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Viticulture: Biotic and abiotic stress - Grapevine Defence Mechanism and Grape Development

Objective

A. BACKGROUND

A1. Economical importance

The European Union is the largest producer and exporter of wine in the world, with 156 million hectolitres a year, or approximately 60% of world-wide production. Wine exportation value outside of Europe represents more than 2.4 billions of euros, consisting mainly and practically exclusively of guaranteed vintages or high quality wines (AOC or equivalent). There is also a significant contribution from other developed countries (North America and Asia mainly) to the European market.

However, while Europe was the only exporting region twenty years ago, there is now a considerable competition from new producing countries such as Australia, South Africa, Argentina, Chile, etc., which have already taken a significant share of the world market. Other major markets, although still importers of wine, have since developed their national production (examples being the United States and, more recently, China).

The market has thus become world-wide, and in all developed countries, it is now possible for the consumer to find a broad range of wines from all continents.

Over the last decade, European wines have been much improved in quality as a result of varietal changes and restrictions on yields, made possible through recent technological developments. The move towards high quality products has now become the principal objective of European vine growers, who in this way hope to rejuvenate their historical image and thus maintain export markets.

The challenge for sustainable viticulture is to ensure year-after-year improvements in the organoleptic quality of wines while developing new viticultural and oenological practices such integrated pest management (IPM), control of water supply, etc... which are more healthy and environmentally friendly. It is economically and culturally important for the EU that these future developments do not reduce the diversity of vines and wines available today, because this diversity represents one of the most interesting characteristics of this beverage. From an economical standpoint, maintaining this diversity is also a major interest to answer the broad range of consumers tastes and preferences and to differentiate the typicity of European wines from that of other countries, which are often recognizable by a uniform and strong " woody " taste. The advantage of the high diversity of European wines induces in parallel the drawback of complexity.

A2. Diversity and complexity

Compared with other beverage industries, wine has a highly diversified production, resulting in part from the broad range of environmental constraints encountered in the different areas where it is produced. The optimisation of viticultural practices, starting from the choice of cultivars suited to local conditions and ending with the oenological techniques which reveal the typical quality traits obtained in a defined region, results from 2000 years of history. Thus, the wine types produced constitute an important cultural heritage in many regions, providing them with their image and forming a major element in their cultural identity.

Vineyards must face different problems depending on the location. Septentrional areas often encounter difficulties linked to several fungal diseases, whereas in meridional areas water supply may be limiting for optimal growth and development. In all locations, the developmental processes involve a delicate balance between vegetative and reproductive growth, and this balance in turn depends on a perfect control of source/sink relationships.

To ensure a better link between quality and durable viticultural pratices, it is important to put together the vast empirical expertise gained so far by European grapevine growers during centuries, and to connect it with data from genomics and gene expression which finally control the growth of the vine, as well as its ripening (Coombe, 1992, 1997). For example, these data might give some clues about the expression of one given gene controlling a particular trait of ripening or resistance against stress in various cultivars, and under various environmental conditions and agricultural practices. Given the wide diversity of compounds involved, the ripening of the berries (and their organoleptic properties) depend on a delicate balance of expression of the genes encoding the numerous enzymes controlling the primary and secondary metabolism of plant cells.

A3. Gaps

Complete set of data concerning environmental conditions and their impact on vine growth and berry development have been collected in the past. However, different fields of expertise (bioclimatology, ecophysiology, whole plant approaches, characterisation of berry development) have never been fully linked to analyse grapevine behaviour and berry development in interaction with the various environmental factors. As a consequence, our understanding of the factors controlling berry development and disease resistance is still poor. Most importantly, new markers of ripening are needed to complement the ones presently available (sugar content, acidity level, and for red wines, total phenolic compounds), which do not allow a precise determination of harvest time.

There are major gaps of communication between vine growers, scientists dealing with field measurements on grape vine plants, plant pathologists, and biochemists/molecular biologists working on grape. This feeling led for example to the organization of the recent 6th International Symposium on Grapevine Physiology and Biotechnology (Heraklion, Creete, 2000). All over Europe, there are labs and agronomists involved in grapevine research, but the research action is poorly concerted, and usually covers only part of the events which finally contribute to the ripening of grape berries. This has been the situation so far, for example when it is observed that some climates or treatments seem to rise the sugar content. As the reasons are largely unknown, it is impossible to use this information. Conversely, some markers of ripening, and stress resistance have been observed and identified by molecular and biochemical approaches in lab experiments, but their use remain very much restricted if these data are not conforted by field trials, genetic mapping, and in some cases selection programs concerning non AOC wines.

