Molecular cloning of HSP17 gene (sHSP) and their differential expression under exogenous putrescine and heat shock in wheat (Triticum aestivum)

Polyamines (PAs) are low molecular weight ubiquitous nitrogenous compounds found in all the living  organisms, which have been implicated in the expression of various stress-proteins against the abiotic  stresses. Small heat shock proteins (sHSPs) are of particular importance in the thermotolerance and have  been reported to act as molecular chaperones preventing denaturation or aggregation of the target proteins. Here, we report cloning of a small HSP of ~573 bp from C-306 cultivar of wheat (Triticum aestivum L), having open reading frame of 162 amino acids. In silico analysis showed the presence of an alpha crystalline domain (ACD), the signature domain for small HSPs. Consensus localization prediction (ConLoc) provides 98%  consensus prediction of HSP17 in the nucleus. Quantitative real time polymerase chain reaction (qRT-PCR) analysis of HSP17 gene showed maximum (34 fold) transcript in C-306 and minimum (1.5 fold) in HD2329  cultivars of wheat in response to differential treatment of putrescine (1.5 to 2.5 mM + heat shock of 42°C for 2 h). Putrescine seems to enhance the transcript levels against the heat shock much more pronounced in  thermotolerant than in the susceptible cultivars.Key words: Triticum aestivum, heat stress, small heat shock protein, putrescine, HSP17, polyamine, domain, cloning

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 H2O2 (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 H2O2 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 H2O2 (10 mmole/L) leads to increase in the accumulation of intracellular H2O2 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 H2O2 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 H2O2 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 H2O2, which gives inference that H2O2 has regulatory role to play in controlling the expression and activities of these proteins under abiotic stresses.Key words: Antioxidant enzymes, wheat, heat stress, H2O2, proline, catalase, superoxide dismutase, cell membrane stability, reactive oxygen species

Physiochemical response of papaya genotypes exposed to low temperature regimes

Susceptibility to low temperature stress is the major threat to papaya cultivation. Here, we studied a low temperature stress tolerance in papaya plant. We have investigated the effect of different low temperature regimes, 28°/18°C (day/night) to 16°/06°C (day/night) with a gradual decrease of 2°C on every two days and one set with direct exposure to the low temperature of 18°/08°C (day/night), called the acclimatized plant, in five diverse papaya genotypes (Pusa Nanha, Red Lady P-7-2, P-7-9, and P-7-14) and cold tolerant wild relative of cultivated papaya genotype (Vasconcellea cundinamarcensis V.M. Badillo) under controlled regulated conditions. It was observed that there were significant variations in the physiological and biochemical parameters like photosynthetic gas exchange parameters, chlorophyll content, fluorescence parameters, relative water content (RWC), membrane stability index (MSI), total sugars content, total soluble proteins content, lipid peroxidation, and proline accumulation in leaf tissues. Maximum stomatal conductance, chlorophyll fluorescence, RWC, MSI, total sugars, total soluble proteins, proline and lowest MDA contents were observed in Vasconcellea cundinamarcensis followed by inbred P-7-9 as compared to other genotypes under low temperature stress. Among all the papaya genotypes, P-7-9 showed more adaptability to low temperature stress and it further give new insights for developing low temperature tolerant papaya genotypes, especially under changing climate situations

Heat-responsive microRNAs regulate the transcription factors and heat shock proteins in modulating thermo-stability of starch biosynthesis enzymes in wheat (Triticum aestivum L.) under the heat stress

