Estudio de la implicación de las hormonas en la formación de los óvulos de tomate: papel de las enzimas GA2oxidasas

Abstract

[ES] En nuestro laboratorio hemos iniciado el estudio del papel de la hormonas vegetales en la formación de los óvulos de tomate. En Arabidopsis, las auxinas (IAA), las citoquininas (CKs) y los brasinosteroides (BRs) tienen un papel en la iniciacion y desarrollo de los ovulos. El papel de las giberelinas (GAs) no ha sido aun claramente establecido. Mediante el análisis de mutantes de tomate, con alteraciones en los niveles hormonales o en su respuesta, hemos determinado que las giberelinas influyen negativamente en el número de óvulos que se forman dentro del ovario. Este proyecto pretende estudiar si los genes que codifican los enzimas inactivadores de giberelinas (GA2ox) pueden tener un papel en la iniciacion y desarrollo de los ovulos. Para ello utilizaremos mutantes de tomate, donde estos genes han sido silenciados, para estudiar: 1 posibles alteraciones fenotipicas en el numero o desarrollo de los ovulos, 2 el efecto de estas mutaciones sobre los niveles de giberelinas activas en las fases y tejidos en que los ovulos se están desarrollando 3 y si existen otras hormonas, de las que influyen en la formación de los óvulos,cuyo metabolismo pudieran estar afectadas dentro del ovario Para ello estudiaremos el número y la morfología de los óvulos y los ovarios en los mutantes de RNA de interferencia (GA2ox-RNAi) y la linea isogenica control, mediante cortes frescos con un vibratomo. Además, realizaremos cuantificaciones hormonales por espectrometría de masas acoplada a cromatografía liquida (LC-MS) para determinar si las giberelinas estan alteradas en ovarios en desarrollo y si esta alteracion influye en el metabolismo de otras hormonas como auxinas y citoquininas.

[EN] In plants it is important to understand the mechanisms that control the number of ovules and their development, because ultimately they determine the final number of seeds and thus the crop yield. Over recent decades, hormones have been identified as essential factors in ovule formation, as they play a key role in determining their final number. Especially, hormones such as auxins, brassinosteroids or cytokinins are considered very important in this process, where they have a positive effect on the ovule development. However, although the role of gibberellins (GAs) on the ovule development has not yet been described, there are indications that these hormones are essential in this process, having a negative effect on the final number of ovules, what has been studied by analyzing mutants that have changes in hormone levels of GAs or in its response. In this study, it is intended to affirm the negative role of GAs on the ovule development in tomato plants, using the transgenic lines L1i and L5i, where GA2ox genes, which encode gibberellin inactivating enzymes GA 2-oxidases, have been silenced. To do this, the phenotypic alterations in the number or morphology of the ovules, seeds and ovaries between transgenic lines, L1i and L5i, and the control MT were studied, as well as variations in the levels of gibberellins and other hormones (auxins, abscisic acid and jasmonic acid). The results of these studies show that silencing GA2ox genes leads to increased levels of bioactive gibberellin GA1 and decreased inactive products GA51, GA34, GA29 and GA8 in reproductive tissues of transgenic tomato lines, which was expected as a result of silencing GA 2-oxidases. Regarding the levels of auxins, abscisic acid and jasmonic acid, there were no differences relative to the control, suggesting that GAs do not influence the metabolism of these hormones. Moreover, the phenotypic study revealed significant differences only in the transgenic line L5i, where it was observed that variations in the levels of GAs resulted in smaller ovules, as well as a fewer number of ovules and seeds per fruit. This fact could confirm the negative role of gibberellins on the ovule number and its development. Concerning the L1i line, although variations in their levels of GAs were observed relative to MT, it did not show phenotypic alterations, which could be due to differences between the two transgenic lines in localization of the transgene.