RESUMEN
A-type ATP-binding cassette (ABCA) proteins transport lipids and lipid-based molecules in humans, and their malfunction is associated with various inherited diseases. Although plant genomes encode many ABCA transporters, their molecular and physiological functions remain largely unknown. Seeds are rapidly developing organs that rely on the biosynthesis and transport of large quantities of lipids to generate new membranes and storage lipids. In this study, we characterized the Arabidopsis (Arabidopsis thaliana) ABCA10 transporter, which is selectively expressed in female gametophytes and early developing seeds. By 3 d after flowering (DAF), seeds from the abca10 loss-of-function mutant exhibited a smaller chalazal endosperm than those of the wild-type. By 4 DAF, their endosperm nuclei occupied a smaller area than those of the wild-type. The endosperm nuclei of the mutants also failed to distribute evenly inside the seed coat and stayed aggregated instead, possibly due to inadequate expansion of abca10 endosperm. This endosperm defect might have retarded abca10 embryo development. At 7 DAF, a substantial portion of abca10 embryos remained at the globular or earlier developmental stages, whereas wild-type embryos were at the torpedo or later stages. ABCA10 is likely involved in lipid metabolism, as ABCA10 overexpression induced the overaccumulation of triacylglycerol but did not change the carbohydrate or protein contents in seeds. In agreement, ABCA10 localized to the endoplasmic reticulum (ER), the major site of lipid biosynthesis. Our results reveal that ABCA10 plays an essential role in early seed development, possibly by transporting substrates for lipid metabolism to the ER.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Adenosina Trifosfato/metabolismo , Proteínas de Arabidopsis/metabolismo , Endospermo/metabolismo , Regulación de la Expresión Génica de las Plantas , Humanos , Lípidos/análisis , SemillasRESUMEN
Germination requires sufficient water absorption by seeds, but excessive water in the soil inhibits plant growth. We therefore hypothesized that tolerance mechanisms exist that help young seedlings survive and develop in waterlogged conditions. Many ATP-BINDING CASSETTE TRANSPORTER subfamily G (ABCG) proteins protect terrestrial plants from harsh environmental conditions. To establish whether any of these proteins facilitate plant development under waterlogged conditions, we observed the early seedling growth of many ABCG transporter mutants under waterlogged conditions. abcg5 seedlings exhibited severe developmental problems under waterlogged conditions: the shoot apical meristem was small, and the seedling failed to develop true leaves. The seedlings had a high water content and reduced buoyancy on water, suggesting that they were unable to retain air spaces on and inside the plant. Supporting this possibility, abcg5 cotyledons had increased cuticle permeability, reduced cuticular wax contents, and a much less dense cuticle layer than the wild-type. These results indicate that proper development of plants under waterlogged conditions requires the dense cuticle layer formed by ABCG5 activity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Meristema/metabolismo , Hojas de la Planta/metabolismo , Plantones/metabolismoRESUMEN
KEY MESSAGE: The non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Citocininas/metabolismo , Retículo Endoplásmico/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Secuencias de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Citocininas/genética , Retículo Endoplásmico/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Técnicas de Inactivación de Genes , Luz , Filogenia , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Unión Proteica , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/efectos de la radiaciónRESUMEN
Tip-focused accumulation of reactive oxygen species (ROS) is tightly associated with pollen tube growth and is thus critical for fertilization. However, it is unclear how tip-growing cells establish such specific ROS localization. Polyamines have been proposed to function in tip growth as precursors of the ROS, hydrogen peroxide. The ABC transporter AtABCG28 may regulate ROS status, as it contains multiple cysteine residues, a characteristic of proteins involved in ROS homeostasis. In this study, we found that AtABCG28 was specifically expressed in the mature pollen grains and pollen tubes. AtABCG28 was localized to secretory vesicles inside the pollen tube that moved toward and fused with the plasma membrane of the pollen tube tip. Knocking out AtABCG28 resulted in defective pollen tube growth, failure to localize polyamine and ROS to the growing pollen tube tip, and complete male sterility, whereas ectopic expression of this gene in root hair could recover ROS accumulation at the tip and improved the growth under high-pH conditions, which normally prevent ROS accumulation and tip growth. Together, these data suggest that AtABCG28 is critical for localizing polyamine and ROS at the growing tip. In addition, this function of AtABCG28 is likely to protect the pollen tube from the cytotoxicity of polyamine and contribute to the delivery of polyamine to the growing tip for incorporation into the expanding cell wall.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Tubo Polínico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Tubo Polínico/crecimiento & desarrollo , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
