RESUMEN

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H+ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.

RESUMEN

Solutes are subject of constant release and retrieval processes during long distance transport in higher plants. In the stem, sugars and amino acids are used for storage or for nutrition of parenchymatic tissues. Modification of export/import capacities along the transport phloem might provide a powerful tool to enhance the productivity of crop plants.

Asunto(s)

Floema , Plantas , Aminoácidos/metabolismo , Transporte Biológico , Floema/metabolismo , Plantas/metabolismo , Azúcares/metabolismo

RESUMEN

The quiescent center (QC) of the Arabidopsis (Arabidopsis thaliana) root meristem acts as an organizer that promotes stem cell fate in adjacent cells and patterns the surrounding stem cell niche. The stem cells distal from the QC, the columella stem cells (CSCs), are maintained in an undifferentiated state by the QC-expressed transcription factor WUSCHEL RELATED HOMEOBOX5 (WOX5) and give rise to the columella cells. Differentiated columella cells provide a feedback signal via secretion of the peptide CLAVATA3/ESR-RELATED40 (CLE40), which acts through the receptor kinases ARABIDOPSIS CRINKLY4 (ACR4) and CLAVATA1 (CLV1) to control WOX5 expression. Previously, it was proposed that WOX5 protein movement from the QC into CSCs is required for CSC maintenance, and that the CLE40/CLV1/ACR4 signaling module restricts WOX5 mobility or function. Here, these assumptions were tested by exploring the function of CLE40/CLV1/ACR4 in CSC maintenance. However, no role for CLE40/CLV1/ACR4 in constricting the mobility of WOX5 or other fluorescent test proteins was identified. Furthermore, in contrast to previous observations, WOX5 mobility was not required to inhibit CSC differentiation. We propose that WOX5 acts mainly in the QC, where other short-range signals are generated that not only inhibit differentiation but also promote stem cell division in adjacent cells. Therefore, the main function of columella-derived CLE40 signal is to position the QC at a defined distance from the root tip by repressing QC-specific gene expression via the ACR4/CLV1 receptors in the distal domain and promoting WOX5 expression via the CLV2 receptor in the proximal meristem.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Regulación de la Expresión Génica de las Plantas , Meristema/citología , Meristema/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo

RESUMEN

Sucrose transport across membranes requires the activity of transport proteins. Sucrose-specific SWEET proteins mediate sugar efflux out of the cytosol and SUC proteins catalyze the uptake of sucrose from the apoplast. Both transport processes are involved in phloem loading in source leaves as well as in the post-phloem pathway in sink tissues. An important step during the characterization of new sucrose transporters is to analyze their transport activity. This is usually achieved by heterologous expression of the respective gene in yeast cells or Xenopus oocytes and subsequent uptake measurements. However, in some cases, mistargeting to internal membranes or the lack of protein modifications and/or interaction partners in the heterologous system can interfere with uptake analyses. Therefore, a new in planta method was developed that is based on mesophyll protoplasts as expression system and the fluorescent sucrose analog esculin to monitor uptake activities by confocal microscopy. In this chapter we describe the design of constructs required to analyze sucrose transporters in protoplasts, the experimental setup of the protoplast-esculin assay, and the quantitative evaluation of the obtained data. The quantification of esculin uptake allows the application of the new assay to a variety of questions, e.g., by comparison of point mutants, splice variants, or transporters with and without interaction partners.

Asunto(s)

Bioensayo , Esculina/metabolismo , Protoplastos/metabolismo , Sacarosa/metabolismo , Arabidopsis/metabolismo , Transporte Biológico , Microscopía Confocal , Floema/metabolismo , Hojas de la Planta/metabolismo

RESUMEN

The sucrose carrier AtSUC2 of Arabidopsis thaliana is localized in the phloem, where it catalyzes the uptake of sucrose from the apoplast into companion cells. Imported sucrose moves passively via plasmodesmata from the companion cells into the neighboring sieve elements that distribute this disaccharide to the different sink organs. Phloem loading of sucrose by the AtSUC2 protein is an essential process, and mutants lacking this protein stay tiny, develop no or only few flowers, and have a strongly reduced root system. The promoter of the AtSUC2 gene is active exclusively in companion cells of the phloem. Moreover, it drives very strong expression not only in Arabidopsis, but also in all plant species tested so far, including monocot species. Due to these features, the AtSUC2 promoter has become an important tool in diverse areas of plant research during the last two decades. It was used to study phloem development and function including phloem loading and unloading. Furthermore, it was helpful in analyzing the pathways of posttranscriptional silencing by RNA interference, the regulation of flowering, mechanisms of nutrient withdrawal by phloem-feeding pathogens, and other physiological functions that are related to long distance transport. The present paper gives an overview of different approaches in plant research that utilized the strong and companion cell-specific expression of own or foreign genes driven by the AtSUC2 promoter.

