Genome wide transcriptome analysis reveals vital role of heat responsive genes in regulatory mechanisms of lentil (Lens culinaris Medikus).
ABSTRACT: The present study reports the role of morphological, physiological and reproductive attributes viz. membrane stability index (MSI), osmolytes accumulations, antioxidants activities and pollen germination for heat stress tolerance in contrasting genotypes. Heat stress increased proline and glycine betaine (GPX) contents, induced superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) activities and resulted in higher MSI in PDL-2 (tolerant) compared to JL-3 (sensitive). In vitro pollen germination of tolerant genotype was higher than sensitive one under heat stress. In vivo stressed pollens of tolerant genotype germinated well on stressed stigma of sensitive genotype, while stressed pollens of sensitive genotype did not germinate on stressed stigma of tolerant genotype. De novo transcriptome analysis of both the genotypes showed that number of contigs ranged from 90,267 to 104,424 for all the samples with N50 ranging from 1,755 to 1,844?bp under heat stress and control conditions. Based on assembled unigenes, 194,178 high-quality Single Nucleotide Polymorphisms (SNPs), 141,050 microsatellites and 7,388 Insertion-deletions (Indels) were detected. Expression of 10 genes was evaluated using quantitative Real Time Polymerase Chain Reaction (RT-qPCR). Comparison of differentially expressed genes (DEGs) under different combinations of heat stress has led to the identification of candidate DEGs and pathways. Changes in expression of physiological and pollen phenotyping related genes were also reaffirmed through transcriptome data. Cell wall and secondary metabolite pathways are found to be majorly affected under heat stress. The findings need further analysis to determine genetic mechanism involved in heat tolerance of lentil.
Project description:Physiological parameters and expression levels of drought related genes were analyzed in early vegetative stage of two bread wheat cultivars (Sids and Gmiza) differ in drought tolerance capacity. Both cultivars were imposed to gradual water depletion started on day 17 till day 32 after sowing. Sids, the more tolerant cultivar to drought showed higher fresh and dry weights than the drought sensitive genotype, Gmiza. Under water stress, Sids had higher membrane stability index (MSI), lower accumulated H2O2 and higher activity of the antioxidant enzymes; catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and superoxide dismutase (SOD) than Gmiza. On the other hand, the differential expression patterns of the genes dhn, wcor and dreb were observed due to water deficit intensity according to cultivar's tolerance to drought. The DNA sequence alignment of dun showed high similarity of about 80-92% identities with other related plants. The most striking overall observed trend was the highly induction in the expression of dun, wcor and dreb in leaves of the tolerant genotype, Sids under severe water stress.
Project description:DNA methylation is responsive to various biotic and abiotic stresses. Heat stress is a serious threat to crop growth and development worldwide. Heat stress results in an array of morphological, physiological and biochemical changes in plants. The relationship between DNA methylation and heat stress in crops is relatively unknown. We investigated the differences in methylation levels and changes in the cytosine methylation patterns in seedlings of two rapeseed genotypes (heat-sensitive and heat-tolerant) under heat stress. Our results revealed that the methylation levels were different between a heat-tolerant genotype and a heat-sensitive one under control conditions. Under heat treatment, methylation increased more in the heat-sensitive genotype than in the heat-tolerant genotype. More DNA demethylation events occurred in the heat-tolerant genotype, while more DNA methylation occurred in the heat-sensitive genotype. A large and diverse set of genes were affected by heat stress via cytosine methylation changes, suggesting that these genes likely play important roles in the response and adaption to heat stress in Brassica napus L. This study indicated that the changes in DNA methylation differed between heat-tolerant and heat-sensitive genotypes of B. napus in response to heat stress, which further illuminates the molecular mechanisms of the adaption to heat stress in B. napus.
Project description:Heat stress is a major challenge for crop production. During the reproduction stage, pollen development is the most sensitive process to abnormal environmental conditions. In this project, we have developed a heat treatment system for tomato where plants produce a significantly lower number of pollens and the germination rate of those pollens was also very low. The size of the flower buds and pollen developmental stages were examined under confocal microscope. Pollen cells at different developmental stages were collected from flower buds at corresponding sizes using LCM. The single cell population proteomics analysis was used to identify proteome changes in a distinct group of cells. This information was used to determine the function of proteins that are associated with heat stress on cellular processes/functions during pollen development.
Project description:BACKGROUND: Fluctuations in temperature occur naturally during plant growth and reproduction. However, in the hot summers this variation may become stressful and damaging for the molecular mechanisms involved in proper cell growth, impairing thus plant development and particularly fruit-set in many crop plants. Tolerance to such a stress can be achieved by constitutive gene expression or by rapid changes in gene expression, which ultimately leads to protection against thermal damage. We have used cDNA-AFLP and microarray analyses to compare the early response of the tomato meiotic anther transcriptome to moderate heat stress conditions (32°C) in a heat-tolerant and a heat-sensitive tomato genotype. In the light of the expected global temperature increases, elucidating such protective mechanisms and identifying candidate tolerance genes can be used to improve breeding strategies for crop tolerance to heat stress. RESULTS: The cDNA-AFLP analysis shows that 30 h of moderate heat stress (MHS) alter the expression of approximately 1% of the studied transcript-derived fragments in a heat-sensitive genotype. The major effect is gene down-regulation after the first 2 h of stress. The microarray analysis subsequently applied to elucidate early responses of a heat-tolerant and a heat-sensitive tomato genotype, also shows about 1% of the genes having significant changes in expression after the 2 h of stress. The tolerant genotype not only reacts with moderate transcriptomic changes but also exhibits constitutively higher expression levels of genes involved in protection and thermotolerance. CONCLUSION: In contrast to the heat-sensitive genotype, the heat-tolerant genotype exhibits moderate transcriptional changes under moderate heat stress. Moreover, the heat-tolerant genotype also shows a different constitutive gene expression profile compared to the heat-sensitive genotype, indicating genetic differences in adaptation to increased temperatures. In the heat-tolerant genotype, the majority of changes in gene expression is represented by up-regulation, while in the heat-sensitive genotype there is a general trend to down-regulate gene expression upon MHS. The putative functions associated with the genes identified by cDNA-AFLP or microarray indicate the involvement of heat shock, metabolism, antioxidant and development pathways. Based on the observed differences in response to MHS and on literature sources, we identified a number of candidate transcripts involved in heat-tolerance.
