Charles Romieu

Ectopic Expression of VvMybPA2 Promotes Proanthocyanidin Biosynthesis in Grapevine and Suggests Additional Targets in the Pathway

Grapevine (Vitis vinifera) proanthocyanidins contribute to plant defense mechanisms against biotic stress and also play a critical role in organoleptic properties of wine. In grapevine berry, these compounds are mainly accumulated in exocarps and seeds in the very early stages of development. A previous study has already identified VvMybPA1 as the first transcription factor involved in the regulation of the proanthocyanidin pathway during seed development in grapevine. A novel Myb factor, VvMybPA2, which is described in this study, is in contrast mainly expressed in the exocarp of young berries and in the leaves. This transcription factor shows very high protein sequence homology with other plant Myb factors, which regulate flavonoid biosynthesis. Ectopic expression of either VvMybPA1 or VvMybPA2 in grapevine hairy roots induced qualitative and quantitative changes of the proanthocyanidin profiles. High-throughput transcriptomic analyses of transformed grapevine organs identified a large set of putative targets of the VvMybPA1 and VvMybPA2 transcription factors. Both genes significantly activated enzymes of the flavonoid pathway, including anthocyanidin reductase and leucoanthocyanidin reductase 1, the specific terminal steps in the biosynthesis of epicatechin and catechin, respectively, but not leucoanthocyanidin reductase 2. The functional annotation of the genes whose expression was modified revealed putative new actors of the proanthocyanidin pathway, such as glucosyltransferases and transporters.

Changes in acidity and in proton transport at the tonoplast of grape berries during development.

Abstract. As in many fruits, the induction of grape berry (Vitis vinifera L.) ripening results in intense breakdown of malic acid. Using membrane fractions, we tested the hypothesis that changes in acidity resulted from malate vacuolar decompartmentation. The hydrolytic activities of the two primary vacuolar pumps inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) and vacuolar ATPase (V-ATPase; EC 3.6.1.3) increased throughout development with an acceleration during ripening, as confirmed by Western blotting and analysis of transcript expression. The ratio of V-PPase activity to V-ATPase activity was always in favour of V-PPase and reached its maximum value at véraison. The rate of anion transport strongly increased during ripening. Before ripening, tonoplast passive permeability was low, but rose during ripening. Our data indicate that tonoplast leakage dramatically increased during ripening. This leakage is probably the prime cause of malate decompartmentation, amplified by the incapacity of oxidative phosphorylation to face increased energy demand.

Identification of genes associated with flesh morphogenesis during grapevine fruit development

Fruit morphogenesis is a process unique to the angiosperms, and yet little is known about its developmental control. Following fertilization, fruits typically undergo a dramatic enlargement that is accompanied by differentiation of numerous distinct cell types. To identify genes putatively involved in the early development of grapevine fruit, we used the fleshless berry mutant (Vitis vinifera L. cv Ugni Blanc) that has dramatically reduced fruit size due to a lack of pericarp development. Using oligo-specific arrays, 53 and 50 genes were identified as being down- and up-regulated, respectively, in the mutant. In parallel, Suppression Subtractive Hybridization performed between the mutant and the wild type (WT) allowed the identification of new transcripts differentially expressed during the first stages of mutant and WT pericarp development. From this data, the picture emerged that the mutation promotes the expression of several genes related to ripening and/or to stress and impairs the expression of several regulatory genes. Among those, five genes encoding proteins previously reported to be associated with, or involved in, developmental processes in other species (a specific tissue protein 2, ATHB13, a BURP domain protein, PISTILLATA, and YABBY2), were identified and investigated further using real-time PCR and in situ hybridization. Expression in the pericarp was confirmed, specific spatial and/or temporal patterns were detected and differences were observed between the WT and the mutant during fruit development. Expression of these genes appeared to be affected during young fruit development in the mutant, suggesting that they may play a role in grape berry morphogenesis.

Analysis of cell wall neutral sugar composition, β-galactosidase activity and a related cDNA clone throughout the development of Vitis vinifera grape berries.

