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Technischen Universität München (2015)

Positional cloning and physiological analysis of novel Arabidopsis mutants involved in drought stress signaling

Dwi Setyo Rini

Titre : Positional cloning and physiological analysis of novel Arabidopsis mutants involved in drought stress signaling

Auteur : Dwi Setyo Rini

Université de soutenance : Technischen Universität München

Grade : Doktors der Naturwissenschaften 2015

Drought is a major abiotic stress decreasing plant productivity around the world. Drought stress is perceived on the molecular level by unknown means and subsequently activates a signaling cascade which culminates in stomatal closure and massive transcriptome re-organization. The phytohormone abscisic acid (ABA) is responsible for the major part of the drought stress-induced responses and its formation is strongly stimulated by drought stress. The early events of drought stress signal transduction between stress perception and stimulation of ABA synthesis are completely unknown. Isolation and cloning of drought stress signaling mutants are important steps in search for the unknown signaling components. In this work, Arabidopsis thaliana mutants with lesions in drought stress signal transduction generated by mutagenesis of the transgenic ABA reporter line pAtHB6 ::LUC, were studied. The reporter line exhibits ABA-dependent expression of firefly luciferase which may be visualized by in vivo-imaging. Three mutants isolated from the EMS mutant population due to a hypersensitive ABA reporter activation by reduced water availability which were preliminarily named jbp20, phros13, and rrsc7 and they were characterized with respect to their position in the drought stress signaling cascade. According to this analysis, phros13 is impaired in early drought stress signal transduction whereas jbp20 and rrsc7 carry lesions in the subsequent ABA signal transduction.
A combination of map-based cloning and next generation sequencing identified jbp20 as a novel allele of AtCPL3 (At2g33540) encoding a C-terminal domain phosphatase which is a negative regulator of ABA signaling. The mutation in jbp20 generates an additional stop codon causing a premature termination of translation. In the truncated AtCPL3 version generated in jbp20 the phosphatase catalytic domain is missing thereby abrogating the function of the protein as a negative regulatory element and causing a hypersensitive response to osmotic stress and ABA in the mutant. The ABA-hypersensitive phenotype of jbp20 was complemented by expression of wild type AtCPL3. The ABAhypersensitive phenotype of both jbp20 and a T-DNA insertion line of AtCPL3 shows recessive inheritance. The observation of an ABA hypersensitive response in the F1 generation of a cross of jbp20 to the T-DNA insertion line is thus consistent with the conclusion that JBP20 is AtCPL3. Like in jbp20, the inheritance of the mutation in phros13 is also recessive while inheritance of the mutation in rrsc7 is co-dominant.
In physiological analyses, stomata of phros13 and rrsc7 were insensitive to rootapplied osmotic stress indicating that PHROS13 and RRSC7 are negative regulators of stomatal closure in response to restricted water availability.
As a preliminary result of positional cloning, phros13 was mapped to the distal part of chromosome 2 and a region between 5.705 Mb and 14 Mb whereas rrsc7 is localized in a region between 7.4 Mb and 9.5 Mb on the proximal part of chromosome 3. Cloning of these two mutants could not yet be completed due to epigenetic gene silencing of the luciferase reporter used for mapping in subsequent generation of phros13 and rrsc7. Further mapping is therefore intended in a genetic background where silencing effects are restricted due to specific mutations.


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Page publiée le 6 septembre 2015, mise à jour le 10 janvier 2019