Pierre Coutos-Thévenot

Source-to-sink transport of sugar and regulation by environmental factors

Source-to-sink transport of sugar is one of the major determinants of plant growth and relies on the efficient and controlled distribution of sucrose (and some other sugars such as raffinose and polyols) across plant organs through the phloem. However, sugar transport through the phloem can be affected by many environmental factors that alter source/sink relationships. In this paper, we summarize current knowledge about the phloem transport mechanisms and review the effects of several abiotic (water and salt stress, mineral deficiency, CO2, light, temperature, air, and soil pollutants) and biotic (mutualistic and pathogenic microbes, viruses, aphids, and parasitic plants) factors. Concerning abiotic constraints, alteration of the distribution of sugar among sinks is often reported, with some sinks as roots favored in case of mineral deficiency. Many of these constraints impair the transport function of the phloem but the exact mechanisms are far from being completely known. Phloem integrity can be disrupted (e.g., by callose deposition) and under certain conditions, phloem transport is affected, earlier than photosynthesis. Photosynthesis inhibition could result from the increase in sugar concentration due to phloem transport decrease. Biotic interactions (aphids, fungi, viruses…) also affect crop plant productivity. Recent breakthroughs have identified some of the sugar transporters involved in these interactions on the host and pathogen sides. The different data are discussed in relation to the phloem transport pathways. When possible, the link with current knowledge on the pathways at the molecular level will be highlighted.

From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms

Elicitins and lipid-transfer proteins are small cysteine-rich lipid-binding proteins secreted by oomycetes and plant cells, respectively, that share some structural and functional properties. In spite of intensive work on their structure and diversity at the protein and genetic levels, the precise biological roles of lipid-transfer proteins remains unclear, although the most recent data suggest a role in somatic embryogenesis, in the formation of protective surface layers and in defence against pathogens. By contrast, elicitins are known elicitors of plant defence, and recent work demonstrating that elicitins and lipid-transfer proteins share the same biological receptors gives a new perspective to understand the role played by lipid binding proteins, mainly the early recognition of intruders in plants.

The Endopolygalacturonase 1 from Botrytis cinerea Activates Grapevine Defense Reactions Unrelated to Its Enzymatic Activity

A purified glycoprotein from Botrytis cinerea (strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

Sugar-regulated expression of a putative hexose transport gene in grape.

Different lengths of the promoter of grape (Vitis vinifera) VvHT1 (Hexose Transporter 1) gene, which encodes a putative hexose transporter expressed during the ripening of grape, have been transcriptionally fused to the β-glucuronidase reporter gene. In transgenic tobacco (Nicotiana tabacum) transformed with these constructs,VvHT1 promoters were clearly responsible for the sink organ preferential expression. The potential sugar effectors ofVvHT1 promoter were studied in tobacco cv Bright-Yellow 2 cells transformed with chimeric constructs. Glucose (56 mm), sucrose (Suc; 58 mm), and the non-transported Suc isomer palatinose doubled the β-glucuronidase activity conferred by the VvHT1 promoter, whereas fructose did not affect it. These effects were the strongest with the 2.4-kb promoter, which contains all putative sugar-responsive elements (activating and repressing), but they were also significant with the 0.3-kb promoter, which contains only activating sugar boxes. The induction of VvHT1 expression by both Suc and palatinose was confirmed in the homologous grape berry cell culture. The data provide the first example of a putative sugar transporter, which is induced by both glucose and Suc in higher plants. Although induction ofVvHT1 expression by Suc does not require transport, the presence of glucosyl moiety is necessary for Suc sensing. These results provide new insights into sugar sensing and signaling in plants.

