Tissue-specific accumulation and subcellular localization of chalcone isomerase (CHI) in grapevine

Flavonoids are widely distributed secondary metabolic products with many biological functions in plants. Further elucidation of the accumulation and localization patterns of its biosynthesis enzymes will broaden our understanding of flavonoids biosynthesis and regulation. Chalcone isomerase (CHI, EC 5.5.1.6) is an early-step enzyme in the flavonoids biosynthesis pathway. In this study, using an antibody specifically developed against grapevine CHI enzyme, we found that the accumulation of CHI protein exhibited temporal and spatial specificity. In grape berries, CHI was investigated mainly in the outer hypodermis cells of exocarp tissues, in the vascular bundles of mesocarp; and in the integument and the cells around the raphe of seeds. At the subcellular level, CHI was visualized in the cytoplasm, nucleus, and plastids (chloroplasts) of the exocarp cells, while only in the cytoplasm of mesocarp vascular bundle cells. In grapevine vegetable organs, the leaf mesophyll and phloem of leaf veins, the pith ray and primary phloem of stems, the primary phloem and endoderm of roots, and the young leaves, leaf primordium, and the growth point of leaf buds were CHI signal-positive. In these tissue cells, CHI was primarily observed in the cytoplasm, cell wall, and nucleus. The distinct localization patterns of CHI suggested the complexity of flavonoids biosynthesis in grapevine.

     

Effect of UV-C irradiation on stilbene synthase localization in young grape plants

Stilbene synthase (STS) is a pivotal enzyme that catalyzes the biosynthesis of resveratrol and could be induced by UV-C irradiation. However, at present the effect of UV-C irradiation on tissue and subcellular localization of STS is not studied. In this work, subcellular localization of STS in young grape (Vitis vinifera L. cv. Cabernet Sauvignon) plants exposed to UV-C was examined immunohistochemically using a polyclonal antibody raised against grape berry STS. The immunohistochemical analysis showed that the UV-induced STS occurred in palisade tissues of grape leaves and phloem tissues of grape leaf veins, stems, and roots. At the subcellular level, the enhanced STS stimulated by UV-C light was visualized in the cell walls, chloroplasts (plastids), cytoplasm, and nucleus of the phloem (stems and roots), while only in chloroplasts in mesophyll cells. This distribution pattern of STS arising in response to UV-C irradiation may be closely involved in its defense function, which needs much more in-depth evidence to confirm.     

Cloning, Localization, and Expression Analysis of a New Tonoplast Monosaccharide Transporter from Vitis vinifera L

Tonoplast sugar transporters are important for sugar partitioning, immobilization, and accumulation during fruit development and ripening. Here we report the cloning, localization, and functional analysis of one of these transporters in grape berries (Vitis vinifera L.). This clone, named VvTMT1, encodes a 742-aa protein with a calculated molecular mass of 80.2 kDa. Predicted membrane topology and phylogenetic analysis suggest that VvTMT1 belongs to the major facilitator superfamily of membrane carriers. Semiquantitative RT-PCR suggests that VvTMT1 is a sink-specific transporter, whose expression decreases with berry development. Heterologous expression of VvTMT1 in yeast can partially restore growth of the hxt-null strain in glucose and other monosaccharide media, indicating that VvTMT1 is a functional monosaccharide transporter. Induction of VvTMT1-GFP fusion protein expression in transgenic yeast revealed its tonoplast localization. The subcellular localization of VvTMT1 in plants was shown by immunogold labeling of grape berry mesocarp cells and VvTMT1-GFP transient expression in tobacco epidermis cells. Based on the above analyses of VvTMT1, this is the first report of a functional tonoplast-localized monosaccharide transporter in grapevine.     