Abstract Fruit softening during ripening is accompanied by changes in cell wall composition due to the action of cell wall modifying enzymes. Moreover, the cell walls of grape berries form a barrier to the diffusion of aromatic and polyphenolic compounds which are important for wine quality. Samples of grape berries ( Vitis vinifera L., cv Ugni blanc) were harvested in 1996 and 1997 at twelve different developmental stages. The development of berries was characterized by physical, chemical and biochemical analysis. Isolated cell walls were analysed for their neutral sugar contents. The main changes during grape berry development were a large decrease in galactose parallel with glucose accumulation, while other neutral sugars (arabinose, rhamnose, xylose, fucose and mannose) showed no significant variations. For individual berries, galactose loss seemed to be softening-related, while galactose removal per mg of cell wall material was involved in a more general ripening process. β-Galactosidase (EC 3.2.1.23) activity was temporally associated with the loss of cell wall-linked galactosyl residues. A 545-base long partial cDNA ( ϐ-gal 10, accession No. AF159124, GenBank) was isolated from first strand cDNA, and shared significant similarities with several β-gals in data banks. The pattern of transcript expression showed that β-gal 10 was only detectable in the early stages of development, suggesting that β-gal 10 may encode for a β-galactosidase active on cell walls during the early development of grape berries. Relationships between galactose content of the cell wall, β-galactosidase activity and expression of the corresponding transcripts, and their possible involvement in grape berry softening and ripening are discussed.

Identification and functional expression in yeast of a grape berry sucrose carrier

Abstract A large increase in hexose accumulation is typical of grape berry ripening. To analyse sugar transport processes during grape berry ( Vitis vinifera L. cv Ugni Blanc) development, a cDNA encoding for a sucrose transporter, designated VvSUT1 was cloned. Sucrose transport activity of the VvSUT1 protein was demonstrated by heterologous expression in yeast. A study of gene expression along berry development showed that VvSUT1 transcripts are present at all stages but accumulate with the onset of ripening. This work shows that sucrose transport at the plasma membrane of grape berry cells may play a role in the accumulation of sugars during maturation.

The Grapevine fleshless berry Mutation. A Unique Genotype to Investigate Differences between Fleshy and Nonfleshy Fruit

In flowering plants, fruit morphogenesis is a distinct process following fertilization resulting in the formation of a specialized organ associated with seeds. Despite large variations in types and shapes among species, fleshy fruits share common characteristics to promote seed dispersal by animals such as organ growth and metabolite accumulation to attract animal feeding. The molecular biology of fruit ripening has received considerable attention, but little is known about the determinism of early fruit morphogenesis and why some fruits are fleshy while others lack flesh. We have identified in grapevine (Vitis vinifera) a mutation we have named fleshless berry (flb) that reduces by 20 times the weight of the pericarp at ripening without any effect on fertility or seed size and number. The flb mutation strongly impaired division and differentiation of the most vacuolated cells in the inner mesocarp. The timing of ripening was not altered by the mutation although the accumulation of malic acid in the green stage was noticeably reduced while sucrose content (instead of hexoses) increased during ripening. The mutation segregates as a single dominant locus. These results indicate that the Flb− mutant is suitable material to advance our understanding of the genetic and developmental processes involved in the differentiation of an ovary into a fruit.

A Grapevine Gene Encoding a Guard Cell K Channel Displays Developmental Regulation in the Grapevine Berry

SIRK is a K+ channel identified in grapevine (Vitis vinifera), belonging to the so-called Shaker family. The highest sequence similarities it shares with the members of this family are found with channels of the KAT type, although SIRK displays a small ankyrin domain. This atypical feature provides a key to understand the evolution of the plant Shaker family. Expression inXenopus laevis oocytes indicated that SIRK is an inwardly rectifying channel displaying functional properties very similar to those of KAT2. The activity of SIRK promoter region fused to the GUS reporter gene was analyzed in both grapevine and Arabidopsis. Like other KAT-like channels,SIRK is expressed in guard cells. In Arabidopsis, the construct is also expressed in xylem parenchyma. Semiquantitative reverse transcriptase-polymerase chain reaction experiments indicated that SIRK transcript was present at low levels in the berry, during the first stages of berry growth. After veraison, the period of berry development that corresponds to the inception of ripening and that is associated with large biochemical and structural modifications, such as evolution of stomata in nonfunctional lenticels and degeneration of xylem vasculature, the transcript was no longer detected. The whole set of data suggests that in the berriesSIRK is expressed in guard cells and, possibly, in xylem tissues. The encoded channel polypeptide could therefore play a role in the regulation of transpiration and water fluxes in grapevine fruits.

