Flowering onset time is regulated by microRNA-mediated trehalose-6-phosphate signaling in Cajanus cajan L. under elevated CO2.

CO2 levels are known to have an impact on plant development and physiology. In the current study, we have investigated the effect of elevated CO2 on flowering and its regulation through miRNA mediated sugar signaling. We also unraveled small RNA transcriptome of pigeonpea under ambient and elevated CO2 conditions and predicted the targets for crucial miRNAs through computational methods. The results have shown that the delayed flowering in pigeonpea under elevated CO2 was due to an imbalance in C:N stoichiometry and differential expression pattern of aging pathway genes, including SQUAMOSA PROMOTER BINDING PROTEIN-LIKE. Furthermore, qRT PCR analysis has revealed the role of miR156 and miR172 in mediating trehalose-6-phosphate dependent flowering regulation. The current study is crucial in understanding the responses of flowering patterns in a legume crop to elevated CO2 which showed a significant impact on its final yields. Also, these findings are crucial in devising effective crop improvement strategies for developing climate resilient crops, including pigeonpea. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01434-9.

Pod-wall proteomics provide novel insights into soybean seed-filling process under chemical-induced terminal drought stress.

BACKGROUND: Drought is very detrimental when it occurs during the reproductive phase of soybeans, leading to considerable yield loss due to the disproportionate allocation of photo-assimilates to competing sinks. As pod walls are known to play a crucial role in regulating carbon partitioning during seed filling under stress conditions, the present study aims to analyze the stage-specific carbon allocation pattern during potassium iodide (KI)-simulated terminal drought, and to provide an insight into the pod-wall proteome responses during drought onset. RESULTS: A comparative proteomics approach was adopted to visualize the differential protein expression in soybean pod-wall at stage R5 (seed initiation). Sugar status was analyzed using high-performance liquid chromatography (HPLC) and biochemical methods. Potassium iodide-simulated terminal drought during reproductive stages 4, 5 and 6 (R4, R5, and R6) caused a significant decline in starch, total carbohydrate, and reducing sugar in the leaves; however, the pod-wall and seeds showed a reduction only in the total carbohydrate content, whereas starch and reducing sugar levels remained unchanged. A pod-wall proteome at stage R5 showed immediate induction of proteins belonging to stress signaling / regulation, protein folding / stabilization, redox-homeostasis, cellular energy, and carbon utilization and down-regulation of negative regulators of drought stress and protein degradation-related proteins. CONCLUSIONS: A KI spray effectively simulated terminal drought stress and caused around 50% yield loss when compared to controls. Our results indicate that, at the very onset of desiccation stress, the pod wall (stage R5) activates strong protective responses to maintain the carbon allocation to the surviving seeds. © 2018 Society of Chemical Industry.

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.

Unravelling molecular mechanisms from floral initiation to lipid biosynthesis in a promising biofuel tree species, Pongamia pinnata using transcriptome analysis.

Pongamia pinnata (L.) (Fabaceae) is a promising biofuel tree species which is underexploited in the areas of both fundamental and applied research, due to the lack of information either on transcriptome or genomic data. To investigate the possible metabolic pathways, we performed whole transcriptome analysis of Pongamia through Illumina NextSeq platform and generated 2.8 GB of paired end sequence reads. The de novo assembly of raw reads generated 40,000 contigs and 35,000 transcripts, representing leaf, flower and seed unigenes. Spatial and temporal expression profiles of photoperiod and floral homeotic genes in Pongamia, identified GIGANTEA (GI) - CONSTANS (CO) - FLOWERING LOCUS T (FT) as active signal cascade for floral initiation. Four prominent stages of seed development were selected in a high yielding Pongamia accession (TOIL 1) to follow the temporal expression patterns of key fatty acid biosynthetic genes involved in lipid biosynthesis and accumulation. Our results provide insights into an array of molecular events from flowering to seed maturity in Pongamia which will provide substantial basis for modulation of fatty acid composition and enhancing oil yields which should serve as a potential feedstock for biofuel production.

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water-use efficiency and abiotic stress tolerance in plants.

We have generated 3900 enhancer-based activation-tagged plants, in addition to 1030 stable Dissociator-enhancer plants in a widely cultivated indica rice variety, BPT-5204. Of them, 3000 were screened for water-use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield-related attributes under water-limiting conditions that identified 200 activation-tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ13 C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up-regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating-tagging. The high up-regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis-regulatory elements in their promoter regions along with the high up-regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance.

Ectopic Expression of an Atypical Hydrophobic Group 5 LEA Protein from Wild Peanut, Arachis diogoi Confers Abiotic Stress Tolerance in Tobacco.

