RESUMO
Nitrate (NO3- ) is a source of plant nutrients and osmolytes, but its delivery machineries under osmotic and low-nutrient stress remain largely unknown. Here, we report that AtICln, an Arabidopsis homolog of the nucleotide-sensitive chloride-conductance regulatory protein family (ICln), is involved in response to osmotic and low-NO3- stress. The gene AtICln, encoding plasma membrane-anchored proteins, was upregulated by various osmotic stresses, and its disruption impaired plant tolerance to osmotic stress. Compared with the wild type, the aticln mutant retained lower anions, particularly NO3- , and its growth retardation was not rescued by NO3- supply under osmotic stress. Interestingly, this mutant also displayed growth defects under low-NO3 stress, which were accompanied by decreases in NO3- accumulation, suggesting that AtICln may facilitate the NO3- accumulation under NO3- deficiency. Moreover, the low-NO3- hypersensitive phenotype of aticln mutant was overridden by the overexpression of NRT1.1, an important NO3- transporter in Arabidopsis low-NO3- responses. Further genetic analysis in the plants with altered activity of AtICln and NRT1.1 indicated that AtICln and NRT1.1 play a compensatory role in maintaining NO3- homeostasis under low-NO3- environments. These results suggest that AtICln is involved in cellular NO3- accumulation and thus determines osmotic adjustment and low-NO3- tolerance in plants.
Assuntos
Adaptação Fisiológica , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitratos/metabolismo , Osmose , Homologia de Sequência de Aminoácidos , Proteínas de Transporte de Ânions/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Teste de Complementação Genética , Mutação/genética , Concentração Osmolar , Pressão Osmótica , Fenótipo , Proteínas de Plantas/metabolismo , Frações Subcelulares/metabolismoRESUMO
Magnesium (Mg2+) is an essential nutrient in all organisms. However, high levels of Mg2+ in the environment are toxic to plants. In this study, we identified the vacuolar-type Hâº-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsis avp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg2+. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg2+ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar Hâº-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg2+ homeostasis. Taken together, our results suggest AVP1 is required for cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Pirofosfatase Inorgânica/metabolismo , Magnésio/toxicidade , Vacúolos/enzimologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Teste de Complementação Genética , Homeostase , Pirofosfatase Inorgânica/genética , Mutação/genética , Fenótipo , Vacúolos/efeitos dos fármacosRESUMO
Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are highly conserved endoplasmic reticulum (ER)-resident proteins that establish ER contacts with multiple membrane compartments in many eukaryotes. However, VAP-mediated membrane-tethering mechanisms remain ambiguous. Here, focusing on fission yeast ER-plasma membrane (PM) contact formation, using systematic interactome analyses and quantitative microscopy, we predict a non-VAP-protein direct binding-based ER-PM coupling. We further reveal that VAP-anionic phospholipid interactions may underlie ER-PM association and define the pH-responsive nature of VAP-tethered membrane contacts. Such conserved interactions with anionic phospholipids are generally defective in amyotrophic lateral sclerosis-associated human VAPB mutant. Moreover, we identify a conserved FFAT-like motif locating at the autoinhibitory hotspot of the essential PM proton pump Pma1. This modulatory VAP-Pma1 interaction appears crucial for pH homeostasis. We thus propose an ingenious strategy for maintaining intracellular pH by coupling Pma1 modulation with pH-sensory ER-PM contacts via VAP-mediated interactions.
Assuntos
Membrana Celular , Retículo Endoplasmático , Homeostase , Schizosaccharomyces , Retículo Endoplasmático/metabolismo , Concentração de Íons de Hidrogênio , Membrana Celular/metabolismo , Humanos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Ligação Proteica , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Mutação , Esclerose Lateral Amiotrófica/metabolismoRESUMO
Chlorophyll (Chl) is essential for photosynthetic reactions and chloroplast development. While the enzymatic pathway for Chl biosynthesis is well established, the regulatory mechanism underlying the homeostasis of Chl levels remains largely unknown. In this study, we identified CBD1 (Chlorophyll Biosynthetic Defect1), which functions in the regulation of chlorophyll biosynthesis. The CBD1 gene was expressed specifically in green tissues and its protein product was embedded in the thylakoid membrane. Furthermore, CBD1 was precisely co-expressed and functionally correlated with GUN5 (Genome Uncoupled 5). Analysis of chlorophyll metabolic intermediates indicated that cbd1 and cbd1gun5 mutants over-accumulated magnesium protoporphyrin IX (Mg-Proto IX). In addition, the cbd1 mutant thylakoid contained less Mg than the wild type not only as a result of lower Chl content, but also implicating CBD1 in Mg transport. This was supported by the finding that CBD1 complemented a Mg2+ uptake-deficient Salmonella strain under low Mg conditions. Taken together, these results indicate that CBD1 functions synergistically with CHLH/GUN5 in Mg-Proto IX processing, and may serve as a Mg-transport protein to maintain Mg homeostasis in the chloroplast.