When seeds are exposed to drought and salinity during germination, newly germinated embryos stop growth and enter a quiescent state called postgerminative growth (PGG) arrest. PGG arrest protects embryos from the stress, but it is not known how PGG is restored from the arrest when stress is eased. In this study, we show that under stress- or abscisic acid-induced PGG arrest conditions, Arabidopsis thaliana Raf-type protein kinase 22 (AtRaf22) overexpression accelerated photoautotrophic seedling establishment, whereas atraf22 knockout mutations enhanced the arrest. Furthermore, when the stress intensity was reduced subsequently, AtRaf22 overexpression plants resumed growth and accomplished photoautotrophic transition much faster than the knockout or wild-type plants. These results suggest that AtRaf22 activity is important for maintaining growth capacity during stress-induced PGG arrest, which is most likely critical for competitive growth when the stress subsides and plants resume growth. Such a factor has not been reported before.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Técnicas de Silenciamiento del Gen , Germinación , Plantones/enzimología , Plantones/crecimiento & desarrollo , Plantones/fisiología , Estrés FisiológicoRESUMEN
Seed oil is important not only for human and animal nutrition, but also for various industrial applications. Numerous genetic engineering strategies have been attempted to increase the oil content per seed, but few of these strategies have involved manipulating the transporters. Pyruvate is a major source of carbon for de novo fatty acid biosynthesis in plastids, and the embryo's demand for pyruvate is reported to increase during active oil accumulation. In this study, we tested our hypothesis that oil biosynthesis could be boosted by increasing pyruvate flux into plastids. We expressed the known plastid-localized pyruvate transporter BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2 (BASS2) under the control of a seed-specific soybean (Glycine max) glycinin-1 promoter in Arabidopsis thaliana. The resultant transgenic Arabidopsis plants (OEs), which expressed high levels of BASS2, produced seeds that were larger and heavier and contained 10-37% more oil than those of the wild type (WT), but were comparable to the WT seeds in terms of protein and carbohydrate contents. The total seed number did not differ significantly between the WT and OEs. Therefore, oil yield per plant was increased by 24-43% in the OE lines compared to WT. Taken together, our results demonstrate that seed-specific overexpression of the pyruvate transporter BASS2 promotes oil production in Arabidopsis seeds. Thus, manipulating the level of specific transporters is a feasible approach for increasing the seed oil content.
RESUMEN
The Arabidopsis ATP-Binding Cassette (ABC) transporter ABCC1 sequesters arsenic (As)-phytochelatin conjugates into the vacuole, thereby conferring As resistance. Here, we report the results of a screen for phosphorylation-dependent regulation sites of AtABCC1. Variants of AtABCC1 harboring mutations that replaced amino acid residues Tyr682 , Tyr709 , Tyr822 , Ser846 , Ser1278 , or Thr1408 with alanine confer reduced resistance and decrease the intracellular As content relative to wild-type AtABCC1 when heterologously expressed in the SM7 yeast strain. This suggests that these mutations compromise the vacuolar sequestration of As by AtABCC1. Furthermore, through a phosphomimic mutant study, we found that phosphorylation of Ser846 is required for the As resistance function of AtABCC1. Our analysis provides a first clue as to the phosphorylation-mediated regulation of AtABCC1 activity.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Aminoácidos/genética , Proteínas de Arabidopsis/genética , Arsénico/toxicidad , Resistencia a Medicamentos/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Western Blotting , Mutación Missense , Fosforilación , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismoRESUMEN
Plants reorganize their root architecture to avoid growth into unfavorable regions of the rhizosphere. In a screen based on chimeric repressor gene-silencing technology, we identified the Arabidopsis thaliana GeBP-LIKE 4 (GPL4) transcription factor as an inhibitor of root growth that is induced rapidly in root tips in response to cadmium (Cd). We tested the hypothesis that GPL4 functions in the root avoidance of Cd by analyzing root proliferation in split medium, in which only half of the medium contained toxic concentrations of Cd. The wild-type (WT) plants exhibited root avoidance by inhibiting root growth in the Cd side but increasing root biomass in the control side. By contrast, GPL4-suppression lines exhibited nearly comparable root growth in the Cd and control sides and accumulated more Cd in the shoots than did the WT. GPL4 suppression also altered the root avoidance of toxic concentrations of other essential metals, modulated the expression of many genes related to oxidative stress, and consistently decreased reactive oxygen species concentrations. We suggest that GPL4 inhibits the growth of roots exposed to toxic metals by modulating reactive oxygen species concentrations, thereby allowing roots to colonize noncontaminated regions of the rhizosphere.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metales Pesados/toxicidad , Raíces de Plantas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Transporte Biológico/efectos de los fármacos , Biomasa , Recuento de Células , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Glutatión/farmacología , Meristema/citología , Meristema/efectos de los fármacos , Meristema/metabolismo , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