Localization of the type I ribosome inactivating protein, luffin, in adult and embryonic tissues of Luffa cylindrica L. Roem

The subcellular localization of the type I ribosome-inactivating protein, luffin, has been investigated by means of immunofluorescence light microscopy. A different pattern of protein distribution has been observed in embryonic and somatic tissues. In mature seeds luffin is accumulated within protein bodies in the storage tissue; vacuolar compartmentation in cells of the cotyledonary leaves is maintained during germination of the seedlings. In adult tissues, such as mature leaves and stems, the targeting of the protein is different, since luffin is found in the extracellular spaces. This localization outside the plasma membrane has been confirmed by enzymatic activity determination on the intercellular fluid present in the apoplastic space. Results on luffin localization are discussed with respect to the putative function(s) of this enzyme.Keywords: Luffa cylindrica L. Roem., luffin, ribosome-inactivating proteins (RIPs), secretory proteins, subcellular compartmentation.      

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (β::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed.     

冬性植物红菜薹在不同温度处理下花青素积累的分子机制

芸薹属植物红菜薹（Brassica rapa）是一种常见的蔬菜,它的花茎和叶柄表皮中均积累有花青素。为了解红菜薹中花青素合成的分子机制,进行了花青素含量的测定和花青素合成相关基因的表达分析。研究结果表明,叶柄表皮中的花青素含量显著高于叶片（去主脉）的花青素含量。同时,叶柄表皮花青素合成相关基因的表达水平高于叶柄（去表皮）和叶片（去主脉）的表达水平,这表明红菜薹中花青素的合成调控发生在转录水平。BrMYBA1仅在叶柄表皮中表达,但BrbHLH1和BrWD40在叶片和叶柄表皮中均能检测到表达。因此,BrMYBA1的转录激活可能与红菜薹的花青素合成相关。连续低温处理时,红菜薹叶柄表皮中的花青素含量逐渐增加,而该组织中花青素合成的结构基因表达水平逐渐降低。     

Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues,Flowers, and Developing Seeds

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.     

Transient expression assays in grapevine: a step towards genetic improvement

In the past few years, the usefulness of transient expression assays has continuously increased for the characterization of unknown gene function and metabolic pathways. In grapevine (Vitis vinifera L.), one of the most economically important fruit crops in the world, recent systematic sequencing projects produced many gene data sets that require detailed analysis. Due to their rapid nature, transient expression assays are well suited for large‐scale genetic studies. Although genes and metabolic pathways of any species can be analysed by transient expression in model plants, a need for homologous systems has emerged to avoid the misinterpretation of results due to a foreign genetic background. Over the last 10 years, various protocols have thus been developed to apply this powerful technology to grapevine. Using cell suspension cultures, somatic embryos, leaves or whole plantlets, transient expression assays enabled the study of the function, regulation and subcellular localization of genes involved in specific metabolic pathways such as the biosynthesis of phenylpropanoids. Disease resistance genes that could be used for marker‐assisted selection in conventional breeding or for stable transformation of elite cultivars have also been characterized. Additionally, transient expression assays have proved useful for shaping new tools for grapevine genetic improvement: synthetic promoters, silencing constructs, minimal linear cassettes or viral vectors. This review provides an update on the different tools (DNA constructs, reporter genes, vectors) and methods (Agrobacterium‐mediated and direct gene transfer methods) available for transient gene expression in grapevine. The most representative results published thus far are then described.     

Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp

In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.     

Identification and quantification of the major endogenous cytokinins in pistachio seedlings