†Equal contribution made by both the authors. Abstract Heat-responsive miRNAs regulate the expression of the transcription factors (HSFs) and majority of the heat stress-associated genes (HSPs). Here we report identification of few heats -responsive miRNAs in wheat (Triticum aestivum L.)through de novo sequencing on Illumina Hiseq 2000. Validation of identified miRNAs in endospermic tissues of thermotolerant (HD2985) and thermosusceptible (NIAW-34-34) wheat cultivars using real time PCR showed up-regulation of 4 micro RNAs (tae-miR156, tae-miR167, tae-miR395b and tae-miR398) and down-regulation of 6 micro RNAs (tae-miR159a, tae-miR159b, tae-miR160, tae-miR171a, tae-miR319, and tae-miR1117) in response to the heat stress. Target analysis of identified miRNAs showed HSF3, HSFA4a, HSP17, HSP70 and superoxide dismutase (SOD) as most probable target genes. Expression profiling of identified target genes under heat stress (42°C, 2 h) showed 2.34, 1.33 fold (HSF3), 2.45, 1.44 fold (HSFA4a), 3.9, 1.9 fold (HSP17), 5.6, 2.4 fold (HSP70), 1.9, 1.2 fold (SOD) and 2.7, 1.6 fold (catalase) increase in the expression in HD2985 and NIAW-34-34 cultivars of wheat compared to control. A defragmented, small and pleated starch granule structure was observed in sample with low expression of target genes (NIAW-34-34) compared to intact, robust and globular starch granules in samples with high expression of target genes (HD2985). Transcript profiling and activity assay of soluble starch synthase (SSS) showed less transcript accumulation and activity in heat shock treated samples compared to control sample in both the cultivars

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Not AvailablemiRNAs 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.Not Availabl

Interference in plant defense and development by nonstructural protein NSs of Groundnut bud necrosis virus

6 páginas, 3 figuras, 1 figura suplementaria -- PAGS nros. 368-373Groundnut 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 pathwaysWe acknowledge the financial support by Department of Science and Technology, Govt. of India (Grant DST/INT/SPAIN/P-9/2009); Ministerio de Ciencia e Innovacion Govt. of Spain (Grant ACI 2009-0855). We are also thankful to Indian Agricultural Research Institute, New Delhi, for providing fellowship to Suneha Goswami, Nandita Sahana and Vanita PandeyPeer reviewe

Interference in plant defense and development by nonstructural protein NSs of Groundnut bud necrosis virus

6 páginas, 3 figuras, 1 figura suplementaria -- PAGS nros. 368-373Groundnut 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 pathwaysWe acknowledge the financial support by Department of Science and Technology, Govt. of India (Grant DST/INT/SPAIN/P-9/2009); Ministerio de Ciencia e Innovacion Govt. of Spain (Grant ACI 2009-0855). We are also thankful to Indian Agricultural Research Institute, New Delhi, for providing fellowship to Suneha Goswami, Nandita Sahana and Vanita PandeyPeer reviewe

Characterization of differentially expressed stress-associated proteins in starch granule development under heat stress in wheat (<i>Triticum aestivum </i>L.)

126-138Abiotic stress causes abrupt increase in the expression of stress-associated proteins, which provide tolerance by modulating the defense mechanism of plants. Small heat shock proteins (sHSPs) and anti-oxidant enzymes are important for environmental stress tolerance of the plants. In this study, two full-length cDNAs encoding small heat shock protein (sHSP) and superoxide dismutase (SOD), designated as TasHSP and SODI were identified and characterized from C-306 (thermotolerant) and PBW343 (thermosusceptible) cultivars of wheat (Triticum aestivum L.). An alpha crystalline domain was observed in TasHSP and manganese/iron binding domain in case of SODI. Quantitative real-time PCR showed very high transcript level of TasHSP and SOD in C-306 compared to PBW343 at different stages of growth and against differential heat stress (HS). Under differential HS at milky-dough stage, the fold change in transcript of both TasHSP and SOD was observed maximum in C-306, compared to PBW343. Protein profiling and isoenzymes analysis showed the expression of several heat-stable proteins and prominent isoenzymes of SOD in C-306, compared to PBW343. Scanning electron microscopy (SEM) of starch granules showed globular, well-shaped and more numbers of endospermic cells in C-306, compared to defragmented, irregular shaped and shrunken granules in case of PBW343 under HS treatment (42°C for 2 h). Diurnal change in soluble starch synthase (SSS) activity showed an increase in the activity during afternoon (35°C), compared to morning (29°C) and evening (32°C) in both the cultivars. Under heat stress (42°C for 2 h), a drastic decrease in the SSS activity was observed, due to the thermal denaturation of the enzyme. Thermotolerance capacity analyzed using cell membrane stability (CMS) showed significantly higher CMS in case of C-306, compared to PBW343 at different stages of growth. Findings suggest that abundance of TasHSP and SODI during milky-dough stage plays a very important role in starch granule biosynthesis. The mechanism may be further exploited to develop tolerant wheat cultivar with high quality seeds