KEY MESSAGE: Two Arabidopsis ABC transporters, ABCG1 and ABCG16, are expressed in the tapetal layer, specifically after postmeiotic microspore release, and play important roles in pollen surface development. The male gametophytic cells of terrestrial plants, the pollen grains, travel far before fertilization, and thus require strong protective layers, which take the form of a pollen coat and a pollen wall. The protective surface structures are generated by the tapetum, the tissue surrounding the developing gametophytes. Many ABC transporters, including Arabidopsis thaliana ABCG1 and ABCG16, have been shown to play essential roles in the development of such protective layers. However, the details of the mechanism of their function remain to be clarified. In this study, we show that ABCG1 and ABCG16 are localized at the plasma membrane of tapetal cells, specifically after postmeiotic microspore release, and play critical roles in the postmeiotic stages of male gametophyte development. Consistent with this stage-specific expression, the abcg1 abcg16 double knockout mutant exhibited defects in pollen development after postmeiotic microspore release; their microspores lacked intact nexine and intine layers, exhibited defects in pollen mitosis I, displayed ectopic deposits of arabinogalactan proteins, failed to complete cytokinesis, and lacked sperm cells. Interestingly, the double mutant exhibited abnormalities in the internal structures of tapetal cells, too; the storage organelles of tapetal cells, tapetosomes and elaioplasts, were morphologically altered. Thus, this work reveals that the lack of ABCG1 and ABCG16 at the tapetal cell membrane causes a broad range of defects in pollen, as well as in tapetal cells themselves. Furthermore, these results suggest that normal pollen surface development is necessary for normal development of the pollen cytoplasm.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Meiosis , Proteínas de la Membrana/metabolismo , Polen/citología , Polen/crecimiento & desarrollo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Membrana Celular/metabolismo , Pared Celular/metabolismo , Mitosis , Mucoproteínas/metabolismo , Mutación/genética , Proteínas de Plantas/metabolismo , Polen/ultraestructuraRESUMEN
Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plant's lifestyle, by transporting various compounds across specific membranes of the plant.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Adaptación Fisiológica , Animales , Fenómenos Fisiológicos de las PlantasRESUMEN
Stomata are the tiny valves on the plant surface that mediate gas exchange between the plant and its environment. Stomatal opening needs to be tightly regulated to facilitate CO2 uptake and prevent excess water loss. Plant Rho-type (ROP) GTPase 2 (ROP2) is a molecular component of the system that negatively regulates light-induced stomatal opening. Previously, ROP-interactive Cdc42- and Rac-interactive binding motif-containing protein 7 (RIC7) was suggested to function downstream of ROP2. However, the underlying molecular mechanism remains unknown. To understand the mechanism by which RIC7 regulates light-induced stomatal opening, we analyzed the stomatal responses of ric7 mutant Arabidopsis plants and identified the target protein of RIC7 using a yeast two-hybrid screen. Light-induced stomatal opening was promoted by ric7 knockout, whereas it was inhibited by RIC7 overexpression, indicating that RIC7 negatively regulates stomatal opening in Arabidopsis. RIC7 interacted with exocyst subunit Exo70 family protein B1 (Exo70B1), a component of the vesicle trafficking machinery. RIC7 and Exo70B1 localized to the plasma membrane region under light or constitutively active ROP2 conditions. The knockout mutant of Exo70B1 and ric7/exo70b1 exhibited retarded light-induced stomatal opening. Our results suggest that ROP2 and RIC7 suppress excess stomatal opening by inhibiting Exo70B1, which most likely participates in the vesicle trafficking required for light-induced stomatal opening.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/metabolismo , Estomas de Plantas/fisiología , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Proteínas de Unión al GTP/genética , Técnicas de Inactivación de Genes , Aparato de Golgi/metabolismo , Luz , Plantas Modificadas Genéticamente , Transducción de Señal , Proteínas de Transporte Vesicular/genéticaRESUMEN
Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)-induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H(+)-ATPase vha-a2 vha-a3 and vacuolar H(+)-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution.