Late embryogenesis abundant (LEA) proteins are a group of hydrophilic proteins, which accumulate in plants under varied stress conditions like drought, salinity, extreme temperatures and oxidative stress suggesting their role in the protection of plants against these stresses. A transcript derived fragment (TDF) corresponding to LEA gene, which got differentially expressed in wild peanut, Arachis diogoi against the late leaf spot pathogen, Phaeoisariopsis personata was used in this study. We have cloned its full length cDNA by RACE-PCR, which was designated as AdLEA. AdLEA belongs to the atypical Group 5C of LEA protein family as confirmed by sequence analysis. Group 5C LEA protein subfamily contains Pfam LEA_2 domain and is highly hydrophobic. In native conditions, expression of AdLEA was upregulated considerably upon hormonal and abiotic stress treatments emphasizing its role in abiotic stress tolerance. Subcellular localization studies showed that AdLEA protein is distributed in both nucleus and cytosol. Ectopic expression of AdLEA in tobacco resulted in enhanced tolerance of plants to dehydration, salinity and oxidative stress with the transgenic plants showing higher chlorophyll content and reduced lipid peroxidation as compared to wild type plants. Overexpressed AdLEA tobacco plants maintained better photosynthetic efficiency under drought conditions as demonstrated by chlorophyll fluorescence measurements. These plants showed enhanced transcript accumulation of some stress-responsive genes. Our study also elucidates that ROS levels were significantly reduced in leaves and stomatal guard cells of transgenic plants upon stress treatments. These results suggest that AdLEA confers multiple stress tolerance to plants, which make it a potential gene for genetic modification in plants.

Proline over-accumulation alleviates salt stress and protects photosynthetic and antioxidant enzyme activities in transgenic sorghum [Sorghum bicolor (L.) Moench].

Shoot-tip derived callus cultures of Sorghum bicolor were transformed by Agrobacterium tumefaciens as well as by bombardment methods with the mutated pyrroline-5-carboxylate synthetase (P5CSF129A) gene encoding the key enzyme for proline biosynthesis from glutamate. The transgenics were selfed for three generations and T4 plants were examined for 100 mM NaCl stress tolerance in pot conditions. The effect of salt stress on chlorophyll and carotenoid contents, photosynthetic rate, stomatal conductance, internal carbon dioxide concentration, transpiration rates, intrinsic transpiration and water use efficiencies, proline content, MDA levels, and antioxidant enzyme activities were evaluated in 40-day-old transgenic lines and the results were compared with untransformed control plants. The results show that chlorophyll content declines by 65% in untransformed controls compared to 30-38% loss (significant at P < 0.05) in transgenics but not carotenoid levels. Photosynthetic rate (PSII activity) was reduced in untransformed controls almost completely, while it declined by 62-88% in different transgenic lines. Salinity induced ca 100% stomatal closure in untransformed plants, while stomatal conductance was decreased only by 64-81% in transgenics after 4 days. The intercellular CO2 decreased by ca 30% in individual transgenic lines. Malondialdehyde (MDA) content was lower in transgenics compared to untransformed controls. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and glutathione reductase (GR; EC1.8.1.7) were quantified in leaves exposed to 100 mM NaCl stress and found higher in transgenics. The results suggest that transgenic lines were able to cope better with salt stress than untransformed controls by protecting photosynthetic and antioxidant enzyme activities.

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.

Growth, reproductive phenology and yield responses of a potential biofuel plant, Jatropha curcas grown under projected 2050 levels of elevated CO2.

Jatropha (Jatropha curcas) is a non-edible oil producing plant which is being advocated as an alternative biofuel energy resource. Its ability to grow in diverse soil conditions and minimal requirements of essential agronomical inputs compared with other oilseed crops makes it viable for cost-effective advanced biofuel production. We designed a study to investigate the effects of elevated carbon dioxide concentration ([CO(2)]) (550 ppm) on the growth, reproductive development, source-sink relationships, fruit and seed yield of J. curcas. We report, for the first time that elevated CO(2) significantly influences reproductive characteristics of Jatropha and improve its fruit and seed yields. Net photosynthetic rate of Jatropha was 50% higher in plants grown in elevated CO(2) compared with field and ambient CO(2) -grown plants. The study also revealed that elevated CO(2) atmosphere significantly increased female to male flower ratio, above ground biomass and carbon sequestration potential in Jatropha (24 kg carbon per tree) after 1 year. Our data demonstrate that J. curcas was able to sustain enhanced rate of photosynthesis in elevated CO(2) conditions as it had sufficient sink strength to balance the increased biomass yields. Our study also elucidates that the economically important traits including fruit and seed yield in elevated CO(2) conditions were significantly high in J. curcas that holds great promise as a potential biofuel tree species for the future high CO(2) world.

