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Molecular responses of grapevine to environmental stress
AdvisorCramer, Grant R.
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In the past decade, advances in sequencing and transcript quantification technology have progressed science through the genomic era into the big data era, yielding thousands of studies examining plant gene expression in response to a vast assortment of conditions. Understanding and characterization of Vitis (grapevine) has been vastly improved with modern technology. Grapevines are a culturally and economically important crop susceptible to abiotic and biotic stresses. The Vitis genus consists of tens of thousands of varieties each belonging on a spectrum of tolerance and susceptibility to different stress conditions. Two genes (VviERF6L1 and NCED3) were previously identified from microarray and RNA-Sequencing experiments and implicated in abiotic stress response but required further investigation and characterization in grapevine. The examination of these genes as hub genes in ABA signaling and stress response employed multi-level analyses of DNA, RNA, protein, and metabolite quantification in diverse Vitis species including Vitis vinifera cv. Cabernet Sauvignon (CS), Vitis champinii cv. Ramsey (RA), Vitis riparia cv. Riparia Gloire (RI), and Vitis vinifera x Vitis girdiana cv. SC2 (SC). First, a bioinformatics approach was used to annotate ABA response elements (ABREs) across all promoter regions in the PN40024 reference genome. ABREs were highly abundant and in the majority of PN40024 promoter regions. Various ABREs were identified in the ERF6L1 and NCED3 promoter regions contributing to the understanding of previous transcriptional changes observed in response to abiotic stresses. Many novel and uncharacterized genes were also identified with high numbers of ABREs in respective promoter regions that may provide valuable targets for future studies to improve grapevine breeding programs and abiotic stress tolerance. Meta-data analysis of publicly available microarray and RNA-Sequencing data identified the VviERF6L clade to transcriptionally respond to numerous stimuli including water deficit, cold, salinity, pathogen infection, wounding, and berry ripening. VviERF6Ls were expressed in many tissues including leaves, roots, and berries. Although VviERF6L1 overexpression vines did not have any obvious quantifiable morphological phenotype, the majority of genes differentially expressed in response to VviERF6L1 overexpression were involved in pathogen response. Cis-acting elements like the WBOXATNPR1 in the VviERF6L1 promoter region further implicated a role of VviERF6L1 in pathogen response. This hypothesized function was also supported by known effects of ERF5 and ERF6 Arabidopsis thaliana orthologs on pathogen susceptibility. To test the functional role of VviERF6L1 in pathogen response, a grapevine-optimized P. syringae infection assay was established. VviERF6L1 was demonstrated to have significantly higher transcript abundance in response to P. syringae infection than mock infection. Additionally, VviERF6L1 overexpression vines had significantly fewer colony-forming units during P. syringae infection and thereby higher resistance to the pathogen than empty vector control vines. NINE-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED3) transcripts, NCED3 protein, and abscisic acid (ABA) concentration were quantified using RNA-Sequencing and RT-qPCR, western blots, and HPLC-MS/MS, respectively, in the leaves and roots of the four Vitis species in response to three different water deficit severities. NCED3 was identified as the only ABA metabolism gene that was a hub gene during water deficit response. NCED3 and ABA metabolism were validated as a major part of the core water deficit responses in the four Vitis species. However, ABA metabolism was highly dependent on species, organ, stress severity, and stress duration during water deficit. Interestingly, NCED3 transcript abundance paralleled ABA concentration, but this similarity was not maintained for NCED3 protein, concentrations of which did not significantly change in response to water deficit. Overall, the Texan grapevine, RA, was found to respond earlier and more sensitively during longer-term moderate and severe water deficits than the other more water deficit sensitive species. Altogether, this work furthered the understanding of two genes involved in stress response in grapevine. VviERF6L1 was identified to have a role in abiotic and biotic stress response, but the mechanism of VviERF6L1 in pathogen response requires further investigation. NCED3 was confirmed as an ABA signaling hub during water deficit with specific expression and downstream ABA concentrations being highly dependent upon species, organ, and stress conditions. However, NCED3 protein abundance response to water deficit requires further examination. These genes provide useful targets for future studies and may have applications in breeding programs to improve grapevine stress tolerance.