FUSCA3Functions In Root Regeneration Through Mediating Auxin Biosynthesis And Polar Transport In Arabidopsis

Plant growth and development derives from embryo in Arabidopsis. It has been provedFUSCA3(FUS3), LEAFY COTYLEDON1(LEC1) and LEAFY COTYLEDON2(LEC2) whichare called embryo-identity genes, play an important role in embryogenesis. In somaticembryogenesis, over-expression of LEC1and LEC2could promote induction of somaticembryos without treatment of exogenous hormones, suggesting that these genes regulate theprocess of somatic embryo induction. FUS3and genes involved in embryogenesis have beenproved to have close relationship with plant hormone. Studies on the interactions betweenFUS3and auxin will have important theoretical significance on the mechanism ofembryogenesis and organogenesis, as well as organogenesis. In this study, we focused onstudying the interactions between FUS3and auxin. We first constructed the inducible FUS3over-expression and FUS3antisense-expression vectors to get the transgenic plants. Then weused the root of transgenic plants as explants to regenerate organs using the root regenerationsystem. The results show that altering the expression level of FUS3affects the rate andfrequency of root regeneration. Furthermore, FUS3also regulates auxin biosynthesis, polartransport and response. The main results are as follows:(1) Changes of FUS3expression level obviously affect frequency root regeneration.We induced the over-expression and antisense-expression of FUS3in different culturephases of root regeneration, showing that induction of FUS3expression has more obviouslyeffects in root regeneration phase than in callus formation phase. The rate and frequency ofroot regeneration were induced by over-expression of FUS3, while the expression levels ofWUSCHEL RELATED HOMEOBOX5(WOX5), PLETHORA2(PLT2), and SCARECROW(SCR) were also increased. But FUS3antisense-expression slightly inhibits the rootregeneration. The results suggest that FUS3plays an important role in root regeneration.(2) FUS3affects auxin biosynthesis, polar transport and signaling in root regeneration. We used auxin response element DR5connecting GFP reporter gene to indicate auxinresponse in calli. We found that auxin response signals were enhanced and localizedaccumulation of auxin response signals occurred earlier in the FUS3over-expression callithan in the wild type. While the DR5::GFP signals was decreased and locally accumulatedlater in the FUS3antisense-expression calli. The results of QRT-PCR analysis show that theexpression levels of auxin biosynthesis genes YUCCA2(YUC2), YUCCA4(YUC4) and auxintransport genes PIN-FORMED2(PIN2), PIN-FORMED4(PIN4), PIN-FORMED7(PIN7)were enhanced in the FUS3over expression tissues. While the expression levels of thesegenes were down-regulated in the antisense tissues. Using pPIN2::PIN2-GFP to indicateauxin polar transport, we found that the GFP signals emerged earlier in the FUS3over-expression tissues, but later in the FUS3antisense-expression tissues, compared withthat in the wild type.(3) FUS3directly binds to the intron sequence of YUC4to regulate auxin biosynthesis inroot regeneration.First, we analyzed the motifs of YUC genes. We found that an intron sequence of YUC4gene contains RY motif (CATGCA) which can be recognized and bound by FUS3protein.Then the results of ChIP suggest that FUS3can directly bind to that intron sequence of YUC4gene. Moreover, over-expression of FUS3in yuc4mutants did not cause obvious phenotypeof root regeneration. These results show that FUS3directly regulates auxin biosynthetic geneYUC4to impact root regeneration.(4) FUS3affects plant growth and development in Arabidopsis.We found that growth and development of the root and shoot apex were promoted whenFUS3over-expressed. FUS3over-expression plant has2-3true leaves and longer root thanthe wild type. However, the developments of antisense-expression FUS3plant did not shownthese phenotypes.