Early stages of stomatal development in arabidopsis thaliana: a molecular genetics and transcriptomic approach

• Autores: Alberto de Marcos Serrano
• Directores de la Tesis: Carmen Fenoll (dir. tes.), Montaña Mena Marugan (dir. tes.)
• Lectura: En la Universidad de Castilla-La Mancha ( España ) en 2016
• Idioma: español
• Tribunal Calificador de la Tesis: Crisanto Gutiérrez Armenta (presid.), Luis Javier Oñate Sanchez (secret.), Kiril Mihaylov Mishev (voc.)
• Programa de doctorado: Programa de Doctorado en Ciencias Agrarias y Ambientales por la Universidad de Castilla-La Mancha
• Enlaces
  • Tesis en acceso abierto en: RUIdeRA
• Resumen

  • Stomata are bi-celled epidermal structures that function as valves to regulate gas exchange with the atmosphere; together with the acquisition of an impermeable cuticle in the aerial organs, they are key to the adaptation of plants to a terrestrial environment. The gene circuits that control their development in the model plant Arabidopsis thaliana include the consecutive action of three related bHLH transcription factors: SPEECHLESS (SPCH), MUTE y FAMA. Loss of function mutations in any of these factors render stomataless plants. Exploiting the lack of stomata of the mutant genotypes spch-3 y mute-3 and using the wild type for comparison, we have studied the transcriptomes and the physiology of the three genotypes. Combining both approaches we determined that lack of stomata severely compromises secondary metabolism, as well as nitrogen and sulfur metabolisms, while photosynthetic processes are depressed but not overly altered. The transcriptomic analysis unveiled new genes candidates to regulate stomatal development, some of which were validates using mutants or transgenic lines for conditional overexpression.

    In this Thesis, we also present spch-5, a novel hypomorphic allele of SPCH. This mutation produces apparently healthy and fertile plants but with a drastically reduced number of stomata, which in addition are frequently clustered. In vivo tracking of leaf epidermal cell division and differentiation and the study of transgenic marker lines indicate that the spch-5 phenotype results from a very limited capacity for stomata lineage initiation and progress, as well as an altered capacity to impose asymmetry in size and fate of cell division products, and a faulty orientation of such cell divisions both within and between lineages. In these organs, the spch-5 phenotype is highly dose-dependent. However, growth in brassinosteroids-supplemented medium allows the mutant to reach stomata numbers and distribution patterns similar to the wild type. We undertook a transcriptomic analysis to determine the molecular basis and study the mechanisms underlying the spch-5 phenotype and the brassinosteroids effects. We found, among other results, that transcripts for EPIDERMAL PATTERNING FACTOR 2 (EPF2) and BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) are below functional levels and that BASL is required for the brassinosteroids-dependent alleviation of clustering in spch-5. Since SPCH-5 carries an amino acid change that potentially interferes with DNA binding of the protein, our results may inform on the different functions of SPCH, which can be dependent and independent of its binding to DNA.

    Finally, and related to the early events of stomatal lineage progression, we show the results of an in vivo tracking of cell division and differentiation during leaf epidermal development of a mutant in the gene TOO MANY MOUTHS, tmm-1. Our dynamic analysis of this process shows a delay in stomata differentiation in tmm-1 as compared to the wild type. Such a delay takes place in the mutant in spite of its normal capacity to initiate stomatal precursors, and it indicates that intermediate cell stages are transiently arrested although they resume their development to finally produce stomata. A similar behavior was previously described for stomatal precursors in cylindrical organs of this mutant, but in this case the arrest was permanent and stomata were nor formed. In addition to our novel results, we also present and discuss a review of previous data to shed light on the role of TMM in stomatal lineage progression, showing both similarities and differences between flat and cylindrical organs.