Principal Investigator: José Luis Micol.
Investigators: José Manuel Pérez Pérez, Héctor Candela Antón, Almudena Mollá Morales, Raquel Sarmiento Mañús, Almudena Ferrández Ayela, Rubén Casanova Sáez and Tamara Muñoz Nortes.
Instituto de Bioingeniería. Universidad Miguel Hernández.

An analysis of the interplay of epidermal, mesophyll and vascular development in constructing internal leaf structure and overall leaf shape

Large-scale exploratory approaches to understanding gene function laid the foundations for the “Genomics era”. Some years before the dawn of Genomics, however, large-scale exploratory approaches were already furthering our understanding of gene function in the form of saturation mutagenesis experiments aimed at the identification of all genes that mutate to a given phenotype. Forward genetic approaches, conducted on model organisms such as Drosophila melanogaster and Caenorhabditis elegans, have led to the isolation of large collections of mutants affected in specific developmental processes, whose cellular and molecular characterization has unraveled the underlying genetic mechanisms of animal development.

To shed light on the making of plant leaves, in 1993 we initiated an attempt to isolate as many viable and fertile mutants with abnormal leaf morphology as possible, using the Arabidopsis thaliana model organism. We identified hundreds of mutations, which were found to fall into 147 complementation groups. Using a high-throughput gene mapping method that we developed, we have already cloned 25 of these genes identified by mutation. The products of these genes participate in various developmental processes, such as polar cell expansion, transduction of hormonal signals, gene regulation, plastid biogenesis, and chromatin remodeling, among others. The broad spectrum of leaf morphological alterations that we identified is facilitating the dissection of specific leaf developmental processes.

The objective of this project is to increase our understanding of the interplay of epidermal, mesophyll and vascular development in constructing internal leaf structure and whole leaf shape. To this end, we will follow three different approaches:

(a) The functional characterization of the 7 members of the RETICULATA-RELATED (RER) gene family, which we consider good candidates for testing the hypothesis that leaf morphogenesis is controlled by plastid-to-nucleus signalling. We already cloned two members of this family, RE and VEN5, which encode chloroplast proteins required for mesophyll proliferation in the early stages of leaf development.

(b) The completion of functional studies already initiated but still not finished in our laboratory, namely those of the RUGOSA2 (RUG2) gene, whose protein product is involved in chloroplast and mitochodrion gene transcription regulation (RUG2). The rug2 mutant alleles impair leaf development, which might also result from perturbation of morphogenetic signaling from the chloroplast to the nucleus.

(c) A detailed, large-scale morphometric analysis of 122 of our mutants and several tens of their double mutant combinations, in order to determine the extent of the effects of their mutations on cell number, cell size, or both. The parameters to be measured will include number, perimeter, area and several form variables of whole rosettes and individual leaves, petiole, lamina, adaxial and abaxial epidermis, pallisade and spongy mesophyll, venation pattern, hydathodes, stomata and trichomes. The correlation among these parameters will be studied in order to analyze the coordination between cell proliferation and cell expansion, as well as whether or not this coordination is under cellular, local or organ-wide controls.