Despite drought accounting for over 80% of agricultural losses, little progress has been made towards improving drought tolerance in crops. My approach to identifying traits underlying drought tolerance involved a comparison between two accessions of the crucifer, Eutrema salsugineum, that display differential tolerance to water deficits. The accessions, originating from the semi-arid Yukon, Canada, and a monsoonal region of Shandong, China, were subjected to a two-step, water deficit and recovery protocol to identify physiological characteristics that discern their drought-responsive behaviour. Traits that discriminate between the ecotypes were used to screen recombinant inbred lines (RILs) that were generated by crossing Yukon and Shandong parent plants. Selected physiological measurements were: anthocyanin accumulation, cut rosette water loss (CRWL), solute potential, relative water content (RWC), static leaf water content (SLWC), specific leaf area (SLA), and OJIP fluorescence emission. Of the measurements taken, CRWL measurements and anthocyanin content distinguished the Yukon ecotype from the Shandong ecotype during the first drought exposure whereas SLA and fluorescence responses differentiated these accessions better after plants that experienced the first drought were rewatered and recovering or undergoing a second drought treatment. Sixty-eight RILs were screened using SLA and OJIP fluorescence emission. SLA and OJIP measurements varied among the recombinant inbred lines (RILs) with many lines showing responses to water deficit intermediate to those of the parental lines. Evidence of heritability in SLA and/or OJIP responses to water deficits would make them useful phenotypic markers for identifying quantitative trait loci (QTLs) associated with drought tolerance in future work. / Thesis / Master of Science (MSc)

The accumulation of salts in soil is an important agricultural problem that limits crop productivity. Salts containing sodium (Na⁺) are particularly problematic, as cytosolic Na⁺ can interfere with cellular metabolism and lead to cell death. Maintaining low levels of cytosolic Na⁺, therefore, is critical for plant survival during growth in salt. Mechanisms to regulate Na⁺ accumulation in plant cells include extrusion of Na⁺ from the cell and sequestration of Na⁺ into intracellular compartments. Both of these processes are controlled in part through the action of Na⁺/H⁺ exchangers belonging to the Cation/Proton Antiporter-1 (CPA1) gene family. Genes belonging to this family have been identified in both salt-sensitive and salt-tolerant species, suggesting that salt-tolerant species may have evolved salt tolerance through modification of these existing pathways. The research presented here has focused on understanding how salt tolerance has evolved in Brassicaceae species, and particularly on the role that CPA1 genes have played in the adaptation to salinity of Eutrema salsugineum. Specific projects have sought to understand 1) how copy number variation and changes in coding sequences of CPA1 genes contribute to salt tolerance in E. salsugineum and its salt-tolerant relative Schrenkiella parvula, 2) whether functional or regulatory changes in Salt Overly Sensitive 1 (SOS1) from E. salsugineum (EsSOS1) contribute to its enhanced salt tolerance, and 3) whether accessions of Arabidopsis thaliana differ significantly in their response to salt stress.The results indicate that EsSOS1 and SOS1 from S. parvula (SpSOS1) both confer greater salt tolerance in yeast than SOS1 from A. thaliana (AtSOS1) when activated by the complex of the SOS2 kinase and SOS3 calcium-binding protein, whereas only EsSOS1 confers enhanced salt tolerance in the absence of activation. When expressed in A. thaliana, EsSOS1 also confers greater salt tolerance than AtSOS1 through regulatory changes that likely involve differences in expression pattern. Together, the results presented here suggest that mechanisms regulating cellular Na⁺ accumulation that exist in salt-sensitive crop species could be altered to enhance growth in salty soils. In addition, the 19 A. thaliana accessions used to create the MAGIC population were shown to differ significantly in their response to salt stress.

The drought response of the extremophile Eutrema salsugineum (Thellungiella salsuginea) was studied using an experimental protocol involving two progressive drought exposures separated by a recovery period. Accessions from the Yukon Territory, Canada, and Shandong Province, China, were distinguished with respect to their responses to the initial drought, their recovery from wilting, and their response to a subsequent drought following recovery. Eutrema cauline leaves and rosettes were sampled at different stages of the drought treatment for water status and biomass measurements and this information guided tissue selection for transcriptome sequencing by RNA-Seq. For Yukon plants, the initial drought led to a 46% reduction in stomatal conductance (from 122.3 to 66.3 mol m-2s-1) and 25% reduction in rosette water loss relative to unstressed control plants, evidence of drought avoidance to conserve water. Yukon leaf solute potentials decreased to -1.83 MPa compared to -1.54 MPa for Shandong leaves indicating that more solutes accumulated in Yukon leaves in response to drought. Upon wilting, Yukon plants re-established turgor at significantly lower leaf solute potentials than the level for well-watered Yukon plants consistent with osmotic adjustment. In contrast, leaf solute potentials in re-watered Shandong plants returned to pre-drought levels (-1.6 MPa). During the second drought exposure, leaf water content and specific leaf area measurements were significantly higher in Yukon plants compared to plants experiencing the initial drought and wilting was delayed relative to Shandong plants. At the transcriptional level, the initial drought exposure resulted in over 2000 differentially expressed genes in leaves of Yukon plants compared to only two in Shandong plants. Following exposure to a second drought only 45 genes were differentially expressed in leaves of Yukon plants while Shandong plants underwent substantial transcriptional re-programming with nearly 500 genes showing differential expression. Studies of Eutrema grown under controlled conditions were supplemented by physiological measurements made using Eutrema plants found on saline soils in the Yukon. The average stomatal conductance for field plants was 84.8 mol m-2s-1, a rate similar to that of drought-treated Yukon plants in the cabinet. Leaf solute potentials of field plants ranged from -2.0 MPa to -3.5 MPa. RT-qPCR showed the relative expression of four dehydrin-encoding genes, EsRAB18, EsRD22, EsRD29A, and EsERD1, was high in the field plants and levels of expression were comparable to drought-stressed cabinet plants. In summary, Eutrema salsugineum has a naturally high tolerance to water deficits. Between the two accessions studied, Yukon plants have a superior capacity to withstand drought relative to Shandong plants. The heightened capacity for Yukon plants to recover from drought and tolerate repeated drought exposures makes this accession a particularly valuable model for studying many mechanisms underlying innate and inducible plant tolerance to drought. / Thesis / Doctor of Philosophy (PhD)

