Salt stress, or salinity, is one of the most common environmental stresses affecting crop yield worldwide. Due to the prevalence of salinity stress, it is not surprising that plants have evolved mechanisms to tolerate osmotic and ionic stress caused by salinity. Dehydrins are intrinsically unstructured proteins that accumulate in photosynthetic organisms under dehydrating conditions, such as salinity, and are thought to confer stress tolerance through the stabilization of cellular membranes. <i>Thellungiella salsuginea</i>, a close relative of <i>Arabidopsis thaliana</i>, is a halophyte that thrives in the Canadian sub-Arctic (Yukon Territory), that is able to tolerate extreme conditions, including high salinity. TsDHN-2 is a basic dehydrin from <i>Thellungiella</i> whose transcript increases over 10-fold in response to salinity treatment. Using RNA interference (RNAi) methodology, TsDHN-2 has been silenced and these lines were used in this study to investigate the role TsDHN-2 may play in the salt tolerance of <i>Thellungiella</i>. RNAi line 7-8 presented a 41% reduced expression of TsDHN-2 in comparison to wild-type (WT). Seed of this line showed a 15% germination rate compared to 40% in WT in the presence of 100 mM NaCl. Salinity stress experiments were performed by treating the RNAi lines and WT plants with 300 mM NaCl for up to two weeks. Line 7-8 exhibited a 6.2% greater decrease in photochemical efficiency of photosystem II (PSII) as estimated by the variable to maximal fluorescence ratio (F_v/F_m) and showed 5% greater phenotypic damage than WT when estimated visually. Concentrations of the compatible osmolyte proline increased in response to salt treatment by 3.4-fold in WT and 8.1-fold in line 7-8, suggesting this compound may be a marker for salinity tolerance. Collectively, these data support the notion that TsDHN-2 plays a role in the salinity tolerance mechanisms of <i>Thellungiella</i>.

Salt stress, or salinity, is one of the most common environmental stresses affecting crop yield worldwide. Due to the prevalence of salinity stress, it is not surprising that plants have evolved mechanisms to tolerate osmotic and ionic stress caused by salinity. Dehydrins are intrinsically unstructured proteins that accumulate in photosynthetic organisms under dehydrating conditions, such as salinity, and are thought to confer stress tolerance through the stabilization of cellular membranes. <i>Thellungiella salsuginea</i>, a close relative of <i>Arabidopsis thaliana</i>, is a halophyte that thrives in the Canadian sub-Arctic (Yukon Territory), that is able to tolerate extreme conditions, including high salinity. TsDHN-2 is a basic dehydrin from <i>Thellungiella</i> whose transcript increases over 10-fold in response to salinity treatment. Using RNA interference (RNAi) methodology, TsDHN-2 has been silenced and these lines were used in this study to investigate the role TsDHN-2 may play in the salt tolerance of <i>Thellungiella</i>. RNAi line 7-8 presented a 41% reduced expression of TsDHN-2 in comparison to wild-type (WT). Seed of this line showed a 15% germination rate compared to 40% in WT in the presence of 100 mM NaCl. Salinity stress experiments were performed by treating the RNAi lines and WT plants with 300 mM NaCl for up to two weeks. Line 7-8 exhibited a 6.2% greater decrease in photochemical efficiency of photosystem II (PSII) as estimated by the variable to maximal fluorescence ratio (F_v/F_m) and showed 5% greater phenotypic damage than WT when estimated visually. Concentrations of the compatible osmolyte proline increased in response to salt treatment by 3.4-fold in WT and 8.1-fold in line 7-8, suggesting this compound may be a marker for salinity tolerance. Collectively, these data support the notion that TsDHN-2 plays a role in the salinity tolerance mechanisms of <i>Thellungiella</i>.