Asunto(s)
Arabidopsis/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Estomas de Plantas/metabolismo , Vacuolas/metabolismo , Ácido Abscísico/farmacología , Aminopiridinas/farmacología , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Butiratos/farmacología , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Microscopía Confocal , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/química , Vacuolas/efectos de los fármacos , Vicia faba/citología , Vicia faba/genética , Vicia faba/metabolismoRESUMEN
Auxin and abscisic acid (ABA) modulate numerous aspects of plant development together, mostly in opposite directions, suggesting that extensive crosstalk occurs between the signalling pathways of the two hormones. However, little is known about the nature of this crosstalk. We demonstrate that ROP-interactive CRIB motif-containing protein 1 (RIC1) is involved in the interaction between auxin- and ABA-regulated root growth and lateral root formation. RIC1 expression is highly induced by both hormones, and expressed in the roots of young seedlings. Whereas auxin-responsive gene induction and the effect of auxin on root growth and lateral root formation were suppressed in the ric1 knockout, ABA-responsive gene induction and the effect of ABA on seed germination, root growth and lateral root formation were potentiated. Thus, RIC1 positively regulates auxin responses, but negatively regulates ABA responses. Together, our results suggest that RIC1 is a component of the intricate signalling network that underlies auxin and ABA crosstalk.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Ácidos Indolacéticos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Transducción de Señal , Ácido Abscísico/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Germinación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ácidos Indolacéticos/farmacología , Proteínas Asociadas a Microtúbulos/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Mapeo de Interacción de Proteínas , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Semillas/genética , Semillas/metabolismoRESUMEN
ROP GTPases function as molecular switches in diverse cellular processes. Previously, we showed that ROP2 GTPase is activated upon light irradiation, and thereby negatively regulates light-induced stomatal opening. Here we studied the role of ROP2 during stomatal closure. The expression of a constitutively active form of ROP2 (CA-rop2) in Arabidopsis thaliana and Vicia faba resulted in slower and reduced stomatal closure in response to abscisic acid (ABA) and CO(2) . In contrast, the expression of a dominant-negative form of ROP2 (DN-rop2) and the knockout mutation of ROP2 (rop2 KO) promoted ABA-induced stomatal closure in Arabidopsis. As early as 10 min after ABA treatment, ROP2 was inactivated and translocated to the cytoplasm of the stomatal guard cells. To elucidate the mechanism by which active ROP2 suppresses stomatal closure, we monitored endocytotic membrane trafficking, which is regulated by Rho GTPases in animal cells. We found that the endocytosis of plasma membrane (PM), as tracked by FM4-64, was lower in CA-rop2-expressing guard cells than in those of wild-type plants, which suggests that active ROP2 suppresses the endocytotic internalization of PM, a process required for stomatal closure. Together, our results suggest that ROP2 is inactivated by ABA, and that this inactivation is required for the timely stomatal closure.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Dióxido de Carbono/metabolismo , Proteínas de Unión al GTP/metabolismo , Estomas de Plantas/fisiología , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Citoplasma/metabolismo , Sequías , Endocitosis , Proteínas de Unión al GTP/genética , Técnicas de Inactivación de Genes , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Vicia faba/enzimología , Vicia faba/genética , Vicia faba/fisiología , Agua/fisiologíaRESUMEN
The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Biopolímeros/metabolismo , Carotenoides/metabolismo , Polen/crecimiento & desarrollo , Transportador de Casetes de Unión a ATP, Subfamilia G , Transportadoras de Casetes de Unión a ATP/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Infertilidad Vegetal , Polen/genética , Polen/metabolismo , Polen/ultraestructura , ARN de Planta/genética , Eliminación de SecuenciaRESUMEN
Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Membrana Celular/metabolismo , Cartilla de ADN/genética , Sequías , Genes de Plantas , Prueba de Complementación Genética , Mutación , Estomas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estrés FisiológicoRESUMEN
