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Doctor of Philosophy / Rice (Oryza sativa L.) is very susceptible to water deficit at any time during its life cycle as a semi-aquatic cereal crop. However, the consequential damage is particularly severe if water deficit occurs during reproductive phases. The conspicuous injury often observed in rice plants exposed to water stress during meiosis of the pollen mother cell is the reduction of grain set, which is attributed to the decline of male fertility. In spite of much research on drought-induced male sterility in rice, the underlying mechanisms of the problem are poorly understood. This project was therefore conducted to investigate the molecular mechanisms of water deficit-induced pollen sterility in rice. In this study three consecutive days of water deficit treatment at -0.5 MPa osmotic potential during anther development effectively reduced the leaf water potential (leaf) and the number of viable pollen which later led to a decrease in grain set. Moreover, this thesis demonstrates that the immediate deleterious effects of water deficit to plant fertility could be estimated using a young microspore viability index, which showed a strong correlation with mature viable pollen and grain set. The present work has also illustrated that oxidative stress appears to be a plausible cause for the decline of male fertility and grain set. Water deficit has induced the excessive production of reactive oxygen species (ROS) above the redox balance, which in turn caused detrimental effects to cellular DNA and might result in programmed cell death (PCD) in the anthers. Moreover, ROS accumulation effectively influenced ATP synthesis leading to a decrease in the level of ATP in the anthers. Excessive ROS accumulation after drought could be the consequence of insufficient activity of the antioxidant system, which has been illustrated by qRT-PCR expression analysis of major antioxidant genes. Down-regulation of those genes would increase the incidence of oxidative damage. In contrast, stable or up-regulated expression of these genes resulted in less oxidative damage. Detailed investigations of sugar metabolism in anthers has provided supplemental data to develop a model of sugar unloading and transport within anther using in situ hybridisation to mRNA techniques. Analysis of sugar transportation within the cellular compartments of anther has unveiled the role of sugar metabolism on pollen sterility in rice. qRT-PCR assays of genes associated with the sugar metabolic pathway has demonstrated that the supply of both sucrose and hexoses from the anther walls to the locules was not restricted after water deficit stress. The results indicate that water deficit might not cause sugar starvation for developing microspores as previously thought, nor inhibit the initial steps of sugar utilisation such as glycolysis. This thesis has suggested new ideas regarding the role of rising sugar levels to cope with oxidative stress in anthers. Sugar accumulation might have provided protection against oxidant damage by strengthening the antioxidant system. However, the interplay between sugar and oxidative stress is not straightforward and needs to be further characterised. In-depth investigations on the interaction between sugar signalling and oxidative stress responses may help indentify the role of sugars in protecting anthers under water deficit. Although many studies on drought and chilling stresses in rice anthers have been performed, the causal mechanism of male sterility still remains to be elucidated. Findings presented in this thesis may contribute to understanding molecular mechanisms of male sterility in rice as a response to drought stress. A more detailed investigation of mitochondrial respiration in rice anthers is required to further examine this problem. Finally, this thesis suggests that signalling molecules such as 14-3-3 proteins and abscisic acid (ABA) might act upstream of ROS production and antioxidant defence in plants. Further work on these molecules might therefore further illustrate how they influence plant fertility under water shortage conditions.

Doctor of Philosophy / Rice (Oryza sativa L.) is very susceptible to water deficit at any time during its life cycle as a semi-aquatic cereal crop. However, the consequential damage is particularly severe if water deficit occurs during reproductive phases. The conspicuous injury often observed in rice plants exposed to water stress during meiosis of the pollen mother cell is the reduction of grain set, which is attributed to the decline of male fertility. In spite of much research on drought-induced male sterility in rice, the underlying mechanisms of the problem are poorly understood. This project was therefore conducted to investigate the molecular mechanisms of water deficit-induced pollen sterility in rice. In this study three consecutive days of water deficit treatment at -0.5 MPa osmotic potential during anther development effectively reduced the leaf water potential (leaf) and the number of viable pollen which later led to a decrease in grain set. Moreover, this thesis demonstrates that the immediate deleterious effects of water deficit to plant fertility could be estimated using a young microspore viability index, which showed a strong correlation with mature viable pollen and grain set. The present work has also illustrated that oxidative stress appears to be a plausible cause for the decline of male fertility and grain set. Water deficit has induced the excessive production of reactive oxygen species (ROS) above the redox balance, which in turn caused detrimental effects to cellular DNA and might result in programmed cell death (PCD) in the anthers. Moreover, ROS accumulation effectively influenced ATP synthesis leading to a decrease in the level of ATP in the anthers. Excessive ROS accumulation after drought could be the consequence of insufficient activity of the antioxidant system, which has been illustrated by qRT-PCR expression analysis of major antioxidant genes. Down-regulation of those genes would increase the incidence of oxidative damage. In contrast, stable or up-regulated expression of these genes resulted in less oxidative damage. Detailed investigations of sugar metabolism in anthers has provided supplemental data to develop a model of sugar unloading and transport within anther using in situ hybridisation to mRNA techniques. Analysis of sugar transportation within the cellular compartments of anther has unveiled the role of sugar metabolism on pollen sterility in rice. qRT-PCR assays of genes associated with the sugar metabolic pathway has demonstrated that the supply of both sucrose and hexoses from the anther walls to the locules was not restricted after water deficit stress. The results indicate that water deficit might not cause sugar starvation for developing microspores as previously thought, nor inhibit the initial steps of sugar utilisation such as glycolysis. This thesis has suggested new ideas regarding the role of rising sugar levels to cope with oxidative stress in anthers. Sugar accumulation might have provided protection against oxidant damage by strengthening the antioxidant system. However, the interplay between sugar and oxidative stress is not straightforward and needs to be further characterised. In-depth investigations on the interaction between sugar signalling and oxidative stress responses may help indentify the role of sugars in protecting anthers under water deficit. Although many studies on drought and chilling stresses in rice anthers have been performed, the causal mechanism of male sterility still remains to be elucidated. Findings presented in this thesis may contribute to understanding molecular mechanisms of male sterility in rice as a response to drought stress. A more detailed investigation of mitochondrial respiration in rice anthers is required to further examine this problem. Finally, this thesis suggests that signalling molecules such as 14-3-3 proteins and abscisic acid (ABA) might act upstream of ROS production and antioxidant defence in plants. Further work on these molecules might therefore further illustrate how they influence plant fertility under water shortage conditions.