The major endogenous cytokinins, Z, ZR, DHZ, DHZR, iP and iPR in pistachio seedlings (Pistacia vera L. cv. Ohadi) were purified by HPLC and their identities confirmed using GC-MS. The aerial parts of two-year old pistachio seedlings including mature leaves, young leaves, lateral buds, debarked stems and bark were subjected to analysis. All of the above mentioned cytokinins were identified in the aerial parts except DHZ which was only present in mature leaves. Z-type cytokinins contributed almost 43% of the total cytokinins. ZR and DHZR were identified as the major ribosides and iP as the main base. The greatest concentration of ZR was detected in the bark, amounting to about 48%. DHZR and ZR constituted the major portion of the total cytokinins detected in both young and mature leaves while Z was detected as a minor cytokinin in leaves. The sharp increase of iP concentration during leaf maturation indicates that mature leaves are probably capable of de novo biosynthesis of cytokinins. The absence of DHZ (except in mature leaves) and the presence of considerable concentrations of DHZR in pistachio stems suggest that these tissues are able to metabolize DHZ to DHZR. The large amount of ZR in pistachio leaves suggests that root-derived ZR is transported into the leaves after loading into the xylem. The presence of high amounts of iP in pistachio lateral buds indicates that iP has been accumulated in these parts. The occurrence of a totally different cytokinin distribution pattern in buds, as compared with the other aerial parts, possibly results from their different metabolism.     

Relative importance of nodal roots and apical buds in the control of branching in Trifolium repens L.

Clonal species are characterised by having a growth form in which roots and shoots originate from the same meristem so that adventitious nodal roots form close to the terminal apical bud of stems. The nature of the relationship between nodal roots and axillary bud growth was investigated in three manipulative experiments on cuttings of a single genotype of Trifolium repens. In the absence of locally positioned nodal roots axillary bud development within the apical bud proceeded normally until it slowed once the subtending leaf had matured to be the second expanded leaf on the stem. Excision of apical tissues indicated that while there was no apical dominance apparent within fully rooted stems and very little in stems with 15 or more unrooted nodes, the outgrowth of the two most distal axillary buds was stimulated by decapitation in stems with intermediate numbers of unrooted nodes. Excision of the basal branches from stems growing without local nodal roots markedly increased the length and/or number of leaves on 14 distally positioned branches. The presence of basal branches therefore prevented the translocation of root-supplied resources (nutrients, water, phytohormones) to the more distally located nodes and this caused the retardation in the outgrowth of their axillary buds. Based on all three experiments we conclude that the primary control of bud outgrowth is exerted by roots via the acropetal transport of root-supplied resources necessary for axillary bud outgrowth and that apical dominance plays a very minor role in the regulation of axillary bud outgrowth in T. repens.     

Stress effects of flumioxazin herbicide on grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) grown in vitro

Among the herbicides used in vineyards, the pre-emergence soil-applied flumioxazin (FMX) is a recently synthesized molecule that inhibits chlorophyll biosynthesis in weed species. The aim of this work was to characterize the effects of FMX on non-target grapevine (Vitis vinifera L. cv. Chardonnay) plantlets grown in vitro. FMX treatment (from 1 to 100 M) represented a stress, as revealed by measurement of several parameters. Stem and leaves underwent dehydration and a decrease in both water- and osmotic-potential. Treated plantlets exhibited concomitant accumulation of soluble carbohydrates in all tissues and of free proline in stems and leaves. Moreover, FMX caused lipid peroxidation and electrolyte leakage in leaf tissues. These results indicate that the herbicide FMX is toxic for grapevine grown in vitro. In addition to inhibiting protoporphyrinogen IX oxidase, it causes water stress and membrane alteration in tissues and, as a consequence, generates the accumulation of carbohydrates and free proline.Abbreviations FMX Flumioxazin - MDA Malondialdehyde - PROTOX Protoporphyrinogen IX oxidase - TBARS Thiobarbituric acid reactive substances - TFAA Total free amino acids - TSS Total soluble sugarsCommunicated by S. Gleddie     

