RESUMEN
In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Estradiol/farmacología , GTP Fosfohidrolasas/clasificación , GTP Fosfohidrolasas/genética , Genoma de Planta , Factores de Intercambio de Guanina Nucleótido/clasificación , Factores de Intercambio de Guanina Nucleótido/genética , Membranas Intracelulares/metabolismo , Modelos Biológicos , Filogenia , Plantas Modificadas Genéticamente , Transporte de Proteínas , Transducción de Señal , Regulación hacia Arriba/efectos de los fármacos , Red trans-Golgi/metabolismoRESUMEN
Membrane vesicles delivered to the cell-division plane fuse with one another to form the partitioning membrane during plant cytokinesis, starting in the cell center. In Arabidopsis, this requires SNARE complexes involving the cytokinesis-specific Qa-SNARE KNOLLE. However, cytokinesis still occurs in knolle mutant embryos, suggesting contributions from KNOLLE-independent SNARE complexes. Here we show that Qa-SNARE SYP132, having counterparts in lower plants, functionally overlaps with the flowering plant-specific KNOLLE. SYP132 mutation causes cytokinesis defects, knolle syp132 double mutants consist of only one or a few multi-nucleate cells, and SYP132 has the same SNARE partners as KNOLLE. SYP132 and KNOLLE also have non-overlapping functions in secretion and in cellularization of the embryo-nourishing endosperm resulting from double fertilization unique to flowering plants. Evolutionarily ancient non-specialized SNARE complexes originating in algae were thus amended by the appearance of cytokinesis-specific SNARE complexes, meeting the high demand for membrane-fusion capacity during endosperm cellularization in angiosperms.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citocinesis/fisiología , Magnoliopsida/metabolismo , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Magnoliopsida/genética , Magnoliopsida/crecimiento & desarrollo , Mutación , Transporte de Proteínas , Proteínas SNARE/genéticaRESUMEN
Membrane trafficking is essential to fundamental processes in eukaryotic life, including cell growth and division. In plant cytokinesis, post-Golgi trafficking mediates a massive flow of vesicles that form the partitioning membrane but its regulation remains poorly understood. Here, we identify functionally redundant Arabidopsis ARF guanine-nucleotide exchange factors (ARF-GEFs) BIG1-BIG4 as regulators of post-Golgi trafficking, mediating late secretion from the trans-Golgi network but not recycling of endocytosed proteins to the plasma membrane, although the TGN also functions as an early endosome in plants. In contrast, BIG1-4 are absolutely required for trafficking of both endocytosed and newly synthesized proteins to the cell-division plane during cytokinesis, counteracting recycling to the plasma membrane. This change from recycling to secretory trafficking pathway mediated by ARF-GEFs confers specificity of cargo delivery to the division plane and might thus ensure that the partitioning membrane is completed on time in the absence of a cytokinesis-interphase checkpoint. DOI: http://dx.doi.org/10.7554/eLife.02131.001.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , División Celular , Endocitosis , Aparato de Golgi/metabolismo , Transporte de ProteínasRESUMEN
In Arabidopsis thaliana, the phytohormone auxin is an important patterning agent during embryogenesis and post-embryonic development, exerting effects through transcriptional regulation. The main determinants of the transcriptional auxin response machinery are AUXIN RESPONSE FACTOR (ARF) transcription factors and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) inhibitors. Although members of these two protein families are major developmental regulators, the transcriptional regulation of the genes encoding them has not been well explored. For example, apart from auxin-linked regulatory inputs, factors regulating the expression of the AUX/IAA BODENLOS (BDL)/IAA12 are not known. Here, it was shown that the HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) transcription factor HOMEOBOX PROTEIN 5 (HB5) negatively regulates BDL expression, which may contribute to the spatial control of BDL expression. As such, HB5 and probably other class I HD-ZIP proteins, appear to modulate BDL-dependent auxin response.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Ácidos Indolacéticos/metabolismo , Leucina Zippers , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The cell types of the plant root are first specified early during embryogenesis and are maintained throughout plant life. Auxin plays an essential role in embryonic root initiation, in part through the action of the ARF5/MP transcription factor and its auxin-labile inhibitor IAA12/BDL. MP and BDL function in embryonic cells but promote auxin transport to adjacent extraembryonic suspensor cells, including the quiescent center precursor (hypophysis). Here we show that a cell-autonomous auxin response within this cell is required for root meristem initiation. ARF9 and redundant ARFs, and their inhibitor IAA10, act in suspensor cells to mediate hypophysis specification and, surprisingly, also to prevent transformation to embryo identity. ARF misexpression, and analysis of the short suspensor mutant, demonstrates that lineage-specific expression of these ARFs is required for normal embryo development. These results imply the existence of a prepattern for a cell-type-specific auxin response that underlies the auxin-dependent specification of embryonic cell types.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Arabidopsis/metabolismo , Linaje de la Célula , Ácidos Indolacéticos/farmacología , Raíces de Plantas/embriología , Semillas/crecimiento & desarrollo , Factor 1 de Ribosilacion-ADP/metabolismo , Arabidopsis/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hibridación in Situ , Reguladores del Crecimiento de las Plantas/farmacología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Semillas/efectos de los fármacos , Semillas/metabolismo , Transducción de SeñalRESUMEN
