RESUMEN
Auxins are hormones involved in many cellular, physiological and developmental processes in seed plants and in mosses such as Physcomitrella patens. Control of auxin levels is achieved in higher plants via synthesis of auxin conjugates by members of the GH3 family. The role of the two GH3-like proteins from P. patens for growth and auxin homeostasis was therefore analysed. The in vivo-function of the two P. patens GH3 genes was investigated using single and double knockout mutants. The two P. patens GH3 proteins were also heterologously expressed to determine their enzymatic activity. Both P. patens GH3 enzymes accepted the auxin indole acetic acid (IAA) as substrate, but with different preferences for the amino acid to which it is attached. Cytoplasmic localization was shown for PpGH3-1 tagged with green fluorescent protein (GFP). Targeted knock-out of either gene exhibited an increased sensitivity to auxin, resulting in growth inhibition. On plain mineral media mutants had higher levels of free IAA and less conjugated IAA than the wild type, and this effect was enhanced when auxin was supplied. The DeltaPpGH3-1/DeltaPpGH3-2 double knockout had almost no IAA amide conjugates but still synthesized ester conjugates. Taken together, these data suggest a developmentally controlled involvement of P. patens GH3 proteins in auxin homeostasis by conjugating excess of physiologically active free auxin to inactive IAA-amide conjugates.
Asunto(s)
Bryopsida/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Bryopsida/genética , Ciclopentanos/metabolismo , ADN de Plantas , Expresión Génica , Técnicas de Inactivación de Genes , Homeostasis , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/genética , Proteínas de Plantas/genéticaRESUMEN
Auxin-induced gene expression is described for a variety of different genes including the SAUR-, Aux/IAA- and GH3-families, members of which have been found in seed plants. The precise function of GH3-like proteins in plant development is not well characterised yet. Mutant analysis in Arabidopsis thaliana indicates a possible role for GH3-like proteins in connecting auxin and light signal transduction. Here, we report the isolation of three different GH3-like homologues from a lower land plant, the moss Physcomitrella patens. Two of the GH3-like homologues were chosen for further characterisation. Both genes are expressed in gametophytic tissues, with expression starting very early in moss development. Knockout plants were generated and analysed. In comparison to white-light growth, cultivation of the wild type and knockout plants under red-light conditions resulted in a delay in gametophytic tissue development. The leafy moss plants displayed an elongated phenotype. Growth delay and elongation were even stronger under far-red light conditions. No obvious differences between wild type and knockout plants could be detected under the examined conditions, indicating functional redundancy of the two genes.
Asunto(s)
Bryopsida/genética , Fitocromo/metabolismo , Secuencia de Bases , Bryopsida/crecimiento & desarrollo , Bryopsida/efectos de la radiación , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutación , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Reproducción/efectos de la radiación , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Plant body plans arise by the activity of meristematic growing tips during development and radiated independently in the gametophyte (n) and sporophyte (2n) stages of the life cycle during evolution. Although auxin and its intercellular transport by PIN family efflux carriers are primary regulators of sporophytic shoot development in flowering plants, the extent of conservation in PIN function within the land plants and the mechanisms regulating bryophyte gametophytic shoot development are largely unknown. RESULTS: We have found that treating gametophytic shoots of the moss Physcomitrella patens with exogenous auxins and auxin transport inhibitors disrupts apical function and leaf development. Two plasma membrane-targeted PIN proteins are expressed in leafy shoots, and pin mutants resemble plants treated with auxins or auxin transport inhibitors. PIN-mediated auxin transport regulates apical cell function, leaf initiation, leaf shape, and shoot tropisms in moss gametophytes. pin mutant sporophytes are sometimes branched, reproducing a phenotype only previously seen in the fossil record and in rare natural moss variants. CONCLUSIONS: Our results show that PIN-mediated auxin transport is an ancient, conserved regulator of shoot development.