Water scarcity induced by drought, temperature, and salinity has plagued agricultural sustainability in recent years with unprecedented revenue losses, raising concerns for worldwide food security. Recent studies have revealed unique botanical response mechanisms to combat water related stress, namely the expression of proteins known as the dehydrins. Dehydrin proteins have been shown to serve various intracellular protective functions. The gene for a SK5 type dehydrin from the arctic plant Cerastium arcticum (CaDHN) was introduced into tobacco plants and water deficit tolerance was evaluated. Plants overexpressing CaDHN displayed improved tolerance to salt stress, but no improvement was observed under drought stress. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Orientador: Regiane Cristina Oliveira de Freitas Bueno / Banca: Thiago Assis Rodrigues Nogueira / Banca: Marcelo de Almeida Silva / Banca: Simone de Souza Prado / Resumo: As mudanças climáticas previstas para os próximos anos indicam incremento na temperatura em 1,4°C a 5,8°C em decorrência, principalmente, do aumento da concentração dos gases de efeito estufa, além de alterações na umidade relativa e taxas de CO2 da atmosfera terrestre. Essa mudança influenciará diretamente nos regimes de chuvas, promovendo veranicos e/ou excessos em determinadas regiões. Dessa forma, o objetivo da dissertação foi avaliar os efeitos do estresse hídrico em plantas de milho Bt sob situações de estresse frente ao ataque de S. frugiperda. O delineamento experimental utilizado foi em esquema fatorial triplo em blocos casualizados com quatro lâminas de irrigação (estimadas a partir da tensão do solo): L1- 50% da capacidade de campo (CC) considerada estresse severo, L2- 75% CC estresse moderado, L3- 100% CC controle e L4-125% CC excesso de água; três eventos de milho: PowerCore® (Cry1A.105 Cry2Ab2 Cry1F PAT CP4-ESPS), Herculex ® (Cry1F PAT) e Optimum® Intrasect® (Cry1Ab Cry1F PAT CP4-EPSPS) e três infestações (sem infestação, população S. frugiperda suscetível ao milho Bt e população S. frugiperda resistente ao Bt). Quando as plantas atingiram o estádio fenológico de V8 (fase considerada mais suscetível ao ataque de S. frugiperda e de maior atuação dos mecanismos de ativação de sistema enzimático) foram infestadas artificialmente, por 24h e após este período folhas do terço médio da planta foram coletas para análise enzimática. Os híbridos que ficaram sob estresses... / Abstract: The climate changes foreseen for the next years show an increase in temperature of 1.4°C to 5.8°C, mainly due to the increase in the concentration of greenhouse gases, besides relative humidity alterations and CO2 rates changes of the Earth's atmosphere. This change will directly influence rainfall regimes, promoting summer and / or excess in certain regions. Thus, the objective of the dissertation was to evaluate the effects of water stress in Bt maize plants under stress situations facing the attack of S. frugiperda. The experimental design used was a randomized triple block design with four irrigation slides (estimated from soil tension): L1- 50% of field capacity (CC) severe stress, L2-75% CC moderate stress, L3 -100% CC control and L4-125% CC water excess; (Cry1A.10 Cry1A PAT C4-EPSPS), Herculex® (Cry1F PAT) and Optimum® Intrasect® (Cry1Ab Cry1F PAT CP4-EPSPS) and three infestations (non-infestation, susceptible S. frugiperda population to Bt maize and Bt resistant S. frugiperda population). When plants reached the phenological stage of V8 (phase considered more susceptible to S. frugiperda attack and of greater performance of the mechanisms of activation of enzymatic system), they were infested artificially for 24 hours. After this period, leaves of the middle third of the plant were collected for enzymatic analysis. The development of the hybrids that were under water stresse was slower than other hybrid studied. On the other hand, the analyzes and determinations of e ... / Mestre