Generation of ESTs from grape berry at various developmental stages

Summary Expressed sequence tags (ESTs) are a main tool in functional genomics. In this paper, we report the construction of 3 unidirectional cDNA libraries from green, softening, and ripening grape berries. For each library, a set of 100 randomly selected clones were sequenced at their 3′ end. Two hundred seventy-five expressed sequence tags (ESTs) were thus generated, representing around 187,000 bp of independent cDNA. 78 % of clones demonstrated significant homology to previously deposited nucleotide and/or polypeptide sequences. Transcripts homologous to proteins with known or putative functions were classified into functional categories. Highly represented protein classes were cell wall proteins, ribosomal proteins, proteins involved in signal transduction, and ripening-related proteins. A significant proportion of the ESTs (12 %) demonstrated no significant homology to any deposited sequence. This proportion of novel plant genes was highest (19 %) at veraison, the transient period when the berry softens and irreversibly switches from an organic acid to a sugar-accumulating organ. Expression analysis of these ESTs will define the range of genes involved in the development of grape berry as a model of fleshy, acidic, phenols-rich, and non-climacteric fruit.

New stable QTLs for berry weight do not colocalize with QTLs for seed traits in cultivated grapevine ( Vitis vinifera L.)

BackgroundIn grapevine, as in other fruit crops, fruit size and seed content are key components of yield and quality; however, very few Quantitative Trait Loci (QTLs) for berry weight and seed content (number, weight, and dry matter percentage) have been discovered so far. To identify new stable QTLs for marker-assisted selection and candidate gene identification, we performed simultaneous QTL detection in four mapping populations (seeded or seedless) with various genetic backgrounds.ResultsFor berry weight, we identified five new QTLs, on linkage groups (LGs) 1, 8, 11, 17 and 18, in addition to the known major QTL on LG 18. The QTL with the largest effect explained up to 31% of total variance and was found in two genetically distant populations on LG 17, where it colocalized with a published putative domestication locus. For seed traits, besides the major QTLs on LG 18 previously reported, we found four new QTLs explaining up to 51% of total variance, on LGs 4, 5, 12 and 14. The previously published QTL for seed number on LG 2 was found related in fact to sex. We found colocalizations between seed and berry weight QTLs only for the major QTL on LG 18 in a seedless background, and on LGs 1 and 13 in a seeded background. Candidate genes belonging to the cell number regulator CNR or cytochrome P450 families were found under the berry weight QTLs on LGs 1, 8, and 17. The involvement of these gene families in fruit weight was first described in tomato using a QTL-cloning approach. Several other interesting candidate genes related to cell wall modifications, water import, auxin and ethylene signalling, transcription control, or organ identity were also found under berry weight QTLs.ConclusionWe discovered a total of nine new QTLs for berry weight or seed traits in grapevine, thereby increasing more than twofold the number of reliable QTLs for these traits available for marker assisted selection or candidate gene studies. The lack of colocalization between berry and seed QTLs suggests that these traits may be partly dissociated.

Proton pumps and anion transport in Vitis vinifera: The inorganic pyrophosphatase plays a predominant role in the energization of the tonoplast

Abstract In order to gain insight into vacuolar changes that are associated with fruit specialization regarding the accumulation of organic acids, the properties of tonoplast vesicles extracted from grape cell culture and ripe berries were compared. The two primary pumps, PPase (Pyrophosphatase) and V-ATPase (V-Adenosine Triphosphatase), which were found on both fruit and cell culture tonoplast vesicles, exhibited classical behaviour for known inhibitors and activators. However, the 3-fold higher activity of the PPase in comparison to the V-ATPase was unexpected in a fruit exhibiting a vacuolar pH of 3.5. Surprisingly, the hydrolytic activities of both pumps were stimulated by temperatures up to 65 °C. Anion transport was studied by measuring simultaneously proton entry and membrane potential on the two types of tonoplast vesicles. Maximal acidification rates could be observed upon simultaneous energization by ATP and PPi, and were abolished by imidodiphosphate and bafilomycin, while vanadate had little effect. Comparison of the sequence of transport rates observed with mineral and organic anions on fruit or cultured cell tonoplast vesicles suggested that the fruit vacuole was more specialized in the transport of organic anions than the cultured cells. The transport of malate and tartrate was completely inhibited by 4,4′-diisothio-cyanato-stilbene-2,2′disulphonic acid while chloride transport was less affected. Km values for malate and tartrate transports were similar for vesicles extracted from cultured cell and fruits, around 14 mM. The transport rates of malate and tartrate were not additive, suggesting that a similar transporter was involved in the vacuolar uptake of the two major acids of grape berries.