Title : Functional characterization of ABC transporters in chloroplast
Abstract:
Stress in plants is caused by sudden and restrictive changes in their environment, which disrupts the plant homeostasis and leads to worldwide yield losses. The deleterious effects of stress on plants physiology cause growth retardation, reduced metabolism and altered photosynthesis. Plant hormone auxin plays an important role in plant stress responses. Exogenous auxin treatments affect chloroplast structure, chlorophyll synthesis, nuclear and chloroplastic photosynthetic genes expression, and chloroplast protein levels. Auxin hypothetically has a protective function against photo-oxidative inhibition 1. Approximately 30%–40% of the total free IAA pool is located in the chloroplast2. Moreover, besides providing Tryptophan-derived precursors for Indole-acetic acid (IAA) biosynthesis, chloroplasts were shown to actively biosynthesize auxin 2. Recently, it was shown that chloroplast redox state modulates auxin homeostasis 3. These data suggest a strong connection between chloroplast processes and auxin homeostasis. In Arabidopsis, five members of the ABCB subfamily of transporters have been reported to mediate cellular transport of auxin or auxin derivatives4. Based on AT_CHLORO database5 and sequences analysis6, we identified two chloroplast predicted ABCB transporters ABCB28 and ABCB29 in Arabidopsis
Our main goal is to uncover whether ABCB28 and ABCB29 participate in auxin transport across the chloroplast envelope and their roles in plant development and stress responses. In silico promoter gene analysis showed that motifs in the promoter of our target genes shared some similarities with the promoters of known auxin ABC transporters. Chloroplast subcellular localization was confirmed using 35S:ABCB28:GFP and 35S:ABCB29:CFP constructs and confocal microscopy. ABCB28 and ABCB29 overexpressors, abcb29 knock out and abcb28 knockdown mutants showed altered photosynthesis during early and late seedling development. ABCB28 and ABCB29 over expressing lines exhibited increased auxin induced lateral root development and reduced auxin induced root growth retardation compared to wild type plants. Interestingly, overexpressors showed increased number of siliques and seed production per plant. Moreover, in response to osmotic stress, lateral root development in these lines is stimulated. To find out the substrate specificity of our proteins, we are trying to implement different techniques by using isolated protoplast and chloroplast.