Project description:Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.
Project description:Pollen tube elongation in the pistil is a key step for pollination success in plants, and auxins play an important role in this process. However, the function of auxins in pollen tube elongation in the pistil of rice under heat stress has seldom been previously reported.Two rice genotypes differing in heat tolerance were subjected to heat stress of 40 °C for 2 h after flowering. A sharp decrease in spikelet fertility was found in the Nipponbare (NPB) and its mutant High temperature susceptible (HTS) under heat stress, but the stress-induced spikelet sterility was reversed by 1-naphthaleneacetic acid (NAA), especially the HTS. Under heat stress, the pollen tubes of NPB were visible in ovule, while those of HTS were invisible. However, we found the pollen tubes in ovule when sprayed with NAA. During this process, a significant increase in indole-3-acetic acid (IAA) and reactive oxygen species (ROS) levels was found in the pistil of heat-stressed NPB, while in heat-stressed HTS they were obviously decreased. Additionally, the peroxidase (POD) activity in pistil of NPB was significantly decreased by heat stress, whereas there was no difference between the heat-stressed and non-heat-stressed pistils of HTS.It was concluded that the enhancement of heat tolerance in plants by NAA was achieved through the increase of the levels of auxins, which prevented the inhibition of pollen tube elongation in pistil, and the crosstalk between auxins and ROS, which might be involved in this process. In addition, POD might be a negative mediator in pollen tube elongation under heat stress due to its ability to scavenge ROS and degrade auxin.
Project description:Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).
Project description:Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.
Project description:We investigated the role of ethylene in the response of cotton to high temperature using cotton genotypes with genetically interrupted ethylene metabolism. In the first experiment, Sicot 71BRF and 5B (a lintless variant with compromised ethylene metabolism) were exposed to 45°C, either by instantaneous heat shock or by ramping temperatures by 3°C daily for 1 week. One day prior to the start of heat treatment, half the plants were sprayed with 0.8 mM of the ethylene synthesis inhibitor, aminoethoxyvinylglycine (AVG). In a subsequent experiment, Sicot 71BRF and a putatively heat-tolerant line, CIM 448, were exposed to 36 or 45°C for 1 week, and half the plants were sprayed with 20 ?M of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, (ACC). High temperature exposure of plants in both experiments was performed at the peak reproductive phase (65-68 days after sowing). Elevated temperature (heat shock or ramping to 45°C) significantly reduced production and retention of fruits in all cotton lines used in this study. At the termination of heat treatment, cotton plants exposed to 45°C had at least 50% fewer fruits than plants under optimum temperature in all three genotypes, while plants at 36°C remained unaffected. Heat-stressed plants continued producing new squares (fruiting buds) after termination of heat stress but these squares did not turn into cotton bolls due to pollen infertility. In vitro inhibition of pollen germination by high temperatures supported this observation. Leaf photosynthesis (Pn) of heat-stressed plants (45°C) measured at the end of heat treatments remained significantly inhibited, despite an increased leaf stomatal conductance (gs), suggesting that high temperature impairs Pn independently of stomatal behavior. Metabolic injury was supported by high relative cellular injury and low photosystem II yield of the heat-stressed plants, indicating that high temperature impaired photosynthetic electron transport. Both heat shock and ramping of heat significantly reduced ethylene release from cotton leaf tissues measured at the end of heat treatment but modulating ethylene production via AVG or ACC application had no significant effect on fruit production or retention in heat-stressed cotton plants. Instead, high temperature accelerated fruit abortion by impairing pollen development and/or restricting leaf photosynthesis.
Project description:High temperature (HT) decreases seed set percentage in sorghum (Sorghum bicolor [L.] Moench). The relative sensitivity of pollen and particularly pistil and the mechanistic response that induces tolerance or susceptibility to HT are not well known and hence are the major objectives of this research. The male sterile (ATx399) and fertile (RTx430) lines were exposed to 30/20 °C (optimum temperature), 36/26 °C (HT1 ), and 39/29 °C (HT2 ) from the start of booting to seed set in a controlled environment. Similarly, in the field, HT stress was imposed using heat tents. HT stress decreased pollen germination. Relatively high levels of reactive oxygen species and decreased antioxidant enzyme activity and phospholipid unsaturation were observed in pollen compared to pistil under HT. The severe cell organelle damage was observed in pollen and pistil at 36/26 and 39/29 °C, respectively. The seed set percentage was higher in HT-stressed pistil pollinated with optimum-temperature pollen. Direct and reciprocal crosses indicate that pollen was more sensitive with larger decreases in seed set percentage than pistil under HT stress. The negative impact was greater in pollen than pistil at lower temperatures. Overall, pollen was more sensitive than pistil to HT stress because it is more susceptible to oxidative damage than pistil.