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.

Paclobutrazol treatment as a potential strategy for higher seed and oil yield in field-grown Camelina sativa L. Crantz.

BACKGROUND: Camelina (Camelina sativa L. Crantz) is a non-food oilseed crop which holds promise as an alternative biofuel energy resource. Its ability to grow in a variety of climatic and soil conditions and minimal requirements of agronomical inputs than other oilseed crops makes it economically viable for advanced biofuel production. We designed a study to investigate the effect of paclobutrazol [2RS, 3RS)-1-(4-Chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol] (PBZ), a popular plant growth regulator, on the seed and oil yield of Camelina sativa (cv. Celine). RESULTS: A field-based micro-trial setup was established in a randomized block design and the study was performed twice within a span of five months (October 2010 to February 2011) and five different PBZ treatments (Control: T0; 25 mg l-1: T1; 50 mg l-1: T2; 75 mg l-1: T3; 100 mg l-1: T4; 125 mg l-1: T5) were applied (soil application) at the time of initiation of flowering. PBZ at 100 mg l-1 concentration (T4) resulted in highest seed and oil yield by 80% and 15%, respectively. The seed yield increment was mainly due to enhanced number of siliques per plant when compared to control. The PBZ - treated plants displayed better photosynthetic leaf gas exchange characteristics, higher chlorophyll contents and possessed dark green leaves which were photosynthetically active for a longer period and facilitated higher photoassimilation. CONCLUSION: We report for the first time that application of optimized PBZ dose can be a potential strategy to achieve higher seed and oil yield from Camelina sativa that holds great promise as a biofuel crop in future.

Differential degradation of photosystem I subunits under iron deficiency in rice.

Rice (Oryza sativa) is one of the staple foods of the world. Iron (Fe) deficiency is a major abiotic stress factor that contributes world-wide to losses in crop yield and decline in nutritional quality. As cofactor for many enzymes and proteins, iron is an essential element. It plays a pivotal role in chlorophyll (Chl) biosynthesis, and iron deficiency may result in decreased Chl production and, thus, reduced photosynthetic capacity. Photosystem I (PSI) is a prime target of iron deficiency because of its high iron content (12 Fe per PS). To understand the protein level changes in the light-harvesting complex (LHC) of PSI (LHCI) under iron deficiency, rice seedlings were grown in Hoagland's nutrient medium with and without Fe. Chlorophyll content and photosynthetic efficiency decreased under iron deficiency. Protein gel blots probed with antibodies against the PSI core and Lhca 1-4 proteins revealed that the core subunits PsaA and PsaB remained stable under iron deficiency, whereas PsaC and PsaD decreased by about 50%, and PsaE was completely degraded. Among the LHCI subunits, Lhca1 and Lhca2 decreased by 40 and 50%, respectively, whereas Lhca3 and Lhca4 were completely degraded. We propose that the dissociation of LHCI subunits may be due to increased levels of reactive oxygen species, which is suggested by the increased activity of superoxide dismutase.

Responses of Gmelina arborea, a tropical deciduous tree species, to elevated atmospheric CO2: growth, biomass productivity and carbon sequestration efficacy.

The photosynthetic response of trees to rising CO(2) concentrations largely depends on source-sink relations, in addition to differences in responsiveness by species, genotype, and functional group. Previous studies on elevated CO(2) responses in trees have either doubled the gas concentration (>700 µmol mol(-1)) or used single large addition of CO(2) (500-600 µmol mol(-1)). In this study, Gmelina arborea, a fast growing tropical deciduous tree species, was selected to determine the photosynthetic efficiency, growth response and overall source-sink relations under near elevated atmospheric CO(2) concentration (460 µmol mol(-1)). Net photosynthetic rate of Gmelina was ~30% higher in plants grown in elevated CO(2) compared with ambient CO(2)-grown plants. The elevated CO(2) concentration also had significant effect on photochemical and biochemical capacities evidenced by changes in F(V)/F(M), ABS/CSm, ET(0)/CSm and RuBPcase activity. The study also revealed that elevated CO(2) conditions significantly increased absolute growth rate, above ground biomass and carbon sequestration potential in Gmelina which sequestered ~2100 g tree(-1) carbon after 120 days of treatment when compared to ambient CO(2)-grown plants. Our data indicate that young Gmelina could accumulate significant biomass and escape acclimatory down-regulation of photosynthesis due to high source-sink capacity even with an increase of 100 µmo lmol(-1) CO(2).

