RESUMO
It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Homeostase , Concentração de Íons de Hidrogênio , Immunoblotting , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mutação , Osmose , Fotossíntese/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/ultraestrutura , Antiportadores de Potássio-Hidrogênio/classificação , Antiportadores de Potássio-Hidrogênio/genética , Tilacoides/química , Tilacoides/metabolismoRESUMO
The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K(+) content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K(+)/H(+) exchange activity but very little Na(+/)H(+) exchange compared with controls transformed with the empty vector; Na(+)/H(+) exchange was not detected with concentrations of less than 37.5 mM Na(+) in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+)/H(+) antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+) homeostasis.
Assuntos
Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Potássio/metabolismo , Prótons , Triticum/metabolismo , Adaptação Fisiológica , Arabidopsis/genética , Arabidopsis/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Raízes de Plantas/genética , Antiportadores de Potássio-Hidrogênio/classificação , Antiportadores de Potássio-Hidrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tolerância ao Sal , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologia , Triticum/genéticaRESUMO
The Saccharomyces cerevisiae genome contains three genes encoding alkali metal cation/H+ antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Systematic genome sequencing of other yeast species revealed highly conserved homologous ORFs in all of them. We compared the yeast sequences both at DNA and protein levels. The subfamily of yeast endosomal/prevacuolar Nhx1 antiporters is closely related to mammalian plasma membrane NHE proteins and to both plasma membrane and vacuolar plant antiporters. The high sequence conservation within this subfamily of yeast antiporters suggests that Nhx1p is of great importance in cell physiology. Yeast Kha1 proteins probably belong to the same subfamily as bacterial antiporters, whereas Nhal proteins form a distinct subfamily.