Submitted by Verena Pereira (verenagoncalves@uefs.br) on 2018-07-03T23:23:34Z No. of bitstreams: 1 TESE 2018 - Doutorado RGV_UEFS - Carla Maria de Jesus Silva.pdf: 1687907 bytes, checksum: 3e9fe14e189c00f951079ca2a71e3c66 (MD5) / Made available in DSpace on 2018-07-03T23:23:34Z (GMT). No. of bitstreams: 1 TESE 2018 - Doutorado RGV_UEFS - Carla Maria de Jesus Silva.pdf: 1687907 bytes, checksum: 3e9fe14e189c00f951079ca2a71e3c66 (MD5) Previous issue date: 2018-03-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The watermelon (Citrullus lanatus) is a very important vegetable for a region of Northeast Brazil due to its adaptation as the natural conditions and the good characteristics of the fruit that are obtained. The aims of the current study are to assess watermelon genotype responses to calluses in anthers by using growth and temperature regulators, and to induce polyploidy through colchicine use (at different concentrations), exposure time, mechanical scarification and application methods. Anthers of Smile and Sugar Baby lines were inoculated in MS medium at different concentrations of 2.4-D or of BAP with 2.4-D, in combination with the pre-treatment (low temperature). Crimson Sweet cultivar seedlings were treated with different colchicine concentrations at two different times, with and without scarification, in order to induce polyploidy. Line LDRO was subjected to different colchicine concentrations at two different times and application methods: a) direct in the seed method (with, and without, scarification) (DSM, WE and WOE), b) Radicle emission method (ERM), c) Hypocotyl and root insertion point method (HRIM), d) At the apex of the seedling method (ASM) and e) Inverted hypocotyl method (IHM). Crimson Sweet flower buds were subjected to colchicine, at two different times, in order to induce polyploidy. Results have shown that 2.4-D often induced callus formation in both lines, but BAP/2.4-D interaction and pre-treatment did not increase the induction frequency. Crimson Sweet showed higher induction rate at 0.2% colchicine for 48h, WE. Line LDRO presented plants with tetraploid cells at 0.2% colchicine for 24 and 48h. The method 0.2% DSM, WE and WOE also generated plants with tetraploid cells. The diameter of treated pollen grains in flower buds have increased; the higher rate of non-reduced gametes induction was 16.07% and flower bud diameter (1.5mm) was estimated as adequate for induction / A melancia (Citrullus lanatus) ? uma hortali?a muito importante para a regi?o Nordeste do Brasil por sua adapta??o as condi??es clim?ticas e pelas boas caracter?sticas de fruto que se obt?m. Os objetivos desse trabalho foram avaliar as respostas de gen?tipos de melancia quanto ? indu??o de calos em anteras utilizando reguladores de crescimento e temperatura; e induzir a poliploidia mediante o uso da colchicina em diferentes concentra??es, tempos de exposi??o, escarifica??o mec?nica e m?todos de aplica??o. Anteras das linhagens de Smile e Sugar Baby foram inoculadas em meio MS sob diferentes concentra??es de 2,4-D ou BAP com 2,4-D, associado ao pr?-tratamento (baixa temperatura). Para indu??o de poliploidia, sementes da cultivar Crimson Sweet foram tratadas com diferentes concentra??es de colchicina, em dois tempos, com e sem escarifica??o. Para a linhagem LDRO, utilizaram-se diferentes concentra??es de colchicina em dois tempos e m?todos de aplica??o: a) M?todo direto na semente (com escarifica??o e sem escarifica??o) (MDS, CE e SE), b) M?todo da semente com emiss?o da rad?cula (MER), c) M?todo no ponto de inser??o do hipoc?tilo e raiz (MIHR), d) M?todo no ?pice da pl?ntula (MAP) e e) M?todo do hipoc?tilo invertido (MHI). Na indu??o de poliploidia em bot?es florais de Crimson Sweet utilizou-se colchicina em dois tempos. Os resultados mostraram que o 2,4-D induziu a maior frequ?ncia de calos nas duas linhagens, a intera??o BAP com 2,4-D e o pr?-tratamento, n?o aumentaram a frequ?ncia de indu??o. Para Crimson Sweet, o maior percentual de indu??o foi obtido com colchicina a 0,2% por 48 h, SE. Para LDRO, observou-se uma frequ?ncia de plantas com c?lulas tetraploides com colchicina a 0,2% por 24 h e 48 h. Nos m?todos 0,2% MDS CE e MHI observou-se tamb?m plantas com c?lulas tetraploides. Em bot?es florais, o di?metro dos gr?os de p?len tratados aumentou; o maior percentual de indu??o de gametas n?o reduzidos foi de 16,07% e; o di?metro em torno de 1,5 mm do bot?o floral foi estimado como adequado para indu??o