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.

A root proteomics-based insight reveals dynamic regulation of root proteins under progressive drought stress and recovery in Vigna radiata (L.) Wilczek.

To understand the complex drought response mechanism in crop plants, a systematic root proteomics approach was adopted to identify and analyze the expression patterns of differentially expressed major root proteins of Vigna radiata during short-term (3 days) and consecutive long-term water-deficit (6 days) as well as during recovery (6 days after re-watering). Photosynthetic gas exchange parameters of the plant were measured simultaneously during the stress treatment and recovery period. A total of 26 major protein spots were successfully identified by mass spectrometry, which were grouped according to their expression pattern during short-term stress as significantly up-regulated (9), down-regulated (10), highly down-regulated, beyond detection level of the software (2) and unchanged (5). The subsequent changes in the expression patterns of these proteins during long-term stress treatment and recovery period was analyzed to focus on the dynamic regulation of these functionally important proteins during progressive drought and recovery period. Cytoskeleton-related proteins were down-regulated initially (3d) but regained their expression levels during subsequent water-deficit (6d) while glycoprotein like lectins, which were primarily known to be involved in legume-rhizobia symbiosis, maintained their enhanced expression levels during both short and long-term drought treatment indicating their possible role in drought stress response of legumes. Oxidative stress-related proteins including Cu/Zn superoxide dismutase, oxidoreductase and aldehyde reductase were also up-regulated. The analyses of the dynamic regulation of these root proteins during short- and long-term water-deficit as well as recovery period may prove crucial for further understanding of drought response mechanisms in food legumes.

The antioxidant and antiproliferative activities of methanolic extracts from Njavara rice bran.

BACKGROUND: Free radical-induced oxidative stress is the root cause for many human diseases. Naturally occurring antioxidant supplements from plants are vital to counter the oxidative damage in cells. The main objective of the present study was to characterize the antioxidant and antiproliferative potential of rice bran extracted from an important Indian rice variety, Njavara and to compare the same with two commercially available basmati rice varieties: Vasumathi, Yamini and a non medicinal variety, Jyothi. METHODS: Methanolic extracts of rice bran from four varieties; Vasumathi, Yamini, Jyothi and Njavara were used to study their total phenolic and flavonoid contents, in vitro antioxidant activities including total antioxidant activity, scavenging of nitric oxide and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical, reducing power and cytotoxic activity in C6 glioma cells. Correlation coefficient and regression analysis were done by using Sigmastat version 3.1 and Stata statistical package respectively. RESULTS: Rice bran methanolic extract from Njavara showed the highest antioxidant and cell cytotoxic properties compared to the other three rice varieties. IC50 values for scavenging DPPH and nitric oxide were in the range of 30.85-87.72 microg/ml and 52.25-107.18 microg/ml respectively. Total antioxidant activity and reducing power were increased with increasing amounts of the extract. Total phenolic and flavonoid contents were in the range of 3.2-12.4 mg gallic acid-equivalent (GAE)/g bran and 1.68-8.5 mg quercetin-equivalent (QEE)/g bran respectively. IC50 values of cytotoxic assay (MTT assay) were 17.53-57.78 microg/ml. Correlation coefficient and regression analysis of phenolic content with DPPH and NO scavenging, MTT (-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, total antioxidant assay and reducing power showed a highly significant correlation coefficient values (96-99%) and regression values (91-98%). CONCLUSION: The results of the present study show that the crude methanolic extract from Njavara rice bran contains significantly high polyphenolic compounds with superior antioxidant activity as evidenced by scavenging of free radicals including DPPH and NO. Njavara extracts also showed highest reducing power activity, anti-proliferative property in C6 glioma cells. In conclusion, it is conceivable that the Njavara rice variety could be exploited as one of the potential sources for plant--based pharmaceutical products.

BpaI and BpnI: novel type II restriction endonucleases from Bacillus pasteurii and Bacillus pantothenticus.

Two novel type II restriction endonucleases, designated as BpaI and BpnI, were isolated from Bacillus pasteurii strain1761 and Bacillus pantothenticus strain1639, respectively. They were partially purified and SDS-PAGE indicated Mr values of 28 and 67 kDa for BpaI, 28 and 48 kDa for BpnI. The partially purified endonucleases hydrolyzed DNA into discrete fragments: pUC18 (2.6 kb for BpaI; 1.8 and 0.8 kb for BpnI), pBR322 (2.5 and 1.8 kb for BpaI; 2.6 and 1.7 kb for BpnI) and phix174 DNA (3.2 and 2.1 kb for BpaI; 4 and 1.3 kb for BpnI).