Characterization of mitochondrial dicarboxylate/tricarboxylate transporters from grape berries

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.     

Vitis vinifera root and leaf metabolic composition during fruit maturation: implications of defoliation

Grapevine (Vitis vinifera) roots and leaves represent major carbohydrate and nitrogen (N) sources, either as recent assimilates, or mobilized from labile or storage pools. This study examined the response of root and leaf primary metabolism following defoliation treatments applied to fruiting vines during ripening. The objective was to link alterations in root and leaf metabolism to carbohydrate and N source functioning under conditions of increased fruit sink demand. Potted grapevine leaf area was adjusted near the start of véraison to 25 primary leaves per vine compared to 100 leaves for the control. An additional group of vines were completely defoliated. Fruit sugar and N content development was assessed, and root and leaf starch and N concentrations determined. An untargeted GC/MS approach was undertaken to evaluate root and leaf primary metabolite concentrations. Partial and full defoliation increased root carbohydrate source contribution towards berry sugar accumulation, evident through starch remobilization. Furthermore, root myo‐inositol metabolism played a distinct role during carbohydrate remobilization. Full defoliation induced shikimate pathway derived aromatic amino acid accumulation in roots, while arginine accumulated after full and partial defoliation. Likewise, various leaf amino acids accumulated after partial defoliation. These results suggest elevated root and leaf amino N source activity when leaf N availability is restricted during fruit ripening. Overall, this study provides novel information regarding the impact of leaf source restriction, on metabolic compositions of major carbohydrate and N sources during berry maturation. These results enhance the understanding of source organ carbon and N metabolism during fruit maturation.     

Treatment of Grape Berries,a Nonclimacteric Fruit with a Synthetic Auxin,Retards Ripening and Alters the Expression of Developmentally Regulated Genes

Treatment of grape (Vitis vinifera L.) berries with the synthetic auxin-like compound benzothiazole-2-oxyacetic acid (BTOA) caused a delay in the onset of ripening of approximately 2 weeks. This was manifested as a retardation of the increases in berry weight, color, deformability, and hexose concentration. BTOA treatment also delayed by 2 weeks the increase in abscisic acid level that normally accompanies ripening and altered the expression of a number of developmentally regulated genes. A putative vacuolar invertase, which is normally expressed from berry set until ripening and turned off after ripening commences, remained expressed throughout development in BTOA-treated grape berries. This elevated expression resulted in increased levels of invertase activity. In contrast, the up-regulation of four other genes normally switched on at the time of ripening was delayed in BTOA-treated fruit. These included chalcone synthase and UDP-glucose-flavonoid 3-O-glucosyl transferase, both of which are involved in anthocyanin synthesis, a chitinase, and a ripening-related gene of an unknown function. These observations support the view that auxins (perhaps in conjunction with abscisic acid) may have a role in the control of grape berry ripening by affecting the expression of genes involved in the ripening process.     

植物ASR基因研究进展

ASR（abscisic acid,stress,ripening-induced）基因是近年来从植物中发现的一类受ABA、胁迫和成熟诱导表达的基因,具有保守的ABA/WDS结构域。ASR基因不仅参与植物对干旱、高盐、低温以及脱落酸的胁迫应答,而且参与植物生命活动的许多过程,如果实发育、成熟和糖代谢等。本文综述了近年来国内外ASR基因的研究进展,主要包括ASR基因和蛋白结构特点、ASR基因家族的进化、ASR基因的表达及可能具有的功能,为植物ASR基因研究提供参考。     

脱落酸、胁迫、成熟诱导基因研究进展

近年来从植物中发现了越来越多的受脱落酸、胁迫、成熟诱导表达的基因,这些 ASR(Abscisic acid, Stress and Ripening inducible)基因参与植物对冷、渗透压、脱落酸处理的胁迫应答已被证实,该类基因也参与植物生命活动的许多方面如果实发育、成熟等．对 ASR 基因的克隆鉴定,以及该基因在胁迫应答和果实成熟方面的作用进行了综述．     

