RESUMEN
Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfolípidos/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Citoplasma/metabolismo , Exocitosis , Proteómica/métodosRESUMEN
Localized delivery of plasma-membrane and cell-wall components is a crucial process for plant cell growth. One of the regulators of secretory-vesicle targeting is the exocyst tethering complex. The exocyst mediates first interaction between transport vesicles and the target membrane before their fusion is performed by SNARE proteins. In land plants, genes encoding the EXO70 exocyst subunit underwent an extreme proliferation with 23 paralogs present in the Arabidopsis (Arabidopsis thaliana) genome. These paralogs often acquired specialized functions during evolution. Here, we analyzed functional divergence of selected EXO70 paralogs in Arabidopsis. Performing a systematic cross-complementation analysis of exo70a1 and exo70b1 mutants, we found that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2. In contrast, none of the EXO70 isoforms tested were able to substitute EXO70B1, including its closest relative, EXO70B2, pointing to a unique function of this isoform. The presented results document a high degree of functional specialization within the EXO70 gene family in land plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Exocitosis , Regulación de la Expresión Génica de las Plantas , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
In angiosperms, the process of pollination relies on species-specific interaction and signaling between the male (pollen) and female (pistil) counterparts where the interplay between several pollen and stigma proteins decides the fate of the pollen. In Brassicaceae, the dry stigmatic papillary cells control pollen germination by releasing resources only to compatible pollen thereby allowing pollen to hydrate and germinate. Despite the identification of a number of stigmatic proteins that facilitate pollination responses, the signaling mechanisms that regulate functions of these proteins have remained unknown. Here, we show that, in Arabidopsis, an extremely functionally redundant mitogen-activated protein kinase (MAPK) cascade is required for maintaining stigma receptivity to accept compatible pollen. Our genetic analyses demonstrate that in stigmas, five MAPK kinases (MKKs), MKK1/2/3/7/9 are required to transmit upstream signals to two MPKs, MPK3/4, to mediate compatible pollination. Compromised functions of these five MKKs in the quintuple mutant (mkk1/2/3RNAi/mkk7/9) phenocopied pollination defects observed in the mpk4RNAi/mpk3 double mutant. We further show that this MAPK nexus converges on Exo70A1, a previously identified stigma receptivity factor essential for pollination. Given that pollination is the crucial initial step during plant reproduction, understanding the mechanisms that govern successful pollination could lead to development of strategies to improve crop yield.
Asunto(s)
Arabidopsis/metabolismo , Flores/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Fosforilación , Polinización/fisiología , Serina/metabolismoRESUMEN
Erigeron breviscapus (Vant.) Hand.-Mazz. is a famous traditional Chinese medicine that has positive effects on the treatment of cardiovascular and cerebrovascular diseases. With the increase of market demand (RMB 500 million per year) and the sharp decrease of wild resources, it is an urgent task to cultivate high-quality and high-yield varieties of E. breviscapus. However, it is difficult to obtain homozygous lines in breeding due to the self-incompatibility (SI) of E. breviscapus. Here, we first proved that E. breviscapus has sporophyte SI (SSI) characteristics. Characterization of the ARC1 gene in E. breviscapus showed that EbARC1 is a constitutive expression gene located in the nucleus. Overexpression of EbARC1 in Arabidopsis thaliana L. (Col-0) could cause transformation of transgenic lines from self-compatibility (SC) into SI. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that EbARC1 and EbExo70A1 interact with each other in the nucleus, and the EbARC1-ubox domain and EbExo70A1-N are the key interaction regions, suggesting that EbARC1 may ubiquitinate EbExo70A to regulate SI response. This study of the SSI mechanism in E. breviscapus has laid the foundation for further understanding SSI in Asteraceae and breeding E. breviscapus varieties.
Asunto(s)
Arabidopsis , Erigeron/genética , Proteínas de Plantas , Plantas Modificadas Genéticamente , Ubiquitina-Proteína Ligasas , Arabidopsis/enzimología , Arabidopsis/genética , Erigeron/enzimología , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
ROOT HAIR DEFECTIVE SIX-LIKE4 (RSL4) is necessary and sufficient for root hair elongation in Arabidopsis thaliana. Root hair length is determined by the duration for which RSL4 protein is present in the developing root hair. The aim of this research was to identify genes regulated by RSL4 that affect root hair growth. To identify genes regulated by RSL4, we identified genes whose expression was elevated by induction of RSL4 activity in the presence of an inhibitor of translation. Thirty-four genes were identified as putative targets of RSL transcriptional regulation, and the results suggest that the activities of SUPPRESSOR OF ACTIN (SAC1), EXOCSYT SUBUNIT 70A1 (EXO70A1), PEROXIDASE7 (PRX7) and CALCIUM-DEPENDENT PROTEIN KINASE11 (CPK11) are required for root hair elongation. These data indicate that RSL4 controls cell growth by controlling the expression of genes encoding proteins involved in cell signalling, cell wall modification and secretion.