Asunto(s)

Arabidopsis/fisiología , Proteínas de Transporte de Membrana/genética , Floema/fisiología , Desarrollo de la Planta/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Transporte Biológico , Biomarcadores , Productos Agrícolas , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Interacciones Huésped-Patógeno , Plasmodesmos/genética , Plasmodesmos/metabolismo , Interferencia de ARN , Transducción de Señal

RESUMEN

Pollen tube growth requires a high amount of metabolic energy and precise targeting toward the ovules. Sugars, especially glucose, can serve as nutrients and as signaling molecules. Unexpectedly, in vitro assays revealed an inhibitory effect of glucose on pollen tube elongation, contradicting the hypothesis that monosaccharide uptake is a source of nutrition for growing pollen tubes. Measurements with Förster resonance energy transfer-based nanosensors revealed that glucose is taken up into pollen tubes and that the intracellular concentration is in the low micromolar range. Pollen tubes of stp4-6-8-9-10-11 sextuple knockout plants generated by crossings and CRISPR/Cas9 showed only a weak response to glucose, indicating that glucose uptake into pollen tubes is mediated mainly by these six monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family. Analyses of HEXOKINASE1 (HXK1) showed a strong expression of this gene in pollen. Together with the glucose insensitivity and altered semi-in vivo growth rate of pollen tubes from hxk1 knockout lines, this strongly suggests that glucose is an important signaling molecule for pollen tubes, is taken up by STPs, and detected by HXK1. Equimolar amounts of fructose abolish the inhibitory effect of glucose indicating that only an excess of glucose is interpreted as a signal. This provides a possible model for the discrimination of signaling and nutritional sugars.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Tubo Polínico/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Transporte Biológico/fisiología , Regulación de la Expresión Génica de las Plantas , Hexoquinasa , Proteínas de Transporte de Monosacáridos/genética , Tubo Polínico/crecimiento & desarrollo , Polinización/genética , Polinización/fisiología

RESUMEN

The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in Arabidopsis thaliana. Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that AtSUC6Col-0 is a high-affinity H+-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of AtSUC6Col-0 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of AtSUC6 did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7Col-0 is regulated by intragenic regions. Transport activity of AtSUC7Col-0 could not be analyzed in baker's yeast or Xenopus oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters in planta was developed. Plasma membrane localized SUCs including AtSUC6Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7Col-0 might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an in planta expression system.

RESUMEN

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell-to-cell transport. Here we report on an elegant non-invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA-s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA-s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA-s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA-s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA-s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA-s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.

Asunto(s)

Proteínas Luminiscentes/metabolismo , Células Vegetales/fisiología , Arabidopsis/metabolismo , Arabidopsis/fisiología , Transporte Biológico/fisiología , Pared Celular/metabolismo , Fluorescencia , Meristema/citología , Microscopía Confocal , Células Vegetales/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Plantas Modificadas Genéticamente , Plasmodesmos/metabolismo , Plasmodesmos/fisiología

RESUMEN

The controlled distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is a key element for plant growth and development. Monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family contribute to the uptake of sugars into sink cells. Here, we report on the characterization of STP7, STP8, and STP12, three previously uncharacterized members of this family in Arabidopsis (Arabidopsis thaliana). Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that STP8 and STP12 catalyze the high-affinity proton-dependent uptake of glucose and also accept galactose and mannose. STP12 additionally transports xylose. STP8 and STP12 are highly expressed in reproductive organs, where their protein products might contribute to sugar uptake into the pollen tube and the embryo sac. stp8.1 and stp12.1 T-DNA insertion lines developed normally, which may point toward functional redundancy with other STPs. In contrast to all other STPs, STP7 does not transport hexoses but is specific for the pentoses l-arabinose and d-xylose. STP7-promoter-reporter gene plants showed an expression of STP7 especially in tissues with high cell wall turnover, indicating that STP7 might contribute to the uptake and recycling of cell wall sugars. Uptake analyses with radioactive l-arabinose revealed that 11 other STPs are able to transport l-arabinose with high affinity. Hence, functional redundancy might explain the missing-mutant phenotype of two stp7 T-DNA insertion lines. Together, these data complete the characterization of the STP family and present the STPs as new l-arabinose transporters for potential biotechnological applications.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabinosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Xilosa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN Bacteriano , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Plantas Modificadas Genéticamente , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H(+)symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10 qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Glucosa/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Tubo Polínico/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/efectos de los fármacos , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes Reporteros , Glucuronidasa/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Mutagénesis Insercional/genética , Tubo Polínico/efectos de los fármacos , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de Proteína , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo

