RESUMEN
Arabidopsis thaliana has seven genes for functionally active sucrose transporters. Together with sucrose transporters from other dicot and monocot plants, these proteins form four separate phylogenetic groups. Group-IV includes the Arabidopsis protein SUC4 (synonym SUT4) and related proteins from monocots and dicots. These Group-IV sucrose transporters were reported to be either tonoplast- or plasma membrane-localised, and in heterologous expression systems were shown to act as sucrose/H(+) symporters. Here, we present comparative analyses of the subcellular localisation of the Arabidopsis SUC4 protein and of several other Group-IV sucrose transporters, studies on tissue specificity of the Arabidopsis SUC4 promoter, phenotypic characterisations of Atsuc4.1 mutants and AtSUC4 overexpressing (AtSUC4-OX) plants, and functional comparisons of Atsuc4.1 and AtSUC4-OX vacuoles. Our data show that SUC4-type sucrose transporters from different plant families (Brassicaceae, Cucurbitaceae and Solanaceae) localise exclusively to the tonoplast, demonstrating that vacuolar sucrose transport is a common theme of all SUC4-type proteins. AtSUC4 expression is confined to the stele of Arabidopsis roots, developing anthers and meristematic tissues in all aerial parts. Analyses of the carbohydrate content of WT and mutant seedlings revealed reduced sucrose content in AtSUC4-OX seedlings. This is in line with patch-clamp analyses of AtSUC4-OX vacuoles that characterise AtSUC4 as a sucrose/H(+) symporter directly in the tonoplast membrane.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Sacarosa/metabolismo , Vacuolas/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Flores/citología , Flores/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Genes Reporteros , Hipocótilo/citología , Hipocótilo/genética , Hipocótilo/metabolismo , Proteínas de Transporte de Membrana/genética , Meristema/citología , Meristema/genética , Meristema/metabolismo , Mutagénesis Insercional , Especificidad de Órganos , Filogenia , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión , Plantones/citología , Plantones/genética , Plantones/metabolismo , Simportadores/genética , Simportadores/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
A protocol for the detection of gene transcripts from single plant cells in living, undamaged plant tissue is described. Samples of leaf epidermal, mesophyll and companion cells were extracted by using glass microcapillaries and directly subjected to RT-PCR without any purification steps nor time consuming construction of cDNA libraries. The procedure is not restricted to surface cells or outer cell layers. Even cells from the central region of leaves could be harvested. For identification, companion cells were labelled by expression of the green fluorescent protein under control of a companion cell specific promoter. The described method is applicable to a wide range of plants and genes with different expression levels.
RESUMEN
Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.
Asunto(s)
Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Transporte Biológico , Carbono/metabolismo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Movimiento Viral en Plantas , Potexvirus/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
Macromolecular trafficking within the sieve element-companion cell complex, phloem unloading, and post-phloem transport were studied using the jellyfish green fluorescent protein (GFP). The GFP gene was expressed in Arabidopsis and tobacco under the control of the AtSUC2 promoter. In wild-type Arabidopsis plants, this promoter regulates expression of the companion cell-specific AtSUC2 sucrose-H+ symporter gene. Analyses of the AtSUC2 promoter-GFP plants demonstrated that the 27-kD GFP protein can traffic through plasmodesmata from companion cells into sieve elements and migrate within the phloem. With the stream of assimilates, the GFP is partitioned between different sinks, such as petals, root tips, anthers, funiculi, or young rosette leaves. Eventually, the GFP can be unloaded symplastically from the phloem into sink tissues, such as the seed coat, the anther connective tissue, cells of the root tip, and sink leaf mesophyll cells. In all of these tissues, the GFP can traffic cell to cell by symplastic post-phloem transport. The presented data show that plasmodesmata of the sieve element-companion cell complex, as well as plasmodesmata into and within the analyzed sinks, allow trafficking of the 27-kD nonphloem GFP protein. The data also show that the size exclusion limit of plasmodesmata can change during organ development. The results are also discussed in terms of the phloem mobility of assimilates and of small, low molecular weight companion cell proteins.