Rapid tip growth allows for efficient development of highly elongated cells (e.g. neuronal axons, fungal hyphae and pollen tubes) and requires an elaborate spatiotemporal regulation of the growing region. Here, we use the pollen tube as a model to investigate the mechanism regulating the growing region. ROPs (Rho-related GTPases from plants) are essential for pollen tip growth and display oscillatory activity changes in the apical plasma membrane (PM). By manipulating the ROP activity level, we showed that the PM distribution of ROP activity as an apical cap determines the tip growth region and that efficient tip growth requires an optimum level of the apical ROP1 activity. Excessive ROP activation induced the enlargement of the tip growth region, causing growth depolarization and reduced tube elongation. Time-lapse analysis suggests that the apical ROP1 cap is generated by lateral propagation of a localized ROP activity. Subcellular localization and gain- and loss-of-function analyses suggest that RhoGDI- and RhoGAP-mediated global inhibition limits the lateral propagation of apical ROP1 activity. We propose that the balance between the lateral propagation and the global inhibition maintains an optimal apical ROP1 cap and generates the apical ROP1 activity oscillation required for efficient pollen-tube elongation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrollo , Proteínas de Unión al GTP rho/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Microscopía Confocal , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Tubo Polínico/enzimología , Tubo Polínico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rho/genéticaRESUMEN
Cadmium (Cd) is a widespread soil pollutant; thus, the underlying molecular controls of plant Cd tolerance are of substantial interest. A screen for wheat (Triticum aestivum) genes that confer Cd tolerance to a Cd hypersensitive yeast strain identified Heat shock transcription factor A4a (HsfA4a). Ta HsfA4a is most similar to the class A4 Hsfs from monocots. The most closely related rice (Oryza sativa) homolog, Os HsfA4a, conferred Cd tolerance in yeast, as did Ta HsfA4a, but the second most closely related rice homolog, Os HsfA4d, did not. Cd tolerance was enhanced in rice plants expressing Ta HsfA4a and decreased in rice plants with knocked-down expression of Os HsfA4a. An analysis of the functional domain using chimeric proteins constructed from Ta HsfA4a and Os HsfA4d revealed that the DNA binding domain (DBD) of HsfA4a is critical for Cd tolerance, and within the DBD, Ala-31 and Leu-42 are important for Cd tolerance. Moreover, Ta HsfA4a-mediated Cd resistance in yeast requires metallothionein (MT). In the roots of wheat and rice, Cd stress caused increases in HsfA4a expression, together the MT genes. Our findings thus suggest that HsfA4a of wheat and rice confers Cd tolerance by upregulating MT gene expression in planta.
Asunto(s)
Cadmio/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Triticum/genética , Secuencia de Aminoácidos , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen , Biblioteca de Genes , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Metalotioneína/genética , Metalotioneína/metabolismo , Datos de Secuencia Molecular , Mutación , Oryza/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Triticum/metabolismoRESUMEN
BACKGROUND: Highly elongated eukaryotic cells (e.g., neuronal axons, fungal hyphae, and pollen tubes) are generated through continuous apically restricted growth (tip growth), which universally requires tip-localized Rho GTPases. We used the oscillating pollen tube as a model system to determine the function and regulation of Rho GTPases in tip growth. Our previous work showed that the spatiotemporal dynamics of the apical cap of the activated Rho-like GTPase from Plant 1 (ROP1) are critical for tip growth in pollen tubes. However, the underlying mechanism for the generation and maintenance of this dynamic apical cap is poorly understood. RESULTS: A screen for mutations that enhance ROP1-overexpression-induced depolarization of pollen-tube growth identified REN1 (ROP1 enhancer 1) in Arabidopsis, whose null mutations turn elongated pollen tubes into bulbous cells. REN1 encodes a novel Rho GTPase-activating protein (RhoGAP) required for restricting the ROP1 activity to the pollen-tube tip. REN1 was localized to exocytic vesicles accumulated in the pollen-tube apex, as well as to the apical plasma membrane at the site of ROP1 activation. The apical localization of REN1 and its function in controlling growth polarity was compromised by disruption of ROP1-dependent F-actin and vesicular trafficking, which indicates that REN1 targeting and function is regulated by ROP1 downstream signaling. CONCLUSIONS: Our findings suggest that the REN1 RhoGAP controls a negative-feedback-based global inhibition of ROP1. This function provides a critical self-organizing mechanism, by which ROP signaling is spatially limited to the growth site and temporally oscillates during continuous tip growth. Similar spatiotemporal control of Rho GTPase signaling may also play an important role in cell-polarity control in other systems, including tip growth in fungi and cell movement in animals.