Characterization of putative calcium-dependent protein kinase-1 (TaCPK-1) gene: hubs in signalling and tolerance network of wheat under terminal heat.

Calcium-dependent protein kinase (CDPK) is member of one of the most important signalling cascades operating inside the plant system due to its peculiar role as thermo-sensor. Here, we identified 28 full length putative CDPKs from wheat designated as TaCDPK (1-28). Based on digital gene expression, we cloned full length TaCPK-1 gene of 1691 nucleotides with open reading frame (ORF) of 548 amino acids (accession number OP125853). The expression of TaCPK-1 was observed maximum (3.1-fold) in leaf of wheat cv. HD2985 (thermotolerant) under T2 (38 ± 3 °C, 2 h), as compared to control. A positive correlation was observed between the expression of TaCPK-1 and other stress-associated genes (MAPK6, CDPK4, HSFA6e, HSF3, HSP17, HSP70, SOD and CAT) involved in thermotolerance. Global protein kinase assay showed maximum activity in leaves, as compared to root, stem and spike under heat stress. Immunoblot analysis showed abundance of CDPK protein in wheat cv. HD2985 (thermotolerant) in response to T2 (38 ± 3 °C, 2 h), as compared to HD2329 (thermosusceptible). Calcium ion (Ca2+), being inducer of CDPK, showed strong Ca-signature in the leaf tissue (Ca-622 ppm) of thermotolerant wheat cv. under heat stress, whereas it was minimum (Ca-201 ppm) in spike tissue. We observed significant variations in the ionome of wheat under HS. To conclude, TaCPK-1 plays important role in triggering signaling network and in modulation of HS-tolerance in wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03989-6.

Transcriptional Regulation of Small Heat Shock Protein 17 (sHSP-17) by Triticum aestivum HSFA2h Transcription Factor Confers Tolerance in Arabidopsis under Heat Stress.

Heat shock transcription factors (HSFs) contribute significantly to thermotolerance acclimation. Here, we identified and cloned a putative HSF gene (HSFA2h) of 1218 nucleotide (acc. no. KP257297.1) from wheat cv. HD2985 using a de novo transcriptomic approach and predicted sHSP as its potential target. The expression of HSFA2h and its target gene (HSP17) was observed at the maximum level in leaf tissue under heat stress (HS), as compared to the control. The HSFA2h-pRI101 binary construct was mobilized in Arabidopsis, and further screening of T3 transgenic lines showed improved tolerance at an HS of 38 °C compared with wild type (WT). The expression of HSFA2h was observed to be 2.9- to 3.7-fold higher in different Arabidopsis transgenic lines under HS. HSFA2h and its target gene transcripts (HSP18.2 in the case of Arabidopsis) were observed to be abundant in transgenic Arabidopsis plants under HS. We observed a positive correlation between the expression of HSFA2h and HSP18.2 under HS. Evaluation of transgenic lines using different physio-biochemical traits linked with thermotolerance showed better performance of HS-treated transgenic Arabidopsis plants compared with WT. There is a need to further characterize the gene regulatory network (GRN) of HSFA2h and sHSP in order to modulate the HS tolerance of wheat and other agriculturally important crops.

Lipase - The fascinating dynamics of enzyme in seed storage and germination - A real challenge to pearl millet.

Pearl millet is considered as 'nutri-cereal' because of high nutrient density of the seeds. The grain has limited use because of low keeping quality of the flour due to the activities of rancidity causing enzymes like lipase, lox, pox and PPO. Among all the enzymes, lipase is most notorious because of its robust nature and high activity under different conditions. we have identified 2180 putative transcripts showing homology with different variants of lipase precursor through transcriptome data mining (NCBI BioProject acc. no. PRJNA625418). Lipase plays dual role of facilitating the germination of seeds and deteriorating the quality of the pearl millet flour through hydrolytic rancidity. Different physiochemical methods like heat treatment, micro oven, hydrothermal, etc. have been developed to inhibit lipase activity in pearl millet flour. There is further need to develop improved processing technologies to inhibit the hydrolytic and oxidative rancidity in the floor with enhanced shelf-life.

Insights into decontamination of soils by phytoremediation: A detailed account on heavy metal toxicity and mitigation strategies.

