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 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)

<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)

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-27T12:03:27Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_SilviaPereira..pdf: 1700069 bytes, checksum: de95e5af421e528465694aadb5cdee58 (MD5) / Made available in DSpace on 2017-07-27T12:03:27Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_SilviaPereira..pdf: 1700069 bytes, checksum: de95e5af421e528465694aadb5cdee58 (MD5) Previous issue date: 2016-02-23 / CAPES / Adaptações e mecanismos de tolerância à seca são fatores fundamentais para a sobrevivência das espécies vegetais de clima árido ou semiárido. É devido a adaptações como área foliar, profundidade de raízes, manutenção do status hídrico, espessura da cutícula e densidade estomática que tais espécies obtêm sucesso neste ambiente. Contudo, diante das mudanças climáticas há a necessidade de avaliação da eficiência desses mecanismos diante de uma condição ainda mais limitante. Dessa forma, nosso objetivo foi analisar os principais mecanismos fisiológicos de tolerância a seca de Cynophalla flexuosa (L.) J.Presl., uma espécie lenhosa e sempre verde de uma floresta tropical sazonalmente seca, avaliando de que forma a cera epicuticular (CE) das folhas dessa espécie são eficientes diante das condições do seminário em diferentes épocas do ano. O trabalho foi realizado em três períodos (julho de 2014, dezembro de 2014 e fevereiro de 2015), ao longo de um dia em uma área de Caatinga, utilizando folhas intactas e com parte da CE removida mecanicamente. Foi calculado o balanço hídrico do solo e mensurado o potencial hídrico foliar, trocas gasosas, fluorescência da clorofila a, bioquímica e anatomia foliar. Foi observado que C. flexuosa reduz seu potencial hídrico foliar nos horários mais quentes, sendo uma espécie anisohídrica, capaz de manter sua condutância estomática mesmo diante de um balanço hídrico negativo do solo em todos os períodos de avaliação. A concentração da CE foi maior no mês de fevereiro e seu principal componente foram as cadeias de n-alcanos, compostos muito eficientes em manter a impermeabilidade da cutícula e proteger as folhas. O desempenho fotossintético não mostrou alterações ao longo do dia em folhas que tiveram a CE removida, além da manutenção do metabolismo bioquímico, com poucas variações nos diferentes períodos avaliados. Diante disso, C. flexuosa possui eficientes mecanismos de adaptação à seca, podendo suportar ambientes ainda mais limitantes, como o previsto para as próximas décadas devido às mudanças climáticas. / Adaptations and drought tolerance mechanisms are key factors for the survival of plant species in arid or semi-arid climate. It is due to adaptations such as leaf area, depth of roots, maintenance of water status, cuticle thickness and stomatal density these species succeed in this environment. However, given the climate change there is a need of efficiency evaluation of these mechanisms before a further limiting condition. Therefore, our objective was to analyze the main physiological mechanisms of drought tolerance of Cynophalla flexuosa (L.) J.Presl., a woody and evergreen specie in a seasonally dry tropical forest, assessing how the leaves epicuticular wax (EW) of this species are efficient before the semiarid conditions in different seasons. The study was conducted in three periods (July 2014, December 2014 and February 2015), throughout the one day in an area of Caatinga, using intact leaves and the EW removed mechanically. We calculated the the hydric balance of the soil and measured leaf water potential, gas exchange, chlorophyll fluorescence, biochemistry and leaf anatomy. It was observed that C. flexuosa reduces its leaf water potential at the hottest times, one anisohydric species, able to maintain its stomatal conductance in the face of a negative soil water balance in all evaluation periods, which reduced its xylem potential. The concentration of EW was higher in the month of February and its main component was n-alkane chains, very efficient compounds in maintaining a waterproof cuticle and protect the leaves. The photosynthetic performance showed no change throughout the day in leaves that had the EW removed, as well as maintenance the biochemical metabolism, with few variations in different periods. Therefore, C. flexuosa has efficient mechanisms of adaptation to drought and can withstand even more limiting environments, as predicted in the coming decades due to climate change.