Orientador: Sonia Marli Zingaretti / Banca: Tiago Antunes Paz / Banca: Janete Apparecida Desiderio / Resumo: A cana-de-açúcar é acometida por uma doença conhecida por "escaldadura das folhas" causada pela bactéria colonizadora do xilema Xanthomonas albilineans, considerada uma das principais doenças que atingem a cultura da canade-açúcar. A sintomatologia na fase crônica se caracteriza, principalmente, pelo aparecimento de uma faixa branca ao lado da nervura central da folha, a qual evolui para clorose total causando a morte da planta. Uma vez que o patógeno pode ser transmitido de várias maneiras, o seu controle demanda altos custos. Desta maneira, o desenvolvimento de cultivares tolerantes é uma boa opção para o controle efetivo da doença. A tolerância e sensibilidade das plantas aos fatores bióticos está relacionada com a expressão de genes, e dentre estes, os miRNAs (incluindo o miR397 e o miR319) têm sido relatados como importantes reguladores em vários mecanismos de resposta das plantas. O objetivo deste trabalho foi analisar a expressão de dois miRNAs (miR319 e miR397) em duas cultivares de cana-de-açúcar (RB86-7515 - tolerante e SP78-4467 - suscetível), infectadas por uma linhagem de X. albilineans (IACXa11), considerada a mais virulenta do Brasil. Para isto, as plantas foram cultivadas em vasos, inoculadas com X. albilineans e mantidas em casa de vegetação. Amostras de folhas e colmos foram coletadas em cinco períodos (24, 72, 144, 360 e 720 h) e a expressão dos miRNAs foi analisada pela técnica de Stem-loop RT-qPCR. Os miR397 e miR319 apresentaram-se diferencialmente expre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sugarcane is affected by a disease known as "leaf scald" caused by the bacterium Xanthomonas albilineans, which colonizes the xylem. This disease is one of the most important for sugarcane culture. The chronic phase is mainly characterized by the white band emergence along the central leaf vein, which causes total chlorosis of the leaf and plant death. Since the pathogen can be transmitted in many ways, his control demands high costs, and the development of tolerant cultivars is a good option for disease control. The plant tolerance and sensitivity to biotic factors is related to gene expression, and among these, the miRNAs (including miR397 and miR319), have been reported as important regulators in various plant response mechanisms. The aim of this work was to analyze the expression of two miRNAs (miR319 and miR397) in two sugarcane cultivars (RB86-7515 - tolerant, and SP78- 4467 - susceptible), infected by a strain of X. albilineans (IACXa11), the most virulent in Brazil. The plants were grown in vases, inoculated with X. albilineans and kept in a greenhouse. Samples of leaves and stems were collected in five periods (24, 72, 144, 360, and 720 h), and the miRNA expression was analyzed by Stem-loop RT-qPCR. The miR397 and miR319 expression were different between cultivars and tissues. In the susceptible cultivar (SP78-4467), during the first infection periods (24, 72 and 144 h), there was a late defense response when compared to the tolerant cultivar (RB86-7515). The miR319 presented the same expression profile in leaves and stems of the cultivar RB86-7515 (tolerant), suggesting that the pathogen recognition and defense mechanisms activation were modulated in both tissues. In general, miRNAs analyzes demonstrated that miR397 expression is lower when compared to miR319. The sa... (Complete abstract click electronic access below) / Mestre

Bibliography: leaves 187-216.

Bibliography: leaves 98-123. This thesis investigates the symptoms of leaf senescence in response to plant shoot water stress and demonstrates the effect of a non-hydraulic root signal in the senescence response of mature leaves of sunflower. The alleviation of the leaf soluble protein loss rate by excision of the root system in drying soil indicates that this signal originates in roots in dry soil and acts to promote protein loss.

The genome of living organisms is constantly subjected to the environmental influences that result in different negative, negligible or positive impacts. The ability to maintain the genome integrity and simultaneously provide its flexibility is the main determinant for the evolutionary success of any species. One of the important aspects of genome maintenance is the precise regulation of the DNA repair machinery. Results reported here indicate the existence of a tight, age-dependent regulation of homologous recombination, one of the two main DNA double-strand break repair pathways. We show that recombination is influenced by conditions such as the change of temperature (cold or warm), day length, water availability (drought or overwatering stress) and salinity. These stresses not only influence the genome stability of stress-subjected generations but also change the recombination in subsequent generations. This indicates the possible involvement of homologous recombination in plant evolution and development of plant stress tolerance. / xiv, 121 leaves ; 29 cm.

Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Of these, proline is the most extensively studied. Conclusive demonstration that this imino acid acts as a compatible solute which mediates osmotic adjustment has yet to be achieved, although a causal relationship between increased proline synthesis and plant tolerance of hyperosmotic stresses has previously been demonstrated. It is proposed that in many plants, the metabolic implications of the regulated increase in proline synthesis and/or a decline in proline degradation during stress may play a more important role in acclimation to adverse conditions than the simple accumulation of the end-product of these adjustments. In particular, the stress-induced increase in the transfer of reducing equivalents into proline by Δ¹-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) may be a protective mechanism whereby many species ameliorate shifts in cellular redox potential which accompany all biotic and abiotic stresses which cause proline accumulation, including those that do not cause cellular dehydration. The presence of several putative stress-regulated promoter elements in the AtP5CS1, AtP5CS2 and AtP5CR genes of Arabidopsis thaliana strongly implicates an adaptive role for stress-induced increases in proline synthesis in this species. Sequence homologies of several regions within the 5' untranslated regions of these genes to promoter elements which have been shown to participate in redox control of gene expression, the actions of phytochrome and hormones, and tissue-specific regulation of gene expression are also identified. These provide useful indicators both of the mechanisms by which proline synthesis is regulated and how these may relate to its importance in maintaining metabolic homeostasis. In an attempt to resolve the functionality of proline accumulation under stress, chimeric antisense genes comprising 1050 bp and 999 bp fragments of Arabidopsis cDNAs encoding AtP5CS1 and AtP5CR respectively were inserted in the reverse orientation between the CaMV 35S promoter and the GUS gene (encodes β-glucuronidase) in the plant transformation vector pBI121. These constructs were introduced separately into Arabidopsis by cocultivation with Agrobacterium tumefaciens strains carrying the pBI-P5CS1 (AS) and pBI-P5CR(AS) plasmids. Transgenic plants, which were selected on the basis of kanamycin resistance, regenerated at a low frequency in the presence of 1 mM proline. Transformation of 13 pBI-P5CS1(AS) and 7 pBI-P5CR(AS) lines was confirmed by PCR-mediated amplification of gene fragments within the introduced T-DNA. Segregation ratios for kanamycin resistance indicated that most of the lines have multiple T-DNA insertions. Transformants were characterised with respect to their growth rates and free proline content. In at least two pBI-P5CS1(AS) transformants and two pBI-P5CR(AS) transformants, a reduction in root growth rates in the presence of inhibitory concentrations of NaCI correlated with reduced β-glucuronidase activity relative to transgenic lines that were no more sensitive to NaCI than were controls. A reduction in root growth rate both in the absence and presence of hyperosmotic stress was noted in two pBI-P5CS1(AS) transformants, designated A5 and B12. In 14 day-old plants of the T₂ generation of both A5 and B12, free proline levels were significantly lower than in wild-type plants both in the absence of stress and following 24 h incubation in either 250 mM NaCI or 550 mM sorbitol or at 5 °C. In both lines, reduced growth rates in the absence of osmotic stress could be restored by exogenous proline, but not by exogenous glutamate. When used at isosmotic concentrations, sorbitol caused a larger reduction in free proline levels in both A5 and B12 than did NaCI. This observation may relate to an ABM-mediated post-transcriptional effect on AtP5CS1 gene expression which affects NaCI-, but not sorbitol-mediated proline accumulation in Arabidopsis. Post-transcriptional regulation of the expression of the genes involved in proline biosynthesis may account, at least partly, for the absence of dramatic phenotypic effects in any of the pBI-P5CS1(AS) or pBI-P5CR(AS) lines. Under the premise that regulation of shifts in proline metabolism regulate cellular redox potential under conditions of stress may be mirrored by the involvement of proline metabolism in modulating metabolism during normal growth and development, the effects of exogenous proline on Arabidopsis seed germination, seedling growth and in vitro shoot organogenesis were investigated. A dose-dependent inhibition of radicle emergence by millimolar concentrations of proline could be overcome by the artificial oxidants methylene blue and phenazine