In the current era of rapid industrialization, the foremost challenge is the management of industrial wastes. Activities such as mining and industrialization spill over a large quantity of toxic waste that pollutes soil, water, and air. This poses a major environmental and health challenge. The toxic heavy metals present in the soil and water are entering the food chain, which in turn causes severe health hazards. Environmental clean-up and reclamation of heavy metal contaminated soil and water are very important, and it necessitates efforts of environmentalists, industrialists, scientists, and policymakers. Phytoremediation is a plant-based approach to remediate heavy metal/organic pollutant contaminated soil and water in an eco-friendly, cost-effective, and permanent way. This review covers the effect of heavy metal toxicity on plant growth and physiological process, the concept of heavy metal accumulation, detoxification, and the mechanisms of tolerance in plants. Based on plants' ability to uptake heavy metals and metabolize them within tissues, phytoremediation techniques have been classified into six types: phytoextraction, phytoimmobilization, phytovolatilization, phytodegradation, rhizofiltration, and rhizodegradation. The development of research in this area led to the identification of metal hyper-accumulators, which could be utilized for reclamation of contaminated soil through phytomining. Concurrently, breeding and biotechnological approaches can enhance the remediation efficiency. Phytoremediation technology, combined with other reclamation technologies/practices, can provide clean soil and water to the ecosystem.

Characterizing the putative mitogen-activated protein kinase (MAPK) and their protective role in oxidative stress tolerance and carbon assimilation in wheat under terminal heat stress.

Wheat, being sensitive to terminal heat, causes drastic reduction in grain quality and yield. MAPK cascade regulates the network of defense mechanism operated inside plant system. Here, we have identified 21 novel MAPKs through gel-based proteomics and RNA-seq data analysis. Based on digital gene expression, two transcripts (transcript_2834 and transcript_8242) showing homology with MAPK were cloned and characterized from wheat (acc. nos. MK854806 and KT835664). Transcript_2834 was cloned in pET28a vector and recombinant MAPK protein of ∼40.3 kDa was isolated and characterized to have very high in-vitro kinase activity under HS. Native MAPK showed positive correlation with the expression of TFs, HSPs, genes linked with antioxidant enzyme (SOD, CAT, GPX), photosynthesis and starch biosynthesis pathways in wheat under HS. Wheat cv. HD3086 (thermotolerant) having higher expression and activity of MAPK under HS showed significant increase in accumulation of proline, H2O2, starch, and granule integrity, compared with BT-Schomburgk (thermosusceptible).

Heterologous expression and characterization of novel manganese superoxide dismutase (Mn-SOD) - A potential biochemical marker for heat stress-tolerance in wheat (Triticum aestivum).

Heat stress causes oxidative bursts damaging the organelles and nascent proteins. Plants have inherited antioxidant defense system to neutralize the effect of reactive oxygen species. Superoxide dismutase provides first line of defense against the HS by regulating the accumulation of peroxide radicals inside the cells. Here, we report identification and cloning of putative manganese superoxide dismutase (Mn-SOD) gene of ~733 nt from wheat cv. HD2985 through de novo assembly. The gene was observed to localize on Chr 6D with a mitochondrial targeting peptide sequence and iron/manganese domain. We predicted 147 homologs of Mn-SOD in eukaryotes with diverse speciation nodes. A recombinant Mn-SOD protein of ~25.5 kDa was purified through heterologous expression system. Kinetics assay of recombinant protein showed optimum pH of 8.0, optimum temperature of 35 °C and Km and Vmax values of 1.51 µM and 9.45 U/mg proteins, respectively. Maximum expression and activity of Mn-SOD was observed in leaves from Raj3765, as compared to stem and spike during milky-ripe stage under differential HS. In gel activity assay showed the appearance of all the three isoforms of SOD in thermotolerant cv. under HS. Mn-SOD, being active at pivotal position, can be also used as potential biochemical marker in wheat breeding program.

Gamma irradiation protect the developing wheat endosperm from oxidative damage by balancing the trade-off between the defence network and grains quality.

