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>Magister Scientiae - MSc / Food security has always been one of the priority concerns in Africa, and it is mostly threatened by drought stress due to climate change. Drought-induced stress is one of the serious limiting factors of plant production, and it is known to impose oxidative stress as a consequence of excessive reactive oxygen species (ROS) accumulation that lead to lipid peroxidation, which is manifested as increased cell death. Hence, this study investigated the influence of drought stress on two contrasting canola genotypes (Agamax and Garnet), by monitoring their physiological and molecular changes. The results showed that the plant growth and biomass of both genotypes were significantly affected by drought stress as a consequence of excessive ROS accumulation (manifested as H₂O₂ and OH· content). However, under drought stress conditions, the reduction in biomass and shoot length was more pronounced in the Garnet genotype when compared to that of the Agamax genotype. This was further supported by the increase in lipid peroxidation and cell death, which were shown to be significantly higher in the Garnet genotype when compared to the Agamax genotype under drought stress. ROS scavenging ability which prevents oxidative stress and ultimately ROS-induced cellular damage. Hence, given the higher levels of antioxidant activity coupled with the reduction in ROS accumulation that was observed in the Agamax genotype, we suggest that the Agamax genotype might be slightly less susceptible to drought stress, when compared to the Garnet genotype. Furthermore, understanding the proteomic responses of these two contrasting genotypes that showed a marked difference in response to drought stress might help in unlocking complex biological networks of proteins underlying drought stress tolerance. Hence we use two-dimensional (2D) gel electrophoresis coupled with Matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI TOFTOF MS) analysis for this part of the study, in order to detect and analyze those differentially expressed proteins or proteins whose abundance levels were influenced as a consequence of drought stress. To gain additional insight into the leaf proteomes of the two canola genotypes, a protamine sulphate precipitation (PSP) method was used to remove RuBisCo and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. A total of 55 well resolved protein spots were selected for mass spectrometry analysis of which 31 (56%) were positively identified using the selective criteria analysis (SCA). All positively identified proteins were then classified into functional categories including protein folding (3%), photosynthetic (29%), detoxification and protection (20%), and energy related proteins whereas 16% could not be classified into any functional category. Apart from spot 32 (Fe superoxide dismutase) and spot 34 (chloroplast beta-carbonic anhydrase), no further significant difference in protein expression/abundance was observed for all the identified proteins for both genotypes in response to drought stress. Both proteins (spots 32 and 34) have been shown to contain antioxidant activity properties which suggest that they might play a crucial role in improving drought stress tolerance in canola plants.

In semi-arid environments, dry-land farming often exposes crops to drought stress. Although some plant species are well adapted to drought, most crops are not. Drought can reduce plant populations and limit growth and development in ways that have serious yield consequences. Planting at the beginning of the wet season, when rainfalls are often sporadic and unreliable, can expose young maize seedlings to severe drought. Through the use of plant growth regulators (PGR), maize seedlings can perhaps be altered to elicit responses that mimic drought adaptation mechanisms. A series of studies conducted in the laboratory and greenhouse looked at the response of maize seedlings (two hybrids that differed in their reported drought sensitivity) to severe drought and to PGR applications with or without drought. Results showed that drought stress altered plant morphology and key physiological parameters. Applications of three triazoles (paclobutrazol, uniconazole and tetraconazole) altered morphology and physiology in ways that might impart drought resistance. Paclobutrazol and uniconazole increased root:shoot ratio in laboratory studies and in the greenhouse. When compared to non-triazole-treated controls, uniconazole and paclobutrazol treatments caused water conservation in earlier stages of drought stress, and therefore afforded increased transpiration (and presumably less stress) at later stages. Uniconazole and tetraconazole increased photosynthesis of well-watered plants. Proline content was increased to a greater degree by these same two triazoles under drought stress conditions. It is hoped that knowledge obtained from these studies can be extended to drought-prone areas where maize dry-land farming is practiced. / Master of Science