Whatever their quality and coverage, the data concerning either field development (for example sugar content, acidity) or biochemistry, molecular biology and genetics of grapes require the build up of permanent and intensive connections between these areas for a complete understanding and mastering of vine growth .

It is interesting and necessary to organize a network to save time, money, and to improve synergy and efficiency. Clearly, further efforts must be continued to fill these gaps, because improvement of grapevine ripening and defence depends on a thorough knowledge and exploitation of a large body of data including:

-the physiological and developmental characterization of the vine cultivars (rootstocks and scions),
-the growth of the vines and the ripening of the berries, as affected by water status, and agricultural practices affecting water status and ripening (watering, treatment with exogenous growth regulators)
-the growth of the vine and the ripening of the berries as affected by fungal pathogens and agricultural practices affecting resistance against fungi.

All the data concerning post-harvest processes, which also play an important role in wine quality, are out of the scope of the present program, but might be studied in a later program.

A4. Current trends

Although the biological basis of berry development has been considered as a black box until recently (due to the complexity of the metabolic pathways, classical approaches had to be focused on specific hypothesis), it is now possible to directly and simultaneously identify thousands of genes and enzymes involved in berry development by approaches based on the use of DNA chips or macroarrays. The use of macroarrays will allow to determine candidate genes responsive to a particular environmental situation (phytopathogen attack, drought, etc...) and it provides a

preliminary and necessary step before a more thorough study of these genes. This requires first the sequencing and ranking of several thousands ESTs (expressed sequence tags), for the preparation of the arrays. Thanks to constant methodological improvements, this is now possible in a reasonable time, with a reasonable man-power, and at a reasonable cost for a well organized consortium. Although libraries of several thousands ESTs obtained from grape have recently been prepared, these are not freely accessible, due to the participation of an industrial partner in the project (Ablett et al., 2000). It is therefore urgent to develop an organized consortium and project able to use freely these modern tools, in coordination with a physiological and viticultural background. Several of the participants to the present consortium are already developing a program of ESTs expressed during berry ripening. Sequences have begun to be released recently, and several hundreds of sequence will be made available before the end of year 2002. On another hand, a large collection of biological samples (cultivars) and genomic data (RFLP, SSR, RALF, RAPD) are available in different countries involved in this Action (Bulgaria, France, Germany, Greece, Hungary). These efforts need to be coordinated and integrated in a more extensive network that will be open to a wide range of European countries, through data bases accessible to the different participants, and a policy of open publications.

There is an increasing environmental concern about phytochemical treatments and residues, and the admitted levels become more and more restrictive. For example, sodium arsenate, widely used for many years to fight fungal diseases on grapevines, has recently been completely forbidden for this use in EU. Nevertheless, grapevines may still be attacked by several major phytopathogen fungi such as Botrytis, Eutypia and Oidium (Coutos-Th, venot et al., 2001). These diseases may cause considerable loss if they are not prevented or treated. In a context where the use of phytochemicals will be less and less tolerated, the only way to control disease is a thorough knowledge of the resistance mechanisms and of the genes involved in resistance. This will allow the design of viticultural practices able to increase the natural resistance of the plant, and eventually to select and breed appropriate cultivars. With regard to resistance and ripening, the metabolism of secondary compounds in the skin is particularly important, because it is at a crossroad between defense and organoleptic properties. Common precursors from the phenylpropanoid pathway may give birth either to phytoalexins such as stilbene, or flavonoids, tannins and derivatives, which play a key part in the development of the wine aroma during wine-making processes (Boss and Davies, 2001).