Gamma irradiation has been reported to modulate the biochemical and molecular parameters associated with the tolerance of plant species under biotic/ abiotic stress. Wheat is highly sensitive to heat stress (HS), as evident from the decrease in the quantity and quality of the total grains. Here, we studied the effect of pre-treatment of wheat dry seeds with different doses of gamma irradiation (0.20, 0.25 and 0.30 kGy) on tolerance level and quality of developing wheat endospermic tissue under HS (38 °C, 1 h; continuously for three days). Expression analysis of genes associated with defence and starch metabolism in developing grains showed maximum transcripts of HSP17 (in response to 0.25 kGy + HS) and AGPase (under 0.30 kGy), as compared to control. Gamma irradiation was observed to balance the accumulation of H2O2 by enhancing the activities of SOD and GPx in both the cvs. under HS. Gamma irradiation was observed to stabilize the synthesis of starch and amylose by regulating the activities of AGPase, SSS and α-amylase under HS. The appearance of isoforms of gliadins (α, ß, γ, ω) were observed more in gamma irradiated seeds (0.20 kGy), as compared to control. Gamma irradiation (0.25 kGy in HD3118 & 0.20 kGy in HD3086) was observed to have positive effect on the width, length and test seed weight of the grains under HS. The information generated in present investigation provides easy, cheap and user-friendly technology to mitigate the effect of terminal HS on the grain-development process of wheat along with development of robust seeds with high nutrient density.

Quantitative proteomic analysis reveals novel stress-associated active proteins (SAAPs) and pathways involved in modulating tolerance of wheat under terminal heat.

Terminal heat stress has detrimental effect on the growth and yield of wheat. Very limited information is available on heat stress-associated active proteins (SAAPs) in wheat. Here, we have identified 159 protein groups with 4271 SAAPs in control (22 ± 3 °C) and HS-treated (38 °C, 2 h) wheat cvs. HD2985 and HD2329 using iTRAQ. We identified 3600 proteins to be upregulated and 5825 proteins to be downregulated in both the wheat cvs. under HS. We observed 60.3% of the common SAAPs showing upregulation in HD2985 (thermotolerant) and downregulation in HD2329 (thermosusceptible) under HS. GO analysis showed proton transport (molecular), photosynthesis (biological), and ATP binding (cellular) to be most altered under HS. Most of the SAAPs identified were observed to be chloroplast localized and involved in photosynthesis. Carboxylase enzyme was observed most abundant active enzymes in wheat under HS. An increase in the degradative isoenzymes (α/ß-amylases) was observed, as compared to biosynthesis enzymes (ADP-glucophosphorylase, soluble starch synthase, etc.) under HS. Transcript profiling showed very high relative fold expression of HSP17, CDPK, Cu/Zn SOD, whereas downregulation of AGPase, SSS under HS. The identified SAAPs can be used for targeted protein-based precision wheat-breeding program for the development of 'climate-smart' wheat.

Characterization of novel heat-responsive transcription factor (TaHSFA6e) gene involved in regulation of heat shock proteins (HSPs) - A key member of heat stress-tolerance network of wheat.

Heat stress has an adverse effect on the quality and quantity of agriculturally important crops, especially wheat. The tolerance mechanism has not been explored much in wheat and very few genes/ TFs responsive to heat stress is available on public domain. Here, we identified, cloned and characterized a putative TaHSFA6e TF gene of 1.3 kb from wheat cv. HD2985. We observed an ORF of 368 aa with Hsf DNA binding signature domain in the amino acid sequence. Single copy number of TaHSFA6e was observed integrated in the genome of wheat. Expression analysis of TaHSFA6e under differential HS showed maximum transcripts in wheat cv. Halna (thermotolerant) in response to 38 °C for 2 h during pollination and grain-filling stages, as compared to PBW343, HD2329 and HD2985. Putative target genes of TaHSFA6e (HSP17, HSP70 and HSP90) showed upregulation in response to differential HS (30 & 38 °C, 2 h) during pollination and grain-filling stages. Small HSP17 was observed most triggered in Halna under HS. We observed increase in the catalase, guaiacol peroxidase, total antioxidant capacity (TAC), and decrease in the lipid peroxidation in thermotolerant cvs. (Halna, HD2985), as compared to thermosusceptible (PBW343, HD2329) under differential HS. Multiple stresses (heat - 38 °C, 2 h, and drought - 100 mL of 20% polyethylene Glycol 6000) during seedling stage of wheat showed positive correlation between the expression of TaHSFA6e, putative targets (HSP70, HSP90, HSP17) and TAC. Halna (thermotolerant) performed better, as compared to other contrasting cvs. TaHSFA6e TF can be used as promising candidate gene for manipulating the heat stress-tolerance network.

Biochemical Defense Response: Characterizing the Plasticity of Source and Sink in Spring Wheat under Terminal Heat Stress.