益智不同组织多糖含量及其生物合成途径分析

为了解益智（Alpinia oxyphylla）多糖生物合成途径关键酶功能,对其茎、叶、果实中的多糖含量及其单糖组成进行了研究,并采用Real-Time qPCR分析了益智多糖生物合成关键酶基因的表达模式。结果表明,益智多糖含量依次为果实 > 叶 > 茎,主要由葡萄糖、木糖、甘露糖、半乳糖和阿拉伯糖组成;利用益智转录组数据共获得47 690条unigenes,其中31 892条在NR、Swiss-Prot、KEGG、COG、KOG、GO和Pfam数据库获得注释,其中208个unigenes参与益智多糖的生物合成,涉及15个酶。表达分析表明,所筛选的18个基因在茎、叶、果实中均有表达,14个基因在果实中的表达量最高,以糖基转移酶基因和UDP-葡萄糖焦磷酸化酶基因的表达量最高,且其表达模式与不同组织中葡萄糖含量的变化一致。     

An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata,leading to increased induced resistance against grapevine downy mildew

Some β‐1,3‐glucans and particularly sulfated laminarin (PS3) are known as resistance inducers (RIs) in grapevine against the downy mildew. However, their efficacy in vineyard is still often too low, which might be caused by a limited penetration through the leaf cuticle following spray application. We used ¹⁴C‐sucrose uptake experiments with grapevine leaves in order to select a surfactant as saccharide penetration enhancer. Our results showed that although sucrose foliar uptake was low, it was strongly enhanced by Dehscofix CO125 (DE), a highly ethoxylated surfactant. Fluorescent saccharides were then produced and laser scanning microscopy was used to analyze their foliar diffusion pattern in Arabidopsis thaliana and grapevine. Interestingly, sucrose and PS3 were seemingly able to penetrate the leaf cuticle only when formulated with DE. Diffusion could preferentially occur via stomata, anticlinal cell walls and trichomes. In grapevine, PS3 penetration rate was much higher on the stomateous abaxial surface of the leaf than on the adaxial surface. Finally, using DE allowed a higher level of downy mildew control by PS3, which corroborated diffusion observations. Our results have practical consequences for the improvement of treatments with saccharidic inducers on grape. That is, formulation of such RIs plays a critical role for their cuticular diffusion and consequently their efficacy. Also, spray application should preferentially target the abaxial surface of the leaves in order to maximize their penetration.     

Structural and functional characteristics of photosynthetic apparatus of chlorophyll-containing grape vine tissue

Photosynthesis in tissues under periderm of woody stems and shoots of perennial plants occurs in environment that is very different from the internal environment of leaf chloroplasts. These tissues are characterized by high CO₂ and low O₂ concentrations, more acidic surroundings, besides that only light which have passed through periderm reaches photosynthetic antennas. In contrast to leaves of deciduous plants chlorenchyma tissues of wintering plant organs are exposed to temperature fluctuations during all seasons, that is why the photosynthetic apparatus of woody stems has to be able to adapt to a wide range of environmental temperatures. In order to reveal unique features, which enable photosynthetic apparatus of chlorenchyma cells in woody plant organs to implement biological functions under different light and temperature conditions, we studied photosynthetic tissues of stem cortex in grapevine (Vitis vinifera L.) under normal conditions and after exposure to suboptimal temperatures and high light intensity. Comparative analysis of photosynthetic pigment composition and low-temperature chlorophyll fluorescence emission spectrum of leaves, young shoots and chlorenchyma of lignified shoots revealed relatively high level of chlorophyll b and carotenoids, and high photosystem II (PSII) to photosystem I (PSI) ratio in woody shoots. Analysis of parameters of variable chlorophyll fluorescence revealed high PSII activity in grapevine shoot cortex and demonstrated improved freeze tolerance and higher sensitivity to light of photosynthetic apparatus in grape vine in comparison to leaves. It was shown for the first time that photosynthetic apparatus in chlorenchyma cells of vine undergoes so-called “state-transition”–fast rearrangements leading to redistribution of energy between photosystems. Analysis of fatty acid (FA) compositions of lipids in examined tissues showed that the FA unsaturation index in green tissue of vine is lower than in leaves. A distinct feature of FA compositions of lipids in vine cortex was relatively high level of linoleic acid.     

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.