Grape berries are sophisticated biochemical factories of major economical importance. They import and accumulate water, minerals and sugar, and synthesise amino acids, organic acids, as well as flavour and aroma compounds. The most dramatic changes in the composition of grape berries occur after veraison, during the ripening phase. Berries switch from a status where they are small, hard and acidic, with little sugar, to a status where they are larger, softer, much sweeter, less acidic and strongly flavoured and coloured. The flavour that builds up in grapes is mostly the result of the acid/sugar balance, and the synthesis of flavour and aromatic compounds, or precursors taking place during ripening. Substantial scientific progress has been achieved in understanding the physiological, biochemical and molecular aspects of grape berry maturation. Some knowledge has led to the improvement of wine quality through better grape-growing practices, but this area of basic research is still wide open due to the complexity of the biochemical and molecular mechanisms involved in grape development and ripening, and their response to the environment.
Chapter 1 by Tyerman et al. is dedicated to the topic of water relations in the grape berry. This is a hot research topic as the water is the most important constituent of the fruit and, thereby, of wine. Grape berries normally contain 75–85% water, which is the main solvent of solutes including sugars, acids and phenolic compounds. Thus, quality, via the concentration of sugars and flavour compounds, and yield of the vintage are directly affected by berry water content at harvest. The paradigm of water transport across the biological membrane has recently changed, when the molecular basis (aquaporins) was first identified by the team of Peter Agre. The role of aquaporins in the regulation of berry water relations is also discussed in this chapter, with a particular emphasis on the varietal differences observed in aquaporin functioning. This is an interesting area that is still open to further research. Finally, the impact of water status of the parent vine on berry ripening is also discussed.
Chapter 2 by Martins et al. deals with the mineral compounds in the grape berry. Together with water, minerals are imported to grape berries, which is why mineral soil composition has a pivotal influence on grape quality and on the organoleptic properties of wine. Grape berries are extremely rich in K, which is involved in several physiological processes: enzyme activation, cellular membrane transport processes and translocation of assimilates, anion neutralisation, which is essential to the maintenance of membrane potential, and osmotic potential regulation, which controls the plant water relations, turgor maintenance and growth. Thus, the knowledge of the mechanisms mediating the transport of K+ to grape berries and their regulation is of the utmost importance to develop strategies optimising the levels of this cation in berries and improving fruit quality, while maintaining the pH of the wine. In this chapter, the mechanisms of K+ transport in plant cells, and in the grape tissues in particular, are reviewed and discussed. Besides K, the present chapter provides an overview of the dynamics of other important mineral compounds in the grapevine, especially in the berry, namely N, Ca, P, Mg and S, and of the contribution of each element to berry quality and yield.
The grapevine is a good example of a crop where sugar accumulation in the fruit has an important economic role. Massive sugar transport and compartmentation into the grape berry mesocarp cells start at veraison and continue until harvest. This topic is addressed in Chapter 3. How does the root system (rootstock) and the wood of the canes influence the sugar status of the fruit? How does the management of the aerial system affect the berry sugar level? How is sugar imported to the fruit, stored and metabolised? Our understanding of the mechanisms of carbon partitioning and accumulation in grapevines is improving due to the use of molecular techniques. In particular, the cloning of key genes and their expression in both homologous and heterologous systems have proven to be powerful tools, and the sequencing of the grapevine genome and the development of the high throughput techniques, such as microarrays and deep sequencing for gene expression analysis, will enable rapid progress on these subjects in the future. This chapter provides an updated review of the molecular biology of sugar transport and accumulation in the grape. A complex set of transporter proteins, invertases, and finely hormonal regulated mechanisms culminate in the fructose and glucose storage in the vacuole. At the end of the chapter, a model describing the possible routes for sugar import and accumulation during berry ripening is proposed.
