RESUMO
CtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations, and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana, although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN would function in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, precise verification has been lacking. In this paper, we isolated a homologous gene (MAN) of AN from liverwort, Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completely complemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that exists in AN proteins isolated from other plant species. We constructed several plasmids for expressing modified ANs with amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutations in the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examined localization of AN using several techniques. Our results demonstrated that AN localizes on punctuate structures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted an unexpected link between leaf development and membrane trafficking. We should reconsider the roles and evolutionary traits of AN based on these findings.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Marchantia/genética , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Genes de Plantas , Genes Reporter , Teste de Complementação Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Marchantia/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Meristema/metabolismo , Meristema/ultraestrutura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/metabolismo , Fenótipo , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Especificidade da Espécie , Transformação Genética , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.