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Doctor of Philosophy / Department of Agronomy / Allan K. Fritz / Heat and drought stress are two of the most significant abiotic stresses limiting wheat production in the Great Plains and worldwide. Introgression of novel tolerance genes from wild relatives is a strategy which presents promise. This study examined both heat and drought tolerance from the tetraploid species Aegilops geniculata (U[superscript g]U[superscript g]M[superscript g]M[superscript g]). Additional screening for heat tolerance was conducted with the US genome species Aegilops peregrina (Hack) and Aegilops kotschyi (Boiss). A comprehensive screening system for drought tolerance was also constructed to evaluate wheat and its wild relatives. Previous reports suggested that Ae. geniculata accession TA2899 was moderately tolerant to heat stress. It had also previously been used to develop a full set of wheat-Ae. geniculata chromosome addition lines in a Chinese Spring background. To identify the chromosome(s) carrying the heat tolerance, all addition lines, as well as wheat check genotypes, were screened for post-anthesis heat tolerance in two growth chamber experiments. No chromosome addition lines were significantly different (p<0.05) from Chinese Spring, and none were found to have superior performance to the positive check cultivars. Forty-five accessions of Ae. peregrina and its close relative, Ae. kotschyi were screened in a post-anthesis heat experiment. A follow-up experiment compared the genotypes in a split-plot temperature treatment with heat and optimal growth chambers. Many accessions were similar to the control genotypes for grain fill duration, and some exceeded the wheat controls for relative chlorophyll index values on Day 12 and Day 16. TA1889 and TA1904, both Ae. peregrina accessions originating from Israel, had a higher grain fill duration across experiments than the best wheat control, and warrant further investigation. Previous reports suggested drought tolerance in Ae. geniculata. After preliminary screenings, six genotypes were selected for advanced screening and compared with three wheat cultivars. The advanced greenhouse screening system was conducted in 152cm tall PVC growth tubes. The experiment measured multiple plant responses, and had a datalogging system automatically collecting water content and matric potential of the growth media. Multiple accessions warranted further investigation, and showed potentially different modes of drought tolerance, with varying levels of stomatal resistance, biomass, and osmotic adjustment.

Silicon is not considered an essential nutrient, but it is typically abundant in soils and can be taken up in large amounts by plants. Silicon is known to have beneficial effects when added to rice and several other plants. These effects include disease and insect resistance, structural fortification, and regulation of the uptake of other ions. In this study, the effect of silicic acid fertilization on the growth and drought tolerance of four crop plants (corn, wheat, soybean, and rice) was analyzed. Plants were studied using three cultivation techniques: 1) hydroponic solution and subjected to salt stress, 2) low-silicon soil-less medium (peat) and subjected to gradual drought stress, and 3) low-silicon soil-less medium (peat) and subjected to acute drought stress. Silicon was added both as reagent-grade Na2SiO3 and as a siliceous liming agent (PlantTuff). Both forms of Si generally improved drought and salt stress tolerance, but the effects were inconsistent. Silicon increased corn dry mass by up to 18% and the effect was statistically significant (p<0.05) in two out of three techniques. Silicon increased water use efficiency in corn by up to 36% and the effect was statistically significant (p<0.05) in one out of two techniques. In the acute drought stress technique, silicon increased wheat dry mass by 17% and the effect was statistically significant (p<0.05). Silicon increased soybean and rice dry mass by 20 to 30%, but the effect was not statistically significant. Silicon in oldest corn leaves increased from 0.4% to 3% as Si increased from less than 0.01 to 0.8 mM in the hydroponic solution. There was a statistically significant effect of silicon supply on the concentration of some other nutrients, but the effect was often not great enough to be considered biologically important. Rice accumulated the greatest concentration of foliar silicon, corn and wheat were intermediate, and soybean accumulated the least. Collectively, these results indicate an effect of silicon in drought and salinity stress tolerance, but additional studies on the rate and onset of drought are needed to determine interacting factors and better understand the inconsistent results.

