Towards the cloning of tos1, a gene involved in aba responses and drought tolerance in tomato (2008)

Water-stress severely limits plant growth and agricultural productivity. The identification of mutants showing impaired responses to osmotic-stress caused by drought or salinity constitutes a successful strategy for deciphering the molecular basis of stress signalling pathways in plants. The tomato mutant tos1, caused by a single recessive nuclear mutation, is hypersensitive to general osmotic stress and exhibits reduced sensitivity to intracellular abscisic acid (ABA). This reduction in the ABA perception or signaling is a basic cellular trait expressed by the mutant at all developmental stages and it is not caused by a defective biosynthesis of the hormone. Physiological analyses had revealed a gradual decreased in leaves' water potentials of tos1 adult plants in field conditions of high irradiance and low humidity that correlates with the midday depression of stomatal conductance, which occurs even in conditions of water availability. Those lower water potential was related to the appearance of a wilting phenotype and a loss of turgor in the apical leaves, typical of the ABA-deficient mutants (aba) or ABA insensitive (abi), unable to close stomata in response to water loss. Unexpectedly, the tos-1 mutant showed a gradual closure of stomata during the day, indicating that the mechanisms of opening and ABA-dependent closure of stomata are not affected in the mutant. Low values of the hydraulic conductivity found in roots of tos1 could explain the wilty phenotype as a consequence of a reduced activity of the water channels located at the cellular membranes. The work presented here aimed to achieve the molecular identification of the TOS1 locus using a map-based cloning approach. For this purpose, an appropriate mapping population was developed and used to identify DNA markers linked to the locus. Using Bulk Segregant Analysis, we identified AFLP markers closely linked to the target gene which spanned a region of 8.48 cM around TOS1; a low-resolution map was then constructed on the region. The chromosomal location of TOS1, in the short arm of chromosome 3, was determined by means of BAC-library screening, PCR-based marker development and bin-mapping using introgression lines. A high resolution map was developed by highthrough-put analysis of a mapping population of more than 3,000 plants with the flanking markers. We have developed a contig map in the region around TOS1 combining BAC-library screening, BAC-end based marker development, mapping and chromosome walking. Genome-duplications close to TOS1, intra- and inter-chromosomic duplications, and also a region of chromosome 3 not represented in any of the tomato BAC-libraries available for the scientific community have been identified. To overcome problems associated to chromosome walking on regions not represented in the genome, we have developed a new strategy of chromosome landing based on recombinant-Bulk Segregant Analysis. This method has demonstrated to be a powerful tool for chromosome landing in complex genomes as tomato. Based on microarray analyses, we hypothesize that an insufficient water transport related to an impaired root morphogenesis might be the primary cause underlying the pleiotropic phenotype of tos1 under conditions of high irradiance and low water humidity. The identification of TOS1 will shed light on the signalling networks involving water-status perception, root morphogenesis and the physiological-adaptation mechanisms to water deficit.