TWD1 Regulates Early Events Of The Brassinosteroid Signal Transduction Pathway In Arabidopsis

Brassinosteroids(BRs), a class of plant-specific steroid hormones, play crucial roles in various biological processes during normal vegetative and reproductive growth and development. The BRs are perceived by a plasma membrane-localized receptor complex, including the receptor BRI1 and co-receptor BAK1. The BRs signal can then be transduced to two critical transcription factors, BES1 and BZR1, through a phosphorylation/dephosphorylation casscade and regulate the expression of thousands of genes. Like BRI1, BAK1 is also a leucine-rich repeat receptor-like protein kinase(LRR-RLK). BAK1 belongs a member of SERK gene subfamily. Recent studies indicated that BAK1 is involved in various physiological processes including BR signaling, BR-independent cell death pathway, anther development, plant innate immunity and stomatal development. Studies on two novel alleles of BAK1, elg and bak1-5, also emphasize the multiple tasks of BAK1 and/or other SERK members. However, detailed molecular mechanisms of the early events of the BR signaling pathway are poorly understood. Additional studies are necessary.A large member of T-DNA insertion lines in the BAK1 null mutant, bak1-4, background were generated and screened for mutants with altered BR sensitivity. From this screen a typical BR insensitive mutant, 128-12-T01, was obtained. It is renamed as twd1-4 because sequence analysis indicated that it is a novel allele of TWD1. Meanwhile, we isolated another TWD1 allele, twd1-5, from a homemade ethylmethanesulfonate(EMS) population which showed a bri1-like phenotype. twd1-5 was used it as a representative allele for further analyses. twd1-5 is insensitive to exogenously applied brassinolide(BL) and is hypersensitive to a BR specific biosynthesis inhibitor. Genetic experiments indicated that TWD1 does not directly regulate BR biosynthesis, instead modulates BR signaling. Overexpression of TWD1 can not suppress the defective phenotype of BRI1 alleles. The endoplasmic reticulum(ER)-localization and protein abundance of BRI1 are not affected in twd1-5, suggesting TWD1 is not involved in BRI1 ER quality control, although it was shown to localize on ER and modulate trafficing of ABCB1 and ABCB19. In fact, the membrane anchored TWD1 interacts with BRI1 on the plasma membrane as revealed by a series of biochemical assays. The BR signaling are partially blocked in twd1-5, which may be caused by reduced ligand-induced phosphorylation activities of BRI1 and BAK1. Furthermore, loss-of-function of TWD1 significantly blocks the BR-induced interaction between BRI1 and BAK1. We conclude that TWD1 plays an important role in the initiation of BR signaling through its interaction with BRI1.To screen more components in BR signal transduction pathway, 44 BR related genes were selected from previous studies and overexpressed in br6ox2 background. Using this reverse genetic approach, overexpression of BED1 was found to enhance the defective phenotype of br6ox2 which results in smaller leaves, shortened statue and pedicel. Seedling of transgenic plants overexpressing BED1(BED1-OX) is slightly insensitive to BL treatment and the mature BED1-OX plants display visible BR defective phenotypes. The BED1-OX plants also exhibit erecta-like phenotypes including clustered ?ower buds, short pedicel, short and blunt siliques.Hhowever, no erecta-like stomatal defects were found. Loss-of-function mutants of BED1, generated by using an artificial microRNA(amiRNA) approache do not show any phenotypic changes; whereas overexpression of BEDL2 and BEDL3, homologous genes of BED1, resulted in BED1-OX-like phenotypes, implying they are functionally redundant. In addition, consititutively activated components of the MAPK pathway including YODA, MKK4, and MKK5 can completely suppress the defective phenotypes of BED1-OX. These results reveal that BED1 may regulate inflorescence architecture by inhibiting cell proliferation and promoting primordium differentiation in a MAPK-dependent manner, and this process requires BAK1/SERKs, the co-receptors of ERECTA family.Genetic suppressors of bri1-9 was screened utilizing a Fox(Full-length cDNA over-expressing) gene hunting system. In this screen, we obtained a mutant, cli9-s1, showing proliferous shoot apical meristem and abnormal phyllotaxy. Constitutive overexpression of the non-catalytic subunit of nuclear DNA-dependent RNA polymerases, NRP(B/D/E)6A, recapitulates the cli9-s1 phenotype. Overexpression of both NRP(B/D/E)6A and its homologous gene, NRP(B/E)6B, do not influence the physiological function of RNA polymerases(Pol) IV and V, however, they cause diversiform and unstable defective phenotypes similar to those of nrpb2-1, a mutant of Pol II. But unfortunately, these phenotypes seem not to relate to BR biosynthesis or signaling. Single mutant of both NRP(B/D/E)6A and NRP(B/E)6B does not display visiable phenotypes, but their double mutant exhibit delayed embryo development and enlarged suspensor which ultimately leads to embryonic lethality. These results suggest NRP(B/D/E)6A plays crucial roles in meristem and embryo development.