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.

Mitigation of drought-induced oxidative damage by enhanced carbon assimilation and an efficient antioxidative metabolism under high CO2 environment in pigeonpea (Cajanus cajan L.).

In the current study, pigeonpea (Cajanus cajan L.), a promising legume food crop was assessed for its photosynthetic physiology, antioxidative system as well as C and N metabolism under elevated CO2 and combined drought stress (DS). Pigeonpea was grown in open top chambers under elevated CO2 (600 µmol mol-1) and ambient CO2 (390 ± 20 µmol mol-1) concentrations, later subjected to DS by complete water withholding. The DS plants were re-watered and recovered (R) to gain normal physiological growth and assessed the recoverable capacity in both elevated and ambient CO2 concentrations. The elevated CO2 grown pigeonpea showed greater gas exchange physiology, nodule mass and total dry biomass over ambient CO2 grown plants under well-watered (WW) and DS conditions albeit a decrease in leaf relative water content (LRWC). Glucose, fructose and sucrose levels were measured to understand the role of hexose to sucrose ratios (H:S) in mediating the drought responses. Free amino acid levels as indicative of N assimilation provided insights into C and N balance under DS and CO2 interactions. The enzymatic and non-enzymatic antioxidants showed significant upregulation in elevated CO2 grown plants under DS thereby protecting the plant from oxidative damage caused by the reactive oxygen species. Our results clearly demonstrated the protective role of elevated CO2 under DS at lower LRWC and gained comparative advantage of mitigating the DS-induced damage over ambient CO2 grown pigeonpea.

De novo transcriptome analysis of an imminent biofuel crop, Camelina sativa L. using Illumina GAIIX sequencing platform and identification of SSR markers.

Camelina sativa L. is an emerging biofuel crop with potential applications in industry, medicine, cosmetics and human nutrition. The crop is unexploited owing to very limited availability of transcriptome and genomic data. In order to analyse the various metabolic pathways, we performed de novo assembly of the transcriptome on Illumina GAIIX platform with paired end sequencing for obtaining short reads. The sequencing output generated a FastQ file size of 2.97 GB with 10.83 million reads having a maximum read length of 101 nucleotides. The number of contigs generated was 53,854 with maximum and minimum lengths of 10,086 and 200 nucleotides respectively. These trancripts were annotated using BLAST search against the Aracyc, Swiss-Prot, TrEMBL, gene ontology and clusters of orthologous groups (KOG) databases. The genes involved in lipid metabolism were studied and the transcription factors were identified. Sequence similarity studies of Camelina with the other related organisms indicated the close relatedness of Camelina with Arabidopsis. In addition, bioinformatics analysis revealed the presence of a total of 19,379 simple sequence repeats. This is the first report on Camelina sativa L., where the transcriptome of the entire plant, including seedlings, seed, root, leaves and stem was done. Our data established an excellent resource for gene discovery and provide useful information for functional and comparative genomic studies in this promising biofuel crop.

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.

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.

Genome sequencing and analysis uncover the regulatory elements involved in the development and oil biosynthesis of Pongamia pinnata (L.) - A potential biodiesel feedstock.

Due to rapid industrialization, the consumption of petro-products has increased, while fossil fuel resources have been gradually depleted. There has been a resurgence of interest in plant-derived biofuels as a sustainable alternative to fossil fuels for the purpose of reducing greenhouse gas emissions. Pongamia pinnata L., which is also known as Millettia pinnata is an oil-yielding, leguminous tree with a large and complex genome. Despite its multiple industrial applications, this orphan tree species has inconsistent yields and a limited understanding of its functional genomics. We assessed physiological and morphological characteristics of five high-yielding pongamia accessions and deduced important yield descriptors. Furthermore, we sequenced the genome of this potential biofuel feedstock using Illumina HiSeq, NextSeq, and MiSeq platforms to generate paired-end reads. Around 173 million processed reads amounting to 65.2 Gb were assembled into a 685 Mb genome, with a gap rate of 0.02%. The sequenced scaffolds were used to identify 30,000 gene models, 406,385 Simple-Sequence-Repeat (SSR) markers, and 43.6% of repetitive sequences. We further analyzed the structural information of genes belonging to certain key metabolic pathways, including lipid metabolism, photosynthesis, circadian rhythms, plant-pathogen interactions, and karanjin biosynthesis, all of which are commercially significant for pongamia. A total of 2,219 scaffolds corresponding to 29 transcription factor families provided valuable information about gene regulation in pongamia. Similarity studies and phylogenetic analysis revealed a monophyletic group of Fabaceae members wherein pongamia out-grouped from Glycine max and Cajanus cajan, revealing its unique ability to synthesize oil for biodiesel. This study is the first step toward completing the genome sequence of this imminent biofuel tree species. Further attempts at re-sequencing with different read chemistry will certainly improve the genetic resources at the chromosome level and accelerate the molecular breeding programs.