The objective of this thesis was to study the response of Eutrema salsugineum (Yukon) plants to low phosphate (Pi) using seedlings and four- week-old plants grown on media formulated with variable phosphate (Pi). Seedlings showed similar root architecture whether grown with high Pi or without added Pi. Four-week-old plants grown with 0 or 2.5 mM Pi added to the soil had the same shoot biomass and relative growth rates. Confirmation that plants on low Pi were Pi-deficient despite lacking a Pi-starvation phenotype was provided by the increased expression of Pi-starvation-inducible genes (notably EsIPS2) in Pi-deprived plants. We also found that seedling roots on media lacking Pi did not acidify their rhizosphere nor did they show increased phosphatase secretion or phosphatase activity relative to roots of Pi-sufficient seedlings. In soil-grown plants, leaf P remobilization was slower during dark-induced senescence of Eutrema relative to similarly treated, Pi-starved Arabidopsis. Also related to metabolism, in vitro assays showed that the ratio of maximal PPi- and ATP- dependent phosphofructokinase activities approximated 1:1 and 2:1 for Eutrema leaf and root extracts, respectively, with no Pi-responsive changes found and, relative to Arabidopsis, Eutrema phosphoenolpyruvate carboxylase activities were high. The enzyme activities suggest Eutrema operates glycolytic by-passes under Pi sufficient and deficient conditions. Finally, transcripts for the transcription factors Phosphate Starvation Response 1 (PHR1) and WRKY75 were not Pi-starvation-inducible and were more abundant in Eutrema leaves than in leaves of Pi-deprived Arabidopsis. Global gene expression showed the leaf and root transcriptomes to be about 90% similar between 0 and 2.5 mM Pi- treated plants with 2,901 differentially expressed genes detected by DESeq2. In summary, Eutrema displays few Pi-starvation responsive traits whether those traits reflect changes at the level of gene expression or plant morphology, behaviour consistent with a specialist that is continuously primed for Pi starvation. / Thesis / Doctor of Philosophy (PhD)

Comparative sequence analyses were performed with members of the Solanaceae and the Brassicaceae. These studies investigated genomic organization, determined levels of microcolinearity, identified orthologous genes and investigated the molecular basis of trait differences. The first analysis was performed by comparison of tomato (Solanum lycopersicum) genomic sequence (119 kb) containing the JOINTLESS1 (J1) locus with orthologous sequences from two potato species, a diploid, Solanum bulbocastanum (800-900 Mb, 2N=2X=24), and a hexaploid, Solanum demissum (2,700 Mb, 2N=6X=72). Gene colinearity was well maintained across all three regions. Twelve orthologous open reading frames were identified in identical order and orientation and included three putative J1 orthologs with 93-96% amino acid sequence identity in both potato species. Although these regions were highly conserved, several local disruptions were detected and included small-scale expansion/contraction regions with intergenic sequences, non-colinear genes and transposable elements. Three putative Solanaceous-specific genes were also identified in this analysis. The second analysis was performed by comparison of a Thellungiella halophila (T. halophila) genomic sequence (193 kb) containing the SALT OVERLY SENSITIVE1 (SOS1) locus with the orthologous sequence (146 kb) in Arabidopsis thaliana (Arabidopsis). T. halophila is a halophytic relative of Arabidopsis thaliana that exhibits extreme salt tolerance. Twenty-five genes, including the putative T. halophila SOS1 (ThSOS1), showed a high degree of colinearity with Arabidopsis genes in the corresponding region. Although the two sequences were significantly colinear, several local rearrangements were detected which were caused by tandem duplications and inversions. Three major expansion/contraction regions in T. halophila contained five LTR retrotransposons which contributed to genomic size variation in this region. ThSOS1 shares similar gene structure and sequence with Arabidopsis SOS1 (AtSOS1), including 11 transmembrane domains and a cyclic nucleotide-binding domain. Three Simple Sequence Repeats (SSRs) were detected within a 540 bp region upstream of the putative translational start site in ThSOS1. The (CTT)n repeat is present in different copy numbers in ThSOS1 (18 repeats) and AtSOS1 (3 repeats). When present in the 5' UTRs of some Arabidopsis genes, (CTT)n serves as a putative salicylic acid responsive element. These SSRs may serve as cis-acting elements affecting differential mRNA accumulation of SOS1 in the two species.