Epicuticular waxes form a layer on the outer surface of all land plants and play a fundamental role in their interaction with the environment. Development of a rapid and accurate method for the characterization of these waxes could allow the use of wax composition as a novel phenotype during plant breeding and selection. In this study, the suitability of Direct Analysis in Real Time Mass Spectrometry (DART-MS) for the characterization of epicuticular waxes was investigated. This method provides a “fingerprint” of the relative abundance of all constituents in the analyte based on mass, is suitable for very high throughput, requires minimal sample preparation, and is able to provide for the characterization of even complex biological mixtures. Herein, the suitability of DART-MS for analysis of epicuticular wax was investigated through analysis of samples of extracted wax collected from the flag leaves of a 279 line association mapping population grown across four environments with two replicates in both irrigated and drought treatments. Additionally, for a subset of samples, wax was collected from glumes in order to test for differences in wax composition between tissue types. In all, a total of 3,454 wax extracts were analyzed with three technical replicates. The above analysis generated a total of 13,164 mass spectra (“fingerprints”), made at an average rate of 30 seconds each. Multivariate analyses including random forest, principal component analysis, and linear discriminant analysis, were used to identify the presence of differences between the spectra of wax from different tissues (leaves/glumes) and treatment types (irrigated/drought). The peaks best serving as predictors of sample class for each comparison were examined, and tentative identifications were made through comparison of the associated mass with literature and publicly available databases. As a separate test of concept, the ability to distinguish between the epicuticular wax compositions of individual varieties was determined through analysis of a group of closely related lines developed at CIMMYT which differed in agronomic performance.

Orientadora: Sonia Marli Zingaretti Di Mauro / Banca: Maria Inês Tiraboschi Ferro / Banca: Renê de Oliveira Beleboni / Resumo: A cultura da cana-de-açúcar é de grande importância econômica nas regiões tropicais e subtropicais, especialmente para alguns países da América, como o Brasil, que é atualmente o maior produtor mundial. Estresses abióticos, como a seca, podem reduzir os rendimentos das lavouras. Sendo assim, a identificação e a compreensão dos mecanismos de tolerância à seca são fundamentais no desenvolvimento de novas cultivares comerciais mais tolerantes ao déficit hídrico. O objetivo deste trabalho foi identificar, através da técnica de macroarranjos de cDNA, o perfil de expressão de genes pertencentes a diferentes vias metabólicas em folhas de duas cultivares de cana-de-açúcar (Saccharum spp), uma tolerante ao estresse por déficit hídrico (SP83-2847) e outra sensível (SP90-1638) submetidas a dois períodos de restrição no fornecimento de água, ocasionando um estresse por déficit hídrico leve (T1) e severo (T2). Por meio das análises dos resultados foi possível identificar, na cultivar tolerante, a indução de ESTs (etiquetas de seqüências expressas) com similaridade a genes de enzimas de síntese de osmoprotetores, tais como prolina, hidroxiprolina e GABA (ácido g-aminobutírico); de hormônios vegetais como o ácido abscísico (ABA) e o ácido jasmônico (JA) e repressão de ESTs similares aos genes das enzimas de biossíntese de amido, de glicina betaína e de algumas enzimas do sistema de defesa antioxidante. Ao passo que, na cultivar sensível foram induzidas ESTs similares aos genes de enzimas de síntese dos osmoprotetores trealose e glicina betaína; do sistema de defesa antioxidante e reprimidas ESTs com similaridade a genes das enzimas de síntese de prolina, hidroxiprolina e GABA e envolvidas na biossíntese de ABA e de jasmonatos. Em ambas as cultivares, ESTs similares a genes de diferentes enzimas fotossintéticas foram reprimidas. / Abstract: Sugarcane crop is of large economic importance in the tropical and subtropical regions, especially in some countries of Central and South America as Brazil, which is actually the major worldwide producer. Abiotic stress, such as drought, can reduce yield of the farmings. Thus, identification and understanding of the drought tolerance mechanisms is crucial to the development of new commercials cultivars more tolerant to water deficit. The aim of this study was to identify, using cDNA macroarrays technique, expression profile of genes involved in distinct metabolic pathways in leaves of two sugarcane (Saccharum spp) cultivars, one water stress tolerant (SP83-2847) and another water stress sensitive (SP90-1638) which were submitted to periods of withhold watering occasioning a mild (T1) and severe (T2) water deficit stress. Through the analysis of the results, it was identified in the tolerant cultivar up-regulated ESTs similar to genes of enzymes involved in the synthesis of osmoprotectants, such as proline, hydroxyproline, GABA (g-amino butyric acid), of synthesis of plant hormones as abscisic acid (ABA) and jasmonic acid (JA); and down-regulated ESTs similar to genes of enzymes of the biosynthesis of starch, glycine betaine and of some enzymes involved antioxidant defense system. In the other hand, ESTs similar to genes of enzymes involved in the biosynthesis of the osmoprotectants as trehalose and glycine betaine and enzymes from the antioxidant defense system were induced as well as were down-regulated ESTs similar to genes of enzymes of synthesis of proline, hydroxyproline and GABA and involved in biosynthesis of ABA and jasmonates, for the sensitive cultivar. In both cultivars, ESTs with similarity to genes of different photosynthetic enzymes were repressed. / Mestre