ethosulphate. Assays of the rate-limiting dehydrogenases of the oxidative pentose phosphate pathway (OPPP), as well as changes in the contributions of ¹⁴C₁ - and ¹⁴C₆ -labelled glucose to respired CO₂ during germination, are consistent with activation of the OPPP during Arabidopsis seed germination. An approximately four-fold increase in free proline, which peaked at the time of radical emergence, was not parallelled by changes in other amino acids and could not be ascribed to degradation of seed storage proteins. Delayed radical emergence in T₂ generation seeds of the pBI-P5CS1(AS) lines A5 and B12 correlated with an approximately 35% reduction in the maximal concentration of proline accumulated during germination. Millimolar concentrations of exogenous proline had a dose-dependent inhibitory effect on Arabidopsis seedling growth both in the light and in darkness. This reduction in growth arises at least in part from a decline in cell elongation. Accordingly, exogenous proline increased total extractable peroxidase activity in Arabidopsis seedlings through the selective induction of peroxidase isoforms. Histochemical analysis of the hypocotyls of plants grown in the presence of exogenous proline suggested that proline increased the levels of lignin and/or the phenolic precursors thereof. A dose-dependent decrease in extractable chlorophyll and damage to chloroplastic and mitochondrial ultrastructure was observed in 21 day-old Arabidopsis seedlings grown in the presence of millimolar concentrations of exogenous proline. In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mM proline, and to a lesser extent by 5 mM proline, but inhibited by inclusion of 10 mM proline in the hormonallysupplemented regeneration media. The ability of low concentrations of proline analogues (azetidine-2-carboxylate and thioproline) to overcome the stimulatory effect of 1 mM proline, and a slight increase in the stimulative effects of 1 mM proline by D-proline, are consistent with an important role for the interconversions of proline and its precursors in regulating cell division and differentiation. Together, these data strongly support an important role for the interconversions of proline and its precursors in the regulation of intermediary metabolism under both normal and stressful conditions. These findings draw into question the widely accepted, although poorly investigated, hypothesis that proline is an inert compatible solute that can be accumulated to high levels with minimal effects on cellular metabolism. The novel proposal that stress-induced changes in proline metabolism exert a regulatory effect though an influence on the level of reduction of the cellular NADP pool is discussed in relation to recent evidence that a signal related to proline synthesis and/or degradation selectively increases the expression of stress-induced plant genes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

y-Aminonbutyric acid (GABA) is a ubiquitous non-protein amino acid found ill many plants and organisms. GABA accumulation in plants has previously been reported as result of stresses such as water deprivation, high salinity and temperature extremes. It is thought that GABA accumulates as a compatible solute in the cytoplasm where it becomes a major constituent of the free amino acid pool. GABA is synthesised from the decarboxylation of glutamate by glutamate decarboxylase (GDC). In some plants, GDC is activated by the lowering of the cytoplasmic pH and the presence of calmodulin and Ca²+ A calmodulin-induced activation of may be due to the physiological factors and environmental stimuli acting in concert leading to the synthesis and accumulation of GABA. The GABA content of Arabidopsis thaliana var. Columbia (L) Heynh leaves was found to increase by over 130% due to water deprivation. NaCl concentrations of up to 100 mM seemed to cause GABA accumulation due to a decrease in osmotic potential. Concentrations of NaCl above 100 mM probably caused GABA accumulation due to combined hyperosmosis and salt toxicity effects. The high levels of GABA in the leaves were maintained throughout a 24 h stress-application period, consistent with its role as compatible solute. The accumulation of GABA followed by its decline in the dark could be attributed to its rapid metabolism because of an active GABA shunt. This is in contrast to the absence of major variations in the amount of GABA in the light confirming its decreased role as a channel for the glutamate carbon and nitrogen under such conditions. A substantial increase in the GABA content was followed by a dramatic decrease in the last 12 h of incubation. This profile of GABA could support its proposed role as a temporary sink for nitrogen and carbon from glutamate during environmental stress. Glutamate decarboxylase appears to be encoded by a single gene in the genome of Arabidopsis. Sequence analysis reveals that the protein possesses what could be a carboxy-terminal, calmodulin- binding domain, which is consistent with other glutamate decarboxylases. The 30-amino acid peptide contains a TrpLysLys motif found in some calmodulin targets. The secondary structure predictions of this peptide suggest a potential to form an a- helix which is also consistent with proteins known calmodulin- binding domains. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