This short analysis, and the emergence of new tools to analyse gene expression underlines that it will be strongly beneficial to organize the expertise, facilities, and approaches of grape berry ripening and defence at the European level. This organization will allow to take the best advantage of the large flow of information that will be delivered in the near future. Furthermore, synergy will be achieved by a close collaboration between ecophysiologists, physiologists, molecular biologists and geneticists. This Action also complements other COST Actions presently underway, either dealing with similar concerns (Phytodiagnosis) or using similar tools (Multiarrays). Contacts will be established with these Actions if the present Viticulture Action is developed.

B. OBJECTIVES AND BENEFITS

The main objective of the Action is to increase our knowledge of the biological phenomena involved during the key stages of grape ripening, defence against fungal diseases and resistance to drought, thus allowing significant improvement of viticultural practices during vine development and berry ripening.

These objectives require a wide range of expertise and make it necessary to build a project at the European scale. The network to be created is needed to take full advantage of the European geographical space and of its different pedoclimatical conditions. The general strategy is to create an organized network which will generate and organize basic data on vine genomics and vine ecophysiology, comparable to that developed on crops of similar importance (wheat, rice, maize). This network will associate knowhow and expertise from a wide body of researchers including grapevine growers, agronomists, plant physiologists, biochemists, molecular biologists and geneticists.

This general strategy can be detailed as follows.

(i)to define common measurements and standards, for the assessment of grape development and ripening under various internal (genetic) and external (water status, phytopathogen attacks) constraints.

(ii)to collect and to connect all the data and tools concerning the genetical background of rootstocks, scions and expressed sequence tags.

(iii)To assess the expression of genes under the conditions defined in (i), in order to find new markers of ripening, and the most relevant genes involved in resistance to biotic (fungal diseases) and abiotic stress (especially drought).

These strategies are linked since molecular biology tools can generate considerable information on berry growth and vine response to environment. This will lead to the design of new predictive tools and viticultural practices more friendly to the environment, and to the production of safer wines.

The Action will also contribute to the mapping of genes which control grape quality traits. This resource, which is essential for accelerated breeding, is presently critically lacking.

It will provide a basis to develop proteomic approaches which may be envisaged as a continuation of this Action. Indeed, protein translation, protein stability and post-translational modifications may play a significant part to alter the final enzymatic activity resulting from gene expression (transcripts). Although several informal contacts have been made with proteomic platforms, the proteomic approach is not an integral part of the present proposal, to allow a better focus and a realistic size, and because the methodological tools are quite different from that of the transcriptomic approaches.

C. SCIENTIFIC PROGRAMME

The scientific programme involves two levels of approaches which are necessary and complementary, and should be coordinated, that is to say an ecophysiological and physiological approach at the field and plant level, a molecular and genetic approach at the genome level. Both approaches have been disconnected so far. However, they deal with two facets of the same problems, at different scales. The project is meant to have them closely interacting through a systematic study of gene expression under various ecophysiological conditions, using macroarrays.

It will search correlations between the ecophysiological conditions and gene expression. A limited number of specific problems of grapevine ripening and defence will be studied, as detailed below. The general objective may be divided in five tasks requiring five work groups (WGs), organized and strongly interacting as described in Figure 1 and detailed below.

Work group 1. Sampling and ecophysiology.

Management of grapevine is designed to restrict vegetative development and grape yield, in order to favor grape development and express quality traits for the technological process. Grapevine developmental control is obtained by various viticultiral practices)(Chaumont et al., 1994; Edson et al., 1995; Ollat et GaudillSre, 1995). To optimize these practices, the status of the vine plant and of the berries must be assessed along the development stages by biophysical and biochemical indicators (sugar, total acidity, anthocyanins, flavonols...). However, there are major problems due to (i) the heterogeneity of sampling induced by the wide variability of ripening among different berries of a cluster, among the different clusters of a plant, among the different plants of a same vineyard (ii) the different physical, chemical and biochemical methods used for the measurements.

WG1, which includes all participants, will standardize the sampling methods and design a common set of analytical procedures to assess the development of the vine, the ripening of the berries, and their disease resistance. A subgroup of WG1 will also build the network (essentially through e-mail and through a web site) to exchange all the ecophysiological data and information in real time. Various European vineyards differing by their severity of drought or fungal stresses (Atlantic, Mediterranean, Central Europe, Nordic...) will be selected. For the limited number of sampling places selected, a special effort will be made to study gene expression with macroarrays prepared in Work groups 2, 3 and 4 (see below).