Long non-protein coding RNAs (lncRNAs) represent a diverse and enigmatic classification of RNA. With roles associated with development and stress responses, these non-coding gene regulators are essential, and yet remain understudied in plants. Thus far, of just over 430 experimentally validated lncRNAs, only 13 are derived from plant systems and many of which do not meet the classic criteria of the RNA class. Without a solid definition of what makes a lncRNA, and few empirically validated transcripts, methods currently available for prediction fall short. To address this deficiency in lncRNA research, we constructed and applied a machine learning-based lncRNA prediction protocol that does not impose predefined rules, and utilises only experimentally confirmed lncRNAs in its training datasets. Through model evaluation, we found that our novel lncRNA prediction tool had an estimated accuracy of over 96%. In a study that predicted lncRNAs from transcriptomes of evolutionary diverse plant species, we determined that molecular features of lncRNAs display different phylogenetic signal patterns compared to protein-coding genes. Additionally, our analyses suggested that stress-resistant species express fewer lncRNAs than more stress sensitive species. To expand on these results, we used the prediction tool in concert with a transcriptomic study of two natural accessions of the drought tolerant species Eutrema salsugineum. Previously reported to show little physiological differences in a first drought, but differ significantly in a second, we instead demonstrated that the two ecotypes displayed vastly different transcriptomic responses, including the expression of lncRNAs, to a first and second drought treatment. In conclusion, the prediction tool can be applied to studies to further our knowledge of lncRNA evolution and as an additional tool in classic transcriptomic studies. The suggested importance of lncRNAs in drought resistance, and evidence of expression in two natural E. salsugineum accessions, merits further studies on the molecular and evolutionary mechanisms of these putatively regulatory transcripts. / Thesis / Doctor of Philosophy (PhD)

<p><em>Eutrema salsugineum</em> is an extremophile plant native to the Yukon Territory and coastal China. As an extremophile, Yukon <em>Eutrema</em> is tolerant to highly saline, drought conditions and cold temperatures while Shandong <em>Eutrema</em> can survive in highly saline conditions (Griffith et al., 2007; Guevara et al., 2012; Inan et al., 2004). The disease resistance responses of the Yukon and Shandong accessions of <em>Eutrema</em> were investigated to understand how an abiotic stress-tolerant plant responds to biotic stress. A pathosystem was developed using <em>Pseudomonas</em> <em>syringae</em> pv. <em>tomato</em> DC3000 (<em>Pst</em>) to examine <em>Eutrema</em> defense responses. Compared to <em>Arabidopsis </em>(Col-0), both <em>Eutrema</em> accessions exhibited resistance to <em>Pst,</em> with Shandong <em>Eutrema</em> displaying greater resistance than Yukon <em>Eutrema</em>. Resistance to <em>P. syringae</em> pv. <em>maculicola</em> (<em>Psm</em>) was also observed in both accessions. Furthermore, both <em>Eutrema</em> accessions displayed a differential capacity for effector-triggered immunity (ETI). RNA-Seq data of uninoculated Shandong vs. Yukon <em>Eutrema</em> revealed an overrepresentation of defense genes including <em>PR1</em> (<em>pathogenesis-related1</em>; Champigny et al., 2013). Expression of the <em>Eutrema</em> <em>PR1</em> ortholog in uninoculated Shandong leaves combined with enhanced resistance to <em>Pst</em> compared to Yukon <em>Eutrema</em> or Col-0 <em>Arabidopsis</em> suggests that Shandong plants exist in a defense-primed state. Resistance to other pathogens such as <em>Pectobacterium</em> <em>carotovorum</em> ssp. <em>wasabiae</em> (<em>Pcw</em>) further supported the hypothesis that Shandong <em>Eutrema</em> is primed for pathogen tolerance. The <em>Eutrema</em>-<em>P. syringae</em> pathosystem will facilitate future studies to understand how <em>Eutrema</em> deals with multiple or concurrent stresses and this knowledge will contribute to efforts to improve tolerance to both abiotic and biotic stress in crop plants.</p> / Master of Science (MSc)