<p> Abiotic stresses such as extreme temperatures, drought and high salinity severely compromise plant productivity, and have placed selective pressure for the acquisition of traits enabling plants to adjust to and recover from these unfavorable environmental conditions. Thellungiella salsuginea is a plant that is native to highly saline and semiarid environments and exhibits an exceptional ability to tolerate abiotic stress. In this thesis, I report on laboratory and field studies aimed at identifying traits that allow Thellungiella to tolerate harsh environmental conditions. It was found that Thellungiella accumulates organic solutes in response to abiotic stress. Transcript and metabolite profiling approaches were used to identify metabolic pathways important for the accumulation of compatible organic solutes in Thellungiella in response to sub-optimal environmental conditions. The relative abundance of transcripts encoding enzymes associated with the biosynthesis of compatible organic solutes such as proline or galactinol showed stress-responsive increases in cabinet-grown material and these metabolites were accumulated in salt or drought treated plants, respectively. However, proline and galactinol were found to be of low relative abundance in leaves of field plants. In contrast, several carbohydrates including sucrose, glucose, and fructose made a greater relative contribution to the field plant profiles suggesting that carbohydrates play an important role in plant abiotic stress tolerance during growth under field conditions. The identification of stress-specific metabolic changes can be used to identify important biochemical traits underlying environmental stress tolerance in Thellungiella. This information can be used to improve the tolerance of stress -sensitive crops (including a related crucifer species, canola) that are grown in areas where persistent droughts, saline soils and early or late frosts frequently occur. </p> / Thesis / Doctor of Philosophy (PhD)

<p>Phosphorus is a macronutrient required for plant growth and reproduction. Insufficient supplies of phosphate will adversely impact plant growth. In an effort to supply adequate phosphate to crops, large quantities of phosphate-rich fertilizer are applied to fields but much of the phosphate can leach from the soil as run-off, impacting water systems. Therefore, proper management of phosphate and the development of phosphate efficient genotypes of plants are strategies needed for a sustainable agriculture industry.</p> <p>This thesis project focused on the development of biomarkers of phosphate stress in <em>Thellungiella salsuginea, </em>a plant highly tolerant to salt, cold and water deficit. Biomass determinations and real-time quantitative PCR were used to determine the gene expression of several genes selected as known phosphate-responsive genes from studies of phosphate starvation of the related genetic model plant <em>Arabidopsis thaliana.</em></p> <p><em> Thellungiella </em>seedlings were grown on 5 and 500 µM phosphate media. The expression of several genes (<em>RNS1, At4, Pht1;1, Pht1;4, Pht1;5, Siz1, PHR1, WRKY75, </em>and<em> Pht2;1</em>) were assayed for their response to media phosphate content. <em>RNS1</em> and <em>At4 </em>expression was estimated from cDNA prepared from shoot tissues while <em>At4, Pht1;1</em> and <em>Pht1;5</em> expression was determined from root tissues. In all tissue sources, significantly increased expression of <em>RNS1</em>, <em>At4</em>,<em> Pht1;1</em> and <em>Pht1;5</em> was observed under 5 µM phosphate exposure.</p> <p><em> </em>Two natural accessions of <em>Thellungiella</em> were used in this study with one originating from the Yukon Territory, Canada and the second from Shandong Province, China. Seedlings of both ecotypes were grown on defined media plates containing various concentrations of phosphate (0, 25, 125, 250, 500, and 2000 µM). For both accessions, the addition of as little as 25 µM phosphate led to significant increases in root and shoot biomass. Gene expression levels corresponding to <em>RNS1, At4</em> and <em>Pht1;1</em> were the highest in Yukon and Shandong <em>Thellungiella </em>grown on 0 µM phosphate media. The addition of 25 µM phosphate to the media was enough to significantly decrease transcript abundance of <em>RNS1, At4 </em>and <em>Pht1;1. </em>In a test using the transfer of Yukon <em>Thellungiella </em>seedlings from high (500 µM) to low (5 µM) phosphate the expression of <em>At4</em> in roots and shoots increased 30-fold over a five-day period and only <em>Pht1;1</em> expression increased in the roots over the same time period.