Summary
The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes.We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65–1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations.yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4).The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)–glutamic acid (E)–phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization.
γ-Tubulin is required for microtubule (MT) nucleation at MT organizing centers such as centrosomes or spindle pole bodies, but little is known about its noncentrosomal functions. We conditionally downregulated γ-tubulin by inducible expression of RNA interference (RNAi) constructs in Arabidopsis thaliana. Almost complete RNAi depletion of γ-tubulin led to the absence of MTs and was lethal at the cotyledon stage. After induction of RNAi expression, γ-tubulin was gradually depleted from both cytoplasmic and microsomal fractions. In RNAi plants with partial loss of γ-tubulin, MT recovery after drug-induced depolymerization was impaired. Similarly, immunodepletion of γ-tubulin from Arabidopsis extracts severely compromised in vitro polymerization of MTs. Reduction of γ-tubulin protein levels led to randomization and bundling of cortical MTs. This finding indicates that MT-bound γ-tubulin is part of a cortical template guiding the microtubular network and is essential for MT nucleation. Furthermore, we found that cells with decreased levels of γ-tubulin could progress through mitosis, but cytokinesis was strongly affected. Stepwise diminution of γ-tubulin allowed us to reveal roles for MT nucleation in plant development, such as organization of cell files, anisotropic and polar tip growth, and stomatal patterning. Some of these functions of γ-tubulin might be independent of MT nucleation.
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