Acquisition of cell identity in plants relies strongly on positional information, hence cell-cell communication and inductive signalling are instrumental for developmental patterning. During Arabidopsis embryogenesis, an extra-embryonic cell is specified to become the founder cell of the primary root meristem, hypophysis, in response to signals from adjacent embryonic cells. The auxin-dependent transcription factor MONOPTEROS (MP) drives hypophysis specification by promoting transport of the hormone auxin from the embryo to the hypophysis precursor. However, auxin accumulation is not sufficient for hypophysis specification, indicating that additional MP-dependent signals are required. Here we describe the microarray-based isolation of MP target genes that mediate signalling from embryo to hypophysis. Of three direct transcriptional target genes, TARGET OF MP 5 (TMO5) and TMO7 encode basic helix-loop-helix (bHLH) transcription factors that are expressed in the hypophysis-adjacent embryo cells, and are required and partially sufficient for MP-dependent root initiation. Importantly, the small TMO7 transcription factor moves from its site of synthesis in the embryo to the hypophysis precursor, thus representing a novel MP-dependent intercellular signal in embryonic root specification.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/embriología , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Desarrollo Embrionario/genética , Genes de Plantas/genética , Ácidos Indolacéticos/metabolismo , Meristema/citología , Meristema/embriología , Meristema/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Raíces de Plantas/citología , Transducción de SeñalRESUMEN
The Arabidopsis embryonic root meristem is initiated by the specification of a single cell, the hypophysis. This event critically requires the antagonistic auxin response regulators MONOPTEROS and BODENLOS, but their mechanism of action is unknown. We show that these proteins interact and transiently act in a small subdomain of the proembryo adjacent to the future hypophysis. Here they promote transport of auxin, which then elicits a second response in the hypophysis itself. Our results suggest that hypophysis specification is not the direct result of a primary auxin response but rather depends on cell-to-cell signaling triggered by auxin in adjacent cells.
Asunto(s)
Arabidopsis/embriología , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico Activo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Meristema/citología , Meristema/embriología , Meristema/metabolismo , Modelos Biológicos , Mutación , Raíces de Plantas/embriología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The plant hormone auxin elicits many specific context-dependent developmental responses. Auxin promotes degradation of Aux/IAA proteins that prevent transcription factors of the auxin response factor (ARF) family from regulating auxin-responsive target genes. Aux/IAAs and ARFs are represented by large gene families in Arabidopsis. Here we show that stabilization of BDL/IAA12 or its sister protein IAA13 prevents MP/ARF5-dependent embryonic root formation whereas stabilized SHY2/IAA3 interferes with seedling growth. Although both bdl and shy2-2 proteins inhibited MP/ARF5-dependent reporter gene activation, shy2-2 was much less efficient than bdl to interfere with embryonic root initiation when expressed from the BDL promoter. Similarly, MP was much more efficient than ARF16 in this process. When expressed from the SHY2 promoter, both shy2-2 and bdl inhibited cell elongation and auxin-induced gene expression in the seedling hypocotyl. By contrast, gravitropism and auxin-induced gene expression in the root, which were promoted by functionally redundant NPH4/ARF7 and ARF19 proteins, were inhibited by shy2-2, but not by bdl protein. Our results suggest that auxin signals are converted into specific responses by matching pairs of coexpressed ARF and Aux/IAA proteins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/embriología , Hipocótilo/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras GenéticasRESUMEN
Developmental responses to the plant hormone auxin are thought to be mediated by interacting pairs from two protein families: short-lived inhibitory IAA proteins and ARF transcription factors binding to auxin-response elements. monopteros mutants lacking activating ARF5 and the auxin-insensitive mutant bodenlos fail to initiate the root meristem during early embryogenesis. Here we show that the bodenlos phenotype results from an amino-acid exchange in the conserved degradation domain of IAA12. BODENLOS and MONOPTEROS interact in the yeast two-hybrid assay and the two genes are coexpressed in early embryogenesis, suggesting that BODENLOS inhibits MONOPTEROS action in root meristem initiation.