|dc.description.abstract||Field pea (Pisum sativum L.), a cool-season legume crop, is known for poor heat tolerance. Over the last decade, progress has been made in the understanding of the physiological damage of pea plants caused by heat stress (HS), however, knowledge of the hormonal and genetic basis of heat stress response (HSR) is still scarce. This thesis was focused on the characterization of abscisic acid (ABA) metabolism and transcriptome response among pea varieties contrasting in heat tolerance.
In Study I, two heat tolerant pea varieties, CDC Meadow and PR11-2, and two sensitive varieties, Nitouche and PR11-90, were evaluated, whose heat tolerance were previously characterized in field trials. Plants of individual varieties were heat stressed for 3 h, 6 h, 12 h or 24 h at 38°C before pollination. RNA extracted from anthers and stipules on the same flowering node were sampled for transcriptional profiling of two pea heat shock protein (HSP) genes, PsHSP18.1 and PsHSP71.2. Additional stipules were sampled for the quantification of ABA concentration and its five key catabolites from the four major ABA catabolic pathways by liquid chromatography-multiple reaction monitoring mass spectrometry. Both pea HSP genes and ABA metabolism responded rapidly after 3 h at HS. However, PsHSP18.1 and PsHSP71.2 had similar induction levels between heat tolerant and susceptible varieties, suggesting the function of these two genes is conserved in heat response of pea. Heat tolerant varieties had a higher ABA synthesis and turnover rate at 3 h HS, than their respective heat susceptible counterparts.
In Study II, heat tolerant variety, PR11-2, and heat susceptible variety, PR11-90, were selected from Study I to characterize the differential transcription at 3 h HS via RNA-Seq technology. The widely grown and moderately heat tolerant variety, CDC Amarillo, was included as a check. Differentially expressed genes (DEGs) were identified at log2 |fold change (FC)| ≥ 2 between HS and control temperature. The three varieties shared 588 DEGs which were up-regulated and 220 genes which were down-regulated in anthers when subjected to HS. In stipules, 463 upregulated genes and 416 downregulated genes were consistent among varieties. The above heat-induced genes of stipules and anthers were related to several biological processes, i.e., response to heat, protein folding, and DNA templated transcription. Ten gene ontology (GO) terms were over-represented in the consistently down-regulated DEGs of the two organs, and these terms were mainly related to cell wall macromolecule metabolism, lipid transport, lipid localization, and lipid metabolic processes. GO enrichment analysis on distinct DEGs of individual pea varieties suggested that heat affected biological processes were dynamic, and variety distinct responses characterize the heat tolerance variation among pea varieties at the transcriptional level. Several biological processes, e.g., cellular response to DNA damage stimulus in stipule and electron transport chain in anther, that were only observed in heat induced PR11-2 and CDC Amarillo, and their relevance to field pea heat tolerance is worth further validation.
To validate the above transcriptional variation at HS, 39 recombinant inbred lines (RILs) were made from the cross of PR11-2 and CDC Amarillo in Study III, and they were tested in field trials in 2020 and 2021, to investigate the genetic loci associated with heat responsive traits relating to flowering and yield components. In total, four consistent loci were identified to be associated with heat responsive traits over multiple site-years, which were a QTL for days to flowering at chromosome 7, a QTL for pod number at chromosome 2, and one each QTL for reproductive node number and days to maturity at chromosome 5. Notedly, eight genes (5g161560, 5g165160, 5g171400, 5g198960, 7g051680, 7g091560, 7g091680 and 7g093240) within the aforementioned QTLs were differentially expressed between PR11-2 and CDC Amarillo in Study II. As a result, these eight genes were proposed to contribute to the superior heat tolerance of PR11-2 over CDC Amarillo. Collectively, my thesis expands the current understanding of pea heat response at the hormonal, transcriptional and genetic levels.||