| Auxin, which plays a crucial role in plant growth and development, is one of the most important phytohormone in plants. Among all the auxin-related researches, Auxin transport is considered as one of the hottest spot for its determinant effect on realizing the physiological function of auxin. Comparing to the many research results on the polar auxin transport recently, intracellular auxin transport has witness so few achievements in its progress and mechanism, hence, little is known on this important physiological progress till now. Plant height is an important agronomic trait for its effect on grain production. By screening the rice RNAi mutant library, we isolated a dwarf mutant due to the downregulation of OsCOLE1(OsCOVE-LIKE 1). Further analysis indicated that Os COLE1 could regulate Os CLIP(Os COLE1-INTERACTION PROTEIN)-mediated auxin transport from the vacuole to the cytoplasm and affect the plant growth and development. The main results are as follow:1. We generated the RNAi and overexpression transgenic lines of OsCOLE1. Phenotypic observation of transgenic plants between grain filling and maturation stages indicated that upregulation of OsCOLE1 could not only increase the plant height, the stem base(including the first and second internodes) length and free IAA content at grain filling stage, but also increase local cell length and attenuate cell wall lignification in the second internode. However, downregulation of OsCOLE1 could lead to the opposite phenotypes.2. Homologous alignment of Os COLE with BLASTP demonstrated that Os COLE is the homology of AtCOV1 with 72.5% similarity. In addition, protein sequences of COV1 and its homologies are quite variable on the N-terminus, whereas considerably conserved on the C-terminus.3. Subcellular localization of Os COLE1 fusing with m Cherry indicated that Os COLE1 is a tonoplast-localized protein.4. By screening a rice c DNA yeast two-hybrid library with OsCOLE1, the interacting protein of Os COLE1 was found out, named as Os CLIP. Homologous alignment of Os COLE with BLASTP demonstrated that Os COLE and its homologies are quite different among plant species. Prediction of transmembrane helices of Os CLIP indicated that Os CLIP possesses the similar transmembrane structures to At WAT1, a novel tonoplast-localized auxin transporter, suggesting Os CLIP a new auxin transporter. Subcellular localization of Os CLIP fusing with eYFP indicated that Os CLIP is a tonoplast-localized protein.5. By performing the split-ubiquitin yeast two-hybrid assays and BiFC assays, the interaction relation between Os COLE1 and Os CLIP on both N-terminuses was corroborated.6. Exogenous IAA transport assay in yeast verified that Os CLIP could transport IAA in the s imilar manner to AtWAT1. Besides, co-expression Os COLE1 and Os CLIP could significantly elevate the IAA absorptive capacity of yeast implying the regulation ability of Os COLE1 on Os CLIP-mediated auxin transport.7. Transcript levels measurement of known phenotype related genes in OsCOLE1 transgenic plants indicated that transcript levels of OsCOLE1 could affect the expression of endoplasmic reticulum-localized auxin transporter genes: OsPIN5 a, OsPIN8, OsPIN9, and lignin synthesis-related genes: OsPAL4, Os4CL1, Os4CL3, Os4CL4, Os4CL5, suggesting Os COLE1-regulated auxin transport from the vacuole to the cytoplasm affecting the intracellular auxin metabolism and widely inf luencing the plant growth and development.Taking together, we clarified the regulatory mechanism of Os COLE1-regulated auxin transport in plant cell and its function on plant growth and development, revealed a novel auxin transport pathway and expanded our knowledge on auxin transport. |
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