Abstract The aim was to study the effects of drought and indirect drought-related stresses on anthocyanin content in bilberry (Vaccinium myrtillus L.). Anthocyanin content was also studied in relation to developmental stage (juvenile vs. mature leaves, previous vs. current-year stems). It was hypothesised that drought-stressed plants accumulate anthocyanins, but their direct role in osmotic regulation was questioned. Direct drought was created by preventing water supply. Freezing-induced dehydration was accomplished by removal of snow. Effects of summertime chilling (+2 °C during active growth, +5/0 °C during frost hardening) on water status were tested. The trace metal Nickel (Ni) was applied to the soil, as Ni may interfere with plant water relations at the rhizospheric level. Salt (Na+) was added to the soil to observe salt-induced disturbance in osmotic balance and ion homeostasis. Tissue water content (TWC) decreased and anthocyanin level increased under direct drought, especially in the mature leaves. The freezing-stressed plants contained the same TWC and anthocyanin levels in mid-winter compared to plants that overwintered below snow. The freezing-stressed plants had lower TWC and a similar anthocyanin level in early spring, and lower TWC and anthocyanin level in late spring than plants that overwintered below snow. In the summer and autumn following snow removal, current-year stems of freezing-stressed plants had the same TWC, but higher levels of anthocyanins than current-year stems of plants that overwintered below snow. New growth was thus affected by the freezing stress experienced by previous-year stems. Chilling had no effect on anthocyanins. Although TWC decreased and anthocyanin level increased from active growth to the beginning of frost hardening, no increase was observed during frost hardening. Ni did not cause drought stress in the aboveground shoots, but anthocyanin level decreased in the aboveground shoots along with Ni accumulation in the belowground stems. Na+ increased TWC in the belowground stems, but decreased TWC and anthocyanin level in the aboveground stems. It is proposed that anthocyanins do not have direct role in osmotic regulation, or in the development of freezing tolerance. It is suggested that the increase in anthocyanin level under direct drought stress is mainly due to the photoprotection of chlorophylls by anthocyanins. This is supported by two facts: (1) At increased anthocyanin level in the juvenile leaves, chlorophyll a was stabilized despite continuing drought stress, and (2) after the initial peak in the mature leaves, the accumulation of anthocyanins ceased although the drought became more severe. As chlorophyll a decreased in the mature leaves due to senescence, there was less demand for such high levels of anthocyanins.

>Magister Scientiae - MSc / Sugargraze is a moderately drought tolerant sweet sorghum hybrid which is ideal for grazing, winter stand over and pit silage. A major advantage that Sugargraze has over other forages is its very high sugar content which improves feed quality thus increasing palatability and results in significantly reduced feed wastage. This study explored the influence of hyperosmotic stress on plant development, ROS accumulation, antioxidant capacity and the extent of cell death. Heat shock protein (Hsp70) expression immunoblotting assays were used to demonstrate whether the various treatment conditions induced stress within natural physiological parameters for the experimental material. This was coupled with the separation, visualization and identification of abundant proteins in Sugargraze leaves in response to hyperosmotic stress using two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS). The results showed that hyperosmotic stress significantly influences plant development by reducing plant biomass and increasing the levels of ROS accumulation, proline content and subsequently reducing total chlorophyll content. An over accumulation of ROS in the form of hydrogen peroxide and lipid peroxidation was observed in the stressed plants which was supported by the extent of cell death. Although an increase in antioxidant enzyme activity (in the form of total enzymatic activity or individual isoform activity) in response to hyperosmotic stress was observed, this increase was not sufficient to counter the deleterious effects caused by the stress conditions hence the decrease in plant biomass and increase in cell death. Western blotting analysis of Sugargraze leaf tissues using Hsp70 antibodies showed that hyperosmotic stress induced Hsp70 expression to levels significantly higher than observed for the control plants. A total of thirteen CBB stained spots were selected for mass spectrometric identification, owing to their good resolution and abundance levels, and of these, nine were positively identified. Identified proteins were divided into functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of the proteins of interest identified will be subjected to further investigation via bioinformatic and molecular biology approaches.