RESUMEN

UNLABELLED: Polycystic kidney diseases are characterized by the development of numerous bilateral renal cysts that continuously enlarge resulting in a decline of kidney function due to compression of intact nephrons. Cyst growth is driven by transepithelial chloride secretion which depends on both intracellular cAMP and calcium. Mechanisms that are involved in the regulation of the underlying secretory pathways remain incompletely understood. Here we show that glucose concentration has a strong impact on cyst growth of renal tubular cells within a collagen matrix as well as in embryonic kidneys deficient or competent for Pkd1. Glucose-dependent cyst growth correlates with the transcriptional induction of the calcium-activated chloride channel anoctamin 1 (ANO1) and its increased expression in the apical membrane of cyst-forming cells. Inhibition of ANO1 with the specific inhibitor CaCCinh-AO1 significantly decreases glucose-dependent cyst growth in both models. Ussing chamber analyses revealed increased apical chloride secretion of renal tubular cells upon exposure to high glucose medium which can also be inhibited by the use of CaCCinh-AO1. These data suggest that glycemic control may help to reduce renal cyst growth in patients with polycystic kidney disease. KEY MESSAGE: Renal cyst growth depends on glucose concentration in two in vitro cyst models. High glucose leads to upregulation of the calcium-activated chloride channel ANO1. High glucose promotes calcium-activated chloride secretion via ANO1. Glucose-dependent secretion can be inhibited by a specific inhibitor of ANO1.

Asunto(s)

Cloruros/metabolismo , Quistes/patología , Glucosa/farmacología , Túbulos Renales/patología , Riñón Poliquístico Autosómico Dominante/patología , Canales Catiónicos TRPP/genética , Animales , Anoctamina-1 , Calcio/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/biosíntesis , Canales de Cloruro/genética , AMP Cíclico/metabolismo , Perros , Túbulos Renales/citología , Túbulos Renales/embriología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Transcripcional/genética

RESUMEN

Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior.

Asunto(s)

Fraxinus/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Plantas/genética , Sacarosa/metabolismo , Transporte Biológico , Fraxinus/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/metabolismo , Floema/metabolismo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Plant development requires accurate coordination of gene expression, both in actively dividing meristematic cells and differentiated cells. Cell fate establishment and maintenance, among others, are mediated by chromatin organization complexes that determine the stable transcriptional states of specific cell types. Here, we focus on MAIN-LIKE1 (MAIL1), one of three homologs of MAINTENANCE OF MERISTEMS (MAIN), which form a plant-specific gene family in Arabidopsis thaliana. We show that MAIL1 encodes a ubiquitously expressed nuclear protein. A mail1 loss-of-function mutant developed short primary roots, in which the meristematic cells accumulated DNA double-strand breaks and underwent massive cell death. In addition, mail1 mutant showed also cell differentiation defects in root and shoot tissues, and developed disorganized callus-like structures. The genetic interaction between main and mail1 mutants suggests that they act in the same pathway, and that both are essential for maintaining correct cell division acitivity in meristematic cells, while MAIL1 has an additional function in differentiating cells.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Diferenciación Celular , División Celular , Expresión Génica , Genes Reporteros , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/fisiología , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/fisiología

RESUMEN

Sedentary plant parasitic nematodes such as root-knot nematodes and cyst nematodes induce giant cells or syncytia, respectively, in their host plant's roots. These highly specialized structures serve as feeding sites from which exclusively the nematodes withdraw nutrients. While giant cells are symplastically isolated and obtain assimilates by transporter-mediated processes syncytia are massively connected to the phloem by plasmodesmata. To support the feeding sites and the nematode during their development, phloem is induced around syncytia and giant cells. In the case of syncytia the unloading phloem consists of sieve elements and companion cells and in the case of root knots it consists exclusively of sieve elements. We applied immunohistochemistry to identify the cells within the developing phloem that responded to auxin and cytokinin. Both feeding sites themselves did not respond to either hormone. We were able to show that in root knots an auxin response precedes the differentiation of these auxin responsive cells into phloem elements. This process appears to be independent of B-type Arabidopsis response regulators. Using additional markers for tissue identity we provide evidence that around giant cells protophloem is formed and proliferates dramatically. In contrast, the phloem around syncytia responded to both hormones. The presence of companion cells as well as hormone-responsive sieve elements suggests that metaphloem development occurs. The implication of auxin and cytokinin in the further development of the metaphloem is discussed.