</p> <p><em>RNS1</em> and <em>At4</em> share attributes that make them suitable biomarkers for phosphate stress in plants. Both genes are expressed in the shoots making it easier to remove tissue for monitoring gene expression, and both genes show readily discernible increases in transcript levels for determination by qPCR. At present, however, the role for their products in phosphate assimilation by plants is uncertain. This lack of knowledge is a deterrent to adopting these genes for widespread use as biomarkers. In particular, more work needs to be done to characterize factors that elicit their expression to test the specificity of their response to phosphate stress in <em>Thellungiella</em>.</p> / Master of Science (MSc)

<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)

<p> Thellungiella salsuginea (also known as T. halophila), is an emerging model species for studies of plant tolerance to osmotic stress. Currently, Thellungiella plants originating from two geographical locations are being developed for research. Yukon Thellungiella is native to the saline and alkaline soils of the Yukon Territory, Canada. The seeds in this study were collected at the Takhini Salt-Flats near Whitehorse, YT, a sub-arctic and semi-arid region. Plants from the Yukon have been proposed to be a suitable species to study plant tolerance to salinity, cold temperatures, and water deficits. Shandong Thellungiella is native to the saline coast of north-eastern China in Shandong Province where the climate is temperate and affected by summer monsoons. This plant has been proposed as an ideal model for the study of salt tolerance mechanisms but is reported to show little drought tolerance.</p> <p> An objective of this study was to compare Yukon and Shandong Thellungiella plants with respect to their ability to withstand water deficits. Plants were grown simultaneously in controlled environment chambers where watering was withheld until plants visibly wilted. Wilting occurred at a leaf relative water (RWC) content of about 50-60% and then turgor was restored by re-watering. In a second experiment plants allowed to wilt and recover once were then subjected to a second wilting and recovery cycle. The third experimental approach tested the survival capacity of plants after experiencing a wilting episode where leaf RWC dropped to about 30%.</p> <p> With the first drought simulation treatment, both Yukon and Shandong plants took about 6 days to wilt when watering was stopped. After re-watering and recovery, the Yukon plants subjected to a second drought episode took almost two days longer to wilt while the similarly treated Shandong plants showed no change in the days taken to wilt. This indicates that Yukon plants show improved tolerance to stress after a single exposure to a water deficit. Yukon plants were also able to grow and complete their life cycle following exposure to severe water deficit treatment whereas Shandong plants died. Measurements of solute potential showed that the Yukon plants re-established turgor at a lower solute potential of -2.06 +/- 0.03 MPa following recovery from a second drought treatment suggesting that this plant can undergo osmotic adjustment. No evidence of osmotic adjustment was observed for Shandong Thellungiella.</p> <p> Gas chromatography/mass spectrometry (GC/MS) was used to identify the metabolites associated with Thellungiella leaves recovering from water deficits relative to those from unstressed, well-watered controls. For comparison, metabolite profiles were also prepared from leaves of plants harvested at a Yukon field site during a dry year (2003) and a year of higher than normal rainfall (2005). The data was analyzed to identify treatment/sample-specific patterns using ANOVA to test for significance among quantitative and qualitative changes for individual metabolites. Significant changes were then subjected to hierarchical cluster analysis (HCA) and principal component analysis (PCA). Using ANOVA and HCA, we were able to identify the most likely metabolite candidates contributing to the superior tolerance of Thellungiella, and their linkages between broad spectrums of metabolites. Using PCA we were able to assign clusters to the individual plant treatments for each plant source, and identify the most important components contributing to these clusters. Of the ca 289 components detected, only a small subset of components underwent statistically significant changes in abundance. Most of the drought-stress related changes were attributed to sugars: hexoses and disaccharides. Sugars accumulating in the more drought-tolerant Yukon plants and in a dry field season included fructose, glucose and galactose. Of the sugar alcohols, only myo-inositol showed patterns of interest in view of its enrichment in tissues showing superior tolerance to low water conditions. Similar patterns were also shown by the organic acid, threonic acid.