Effect of water stress on gene expression and morphophysiology in Eucalyptus spp.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-06T13:30:02Z No. of bitstreams: 2 inae_marie_araujo_silva.pdf: 2900695 bytes, checksum: b0f455759c609f430fbe59626102155a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5)Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-06T13:30:22Z (GMT) No. of bitstreams: 2 inae_marie_araujo_silva.pdf: 2900695 bytes, checksum: b0f455759c609f430fbe59626102155a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5)Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-06T13:30:40Z (GMT) No. of bitstreams: 2 inae_marie_araujo_silva.pdf: 2900695 bytes, checksum: b0f455759c609f430fbe59626102155a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5)Made available in DSpace on 2015-01-06T13:30:40Z (GMT). No. of bitstreams: 2 inae_marie_araujo_silva.pdf: 2900695 bytes, checksum: b0f455759c609f430fbe59626102155a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2013Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)Atualmente, devido as limita??es de expans?o das tradicionais ?reas de plantio de eucalipto, v?rios grupos de pesquisa t?m buscado estrat?gias para ampliar as fronteiras florestais brasileiras. Entretanto, a baixa disponibilidade h?drica dessas regi?es fronteiri?as tem limitado o desenvolvimento de alguns gen?tipos de eucalipto. ? vista disso, objetivou-se: i) avaliar as respostas morfofisiol?gicas de dois gen?tipos de eucalipto contrastantes quanto ? suas capacidades de tolerar o d?ficit h?drico e ii) construir uma biblioteca g?nica diferencial do gen?tipo tolerante ? seca, de maneira a possibilitar ? compreens?o dos mecanismos subjacentes ? toler?ncia ao d?ficit h?drico do ponto de vista gen?tico-molecular. O experimento foi conduzido, em casa de vegeta??o na UFVJM, em Diamantina, MG. As plantas dos dois gen?tipos, clones 953 (Eucalyptus camaldulensis vs. Eucalyptus grandis) e 224 (Eucalyptus grandis vs. Eucalyptus urophylla), modelo de toler?ncia e sensibilidade ao d?ficit h?drico, respectivamente, foram aclimatadas em casa de sombra, com irriga??o, durante 30 dias. Ap?s esse per?odo, foram submetidos a dois diferentes regimes h?dricos (irrigado e n?o irrigado), quando, ent?o, foram avaliados, ao longo do per?odo experimental, o crescimento em altura e di?metro, ?rea foliar, ?ndice de clorofila total, vari?veis da fluoresc?ncia da clorofila a, vari?veis de trocas gasosas, mat?ria seca de todos os compartimentos da planta e suas rela??es derivadas (rela??o mat?ria seca raiz e parte a?rea, raz?o de massa foliar, ?rea foliar espec?fica e raz?o de ?rea foliar), al?m da sintomatologia do estresse. O experimento foi conduzido em um Delineamento Inteiramente Casualizado, em esquema fatorial 2 (gen?tipos) x 2 (regimes h?dricos), totalizando quatro tratamentos (25 repeti??es cada). As an?lises estat?sticas foram realizadas mediante an?lise de vari?ncia. Para o estudo da express?o g?nica, concomitantemente a aplica??o do estresse h?drico, folhas foram coletadas, congeladas em N2 l?quido e armazenadas a - 80 ?C. Em seguida, procedeu-se com as extra??es e purifica??es do RNA total e, posterior, constru??o da biblioteca subtrativa. A defici?ncia h?drica n?o limitou o crescimento em altura dos gen?tipos estudados, embora, tenha reduzido o di?metro e a produ??o de mat?ria seca total. As demais vari?veis morfofisiol?gicas e todas as vari?veis de trocas gasosas foram negativamente afetadas pelo d?ficit h?drico. O clone 224 mostrou-se menos eficiente em termos fotossint?ticos e mais sens?vel ? restri??o h?drica para a maioria das vari?veis analisadas. Em contrapartida, o 953 apresentou maior toler?ncia ? falta d'?gua. Possivelmente, ?rea foliar reduzida, aumentos no teor de clorofila e aparelho fotossint?tico mais eficiente podem ter sido determinantes nessa maior toler?ncia encontrada no clone 953. Essas estrat?gias, podem, portanto, serem alvos em programas de sele??o de materiais gen?ticos mais tolerantes ? seca. Nesse sentido, a efici?ncia qu?ntica do Fotossistema II mostrou-se uma caracter?stica a ser considerada num processo de sele??o para toler?ncia ? falta d'?gua. A biblioteca de cDNA constru?da permitir? identificar quais genes est?o envolvidos nesse processo de toler?ncia no gen?tipo 953. Futuramente, esses genes candidatos poder?o ser transferidos para gen?tipos de interesse econ?mico, por meio de transgenia.Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013.ABSTRACT Currently, due to the limitations of expanding areas traditional of eucalyptus, several research groups have pursued strategies to expand the boundaries Brazilian forest. However, the low water availability of these border regions has limited the development of some genotypes of eucalyptus. Therefore, we aimed to: i) evaluate the morphological and physiological responses of two contrasting genotypes of eucalyptus as to their ability to tolerate drought and ii) build a library differential gene genotype drought tolerant, in order to enable the understanding molecular mechanisms underlying tolerance to water deficit. The experiment was conducted in a greenhouse at UFVJM in Diamantina, MG. The plants of the two genotypes, 953 clones (Eucalyptus camaldulensis vs. Eucalyptus grandis) and 224 (Eucalyptus grandis vs Eucalyptus urophylla) model of tolerance and sensitivity to water stress, respectively, were acclimated in the shade, irrigated for 30 days. After this period, underwent two different water regimes (irrigated and non-irrigated), when then were evaluated over the experimental period, the growth in height and diameter, leaf area, total chlorophyll index, chlorophyll a fluorescence variables, gas exchange variables, dry matter of all plant parts and their relations derived (shoot/root dry weight ratios, leaf weight ratio, specific leaf area and leaf area ratio) and the symptoms of stress. The experiment was conducted in a completely randomized design in a factorial 2 (genotypes) x 2 (water regimes), totaling four treatments (25 replicates each). Statistical analyzes were performed by analysis of variance. For the study of gene expression, concomitant application of water stress, leaves were collected, frozen in liquid N2 and stored at - 80 ?C. Then proceeded with the extraction and purification of total RNA and subsequent construction of the subtractive library. Water stress did not limit the growth in height of the genotypes studied, although it reduced the diameter and total dry matter production. The other morphophysiological variables and all gas exchange variables were negatively affected by drought. Clone 224 was less efficient photosynthetic and more sensitive to water restriction for most variables. In contrast, the 953 was more tolerant to lack of water. Possibly, reduced leaf area, increases in chlorophyll content and photosynthetic apparatus more efficient may have been determinant to increase tolerance found in clone 953. These strategies can be targeted in selection programs of genetic materials more drought tolerant. In this sense, the quantum efficiency of Photosystem II showed a characteristic to be considered in the selection process for tolerance to water shortage. A cDNA library constructed will allow to identify which genes are involved in the process of tolerance on the genotype 953. In the future, these candidate genes may be transferred to genotypes of economic interest through transgenesis