The standardization will allow a better definition of the grape status as well as a better understanding of the interaction between the genetic background, and the environmental conditions (especially water status and fungal attacks) . These indicators will be compared to the modelling approaches which are able to quantify the most significant environmental traits and grapevine response in the vineyard. The models obtained will be tested using gene expression studies conducted in WG 3 and 4.

A subgroup of WG1 will also determine the practical aspects, responsibilities, and accessibility of the different data bases (ecophysiology, EST, genomic) set up by the program.

Work group 2. Systematic sequencing of EST (Expressed sequence tags)

WG2 will prepare and sequence exhaustive libraries of Expressed Sequence Tags (ESTs) from berries or berries parts grown under various environments i.e.various stages of development under normal conditions or affected by fungal disease (e.g. Botrytis) or water stress. Several EST libraries from whole berries have already been constructed, and additional libraries will be prepared during the course of the program. An EST data base will be set up. The ESTs will be analyzed and organized according to their function, and a resource facility will be set up to organize the clones, and to store and to deliver them, according to an agreement prepared by WG1. At least 10,000 independent EST are expected at the end of the program.

Work group 3. Sugar/acid balance, water balance and ripening

The quality of the must obtained from the berries depends on the sugar, acid and aroma content of the berries. Sugars are imported as sucrose by the phloem and stored in high concentration (more than 1 M) as hexoses in the vacuole of the flesh cells. This process depends on membrane transporters (Fillion et al., 1999; Davies et al., 1999; Ageorges et al., 2000) and on enzymes controlling the sugar metabolism (invertases, sucrose synthases, etc...) (Davies et al., 1996). For osmotic reasons, sugar import must be accompanied by a massive influx of water, which involve the activity of water channels (Delrot et al., 2000; Mas et al, 2000). Both sugar influx and water influxes dramatically increase at a special stage of ripening called veraison (Coombe, 1992; Ollat and GaudillSre, 1995), but the events triggering these processes are unknown. Water supply therefore plays a key role in conjunction with sugar import, since it is necessary to maintain sugar import and increase the final weight of the harvest. However, excess water may be detrimental to the start of sugar accumulation, and may also finally dilute the sugar concentration, therefore decreasing the quality of the must prepared from the berries. Another important reason to understand the role of water in ripening, and the exact water need of the vines is the poor availability of water in the mediterranean vine growing countries.

A marked decrease of organic acids (malate, tartrate) concentration in the berries is triggered at the veraison stage (Terrier et al., 2001). Together with sugars, the organic acids determine the sugar/acid balance, a major parameter for vine quality. Excess acid is detrimental to the taste of the wine, whereas too little acid does not allow a good conservation and evolution of the must. The expression of the channels allowing the accumulation of organic acids in the vacuole, as well as that of the enzymes which synthesize or degrade them should therefore be studied in detail.

Thanks to the macroarrays and the EST data base, the impact of environmental conditions on gene expression can be addressed, as well as the genetic diversity of gene expression during berry ripening. WG3 will identify EST corresponding to membrane proteins and soluble enzymes controlling the metabolism and compartmentation of sugars, organic acids and water, and prepare and use macroarrays bearing these clones. Macroarrays containing only these selected genes will be made and dispatched for further and more precise analysis among different participants and locations. The macroarrays bearing clones of genes involved in the sugar/acid balance and water transport will be used to study the expression of these genes along the development and ripening of the berries under various ecophysiological conditions for the locations selected in WG1. In addition to identifying the more important gene(s) controlling these processes, this study should allow to identify new markers of ripening and resistance to drought.

Work group 4. Secondary metabolites and defence reactions

The quality of the must also depends on the presence of precursors of aromas, flavors and colors in the must. These compounds derive from the pathways of secondary metabolism (mainly the phenylpropanoid pathway and the terpenic pathway). The phenylpropanoid pathway also plays an important role in defence reactions, because it leads to the synthesis of tannins and toxic compounds. In case of fungal attack, defence reactions also involve the synthesis of pathogenesis related proteins (PR-proteins) which mainly include various hydrolases. The expression of some key enzymes of the phenylpropanoid pathway and of some PR-proteins, may be controlled by the sugar content of the cells (Tsukuya et al., 1991; Tatersall et al., 1997). In this respect, it will be interesting to correlate the expression of the corresponding genes with the sugar content of the berries, and hence with the expression of the genes studied in WG 3.