Biochemical and ultrastructural changes associated with chilling injury (CI) in soybean seeds imbibed at 5°C and 25°C were investigated. Soybean seed germination appeared to be affected by chilling temperatures and initial seed moisture content. Seeds with higher moisture contents exhibited 85% germination, while low moisture content seeds had a 32% germination. Leakage rates were greater in chilled seeds, indicating that membrane integrity in the tissues was impaired at chilling. The low rates of potassium ion leakage between 6 and 24 hours of imbibition compared to the high peroxide levels observed during this period led to the suggestion that lipid peroxidation was a better marker of CI than leakage. Transient changes in lipid hydroperoxide levels were observed in chilled and non-chilled seeds and axes. However, in axes, the increase in lipid hydroperoxides after 12 hours of imbibition at chilling temperatures was associated with an 18% decline in linoleic acid levels of total lipid fraction. Similarly, a 10% decline was observed in the polar lipid fraction. These results suggest that the capacity of seeds to control lipid peroxidation may be an important component in CI and that a consequence of peroxidation is likely to be a loss of fatty acid unsaturation. Sugar levels were not affected by chilling and non-chilling temperatures and no relationship could be established with CI. Antioxidant defense enzymes (catalase and superoxide dismutase) were expressed at chilling and non-chilling temperatures and increases were observed after 24 hours of imbibition which showed an apparent correlation with increases to lipid hydroperoxide levels. Enzyme levels decreased after 48 hours of imbibition at a time which coincided with the decline observed in the peroxide levels. Overall, no marked differences were observed in chilled and non-chilled cells at the ultrastructural level, except that vacuolar reserve mobilization was markedly impeded. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

In Arabidopsis thaliana (L.) Heyhn, the size of the pool of free proline increases up to 27-fold in response to osmotic stress. The magnitude of this accumulation is dependent upon the rate of imposition of the stress. Numerous reports have suggested a role for proline accumulation as a general adaptation to environmental stress. However, controversy surrounds the beneficial effect of proline accumulation in plants under adverse environmental conditions. Stress-induced proline accumulation in plants occurs mainly by de novo synthesis from glutamate. The final and only committed step of proline biosynthesis in plants is catalysed by Δ¹-pyrroline-5-carboxylate reductase (P5CR). The sequence of an incomplete 999 bp cDNA encoding P5CR from A. thaliana was determined. This enabled a preliminary molecular study of the structure and function of both the gene and the corresponding enzyme. The 999 bp cDNA insert in the clone Y AP057 was sequenced on the sense and antisense strands following subcloning of four sub-fragments in appropriate orientations. Comparison with known plant P5CR sequences revealed that Y AP057 does not encode the first 23 N-terminal amino acids of P5CR from Arabidopsis. However, it does encode the remaining 253 amino acid residues of Arabidopsis P5CR The cDNA Y AP057 is complete on the 3' end as indicated by the presence of a poly(A) tail. The nucleotide sequence determined shows complete homology to the corresponding exons of the genomic copy of a bona fide gene encoding P5CR in A. thaliana (Verbruggen et al, 1993). The only difference observed between the sequence of Y AP057 and that of a cDNA sequenced by these workers is that polyadenylation was initiated seven nucleotides earlier in Y AP057 than in the sequence of the published cDNA. Genomic Southern analysis suggests the presence of only a single copy of the gene encoding P5CR in Arabidopsis. Restriction mapping and sequencing the ends of another incomplete Arabidopsis P5CR cDNA clone FAFJ25 (664 bp) indicated that the regions sequenced were completely homologous to the corresponding portions of Y AP057. Analysis