases (AKRs) as multi-tasking soldiers involved in diverse plant metabolic processes and stress defense: A structure-function update.

The aldo-keto reductase (AKR) superfamily comprises of a large number of primarily monomeric protein members, which reduce a broad spectrum of substrates ranging from simple sugars to potentially toxic aldehydes. Plant AKRs can be broadly categorized into four important functional groups, which highlight their roles in diverse plant metabolic reactions including reactive aldehyde detoxification, biosynthesis of osmolytes, secondary metabolism and membrane transport. Further, multiple overlapping functional aspects of plant AKRs including biotic and abiotic stress defense, production of commercially important secondary metabolites, iron acquisition from soil, plant-microbe interactions etc. are discussed as subcategories within respective major groups. Owing to the broad substrate specificity and multiple stress tolerance of the well-characterized AKR4C9 from Arabidopsis thaliana, protein sequences of all the homologues of AKR4C9 (A9-like proteins) from forty different plant species (Phytozome database) were analyzed. The analysis revealed that all A9-like proteins possess strictly conserved key catalytic residues (D-47, Y-52 and K-81) and belong to the pfam00248 and cl00470 AKR superfamilies. Based on structural homology of the three flexible loops of AKR4C9 (Loop A, B and C) responsible for broad substrate specificity, A9-like proteins found in Brassica rapa, Phaseolus vulgaris, Cucumis sativus, Populus trichocarpa and Solanum lycopersicum were predicted to have a similar range of substrate specificity. Thus, plant AKRs can be considered as potential breeding targets for developing stress tolerant varieties in the future. The present review provides a consolidated update on the current research status of plant AKRs with an emphasis on important functional aspects as well as their potential future prospects and an insight into the overall structure-function relationships of A9-like proteins.

Polyphasic chlorophyll a fluorescence kinetics and leaf protein analyses to track dynamics of photosynthetic performance in mulberry during progressive drought

Modulation of photosynthesis and the underlying mechanisms were studied in mulberry (Morus indica L. genotype V1) under progressive drought stress conditions. Five months old potted mulberry plants were arranged in a semi-controlled glasshouse chamber in completely randomized block design with four replications. On day 1 (D1), the plants were subjected to two watering treatments: well-watered (WW) and water-stressed (WS). In WS plants, watering was completely withheld for next 10days (D1-D10), whereas the WW plants were maintained at 100% pot water holding capacity. Photosynthetic performance was tracked periodically (from D0 to D10) through measurements of leaf gas exchange and chlorophyll a fluorescence (OJIP) transients and additionally leaf protein analyses were performed on D10. Down-regulation in net CO(2) fixation (P(n)) was primarily mediated through stomatal limitation which concurrently reduced transpiration rate (E), stomatal conductance (g(s)) and intercellular CO(2) concentration (C(i)). The OJIP transients and other associated biophysical parameters elucidated the events of photoacclimatory changes in photosystem II (PSII) with progressive increase in drought stress. Down-regulation of PSII activity occurred predominantly due to increase in inactive reaction centers (RCs), decrease in electron transport per RC (ET(O)/RC) as well as per leaf cross-section (ET(O)/CS(m)) and enhanced energy dissipation. The L and K-bands appeared only in the stage of extreme drought severity indicating the ability of genotype V1 to resist drought-induced damage on structural stability of PSII and imbalance between the electrons at the acceptor and donor sides of PSII, respectively. Drought-induced changes in leaf protein analyses revealed significant up-regulation of important proteins associated to photostability of thylakoid membrane including oxygen evolving enhancer, chlorophyll a/b binding proteins, rubisco and rubisco activase. Further, the antioxidative defense proteins including peroxiredoxin and NADH ubiquinone oxidoreductase were also enhanced. In conclusion, our data demonstrate an integrated down-regulation of the photosynthetic process to maintain intrinsic balance between electron transfer reactions and reductive carbon metabolism without severe damage to PSII structural and functional integrity.

Molecular Cloning and Characterization of γ-Glutamyl Cysteine Synthetase (VrγECS) from Roots of Vigna radiata (L.) Wilczek Under Progressive Drought Stress and Recovery

Glutathione is an essential redox buffer and an antioxidant in majority of higher plants, imparting tolerance against abiotic stress. The rate-limiting enzyme, gamma-glutamyl cysteine synthetase (γECS), plays an important role in regulation of glutathione biosynthesis under adverse environmental conditions including drought. To understand the role of γECS in an economically important food legume, Vigna radiata (L.) Wilczek, under progressive drought stress, we cloned and derived the full-length cDNA sequence and denoted it as VrγECS. Real-time PCR analysis of VrγECS in the roots of V. radiata, during progressive drought stress and recovery, indicated a stable expression pattern of the gene. However, the VrγECS enzyme activity altered differentially during varying water-deficit conditions and recovery period, reflecting the existence of some post-transcriptional or post-translational regulatory system for the enzyme. Linear regression analysis between H2O2 and lipid peroxidation as well as H2O2 and VrγECS enzyme activity during drought stress and recovery demonstrates the delicate inter-relationships and putative regulatory mechanisms operating in the root system under adverse conditions. The present study could contribute towards understanding the complex regulation of γECS in glutathione biosynthesis in an important food legume under drought stress.