Chapter 4 is devoted to the biochemistry of organic acids in the grape. The author of this chapter pioneered the elucidation of the key steps involved in the metabolism of malic and tartaric acid in the grape berry. Both acids may account for over 90% of the total acidity in the berry, and they are also the largest contributors to the pH of juice, must and wine. Despite the close structural similarity of tartaric acid and L-malic acid, it was demonstrated in 1958 that different metabolic pathways led to the formation of these two acids in grapevines. What is the precursor of tartaric acid? How is the precursor metabolised? Besides the L-idonate dehydrogenase enzyme, the identification and analysis of tartaric acid synthesis genes and their encoded enzymes await further discoveries, remaining important research topics. L-malic acid is the second major organic acid that accumulates during grape berry development. In contrast to tartaric acid, malic acid formed in the berry at pre-veraison is broken down during a brief period around veraison. This process, which is regulated by temperature and by developmental cues, results in marked changes in the acid composition of grapes at harvest. As explained in this chapter, the pathways of malic acid synthesis and breakdown, and the details of its participation in a wide range of metabolic processes have been determined for the grape berry, but their dependence on environmental conditions, such as heat and water availability, is not yet fully understood. In other words, the regulation of the well-characterised switch between synthesis and breakdown of the malate at the time of veraison remains largely uncharacterised.
Chapter 5 addresses the subject of grape berry phenolics. It is well known that berry phenolics largely contribute to wine quality and have beneficial effects on many aspects of the human health. In the past decade, significant advances have been made towards a better understanding of the genetics, biochemistry and physiology governing the synthesis of this class of secondary metabolites. Three main classes of phenolic compounds are synthesised in the grape berry: phenolic acids, stilbenes and flavonoids, but phenolic composition is highly diversified among different varieties and the environments where they are grown. This chapter reviews the chemical composition and the pattern of accumulation of phenolics in the grape berry, the diversity in phenolic composition of the grapevine germplasms, and the molecular basis of their synthesis, including the effect of environmental factors. It is well known that anthocyanins are responsible for the red, purple and blue pigmentation of the grape berries. But how are they synthesised and where are they accumulated? Also, how are they transported across the biological membranes?
Chapter 6 deals with the aromas of the grape berry and wine. The subtleness, complexity and uniqueness of aromas are a source of great pleasure for wine lovers. Bouquet and flavour are obviously related to the expertise of the winemaker and the techniques used, but primarily they reflect grape composition, especially varietal character – and its particular expression in a given terroir (climate, soil, and viticultural practices). How can the chemistry of flavours explain what our senses perceive? The study of grape and wine aromas is laborious because the most important grape aroma compounds are often present in very low concentrations. The first studies of grape flavours date back to the early 1950s when gas chromatography made it possible to separate volatile components in the vapour phase. Since then, many volatile compounds have been identified, especially through the coupling of gas chromatography with olfactometry and mass spectrometry. This chapter explores the role and diversity of methoxypyrazines, terpenic compounds, C13-norisoprenoids derivatives and thiols, together with the metabolic pathways involved in their synthesis. Still, many odourless and non-volatile compounds in grapes are the source of odouriferous compounds in wine. For instance, fermentation significantly alters the monoterpene composition of grapes through chemical and microbiological processes. The most major transformation concerns the degradation of nerol and geraniol by the yeast Saccharomyces cerevisiae via an enzymatic reduction.
Polyamines (PAs) (Chapter 7) are the most important biogenic amines found so far in plants, and they are associated with numerous developmental and stress-related processes. The most abundant and well-described Pas are putrescine (Put), spermidine (Spd) and spermine (Spm). As argued in this chapter, these biogenic amines may play a fundamental role in grape berry set and development, solely or in combination with the hormones. For instance, the balance between PA and ethylene synthesis may be one of the major determinantal factors regulating the fruit-set process. Also, in berry abscission, which is largely linked to metabolic and hormonal ‘disorders’ and repression of the nutrient supply to the inflorescence, ABA and ethylene are considered to be among the primary drivers of the procedure, but PAs may act as regulators as well. The translocation under photoperiodic flowering induction of free Spd to the inflorescence seems to be a part of the complex mechanism which occurs during the transition of vegetative buds to flowers. Thus, this chapter illustrates important correlative roles of PAs in grape berry development and summarises the current literature, providing an accurate and updated overview of the ongoing research.