The vascular expression pattern directed by the Eucalyptus gunnii cinnamyl alcohol dehydrogenase EgCAD2 promoter is conserved among woody and herbaceous plant species

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the last step in the synthesis of the monomeric precursors of lignin. Here, we demonstrate that the vascular expression pattern conferred by the Eucalyptus gunniiEgCAD2 promoter in transgenic poplar (Populus tremula × Populus alba) is conserved in another perennial woody angiosperm of economic interest (Vitis vinifera L.), as well as in a model herbaceous plant (Nicotiana tabacum L.). Furthermore, promoter deletion analysis performed in both tobacco and poplar allowed us to identify the proximal region [−340/−124] as essential for vascular cambium/xylem-specific expression whereas the [−124/+117] region was shown to contain cis element-driving activity in phloem fibres. Interestingly, the [−340/−124] fragment contains an AC-rich cis-acting element present in numerous genes of the phenylpropanoid pathway expressed in xylem tissues, and known as a consensus Myb transcription factor binding site, suggesting that common Myb sites may provide a mechanism by which different steps of phenylpropanoid metabolism are coordinately regulated and expressed in vascular tissues. We have also shown in both tobacco and poplar that the EgCAD2 promoter is inducible by wounding and the cis-elements responsible for wounding responsiveness are located in the distal promoter region. Taken together, our data suggest that the mechanisms controlling developmental and wounding inducible expression of the EgCAD2 promoter are conserved among perennial woody and annual herbaceous plant species enabling us now to investigate in depth the transcriptional regulation of the EgCAD2 promoter in tobacco.

The Vitis vinifera sugar transporter gene family: phylogenetic overview and macroarray expression profiling

BackgroundIn higher plants, sugars are not only nutrients but also important signal molecules. They are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. The availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous Arabidopsis thaliana using a phylogenetic analysis.ResultsIn grapevine, one of the most economically important fruit crop in the world, it appeared that sucrose and monosaccharide transporter genes are present in 4 and 59 loci, respectively and that the monosaccharide transporter family can be divided into 7 subfamilies. Phylogenetic analysis of protein sequences has indicated that orthologs exist between Vitis and Arabidospis. A search for cis-regulatory elements in the promoter sequences of the most characterized transporter gene families (sucrose, hexoses and polyols transporters), has revealed that some of them might probably be regulated by sugars. To profile several genes simultaneously, we created a macroarray bearing cDNA fragments specific to 20 sugar transporter genes. This macroarray analysis has revealed that two hexose (VvHT1, VvHT3), one polyol (VvPMT5) and one sucrose (VvSUC27) transporter genes, are highly expressed in most vegetative organs. The expression of one hexose transporter (VvHT2) and two tonoplastic monosaccharide transporter (VvTMT1, VvTMT2) genes are regulated during berry development. Finally, three putative hexose transporter genes show a preferential organ specificity being highly expressed in seeds (VvHT3, VvHT5), in roots (VvHT2) or in mature leaves (VvHT5).ConclusionsThis study provides an exhaustive survey of sugar transporter genes in Vitis vinifera and revealed that sugar transporter gene families in this woody plant are strongly comparable to those of herbaceous species. Dedicated macroarrays have provided a Vitis sugar transporter genes expression profiling, which will likely contribute to understand their physiological functions in plant and berry development. The present results might also have a significant impact on our knowledge on plant sugar transporters.

Nonspecific Lipid-Transfer Protein Genes Expression in Grape (Vitis sp.) Cells in Response to Fungal Elicitor Treatments

Nonspecific lipid transfer proteins (nsLTPs) are small, basic cystein-rich proteins believed to be involved in plant defense mechanisms. Three cDNAs coding nsLTPs from grape (Vitis vinifera sp.) were cloned by reverse-transcriptase-polymerase chain reaction (RT-PCR) and PCR. The expression of nsLTP genes was investigated in 41B-rootstock grape cell suspension, in response to various defense-related signal molecules. Ergosterol (a fungi-specific sterol) and a proteinaceous elicitor purified from Botrytis cinerea strongly and rapidly induced the accumulation of nsLTP mRNAs. Jasmonic acid, cholesterol, and sitosterol also promoted nsLTPs mRNA accumulation, although to a lesser extent, whereas salicylic acid had no effect. High performance liquid chromatography analysis indicated that the amounts of three LTP isoforms (previously named P1, P2, and P4) were increased by ergosterol. None of the four isoforms displayed any significant antifungal properties, with the exception of the P4 isoform, which reduced Botrytis mycelium growth in vitro, but only in calcium-free medium. The results are discussed in the context of plant-pathogen interactions.

Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew.