</p> <p> A complementary approach was used to characterize metabolic traits associated with exposure to cold temperatures. In this study, a higher content of proline and citrate distinguished plants exposed to cold temperatures irrespective of whether the plants were in cabinets or in the field. Proline content, however, did not show drought-responsive accumulation under any drought treatment tested. As such, by comparison with the drought-stress data we can identify possible stress-specific signatures among metabolites undergoing changes. The study of stress-responsive traits could help develop a better understanding of plant systems and their response to specific environmental conditions.</p> / Thesis / Master of Science (MSc)

El trabajo realizado en la Tesis Doctoral llega a las siguientes conclusiones:1.- T. halophila muestra una relación en peso raíz/parte aérea mayor que A. thaliana, y esta diferencia se ve incrementada en condiciones de estrés salino, lo que podría suponer una ventaja para afrontarlo.2.- El estrés salino produce en A. thaliana mayores reducciones en la absorción y en las concentraciones internas de K+ que en T. halophila, a la vez que T. halophila presenta menor absorción de Na+ y transporte a la parte aérea que A. thaliana. Ambas circunstancias resultan en una mayor relación K+/Na+ en T. halophila, lo que puede suponer una mayor tolerancia a la salinidad.3.- El gen ThHAK5 codifica para un transportador que media un transporte de K+ de alta afinidad en levaduras similar al observado en las plantas de T. halophila lo que sugiere que este transportador juega un papel fundamental en la absorción de K+ en el rango de la alta afinidad en esta especie vegetal.4.- Aunque AtHAK5 y ThHAK5 presentan una gran homología de secuencia y unas características funcionales similares, la regulación de los genes que los codifican difieren en condiciones salinas. Así, la salinidad reduce en menor medida la inducción de ThHAK5 por ayuno de K+. En consecuencia, la absorción de K+ de alta afinidad está menos afectada por la presencia de NaCl en el medio externo en T. halophila.5.- La mutagénesis al azar permite encontrar aminoácidos importantes para la función de las proteínas y ésta ha permitido identificar dos versiones mutantes del transportador de K+ de alta afinidad AtHAK5 más eficientes, capaces de transportar K+ a concentraciones externas de Na+ muy elevadas (0.1 mM K+ y 800 mM Na+). / The work done in this Thesis provides some interesting conclusions:1.- Thellungiella halophila show a weight ratio root/shoot bigger than Arabidopsis thaliana, and this difference arise under salt stress, what might provide an effective mechanism of salt tolerance to T. halophila.2.- In A. thaliana, salt stress induces a bigger reduction of K+ uptake and tissue concentrations than in T. halophila, and at the same time T. halophila shows a reduced Na+ uptake and Na+ transport to the shoot. Both properties enable a higher ratio K+/Na+ in T. halophila which might be another mechanism of salt tolerance. 3.- The ThHAK5 gene isolated in this Thesis, encode a K+ transporter that mediates high affinity K+ transport in Saccharomyces cerevisiae similar to the observed in intact plants of T. halophila, which suggest a key role of this transporter in the high affinity range of concentrations.4.- Although AtHAK5 and ThHA5 shows high sequence homology and similar functional properties, gene regulation is different under salt stress. Thus, salinity reduces to a lesser extent the K+-starvation ThHAK5 induction. As a consequence, high affinity K+ uptake is less affected by NaCl in T. halophila. 5.- Random mutagenesis allows the identification of important aminoacids for protein function, and with this technique two more efficient mutant versions of AtHAK5 have been isolated. The evolved AtHAK5 mutant versions are able to transport K+ at high Na+ external concentrations (0.1 mM K+ and 800 mM Na+) in yeast.