of codon usage in the Arabidopsis gene encoding P5CR revealed it to closely resemble the consensus pattern of codon usage in A. thaliana. This suggests that the gene is moderately. expressed. Expression of the gene encoding P5CR in Arabidopsis is not likely to be subject to translational control. Although P5CR from A. thaliana has a fairly high composition of hydrophobic amino acid residues, it does not possess any stretches of hydrophobic amino acids of sufficient length to act as membrane-spanning domains or to anchor the enzyme in a membrane. Neither does it contain an N- terminal leader sequence capable of directing it to either the plastid or mitochondrion. The enzyme therefore appears to be cytosolic. The nucleic acid and deduced amino acid sequences of Arabidopsis P5CR were compared with those from·eleven other organisms for which P5CR sequences are currently available. Except among the three different plants examined, P5CR sequences displayed less identity at the amino acid level than at the nucleotide level. The deduced amino acid sequence of Arabidopsis P5CR exhibits high similarity to the corresponding genes and amino acid sequences of P5CR from soybean and pea. Lower but significant similarity was observed to the amino acid sequences of P5CRs from human, Saccharomyces cerevisiae and the bacteria Escherichia coli, Pseudomonas aeruginosa, Thermus thermophilus, Mycobacterium leprae; Treponema pallidum and Methanobrevibacter smithii. Similarity was also observed to the translational product of a gene from Bacillus subtilis with high homology to the E. coli proC gene. However, construction of a phenogram indicating the relatedness of the various P5CR enzymes suggests that sequence analysis of this enzyme is not a good indicator of evolutionary relatedness of organisms from different biological kingdoms. Multiple alignment of the twelve known P5CR sequences indicated homology between the sequences across their entire lengths. Homology was particularly high in the C-terminal portions of the P5CRs studied. It is speculated that this region may be of importance in binding of the substrate Δ¹-pyrroline-S-carboxylate (P5C). Another region displaying high sequence conservation was found in the central portion of all P5CRs. All P5CRs studied, with the exception of PSCR from T. pallidum contained an N-terminal domain capable of binding a nicotinamide dinucleotide cofactor. Comparison of this region with consensus sequences for NADH and NADPH binding sites in proteins suggests that NADPH is the preferred reductant used by P5CRs from plants and human. In contrast, the N-terrninal domains of P5CRs from S. cerevisiae, M smithii, T. thermophilus and M leprae display greater similarity to a consensus NADH-binding site. The definite preference of plant P5CRs for NADPH in comparison with NADH suggests that P5CR may be involved in regulating the redox potential within plant cells and that this step in proline biosynthesis from glutamate may be of importance in overall metabolic regulation. Three amino acid residues are universally conserved in all P5CRs studied. All are found within blocks of high sequence similarity. These residues are likely to be of importance in the structure or catalytic mechanism of P5CR. A number of other residues are common to several of the enzymes examined. These may also be of importance in subsequent manipulation of Arabidopsis P5CR at the molecular level. Prediction of the putative secondary structures of A. thaliana, soybean, pea, human and E. coli indicated a high degree of similarity between the enzymes. This was particularly evident in the region of the putative P5C-binding domain. Considerable similarity exists in hydrophobicity profiles of P5CRs from these five organisms. Proline levels in reproductive organs of unstressed Arahidopsis plants were considerably higher than those in vegetative tissues. This suggests differential expression of enzymes involved in proline metabolism in these organs. In situ hybridisation studies indicated an increase in levels of mRNA transcripts encoding P5CR in stem tissues in response to water deprivation stress. Regulation of levels of mRNA transcript encoding P5CR in Arabidopsis therefore appears to be an osmotically sensitive process. Furthermore, this accumulation of transcript occurred in a tissue-specific manner. In particular, an increase in levels of transcript encoding P5CR was observed in the cortical parenchyma, phloem, vascular cambium and pith parenchyma in the vicinity of the protoxylem. The significance of these findings in contributing to a better understanding of the role of proline in adaptation to environmental stress is discussed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.