Physiological optimality, allocation trade-offs and antioxidant protection linked to better leaf yield performance in drought exposed mulberry

BACKGROUND Mulberry (Morus spp. L.), usually linked to silkworm rearing, is now considered as a potential forage for livestock feeding and has great potential in world agriculture. Trait-based investigations for leaf yield stability in mulberry under water stress have not been studied extensively. The present study aims to identify candidate traits conferring leaf yield stability in mulberry under drought. RESULTS Four popular, indigenous mulberry cultivars (Morus indica L. cvs AR-12, K-2, M. Local and V-1) were investigated. Low leaf temperature (T(l)), higher internal/ambient CO(2) ratios (C(i)/C(a)), greater stomatal conductance to CO(2) (g(s)) and stability in photosystem II efficiency were associated with better net photosynthetic rates (P(n)) in V-1, generating maximum leaf yield when compared to other drought-exposed cultivars. Increased accumulation of foliar α-tocopherol and ascorbic acid-glutathione pool, associated with higher carotenoids, proline and glycine betaine, facilitated lower lipid peroxidation and better leaf yield in V-1 under drought. CONCLUSION Minimal plasticity in photosynthetic gas exchange traits and better quantitative growth characteristics were attributed to leaf yield stability under drought. Lower photoinhibition, stabilized photochemistry, effective osmoregulation and enhanced activity of foliar antioxidants extensively contributed to drought tolerance and higher leaf yield in mulberry.

Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L., a promising biofuel plant.

In the present study, we have cloned a gene encoding JcMT2a protein from Jatropha curcas L., a promising biofuel tree species. Full length sequence of JcMT2a gene was isolated using RACE PCR. Heterologous expression of JcMT2a in Escherichia coli and its purification has shown distinct bands corresponding to the GST and GST-fused JcMT2a protein. Significant tolerance was observed in E. coli cells expressing recombinant GST-JcMT2a for zinc, copper and cadmium metals compared to cells expressing GST alone. JcMT2a also restored Cu and Cd tolerance in the metal sensitive yeast mutants. Quantitative real time PCR showed a significant increase in JcMT2a transcripts with Cu and Cd in the leaf compared to root tissue. Our Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis clearly demonstrates that J. curcas L. could be a potential candidate for phytoremediation to clean heavy metals from the environment, in addition to its non-edible oil seed yields for biodiesel production.

Interdependence of plant water status with photosynthetic performance and root defense responses in Vigna radiata (L.) Wilczek under progressive drought stress and recovery.

The present study investigates the interdependence of plant water status with foliar and root responses in Vigna radiata L.Wilczek under progressive drought. Vegetatively-mature V. radiata plants were subjected to water withdrawal for 3 and 6days (D3 and D6, respectively) and then re-watered subsequently for 6days (6R) for stress-recovery. Changes in plant water status were expressed in terms of leaf and root moisture contents (LMC and RMC, respectively) and leaf relative water content (LRWC). Progressive drought caused apparent decrease in LRWC, LMC and RMC depicting significant level of dehydration of leaf and root tissues. Stomatal limitation alone could not account for the observed decrease in net CO2 assimilation rates (Pn) due to comparatively less decrease in sub-stomatal CO2 (Ci) concentrations with respect to other gas exchange parameters indicating possible involvement of non-stomatal limitations. Analysis of polyphasic chl a fluorescence kinetics during progressive drought showed decreased energy connectivity among PSII units as defined by a positive L-band with highest amplitude during D6. Efficiency of electron flux from OEC towards PSII acceptor side was not significantly affected during drought conditions as evidenced by the absence of a positive K-band. Increasing root-level water-limitation enforced a gradual oxidative stress through H2O2 accumulation and membrane lipid peroxidation in V. radiata roots exhibiting drastic enhancement of proline content and a significant but gradual increase in ascorbic acid content as well as guaiacol peroxidase activity under progressive drought. Expression analysis of Δ(1) pyrroline-5-carboxylate synthetase (P5CS) through real time PCR and enzyme activity studies showed a strong positive correlation between VrP5CS gene expression, enzyme activity and proline accumulation in the roots of V. radiata under progressive drought and recovery. Drought-induced changes in root moisture content (RMC) showed positive linear correlations with leaf water content, stomatal conductance as well as transpirational water loss dynamics and a significant negative correlation with the corresponding drought-induced expression patterns of ascorbate, guaiacol peroxidase and proline in roots of V. radiata. The study provides new insights into the plant water status-dependent interrelationship between photosynthetic performance and major root defense responses of V. radiata under progressive drought conditions.