WG4 will identify EST corresponding to enzymes and structural proteins involved in defence against fungi, and prepare macroarrays bearing these clones. Some of these EST will be identified through the general libraries made in WG1. However, in addition, WG4 will prepare cDNA libraries from the skin, which are particularly relevant for resistance against fungi, phenylpropanoid metabolism and development of aroma. Indeed, most of the compounds involved in these processes are concentrated in the skin, but poorly abundant in the flesh cells (Boss and Davies, 2001).

The macroarrays prepared with these clones will be used to study the expression of the genes involved in secondary metabolism and defence, along the development and ripening of the berries under various ecophysiological conditions for the locations selected in WG1.

WG5. Grapevine genome database

WG5 will set up a data base for molecular markers and grape genome. Development of better standardized analytical methods concerning grape quality and better knowledge of berry development should accelerate the mapping of genes controlling important quality traits. Segregation of important quality traits in a progeny can also provide very useful plant models for studying berry development and resistance. This data base, which will be an important tool for breeding programs, has already been started in several participants' labs, and will be extended and complemented with new data coming from the program. Links will have to be made with the ecophysiological data base (WG1) and the EST data base (WG2).

These sites will be linked to readily available web sites on grapevine varieties (for example http://www.biology.uoc.gr/gvd/, http://www.dainet.de/eccdb/vitis/ ).

D. ORGANISATION

The organisation and coordination of the COST Action will be assumed by a Management Committee (MC) composed of one or two members from each participating country. The MC will be formally established during a meeting in Brussels organised by the COST secretariat. At the first meeting, the MC will appoint the chair and the deputy chair of the MC and the five WGs. This MC will develop special focus and effort to ensure constant interactions and networking of the different WGs.

This general organisation, as well as the expected output directly results from the objectives and working groups described above. Each work group will gather scientists using different approaches, from the field (ecophysiology) to the gene (molecular biology). The results obtained by the different groups will concur to the final objective which is to improve viticultural practices and wine quality.

E. TIMETABLE

Improvement of plant genotypes by breeding is a long term undertaking. Regularly plant breeders assume a 15 year period to obtain a commercially viable variety. Since this Action is tackling more basic aspects of quality analysis and evaluation, only a six year period is asked for in order to gain maximum scientific progress. However, the perspective of the research and co-operation goes far beyond this period.

The meetings, the calendar, and the milestones for each work group have been forecast. General meetings will be held once a year, preferentially in different countries each time. They will describe the results, plan the next experiments, and foster exchange of scientists which will be managed by the Managing Committee.

In addition to regular meetings allowing the report of the data and the planning of future experiments and work, the MC will promote an active policy of short term exchanges of young and senior scientists, with a priority for Ph.D and young postdoc students, and for the training of participants from less developed European countries.

F. ECONOMIC DIMENSION

The following countries have actively participated in the preparation of the Action or otherwise indicated their interest:

AustriaIsrael
BulgariaItaly
CyprusPortugal
FranceSlovenia
GermanySpain
GreeceSwitzerland

On the basis of the annual estimates provided by the representatives of these countries, the economic dimension of the activities to be carried out under the Action has been estimated, in 2002 prices, at Euro 45 million.

This estimate is valid under the assumption that all the countries mentioned above, but no other countries, will participate in the Action. Any departure from this will change the total cost accordingly.

G. DISSEMINATION PLAN

A subgroup of WG1 will establish a platform for co-ordination and internal dissemination of the accumulated information among the participants, through the creation of an interactive internet homepage. The MC will obtain reports, summaries and discussion contributions from all WG's, to be placed on the web-page.

WG2 will be responsible for the establishment and feeding of the EST data base. WG5 will be responsible for the establishment and linking of the genomic data base.

External dissemination of knowledge and patenting will strictly conform to the policy usually followed in COST Actions. An annual progress report on-line will be delivered, as well as access in real-time to the different databases that may result from the COST Action.

Coordinator

N/A