RESUMEN

Stem cells in the root and shoot apical meristem provide the descendant cells required for growth and development throughout the lifecycle of a plant. We found that mutations in the Arabidopsis MAINTENANCE OF MERISTEMS (MAIN) gene led to plants with distorted stem cell niches in which stem cells are not maintained and undergo premature differentiation or cell death. The malfunction of main meristems leads to short roots, mis-shaped leaves, reduced fertility and partial fasciation of stems. MAIN encodes a nuclear-localized protein and is a member of a so far uncharacterized plant-specific gene family. As main mutant plants are hypersensitive to DNA-damaging agents, expression of genes involved in DNA repair is induced and dead cells with damaged DNA accumulate in the mutant meristems, we propose that MAIN is required for meristem maintenance by sustaining genome integrity in stem cells and their descendants cells.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Inestabilidad Genómica , Meristema/genética , Mutación , Proteínas Nucleares/genética , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/metabolismo , Fenotipo , Raíces de Plantas/genética , Brotes de la Planta/genética , Semillas/genética , Semillas/crecimiento & desarrollo

RESUMEN

The Arabidopsis SUC5 protein represents a classical sucrose/H(+) symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5.

Asunto(s)

Arabidopsis/embriología , Biotina/metabolismo , Proteínas de Transporte de Membrana/fisiología , Proteínas de Plantas/fisiología , Semillas/metabolismo , Triglicéridos/metabolismo , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Grasos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas de Transporte de Membrana/genética , Mutación , Proteínas de Plantas/genética , Semillas/crecimiento & desarrollo , Sacarosa/metabolismo

RESUMEN

Many membrane proteins are involved in the transport of nutrients in plants. While the import of amino acids into plant cells is, in principle, well understood, their export has been insufficiently described. Here, we present the identification and characterization of the membrane protein Siliques Are Red1 (SIAR1) from Arabidopsis (Arabidopsis thaliana) that is able to translocate amino acids bidirectionally into as well as out of the cell. Analyses in yeast and oocytes suggest a SIAR1-mediated export of amino acids. In Arabidopsis, SIAR1 localizes to the plasma membrane and is expressed in the vascular tissue, in the pericycle, in stamen, and in the chalazal seed coat of ovules and developing seeds. Mutant alleles of SIAR1 accumulate anthocyanins as a symptom of reduced amino acid content in the early stages of silique development. Our data demonstrate that the SIAR1-mediated export of amino acids plays an important role in organic nitrogen allocation and particularly in amino acid homeostasis in developing siliques.

Asunto(s)

Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Homeostasis , Semillas/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/genética , Animales , Transporte Biológico , Membrana Celular/metabolismo , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/metabolismo , Familia de Multigenes , Mutación/genética , Oocitos/metabolismo , Especificidad de Órganos , Oryza , Fenotipo , Filogenia , Haz Vascular de Plantas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Fracciones Subcelulares/metabolismo , Xenopus laevis

RESUMEN

Developing flowers are important sinks in Arabidopsis thaliana. Their energy demand is covered by assimilates which are synthesized in source leaves and transported via the vasculature. Assimilates are unloaded either symplastically through plasmodesmata or apoplastically by specific transport proteins. Here we studied the pathway of phloem unloading and post-phloem transport in developing gynoecia. Using phloem-mobile fluorescent tracers, we show that phloem unloading into cells of ovule primordia followed a symplastic pathway. Subsequently, the same tracers could not move out of phloem cells into mature ovules anymore. A further change in the mode of phloem unloading occurred after anthesis. In open flowers as well as in outgrowing siliques, the phloem was again unloaded via the symplast. This observed onset of symplastic phloem unloading was accompanied by a change in frequency of MP17-GFP-labeled plasmodesmata. We could also show that the change in cell-cell connectivity was independent of fertilization and increasing sink demand. The presented results indicate that symplastic connectivity is highly regulated and varies not only between different sink tissues but also between different developmental stages.