Water deficit as a regulatory switch for legume root responses

Plant roots perceive declining soil water potential as an initial signal which further triggers an array of physiological, morphological and molecular responses in the whole plant. Understanding the root responses with parallel insights on protein level changes has always been an area of interest for stress biologists. In a recent study, we reported drought stress-induced changes among certain structural and functional root proteins involved in reactive oxygen species (ROS) detoxification, primary and secondary metabolite biosynthetic pathways as well as proteins associated with cell signalling in an economically important legume crop Vigna radiata (L.) Wilczek. We also demonstrated photosynthetic gas exchange characteristics and root physiology under varying levels of water-deficit and recovery. In this report, we depict a closer analysis of the expression patterns of the identified proteins which were categorized into five major functional groups. These proteins represent a unique coherence and networking with each other as well as with the overall physiological and metabolic machinery in the plant cell.

Detoxification potential and expression analysis of eutypine reducing aldehyde reductase (VrALR) during progressive drought and recovery in Vigna radiata (L.) Wilczek roots.

Generation of reactive oxygen species (ROS) in plants is an inevitable consequence of adverse environmental cues and the ability to detoxify deleterious by-products of ROS-mediated oxidation reactions reflect an important defence strategy to combat abiotic stress. Here, we have cloned the eutypine reducing aldehyde reductase gene (VrALR) from Vigna radiata (L.) Wilczek roots. We have expressed and purified the VrALR protein and analyzed its enzyme kinetic parameters and catalytic efficiency with three different substrates to confirm its identity. The functional characterization of this enzyme was unravelled through heterologous expression of the gene in Escherichia coli BL21 and an oxidative stress-sensitive Saccharomyces cerevisiae mutant strain, W3O3-1-A. Finally, the endogenous VrALR enzyme activity and the mRNA expression patterns of the VrALR gene in the roots of V. radiata in response to progressive drought stress in vivo was studied to correlate the ROS-detoxifying role of this important enzyme under the influence of progressive drought stress. Our results, for the first time, demonstrate that eutypine reducing VrALR provides varying degree of stress tolerance in bacteria, yeast systems and also plays a promising protective role against oxidative stress in V. radiata roots during gradual water deprivation. The present study provides an unequivocal evidence to understand the crucial role of aldehyde reductase ROS-detoxifying system which is highly essential for developing stress tolerance in economically important crop plants.

Physiological and molecular insights into the high salinity tolerance of Pongamia pinnata (L.) pierre, a potential biofuel tree species

Soil salinity is gradually becoming a threat to the global economy by affecting agricultural productivity worldwide. Here, we analyze the salinity tolerance of Pongamia pinnata with an insight into the underlying physiological and molecular responses. Despite a reduction in net photosynthetic rate, P. pinnata efficiently maintained its leaf water potentials even at 500mM NaCl for 15days and displayed no visible stress symptoms. Na+ localization analysis using CoroNa-Green AM revealed effective Na+ sequestration in the roots when compared to leaves. Elemental analysis demonstrated that roots accumulated more of Na+ while K+ content was higher in leaves. At the molecular level, salt stress significantly induced the expression levels of salt overly sensitive1 (SOS1), SOS2, SOS3, high affinity K+ transporter (HKT1), ABA biosynthetic and receptor genes (NCED and PYL4), guaiacol peroxidase (POD) exclusively in roots while tonoplast localized Na+/H+ exchanger (NHX1) was significantly enhanced in leaves. Our results clearly demonstrate that leaves and roots of Pongamia exhibit differential responses under salt stress although roots are more efficient in sequestering the Na+ ions. The present study provides crucial inputs for understanding salt tolerance in a tree species which can be further utilized for developing salt tolerance in higher plants.

Simplifying the root dynamics: from complex hormone–environment interactions to specific root architectural modulation

The recent unveiling of the intriguing interactions among phytohormones and environmental cues in regulating root architecture for optimum plant acclimation has opened new avenues for research. Additional functions of transcriptional as well as protein-level regulators are being identified, uncovering novel interactions between hormonal and environmental signaling pathways, for shaping the root system architecture (RSA). Owing to the importance of root architectural dynamics under constantly encountered external factors, it is crucial to have a regular and comprehensive update of these interactions, affecting RSA, in order to improve crop performance. Moreover, it is equally important to identify and highlight, in crop species, the crucial regulators, which actively mediate hormonal as well as hormone–environment interactions, but have so far been characterized only in model plants such as Arabidopsis. Such updates will open up new research possibilities for plant biologists in extending the present knowledge on root system plasticity from Arabidopsis to economically important crop plants. Here, we provide a consolidated review of the recent findings on novel inter-hormonal and hormone–environment interactions with special emphasis on key downstream regulators and signaling pathways. We conclude by dissecting the gaps and challenges encountered at present, with an outline for future perspectives to channel the enormous information on hormone–environment regulation of RSA, towards a common output in the form of specific modulation of RSA components.