Most WRKY transcription factors activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. We previously identified a WRKY gene, VvWRKY1, which is able to enhance tolerance to fungal pathogens when it is overexpressed in tobacco. The present work analyzes the effects of VvWRKY1 overexpression in grapevine. Microarray analysis showed that genes encoding defence-related proteins were up-regulated in the leaves of transgenic 35S::VvWRKY1 grapevines. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway were overexpressed in the transgenic grapes. The ability of VvWRKY1 to trans-activate the promoters of these genes was demonstrated by transient expression in grape protoplasts. The resistance to the causal agent of downy mildew, Plasmopara viticola, was enhanced in the transgenic plants. These results show that VvWRKY1 can increase resistance of grapevine against the downy mildew through transcriptional reprogramming leading to activation of the jasmonic acid signalling pathway.

Gene expression profiling in susceptible interaction of grapevine with its fungal pathogen Eutypa lata: extending MapMan ontology for grapevine.

BackgroundWhole genome transcriptomics analysis is a very powerful approach because it gives an overview of the activity of genes in certain cells or tissue types. However, biological interpretation of such results can be rather tedious. MapMan is a software tool that displays large datasets (e.g. gene expression data) onto diagrams of metabolic pathways or other processes and thus enables easier interpretation of results. The grapevine (Vitis vinifera) genome sequence has recently become available bringing a new dimension into associated research. Two microarray platforms were designed based on the TIGR Gene Index database and used in several physiological studies.ResultsTo enable easy and effective visualization of those and further experiments, annotation of Vitis vinifera Gene Index (VvGI version 5) to MapMan ontology was set up. Due to specificities of grape physiology, we have created new pictorial representations focusing on three selected pathways: carotenoid pathway, terpenoid pathway and phenylpropanoid pathway, the products of these pathways being important for wine aroma, flavour and colour, as well as plant defence against pathogens. This new tool was validated on Affymetrix microarrays data obtained during berry ripening and it allowed the discovery of new aspects in process regulation. We here also present results on transcriptional profiling of grape plantlets after exposal to the fungal pathogen Eutypa lata using Operon microarrays including visualization of results with MapMan. The data show that the genes induced in infected plants, encode pathogenesis related proteins and enzymes of the flavonoid metabolism, which are well known as being responsive to fungal infection.ConclusionThe extension of MapMan ontology to grapevine together with the newly constructed pictorial representations for carotenoid, terpenoid and phenylpropanoid metabolism provide an alternative approach to the analysis of grapevine gene expression experiments performed with Affymetrix or Operon microarrays. MapMan was first validated on an already published dataset and later used to obtain an overview of transcriptional changes in a susceptible grapevine – Eutypa lata interaction at the time of symptoms development, where we showed that the responsive genes belong to families known to be involved in the plant defence towards fungal infection (PR-proteins, enzymes of the phenylpropanoid pathway).

Expression of Arabidopsis sugar transport protein STP13 differentially affects glucose transport activity and basal resistance to Botrytis cinerea

Botrytis cinerea is the causing agent of the grey mold disease in more than 200 crop species. While signaling pathways leading to the basal resistance against this fungus are well described, the role of the import of sugars into host cells remains to be investigated. In Arabidopsis thaliana, apoplastic hexose retrieval is mediated by the activity of sugar transport proteins (STPs). Expression analysis of the 14 STP genes revealed that only STP13 was induced in leaves challenged with B. cinerea. STP13-modified plants were produced and assayed for their resistance to B. cinerea and glucose transport activity. We report that STP13-deficient plants exhibited an enhanced susceptibility and a reduced rate of glucose uptake. Conversely, plants with a high constitutive level of STP13 protein displayed an improved capacity to absorb glucose and an enhanced resistance phenotype. The correlation between STP13 transcripts, protein accumulation, glucose uptake rate and resistance level indicates that STP13 contributes to the basal resistance to B. cinerea by limiting symptom development and points out the importance of the host intracellular sugar uptake in this process. We postulate that STP13 would participate in the active resorption of hexoses to support the increased energy demand to trigger plant defense reactions and to deprive the fungus by changing sugar fluxes toward host cells.