Asunto(s)

Arabidopsis/crecimiento & desarrollo , Óvulo Vegetal/embriología , Floema/crecimiento & desarrollo , Arabidopsis/embriología , Arabidopsis/metabolismo , Flores/crecimiento & desarrollo , Flores/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Óvulo Vegetal/metabolismo , Floema/metabolismo , Proteínas de Plantas/biosíntesis , Plasmodesmos/metabolismo , Polinización , Proteínas Recombinantes de Fusión/biosíntesis , Semillas/crecimiento & desarrollo , Semillas/metabolismo

RESUMEN

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria uses the type III secretion system (T3SS) to inject effector proteins into cells of its Solanaceous host plants. It is generally assumed that these effectors manipulate host pathways to favor bacterial replication and survival. However, the molecular mechanisms by which type III effectors suppress host defense responses are far from being understood. Based on sequence similarity, Xanthomonas outer protein J (XopJ) is a member of the YopJ/AvrRxv family of SUMO peptidases and acetyltranferases, although its biochemical activity has not yet been demonstrated. Confocal laser scanning microscopy revealed that green fluorescent protein (GFP) fusions of XopJ are targeted to the plasma membrane when expressed in plant cells, which most likely involves N-myristoylation. In contrast to a XopJ(C235A) mutant disrupted in the catalytic triad sequence, the wild-type effector GFP fusion protein was also localized in vesicle-like structures colocalizing together with a Golgi marker protein, suggesting an effect of XopJ on vesicle trafficking. To explore an effect of XopJ on protein secretion, we used a GFP-based secretion assay. When a secreted (sec)GFP marker was coexpressed with XopJ in leaves of Nicotiana benthamiana, GFP fluorescence was retained in reticulate structures. In contrast, in plant cells expressing secGFP alone or along with the XopJ(C235A) mutant, no GFP fluorescence accumulated within the cells. Moreover, coexpressing secGFP together with XopJ led to a reduced accumulation of secGFP within the apoplastic fluid of N. benthamiana leaves, further showing that XopJ affects protein secretion. Transgenic expression of XopJ in Arabidopsis suppressed callose deposition elicited by a T3SS-negative mutant of Pseudomonas syringae pv. tomato DC3000. A role of XopJ in the inhibition of cell wall-based defense responses is discussed.

Asunto(s)

Proteínas Bacterianas/fisiología , Pared Celular/microbiología , Enfermedades de las Plantas/microbiología , Xanthomonas campestris/patogenicidad , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Pared Celular/metabolismo , Proteínas Fluorescentes Verdes/análisis , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/microbiología , Proteínas Recombinantes de Fusión/análisis , Nicotiana/metabolismo , Nicotiana/microbiología , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismo

RESUMEN

Sedentary nematodes are destructive plant pathogens that cause significant yield losses. In the roots of their host plants, cyst nematodes (CNs) and root-knot nematodes (RKNs) induce different, highly specialized feeding sites--syncytia or giant cells (GCs), respectively--to optimize nutrient uptake. We compared the mechanisms by which nutrients are delivered from the model host plant, Arabidopsis, to GCs induced by the RKN Meloidogyne incognita or to syncytia induced by the CN Heterodera schachtii. From previous work, syncytia were known to be symplastically connected to newly formed host phloem composed of sieve elements (SEs) and companion cells. Here we studied the formation of plasmodesmata (PD) during GC and syncytia development by monitoring a viral movement protein that targets branched PD and the development of host phloem during GC formation by applying confocal laser scanning microscopy and immunocytochemistry. Analyses of plants expressing soluble or membrane-anchored green fluorescent protein in their phloem demonstrated symplastic isolation of GCs. GCs were found to be embedded in a tissue that consists exclusively of SEs. These de novo-formed SEs, contained nuclei and were interconnected by secondary PD. A similar interconnection of SEs was observed around syncytia. However, these secondary PD were also present at the SE-syncytium interface, demonstrating the postulated symplastic connection. Our results show that CNs and RKNs, despite their close phylogenetic relatedness, employ fundamentally different strategies to withdraw nutrients from host plants.

Asunto(s)

Células Gigantes/parasitología , Interacciones Huésped-Parásitos , Nematodos/fisiología , Plantas/parasitología , Animales , Arabidopsis/parasitología , Células Gigantes/patología , Plasmodesmos , Tylenchoidea