RESUMO
Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.
Assuntos
Arabidopsis/enzimologia , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Fosfatidilserinas/metabolismo , Pólen/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Nucléolo Celular/metabolismo , DNA Complementar/genética , Retículo Endoplasmático/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/ultraestrutura , Pólen/enzimologia , Pólen/genética , Pólen/ultraestrutura , Regiões Promotoras Genéticas/genética , RNA de Plantas/genética , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Deleção de SequênciaRESUMO
Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.