RESUMO
The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na+ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.
Assuntos
Chenopodiaceae , Proteínas de Plantas , Tolerância ao Sal , Chenopodiaceae/metabolismo , Chenopodiaceae/genética , Chenopodiaceae/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Vacúolos/metabolismo , Salinidade , Cloreto de Sódio/farmacologia , Cloreto de Sódio/metabolismo , Retículo Endoplasmático/metabolismo , Estresse Salino , Proteômica , Nicotiana/metabolismo , Nicotiana/genética , Nicotiana/efeitos dos fármacos , TranscriptomaRESUMO
Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery. hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher K(cat) and lower K(m) values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 null mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Trifosfato de Adenosina/metabolismo , Alelos , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Proteínas de Ciclo Celular/genética , Clonagem Molecular , Ativação Enzimática , Teste de Complementação Genética , Pleiotropia Genética , MicroRNAs/genética , Dados de Sequência Molecular , Fenótipo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genéticaRESUMO
Heritable epigenetic inactivation of the maize Suppressor-mutator (Spm) transposon is associated with promoter methylation, and its reversal is mediated by the transposon-encoded TnpA protein. We have developed an assay that permits demethylation of the Spm sequence to be controlled by inducing the expression of TnpA in plant cells. Using this assay, we show that demethylation is a rapid, active process. TnpA is a weak transcriptional activator, and deletions that abolish its transcriptional activity also eliminate its demethylation activity. We show that cell cycle and DNA synthesis inhibitors interfere with TnpA-mediated Spm demethylation. We further show that TnpA has a much lower affinity for fully methylated than for hemimethylated or unmethylated DNA fragments derived from Spm termini. Based on these observations, we suggest that TnpA binds to the postreplicative, hemimethylated Spm sequence and promotes demethylation either by creating an appropriate demethylation substrate or by itself participating in or recruiting a demethylase.