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Not AvailableMicroRNAs (miRNAs) are small endogenous RNAs of ~22 nucleotides that have been shown to play regulatory role by negatively affecting the expression of genes at the post-transcriptional level. Information of miRNAs on some important crops like soybean, Arabidopsis, and rice, etc. are available, but no study on heat-responsive novel miRNAs has yet been reported in wheat (Triticum aestivum L.). In the present investigation, a popular wheat cultivar HD2985 was used in small RNA library construction and Illumina HiSeq 2000 was used to perform high-throughput sequencing of the library after cluster generation; 110,896,604 and 87,743,861 reads were generated in the control (22 °C) and heat-treated (42 °C for 2 h) samples, respectively. Forty-four precursor and mature miRNAs were found in T. aestivum from miRBase v 19. The frequencies of the miRNA families varied from 2 (taemiR1117) to 60,672 (tae-miR159b). We identify 1052 and 902 mature miRNA sequences in HD2985 control and HStreated samples by mapping on reference draft genome of T. aestivum. Maximum identified miRNAs were located on IWGSC_CSS_3B_scaff (chromosome 3B).We could identify 53 and 46 mature miRNA in the control and HS samples and more than 516 target genes by mapping on the reference genome of Oryza sativa, Zea mays, and Sorghum bicolor. Using different pipelines and plant-specific criteria, 37 novel miRNAs were identified in the control and treated samples. Six novel miRNA were validated using qRT-PCR to be heatresponsive. A negative correlation was, however, observed between the expression of novel miRNAs and their targets. Target prediction and pathway analysis revealed their involvement in the heat stress tolerance. These novel miRNAs are new additions to miRNA database of wheat, and the regulatory network will be made use of in deciphering the mechanism of thermotolerance in wheatNot Availabl

Identification of novel starch synthase genes using <i>de novo</i> assembly and heat-induced expression and activity in developing wheat (<i>Triticum aestivum </i> L.)

254-266Terminal heat stress (HS) reduces quantity and quality of grains. Wheat is highly sensitive to HS and there is a wide diversity among different genotypes for HS-tolerance. The enzymes associated with starch biosynthesis are severely affected by HS, especially starch synthases (SS). Different isoforms of SS have been reported from cereals viz. rice, maize, etc.; however, limited information is available in the wheat. In this study, we investigated the effect of HS on contrasting wheat (Triticum aestivum L.) cultivars — HD2985 (thermotolerant) and HD2329 (thermosusceptible) at grain-filling stage. Here we report the identification and cloning of three putative SS genes from wheat. The putative SS has been characterized in silico with other SS reported from different plant and non-plant sources in order to investigate potential functional and evolutionary relationships between SSs. The correlation between HS and SS activity and other biochemical parameters associated with thermotolerance in two cultivars was also studied. Three putative SS genes (transcript_7326, transcript_24546 and transcript_38472) were cloned from HD2985 cultivar under HS by data mining generated from whole transcriptome analysis. The sequences were in silico characterized and submitted in NCBI GenBank (accession nos. KM206143, KM206144 and KJ854903). Glucosyltransferase domain was observed in all the three sequences. Based on ClustalW alignment, SSs were classified into four distinct families. Expression analysis and SS activity assay showed significantly higher transcript level and SS activity during mealy-ripe stage than at milky-ripe stage in HS-treated HD2985, compared to HD2329 cultivar. Both the cultivars showed decrease in starch content under HS and the overall content was higher in HD2985, compared to HD2329. Similarly, guaiacol peroxidase, catalase, free amino acid and total antioxidant capacity were higher during mealy-ripe stage in HD2985. HS during milky-ripe had more severe impact on the overall physiochemical properties of wheat grain and thus needed to be targeted using different approaches in order to enhance the quality and yield of wheat under HS