FUNCTIONAL GENOMICS, TRANSCRIPTOMICS AND METABOLOMICS OF HEAT STRESS IN BARLEY (HORDEUM VULGARE L.)

Abstract

Adverse impacts of global climate change including extreme high temperature on cereal crop production have been evidenced at the global level. In plants, the elevated temperature above the optimal range dramatically impairs the process of both vegetative and reproductive phases of the development, mainly due to disruption in their vital metabolic processes. However, plants possess an inherent rescue machinery to combat such stressful conditions. Heat shock proteins (HSPs) and heat shock factors (HSFs) are the crucial components of these stress associated rescue mechanisms and also required for normal biological processes. In the present study, a genome wide identification, evolutionary relationship and comprehensive expression analysis of Hsp70, Hsp90 and Hsp100 gene families have been performed in barley. Barley genome was revealed to possess 13 members of Hsp70 gene family, along with 4 members of Hsp110 subfamily, 6 of Hsp90, and 8 members of Hsp100 gene family, and Hsp genes were found to be distributed across all 7 chromosomes of barley. The encoded protein members of these genes were predicted to be localized to cell organelles such as cytosol, mitochondria, chloroplast, and ER. Despite a larger genome size, lesser members of these Hsp genes in barley were observed owing to less duplication events. The variable expression patterns obtained for genes encoding the proteins localized in same subcellular compartment suggest their possible diverse roles and involvement in different cellular responses. Expression profiling of these genes was performed by qRT-PCR in an array of 32 tissues, revealed differential and tissue specific expression of various members of Hsp gene families. We found the upregulation of HvHspc70-4, HvHsp70Mt70-2, HvHspc70-5a, HvHspc70-5b, HvHspc70-N1, HvHspc70-N2, HvHsp110-3, HvHsp90-1, HvHsp100-1, and HvHsp100-2 upon exposure to heat stress during reproductive development. Furthermore, their higher expression during heat stress, heavy metal stress, drought, and salinity stress has also been observed. HSFs of class A are the regulators of several heat shock and other abiotic stress responsive genes. In the present investigation, the constitutive overexpression of class A heat shock factor HvHSFA2c was achieved in barley. Several overexpressing transgenic lines were identified and analyzed for transgene expression by performing histochemical gus assay, genomic PCR, RT and q-RT PCR. The transcriptomics studies of overexpressing transgenic lines through RNA-Seq revealed the potential putative target genes of HvHSFA2c. Here, we inferred that HvHSFA2c may be acting by inducing the transcription of chaperone encoding genes belonging to all five classes of Hsps regulating the protein homeostasis of plants. Apart from chaperones; HvHSFA2c ii may also be regulating the expression of genes associated with calcium-mediated signal transduction pathways, which have been known to be associated with the heat shock response of plants. Also the upregulation of genes involved in biosynthesis of jasmonic acid and its signaling, projected the involvement of JA in heat stress tolerance associated mechanisms. Similarly, the differential expression of early and late auxin responsive genes and gibberellin homeostasis associated genes suggested that HvHSFA2c may be regulating the activity of these phytohormone associated growth mechanisms of plants. Both photosynthetic and respiratory processes are vital for the plants and adversely affected by HS. The overexpression of genes related to light and dark phases of photosynthesis and mitochondrial respiratory electron transport chains suggested that HvHSFA2c may be providing thermotolerance by stabilizing the activity of these pathways. The differential expression (up and downregulation) of several other stress associated transcription factors such as WRKY, Myb, AP2/ERF etc. suggested the convergence of stress associated signaling and transcriptional regulation. We further inferred that HvHSFA2c may also be potentially targeting the ROS scavenging pathways by activating flavonoid biosynthesis, and antioxidative enzymes such as superoxide dismutase catalase and ascorbate peroxidase. The higher activity of these enzymes was further validated in our overexpressing transgenic lines. Here, we concluded that, the genes associated with these described pathways may potentially be observed under the direct or indirect regulation of HvHSFA2c. Furthermore, the comparative metabolic profiling of wild type plants of barley subjected to HS and plants grown under controlled conditions was performed to analyze the metabolic adaptation of these plants during heat stress. The changes were revealed in metabolomics fluxes of non-reducing sugars, several amino acids, and accumulation of secondary metabolites which may be protecting plants during heat stress conditions. This study provided the insights of heat stress tolerance mechanisms in barley plants through comparative genomic studies of high molecular weight heat shock proteins, and functional study of a heat shock factor HvHSFA2c. The metabolomics study suggested the alterations in key metabolic processes such as citric acid cycle, carbohydrate metabolism and secondary metabolism.