Effect Of Auxin TIR1 Signaling Pathway On Stomatal And Pavement Cell Development In Epidermis Of Arabidopsis Thaliana

The auxin transcriptional signaling pathway, which is mediated by the auxin co-receptors of TIR1-AFB family, the auxin signaling repressors of the Aux/IAA family, and the transcription factors of the Auxin response factors ?ARF? family, play key roles in multiple developmental processes, including embryogenesis, root development, patterning of the angiosperm female gametophyte, and so on. But how this signaling module involves in the stoamtal and pavement cell development on the epidermis of leaves needs further study. In this research, dynamic changes of auxin input and output activity during stomatal and PV cell development in the epidermis of cotyledon were monitored by time-lapse imaging using nuclear-localized marker gene 35S:D?-VENUS-N7 ?D?? and pDR5rev3X:VENUS-N7 ?DR5?, combined with membrane-localized auxin transport marker pPIN3:PIN3-GFP ?PIN3?. Then exogenous treatment with auxin signaling inhibitors and transport blockers was used to explore the function of auxin in stomatal and pavement cell development, combining with the phenotype of pavement and stomatal development of multiple tir1afb mutants on their epidermis of cotyledon. The effect of auxin signaling and transport inhibitors on the expression of key genes in stomatal development were also investigated to explore the mechanism of auxin regulation on stoamtal development. The main results are as follows:1. High level of input signal and output transcriptional activity of IAA-SCF^TIR1/AFBs -ARFs patheay are prior to the asymmetric division of proto-epidermis cells and the entry of their daughter cells into stomatal development, while decrease of input signal and AFRs activity is necessary for the transition of stomatal stem cells amplification to terminal cell differential, ie, the final symmetric division of GMC and GC morphogenesis. As for the pavement development, high auxin input and output activities are existed in cells of all stages, including morphogenesis and cell expansion.2. Nuclei localization in the epidermis cells was strictly regulated on the flat surface. In stomatal development pathway, the movement of nuclei to one corner in precursor cell predicts the site of following asymmetric division and thus the position of the future stoma, which may involve in the regulation of the division and daughter cell fate acquisition. At the same time, the position of nuclei in mature pavement cells are mainly in the neck region, with its axis parallel to that of whole cell, which indicates nuclei localization is strictly regulated and may play a part in following morphgenesis and cell expansion.3. Stoma and PV cell development are regulated by Auxin transcriptional pathway mediated by TIRl/AFBs and auxin polar transportation mediated by pinoid proteins ?PINs?. With the treatment of chemical inhibitor of TIR/AFBs signaling and analysis of phenotype of receptor mutants, function of signaling transduction and transportation of auxin in stoma and pavement cell development were studied. Retardation of development stages and patterning defects of stoma distribution on epidermis were found when auxin signaling or/and transportation were blocked chemically or genetically, accompanying by the inhibiton of morphogenesis and extension of PV cells.4. Auxin regulate expression of key regulation gene SPCH and MUTE, while polar auxin transport could affect the expression of TMM, SPCH, MUTE and FAMA in stomatal development pathway. Thus auxin controls the proceeding of stages by regulation of these key genes expressed in different time and space.In conclusion, auxin transcriptional pathway mediated by TIRl/AFBs and its polar transport regulate multiple processes in the epidermis development. Auxin tightly controls the stages progress of stomatal development in time and space, and the morphogenesis and cell extensions of pavement cells, thus determined the distribution of different lineages of cells and their interaction on the epidermis of cotyledon. These results will help us to understand the function of auxin on epidermis development, and lay foundation for further researches on auxinology.