Wheat is highly prone to terminal heat stress (HS) under late-sown conditions. Delayed- sowing is one of the preferred methods to screen the genotypes for thermotolerance under open field conditions. We investigated the effect of terminal HS on the thermotolerance of four popular genotypes of wheat i.e. WR544, HD2967, HD2932, and HD2285 under field condition. We observed significant variations in the biochemical parameters like protein content, antioxidant activity, proline and total reducing sugar content in leaf, stem, and spike under normal (26 ± 2°C) and terminal HS (36 ± 2°C) conditions. Maximum protein, sugars and proline was observed in HD2967, as compared to other cultivars under terminal HS. Wheat cv. HD2967 showed more adaptability to the terminal HS. Differential protein-profiling in leaves, stem and spike of HD2967 under normal (26 ± 2°C) and terminal HS (36 ± 2°C) showed expression of some unique protein spots. MALDI-TOF/MS analysis showed the DEPs as RuBisCO (Rub), RuBisCO activase (Rca), oxygen evolving enhancer protein (OEEP), hypothetical proteins, etc. Expression analysis of genes associated with photosynthesis (Rub and Rca) and starch biosynthesis pathway (AGPase, SSS and SBE) showed significant variations in the expression under terminal HS. HD2967 showed better performance, as compared to other cultivars under terminal HS. SSS activity observed in HD2967 showed more stability under terminal HS, as compared with other cultivars. Triggering of different biochemical parameters in response to terminal HS was observed to modulate the plasticity of carbon assimilatory pathway. The identified DEPs will enrich the proteomic resources of wheat and will provide a potential biochemical marker for screening wheat germplasm for thermotolerance. The model hypothesized will help the researchers to work in a more focused way to develop terminal heat tolerant wheat without compromising with the quality and quantity of grains.

Exploring the heat-responsive chaperones and microsatellite markers associated with terminal heat stress tolerance in developing wheat.

Global warming is a major threat for agriculture and food security, and in many cases the negative impacts are already apparent. Wheat is one of the most important staple food crops and is highly sensitive to the heat stress (HS) during reproductive and grain-filling stages. Here, whole transcriptome analysis of thermotolerant wheat cv. HD2985 was carried out at the post-anthesis stage under control (22 ± 3 °C) and HS-treated (42 °C, 2 h) conditions using Illumina Hiseq and Roche GS-FLX 454 platforms. We assembled ~24 million (control) and ~23 million (HS-treated) high-quality trimmed reads using different assemblers with optimal parameters. De novo assembly yielded 52,567 (control) and 59,658 (HS-treated) unigenes. We observed 785 transcripts to be upregulated and 431 transcripts to be downregulated under HS; 78 transcripts showed >10-fold upregulation such as HSPs, metabolic pathway-related genes, etc. Maximum number of upregulated genes was observed to be associated with processes such as HS-response, protein-folding, oxidation-reduction and photosynthesis. We identified 2008 and 2483 simple sequence repeats (SSRs) markers from control and HS-treated samples; 243 SSRs were observed to be overlying on stress-associated genes. Polymorphic study validated four SSRs to be heat-responsive in nature. Expression analysis of identified differentially expressed transcripts (DETs) showed very high fold increase in the expression of catalytic chaperones (HSP26, HSP17, and Rca) in contrasting wheat cvs. HD2985 and HD2329 under HS. We observed positive correlation between RNA-seq and qRT-PCR expression data. The present study culminated in greater understanding of the heat-response of tolerant genotype and has provided good candidate genes for the marker development and screening of wheat germplasm for thermotolerance.

Identification of Putative RuBisCo Activase (TaRca1)-The Catalytic Chaperone Regulating Carbon Assimilatory Pathway in Wheat (Triticum aestivum) under the Heat Stress.