Chapter 8 details the structure-function of the grape berry vacuoles and the mechanisms of solute transport across the tonoplast. This is a particularly relevant issue since vacuoles are the main reservoirs of sugars, organic acids, aromas, flavours, ions and water in grape berry tissues. At anthesis, most mesocarp cells appear to be univacuolated, but at veraison the large vacuole splits into smaller vacuoles, generating a complex internal membrane structure. This chapter provides interesting images showing a complex array of intact vacuoles occupying much of the cytosol of a mature grape berry cell. Changes in the vacuolation degree may also be involved in the maintenance of turgor pressure, or result from an increase of vacuole storage function. The berry “fattening” that occurs at the ripening stage is mostly due to the massive accumulation of sugars, water and phenolics in the vacuole. Several tonoplast proteins including pumps, carriers, ion channels and receptors were already identified in several plant models at the biochemical and molecular levels, including the most abundant ones, V-ATPase, V-PPase, and water channels (aquaporins). But the molecular mechanisms involved in the accumulation of these solutes, and how they are regulated,is still far from being fully understood in grape. In this context, approaches aiming at the purification of intact vacuoles are developed. The diversity and storage role of the vacuole of grape cells, and the molecular mechanism involved in solute transport across the tonoplast, are discussed in this chapter.
Chapter 9 by Goulao et al. concerns the grape berry cell wall (CW). The CW of mesocarp cells consists of approximately 90% of polysaccharides and less than 10% of a protein fraction rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans are the major constituents. In the exocarp, polysaccharides account for 50% of the cell wall material. Insoluble proanthocyanidins, structural proteins and lignin are also important constituents. Modifications of the grape pulp and skin cell wall during ripening provide the flexibility for the cell to expand during fruit growth and to modulate the final texture, with important repercussions in grape characteristics, wine quality and wine-making methods. For instance, anthocyanin extraction depends directly upon the cell wall degradation in the skin. Nonetheless, the cell wall is also an important source of biologically active signalling molecules, regulating cell-to-cell interactions, and a carbohydrate storage reserve. A number of genes related to primary CW biosynthesis and modification, and to secondary CW biosynthesis were identified in the genome of Vitis vinifera. The public release of the Vitis genome and the annotation of Vitis genes allowed detailed studies of the genes coding for enzymes associated with cell wall biosynthesis, modification during cell growth and fruit ripening, as well as the deposition of secondary wall polymers, providing a better picture of the associated pathways. The grape berry CW transcriptome is thoroughly analysed in this chapter.
The following chapter by Böttcher and Davies (Chapter 10) focuses on the role of hormones during the development of the grape berry. Hormonal coordination is a crucial issue in the development of this non-climacteric fruit, as the changes that occur at both physical and biochemical levels are considerable and rapid, occurring over only a few weeks and involving a range of tissues and cell types. This chapter illustrates the role of ethylene, abscisic acid, brassinosteroids, gibberellins, auxins, salicylic acid and jasmonates in grape berry development and ripening and how they are synthesised/accumulated. These hormones are accumulated to significant levels in flowers, and early in berry development. As extensively discussed in this chapter, hormones have the ability to coordinate changes in a large number of genes in response to both developmental and external cues. Recent technological developments now permit the detection, on a genome wide scale, of the changes in the expression of genes during development, in response to hormone treatments and in hormone mutants. The authors conclude that, despite the expansion of our knowledge on the hormonal control of berry development, there are still many questions to be answered. For instance, the role of ethylene, which is essential to climacteric fruit ripening, is still controversial in grape berry ripening. The same is true for the interactions between the different signalling pathways and how these affect berry development.