Molecular insights into photosynthesis and carbohydrate metabolism in Jatropha curcas grown under elevated CO2 using transcriptome sequencing and assembly.

Jatropha curcas L. (Family - Euphorbiaceae) is a perennial tree of special interest due to its potential as a biofuel plant with high carbon sequestration. In this study, physiological investigations coupled with transcriptomics in relation to photosynthesis were evaluated in Jatropha grown under ambient (395 ppm) and elevated (550 ppm) CO2 atmosphere. Morphophysiological analysis revealed that Jatropha sustained enhanced photosynthesis during its growth under elevated CO2 for one year which might be linked to improved CO2 assimilation physiology and enhanced sink activity. We sequenced and analyzed the leaf transcriptome of Jatropha after one year of growth in both conditions using Illumina HiSeq platform. After optimized assembly, a total of 69,581 unigenes were generated. The differential gene expression (DGE) analysis revealed 3013 transcripts differentially regulated in elevated CO2 conditions. The photosynthesis regulatory genes were analysed for temporal expression patterns at four different growth phases which highlighted probable events contributing to enhanced growth and photosynthetic capacity including increased reducing power, starch synthesis and sucrose mobilization under elevated CO2. Overall, our data on physiological and transcriptomic analyses suggest an optimal resource allocation to the available and developing sink organs thereby sustaining improved photosynthetic rates during long-term growth of Jatropha under CO2 enriched environment.

A novel aldo-keto reductase from Jatropha curcas L. (JcAKR) plays a crucial role in the detoxification of methylglyoxal, a potent electrophile.

Abiotic stress leads to the generation of reactive oxygen species (ROS) which further results in the production of reactive carbonyls (RCs) including methylglyoxal (MG). MG, an α, ß-dicarbonyl aldehyde, is highly toxic to plants and the mechanism behind its detoxification is not well understood. Aldo-keto reductases (AKRs) play a role in detoxification of reactive aldehydes and ketones. In the present study, we cloned and characterised a putative AKR from Jatropha curcas (JcAKR). Phylogenetically, it forms a small clade with AKRs of Glycine max and Rauwolfia serpentina. JcAKR was heterologously expressed in Escherichia coli BL-21(DE3) cells and the identity of the purified protein was confirmed through MALDI-TOF analysis. The recombinant protein had high enzyme activity and catalytic efficiency in assays containing MG as the substrate. Protein modelling and docking studies revealed MG was efficiently bound to JcAKR. Under progressive drought and salinity stress, the enzyme and transcript levels of JcAKR were higher in leaves compared to roots. Further, the bacterial and yeast cells expressing JcAKR showed more tolerance towards PEG (5%), NaCl (200mM) and MG (5mM) treatments compared to controls. In conclusion, our results project JcAKR as a possible and potential target in crop improvement for abiotic stress tolerance.

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.

Persistent stimulation of photosynthesis in short rotation coppice mulberry under elevated CO2 atmosphere.

Current study was undertaken to elucidate the responses of short rotation coppice (SRC) mulberry under elevated CO2 atmosphere (550µmolmol(-1)). Throughout the experimental period, elevated CO2 grown mulberry plants showed significant increase in light saturated photosynthetic rates (A') by increasing intercellular CO2 concentrations (Ci) despite reduced stomatal conductance (gs). Reduced gs was linked to decrease in transpiration (E) resulting in improved water use efficiency (WUE). There was a significant increase in carboxylation efficiency (CE) of Rubisco, apparent quantum efficiency (AQE), light and CO2 saturated photosynthetic rates (AMAX), photosynthetic nitrogen use efficiency (PNUE), chlorophyll a fluorescence characteristics (FV/FM and PIABS), starch and other carbohydrates in high CO2 grown plants which clearly demonstrate no photosynthetic acclimation in turn resulted marked increase in above and below ground biomass. Our results strongly suggest that short rotation forestry (<1year) with mulberry plantations should be effective to mitigate raising CO2 levels as well as for the production of renewable bio-energy.