RuBisCo activase (Rca) is a catalytic chaperone involved in modulating the activity of RuBisCo (key enzyme of photosynthetic pathway). Here, we identified eight novel transcripts from wheat through data mining predicted to be Rca and cloned a transcript of 1.4 kb from cv. HD2985, named as TaRca1 (GenBank acc. no. KC776912). Single copy number of TaRca1 was observed in wheat genome. Expression analysis in diverse wheat genotypes (HD2985, Halna, PBW621, and HD2329) showed very high relative expression of TaRca1 in Halna under control and HS-treated, as compared to other cultivars at different stages of growth. TaRca1 protein was predicted to be chloroplast-localized with numerous potential phosphorylation sites. Northern blot analysis showed maximum accumulation of TaRca1 transcript in thermotolerant cv. during mealy-ripe stage, as compared to thermosusceptible. Decrease in the photosynthetic parameters was observed in all the cultivars, except PBW621 in response to HS. We observed significant increase in the Rca activity in all the cultivars under HS at different stages of growth. HS causes decrease in the RuBisCo activity; maximum reduction was observed during pollination stage in thermosusceptible cvs. as validated through immunoblotting. We observed uniform carbon distribution in different tissues of thermotolerant cvs., as compared to thermosusceptible. Similarly, tolerance level of leaf was observed maximum in Halna having high Rca activity under HS. A positive correlation was observed between the transcript and activity of TaRca1 in HS-treated Halna. Similarly, TaRca1 enzyme showed positive correlation with the activity of RuBisCo. There is, however, need to manipulate the thermal stability of TaRca1 enzyme through protein engineering for sustaining the photosynthetic rate under HS-a novel approach toward development of "climate-smart" crop.

Harnessing Next Generation Sequencing in Climate Change: RNA-Seq Analysis of Heat Stress-Responsive Genes in Wheat (Triticum aestivum L.).

Wheat is a staple food worldwide and provides 40% of the calories in the diet. Climate change and global warming pose a threat to wheat production, however, and demand a deeper understanding of how heat stress might impact wheat production and wheat biology. However, it is difficult to identify novel heat stress associated genes when the genomic information is not available. Wheat has a very large and complex genome that is about 37 times the size of the rice genome. The present study sequenced the whole transcriptome of the wheat cv. HD2329 at the flowering stage, under control (22°±3°C) and heat stress (42°C, 2 h) conditions using Illumina HiSeq and Roche GS-FLX 454 platforms. We assembled more than 26.3 and 25.6 million high-quality reads from the control and HS-treated tissues transcriptome sequences respectively. About 76,556 (control) and 54,033 (HS-treated) contigs were assembled and annotated de novo using different assemblers and a total of 21,529 unigenes were obtained. Gene expression profile showed significant differential expression of 1525 transcripts under heat stress, of which 27 transcripts showed very high (>10) fold upregulation. Cellular processes such as metabolic processes, protein phosphorylation, oxidations-reductions, among others were highly influenced by heat stress. In summary, these observations significantly enrich the transcript dataset of wheat available on public domain and show a de novo approach to discover the heat-responsive transcripts of wheat, which can accelerate the progress of wheat stress-genomics as well as the course of wheat breeding programs in the era of climate change.

Exogenous application of putrescine at pre-anthesis enhances the thermotolerance of wheat (Triticum aestivum L.).

Antioxidant enzymes, besides being involved in various developmental processes, are known to be important for environmental stress tolerance in plants. In this study, the effect of treatment of 2.5 mM putrescine (Put), heat stress (HS -42 degrees C for 2 h) and their combination on the expression and activity of antioxidant enzymes was studied at pre-anthesis in the leaves of two wheat (Triticum aestivum L.) cultivars--HDR77 (thermotolerant) and HD2329 (thermosusceptible). We observed that 2.5 mM Put before HS significantly enhanced the transcript levels of superoxide dismutase (SOD), catalase (CAT), cytoplasmic and peroxisomal ascorbate peroxidase (cAPX, pAPX) in both the cultivars. However, the activities of antioxidant enzymes (SOD, CAT, APX and GR), as well as accumulation of antioxidants (ascorbic acid and total thiol content) were higher in HDR77 than in HD2329 in response to the treatment 2.5 mM Put + HS. No significant change was observed in the proline accumulation in response to HS and combined treatment of 2.5 mM Put + HS. A decrease in the H2O2 accumulation, lipid peroxidation and increase in cell membrane stability (CMS) were observed in response to 2.5 mM Put + HS treatment, as compared to HS treatment alone in both the cultivars; HDR77 was, however, more responsive to 2.5 mM Put + HS treatment. Put (2.5 mM) treatment at pre-anthesis thus modulated the defense mechanism responsible for the thermotolerance capacity of wheat under the heat stress. Elicitors like Put, therefore, need to be further studied for temporarily manipulating the thermotolerance capacity of wheat grown under the field conditions in view of the impending global climate change.