Isolation And Functional Characterization Of Genes Encoding Auxin Signaling Components In Populus Tomentosa Carr

Auxin was one of the earliest discovered and the most classic plant hormones, and involved many growth and development processes of plants. Study of auxin signalin is useful to explaining life phenomena and indicating life mechanism.Populus, with the mature technique of tissue culture, is the model plant in biology study of wood plants. It has little genome with 500 Mbp approximately. Sequencing of P. trichocarpa genome has been finished in 2004.P. tomentosa was chosen as the research object in the paper. In order to study the action of genes encoding auxin signaling components in P. tomentosa development, three related genes were cloned and named PtPIN1, PtIAA1 and PtTIR1 respectively. Protein homology analyses showed PtPIN1 was most similar to Medicago truncatula MtPIN1 and Arabidopsis AtPIN7 with 82% and 79% identity respectively. PtIAA1 had the highest identity with 70% to AtIAA16 of Arabidopsis Aux/IAA gene family. PtTIR1 also had high identity to TIR1 proteins of other plants, especially to Vitis vinifera VvTIR1 with 80% similarity. According to analyses of phylogenetic tree, PtPIN1 gene belonged to PIN-like family of the auxin efflux carrier, while gene PtIAA1 was one of the auxin rapid response factors and belonged to theαsubfamily of Aux/IAA family. PtTIR1, encoding auxin receptor, was grouped to the C4 subfamily of F-box family.To characterize PtPIN1, PtIAA1 and PtTIR1 genes, we constructed their binary vectors, and transformed PtTIR1 into Populus and Arabidopsis. On the base of binary vector of P. tomentosa PtAUX1 constructed by our lab, the auxin influx import carrier PtAUX1 was transformed into Arabidopsis. Both PtAUX1 and PtTIR1 transgenic plants were confirmed by RT-PCR, and phenotypes of transgenic plants and morphologic characters cultured in vivo were analyzed. PtAUX1 influenced myriad developmental processes in Arabidopsis. Transgenic plants were lodged severely, but the stem didn't bend at 90°in the back tropism experiment. Secondly, PtAUX1 regulated root geotropism, promoted root elongation and lateral root initiation, and showed high sensitivity to indole acetic acid. Moreover, the over-expression of PtAUX1 leaded to many organs present universal transparence and hyperplasia. PtAUX1 made trichome number increasing and more two fork branches, and appeared curling and cupule-like leaves. Transgenic plant had abnormal floral organs. For instance, curled calyces with transparent brim produced leaf-like structure, petals with long or quite short figures were transparent and enveloping, and the tetradynamous stamen character was changed. Further more, some stamen had no flower silk and anthes adhibited intogether so that it dispersed difficultly. The transgenic plants also had many figure fruit different to wild type. The above results indicated that PtAUX1 promoted tissues and cells to absorb endogenesis IAA by auxin polar transport, then participated in apex meristem form, and influenced plant morphology, trichomes, roots, stems, leaves, flowers and fruit organs development.Phenotypes of 35S::PtTIR1 transformed into Populus and Arabidopsis showed that PtTIR1 promoted plants'growth, regulated phototropism and geotropism, and modulated root elongation and lateral root initiation. Besides, PtTIR1 affected leaves development. Transgenic buds during Populus transformation were evidently different with wide type buds. The petal-like transgenic buds was pure whilte, but some days later became to mahogany partly. Some transgenic buds were crystal rod-like structure, while some buds can come from root of which plant cultured in root medium. Those Phenotypes were similar to PtAUX1 in some extent, and accorded with auxin acted character. The results proved that PtTIR1 involved in apex meristem form, promoted plants'growth, regulated roots and leaves development by modulating polar auxin distribution in plant. In addition, PtTIR1 may participate in flower development. Therefore, primary functions of PtAUX1 and PtTIR1 were exhibited, and important roles of polar auxin transport in plant were also proved. It was considered PtAUX1 promoted the auxin rapid response factor PtIAA1 combining with the SCFPtTIR1 complex, and PtPIN1 carried IAA out of plant body in time by the vesicle regulate mechanism, then this auxin signal transport modulated plant development.