Suneha Goswami

Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus.

Groundnut bud necrosis virus (GBNV) infects a large number of leguminous and solanaceous plants. To elucidate the biological function of the non-structural protein encoded by the S RNA of GBNV (NSs), we studied its role in RNA silencing suppression and in viral pathogenesis. Our results demonstrated that GBNV NSs functions as a suppressor of RNA silencing using the agroinfiltration patch assay. An in silico analysis suggested the presence of pro-apoptotic protein Reaper-like sequences in the GBNV NSs, which were known to be present in animal infecting bunyaviruses. Utilizing NSs mutants, we demonstrated that a Leu-rich domain was required for RNA silencing suppression activity, but not the non-overlapping Trp/GH3 motif of the Reaper-like sequence. To investigate the role of NSs in symptom development we generated transgenic tomato expressing the GBNV NSs and showed that the expression of NSs in tomato mimics symptoms induced by infection with GBNV, such as leaf senescence and necrosis. As leaf senescence is controlled by miR319 regulation of the transcription factor TCP1, we assessed the accumulation of both RNAs in transgenic NSs-expressing and GBNV-infected tomato plants. In both types of plants the levels of miR319 decreased, while the levels of TCP1 transcripts increased. We propose that GBNV-NSs affects miRNA biogenesis through its RNA silencing suppressor activity and interferes with TCP1-regulated leaf developmental pathways.

Mechanism of action of hydrogen peroxide in wheat thermotolerance - interaction between antioxidant isoenzymes, proline and cell membrane

Terminal heat stress causes an array of physiological, biochemical and morphological changes in plants, which affect plant growth and development. It has very severe effect on the pollen viability and seed setting in wheat. In the present investigation, an altered expression of H 2 O 2 (0.9 μg/g in C-306 and 0.75 μg/g in HD2329) was observed with the highest accumulation at seed hardening stage and against heat shock (HS) of 42°C for 2 h. With the increase in H 2 O 2 accumulation, an increase in the number of isoenzymes of superoxide dismutase and catalase were observed with high activities under differential heat shock. A decrease in the proline accumulation was observed under differential heat shock. Exogenous application of H 2 O 2 (10 mmole/L) leads to increase in the accumulation of intracellular H 2 O 2 and further an increase in the number of isoenzymes of superoxide dismutase (SOD) and catalase (CAT) was observed. The tolerant cultivar was more responsive to exogenous application of H 2 O 2 compared to susceptible cultivar. The percentage decrease in cell membrane stability under differential heat shock was low in H2O2 treated plants compared to non-treated. The results from this study suggest a potential role for H 2 O 2 in regulating the activity of antioxidant enzymes and accumulation of proline inside cells and in turn influence the cell membrane stability under heat stress. All the defense associated genes were observed to be very responsive to intracellular H 2 O 2 , which gives inference that H 2 O 2 has regulatory role to play in controlling the expression and activities of these proteins under abiotic stresses. Key words : Antioxidant enzymes, wheat, heat stress, H 2 O 2 , proline, catalase, superoxide dismutase, cell membrane stability, reactive oxygen species.

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.

SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sangers sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward 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.

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H2O2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H2O2-responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSsS189R) and the other in a non-Trp/GH3 motif (NSsL172R). Tobacco rattle virus (TRV) expressing NSsS189R enhanced the PCD response, but not TRV-NSsL172R, while RNA silencing suppression activity was lost in TRV-NSsL172R, but not in TRV-NSsS189R. Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs.

In planta silencing of NSs and Hc-Pro through RNAi constructs: to develop durable resistance

Groundnut bud necrosis disease and Papaya ring spot disease are devastating disease of tomato and papaya/cucumber. In order to develop transgenic resistance against these viruses, gene silencing approach was followed to target the conserved viral sequences for obtaining wide spectrum of resistance. Viral derived small non coding RNA was used to silence viral gene. To have efficient processing of hairpins, microRNA backbone has been used to harbour viral derived siRNA. An artificial microRNA having miRNA159a backbone harbouring 21-nt siRNA sequences from RNAi suppressor (GBNV–NSs and PRSV–Hc-Pro) genes were constructed. To establish the efficacy of these amiRNA constructs, transient expression assay were performed and attenuation of viral symptoms were observed on expression of artificial microRNA construct in infected plants. Our data demonstrate that expression of virus-specific siRNAs using artificial-miRNA is an effective and predictable approach to silence Groundnut bud necrosis virus (GBNV) and Papaya ringspot virus (PRSV).

miR430: the novel heat-responsive microRNA identified from miRNome analysis in wheat (Triticum aestivum L.)

AbstractmiRNAs are 21–24 nt non-coding RNAs involved in negative regulation of the target gene expression in response to stress and developmental cues. Wheat, the major staple food crop, is highly sensitive to heat stress (HS). Even moderate high temperatures during critical stages causes reduction in yield and quality of the grains. Here, we identified a novel miRNA (candidate miR430) from wheat using de novo assembly, and cloned it from wheat cv. HD2985 using universal adaptor. The identified miRNA was mapped on to the Chromosome 3B and predicted to have more than 30 target genes. Most of the targets identified were associated with heat shock proteins. Expression analysis of miR430 in contrasting wheat cultivars by quantitative real-time PCR revealed significant genotypic variations under HS. Tissue specific expression analysis showed relatively low expression in leaves, as compared with stem and root under HS. A negative correlation was found between the expression of miR430 and their respective target genes under HS. MiR430 can be used to manipulate the expression of target genes under HS towards enhancing HS tolerance for the development of ‘climate-smart’ wheat crop.

Physiological and biochemical alterations due to low temperature stress in papaya genotypes

Papaya (Carica papaya L.) being a typical tropical plant, is highly sensitive to low temperature stress. Thepresent experiment was conducted under completely controlled conditions of National Phytotron Facility, ICAR-IARI, New Delhi to investigate the effect of different low temperature regimes on antioxidant enzymes and otherphysiological and biochemical parameters in five papaya genotypes and one distant relative i.e., cold tolerant genus Vasconcellea cundinamarcensis. The outcomes suggested a genotype-specific substantial increase of antioxidant enzymes activities, viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX) and glutathione reductase (GR) under the cold treatment regimes. The highest percent increase of SOD, APX, GPX and GR activities were found in the tolerant genotype V. cundinamarcensis, while the highest increase in CAT activity was observed in P-7-9 at the 20o/10oC (day/night) temperature regimeover the control. Low temperature regimes led to increase in membrane injury in papaya leaves possibly throughthe phase transition or oxidative damage of cell membrane due to ROS generation in the photosystem becauseof disruption in the photosynthetis process. The highest increase in the membrane injury index was noted ingenotype Red Lady (79.93%). The photosynthetic rate was severely reduced under the low temperature regimes. The total sugars and total soluble proteins content in papaya leaves were observed to increase under the lowtemperature regimes may be due to cold induced osmotic stress.

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.