The transcriptome is the complete set of RNA molecules produced by a cell, tissue or organism. It includes mRNA, rRNA, tRNA and other non-coding RNAs, although in many cases the mRNA profile is the most sought after because it corresponds to the expression of protein-encoding genes. The metabolome, defined by analogy to the transcriptome, is the complete set of metabolites (small molecules, Mr = 1,000 D) produced in a single cell, tissue or organism. Like the transcriptome, the metabolome is complex, dynamic and varies by cell type, developmental stage and in response to internal and external cues. Berry development has been investigated at the transcriptional level to study global expression profiles during formation and ripening, while a smaller number of large-scale metabolite studies have been carried out thus far. This topic is thoroughly addressed by Tornielli et al. in Chapter 11. The availability of the complete grapevine genome sequence makes it much easier to identify candidate genes governing the key processes underlying berry development, particularly those relating to quality traits. This chapter summarizes the wealth of information generated by the grapevine transcriptomic experiments in the last decade according to the principal metabolic pathways and biological processes of berry development, such as photosynthesis, sugar, organic acid and lipid metabolism, secondary metabolism, hormone biosynthesis and regulation, cell wall metabolism and the response to stress. Although the huge amount of information provided by the transcriptomic and metabolomic approaches is frequently difficult to interpret, the investigation of key molecular events underlying grape berry development can provide useful data for the improvement of mature berry quality traits.
Chapter 12 describes the microbial community of the grape berry. How is this topic related to the grape berry biochemistry? Why do grape berry microbionts have important biotechnological repercussions? While the first question is not so easily addressed, the answer to the latter is quite obvious. Important phytopathogens responsible for grapevine diseases worldwide are the oomycete Plasmopara viticola (downy mildew) and the ascomycete Erysiphe necator (powdery mildew). The causal agent of grey rot is the saprophytic mould Botrytis cinerea. A wide diversity of yeast species are also common contaminants of berry surfaces, but the key agent of wine fermentation, Saccharomyces cerevisiae, is rarely recovered from grapes. Bacterial groups include the spoiling acetic acid bacteria and lactic acid bacteria responsible for the malolactic fermentation. As detailed in this chapter, grape berries are able to exudate a variety of compounds through the cuticle and the epicuticular wax layer onto their surfaces, including phenols, sugars, lipids, malic acid, potassium and sodium. Interestingly, grape berries exude onto their surfaces both microbial inhibitory and stimulatory compounds. Also, increasing evidence suggests that the cuticle may be a source of carbon for the berry microflora, particularly the resident community. The capacity of certain pathogens to metabolise cutin and cuticular waxes, as well as pectic and hemicellulosic components of the cell wall, may explain why certain species of the fungi and aerobic yeasts, with a high metabolic diversity, are so frequently found on the sound berry surface. However, the access to the nutrient-rich inner cells through fissures or wounds on the berry skin completely changes the berry microflora. This is a stimulating topic of research and, as explained by the authors, it is somewhat surprising to find only scarce and fragmented information on grape microbiota.
The chemical composition of the grape berry directly affects wine composition and quality. Chapter 13 addresses this topic that has been a matter of passion and controversy both in society and within the scientific community: the health benefits resulting from moderate wine consumption. In the 1990s, people’s attention was increasingly drawn to the positive effects of moderate wine consumption. Indeed, the well-known “French paradox” reports that, although the French diet is relatively rich in saturated lipids compared to that of other countries, the level of mortality due to coronary heart disease is reduced as a result of daily wine consumption. It has been reported that, besides alcohol itself, which in moderate amounts helps blood flow in the body, wine polyphenols contain antioxidant properties, which are also beneficial to health. As thoroughly reviewed in this chapter, epidemiological studies conclude that moderate alcohol and/or wine consumption may protect against the incidence of many diseases of the modern society such as atherosclerosis, hypertension and diabetes. The biochemical mechanisms that may account for these therapeutic properties are also discussed.
It is our firm belief that this eBook, which is written by an international team of leading experts, is the pioneer in offering a focussed and integrated coverage of the biochemistry and molecular biology of the grape berry, emphasising the most important aspects of grape fruit development and ripening. It is a comprehensive and updated eBook for researchers, scientists and biotechnologists, and it can also be used as a reference manual for graduate and undergraduate students as it gathers useful and updated references and original data from leading laboratories worldwide.
Universidade do Minho
M. Manuela Chaves
Universidade Nova de Lisboa
University of Bordeaux