Roles Of Different Components Of Magnesium Chelatase In Abscisic Acid Signal Transduction

In plants and photosynthetic bacteria, Mg-chelatase catalyzes the insertionof Mg2+into protoporphyrin IX. This is the first committed step in chlorophyllbiosynthesis. It has been well established that Mg-chelatase functions incatalyzing Mg-Proto IX production as a hetero-tetramer, which is composed ofMg-chelatase subunits H, I (CHLI), D (CHLD), and a supplementary essentialand component GUN4(Genomes Uncoupled4). We previously reported that themagnesium-protoporphyrin IX (Mg-Proto IX) chelatase large subunit(Mg-chelatase H subunit CHLH/putative ABA receptor ABAR), achloroplast/plastid protein, binds ABA and functions in ABA signaling.However, it remains essentially unknown whether Mg-chelatase heterotetramercomplex or only CHLH function in ABA signaling, and why the Mg-Proto IXproduction process may differ from the CHLH-mediated ABA signaling. In thethesis, we designed biochemical, physiological and genetic experiments toexplore the mechanism.Here we report that, using a newly-developed surface plasmon resonancesystem, ABA-binding to CHLH, but not to the other Mg-chelatasecomponents/subunits CHLI, CHLD and GUN4, was detected.A new rtl1mutant allele of the CHLH gene in Arabidopsis thaliana showedABA-insensitive phenotypes in both stomatal movement and seed germination,which is consistent with the positive role of CHLH in ABA signaling.Upregulation of CHLI1resulted in ABA hypersensitivity in seed germination,while downregulation of CHLI conferred ABA insensitivity in stomatal responsein Arabidopsis, suggesting that CHLI positively regulates the major ABAresponses. Using a tobacco rattle virus (TRV) based virus-induced genesilencing (VIGS) system, we showed that CHLH and CHLI, but not CHLD,regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana).Consistently, the overexpression lines of the CHLD gene showed wild-typeABA sensitivity in Arabidopsis. Both the GUN4-RNA interference andoverexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses, demonstrating that GUN4is not an ABA signaling regulator.These findings provide clear and direct evidence that the Mg-chelatasecatalyzed Mg-Proto IX production is distinct from ABA signaling, givinginformation to understand the mechanism by which the two cellular processesdiffers at the molecular level.