Peroxisomal thiolase mRNA is induced during mango fruit ripening

Fruit ripening is a complex, developmentally regulated process. A series of genes have been isolated from various ripening fruits encoding enzymes mainly involved in ethylene and cell wall metabolism. In order to aid our understanding of the molecular basis of this process in a tropical fruit, a cDNA library was prepared from ripe mango (Mangifera indica L. cv. Manila). By differential screening with RNA poly(A)⁺ from unripe and ripe mesocarp a number of cDNAs expressing only in ripe fruit have been isolated. This paper reports the characterization of one such cDNA (pTHMF 1) from M. indica which codes for a protein highly homologous to cucumber, rat and human peroxisomal thiolase (EC 2.3.1.16), the catalyst for the last step in the -oxidation pathway.The cDNA for the peroxisomal mango thiolase is 1305 bp in length and codes for a protein of 432 amino acids with a predicted molecular mass of 45 532 Da. Mango thiolase is highly homologous to cucumber thiolase (80%), the only other plant thiolase whose cloning has been reported, and to rat and human thiolases (55% and 55% respectively).It is shown by northern analysis that during fruit ripening THMF 1 is up-regulated. A similar pattern of expression was detected in tomato fruit. Wounding and pathogen infection do not appear to affect THMF 1 expression. The possible involvement of thiolase in fatty acid metabolism during fruit ripening will be discussed. To our knowledge this is the first report cloning of a plant gene involved in fatty acid metabolism showing an induction during fruit ripening.     

Expression of ACC synthase is enhanced earlier than that of ACC oxidase during fruit ripening of mume (Prunus mume)

Mume (Japanese apricot: Prunus mume Sieb. et Zucc.) is a climacteric fruit that produces large amounts of ethylene as it ripens. Ripening is accompanied by marked increases in the activities of two ethylene-biosynthetic enzymes, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. To study the molecular aspects of ripening of mume, we isolated cDNA clones for proteins that we considered likely to be involved in the biosynthesis and perception of ethylene during ripening, namely, ACC synthase, ACC oxidase and the ethylene receptor. Northern blotting analysis revealed the markedly increased expression of ACC synthase prior to that of ACC oxidase and the increase in ethylene production during ripening. Overall, the levels of the mRNAs for the genes corresponded closely to the levels of activity of the ethylene-biosynthetic enzymes. Exposure of mature green mume fruit to ethylene for 12 h induced strong expression of ACC synthase, as well as of ACC oxidase. Wounding of the pericarp of mume fruit induced the expression of ACC synthase but not of ACC oxidase. The rate of ethylene production increased only slightly after wounding. These results suggest that expression of the genes for ACC synthase and ACC oxidase must be activated sequentially for maximum production of ethylene during ripening of mume fruit and that several mechanisms regulate the expression of ethylene-biosynthetic genes during ripening.     

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3′4′5′-hydroxylated forms through the differential regulation of F3′H and F3′5′H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.     

Cloning and Expression of a Hexose Transporter Gene Expressed during the Ripening of Grape Berry

The ripening of grape (Vitis vinifera L.) is characterized by massive sugar import into the berries. The events triggering this process and the pathways of assimilate transport are still poorly known. A genomic clone Vvht1 (Vitis vinifera hexose transporter1) and the corresponding cDNA encoding a hexose transporter whose expression is induced during berry ripening have been isolated. Vvht1 is expressed mainly in the berries, with a first peak of expression at anthesis, and a second peak about 5 weeks after véraison (a viniculture term for the inception of ripening). Vvht is strictly conserved between two grape cultivars (Pinot Noir and Ugni-Blanc). The organization of the Vvht1 genomic sequence is homologous to that of the Arabidopsis hexose transporter, but differs strongly from that of the Chlorella kessleri hexose transporter genes. The Vvht1 promoter sequence contains several potential regulating cis elements, including ethylene-, abscisic acid-, and sugar-responsive boxes. Comparison of the Vvht1 promoter with the promoter of grape alcohol dehydrogenase, which is expressed at the same time during ripening, also allowed the identification of a 15-bp consensus sequence, which suggests a possible co-regulation of the expression of these genes. The expression of Vvht1 during ripening indicates that sucrose is at least partially cleaved before uptake into the flesh cells.     

Sequence analysis and transcriptional profiling of two vacuolar H+-pyrophosphatase isoforms in Vitis vinifera

Gene expression of grapevine vacuolar H(+)-pyrophosphatase (V-PPase EC 3.6.1.1.) during fruit ripening has previously been reported. Here we report on putative multiple V-PPase isoforms in grapevine. In this study a full-length cDNA sequence with an open reading frame of 2,295 nucleotides encoding a V-PPase gene (vpp2: acc. nr. AJ557256) was cloned. Sequence analyses of the deduced amino acid residues and RT-PCR experiments indicated that Vitis vinifera L. has at least two distinct isoforms of the V-PPase gene. Bioinformatic analyses of 13 V-PPase protein sequences revealed two highly conserved motifs associated with pyrophosphate (PPi) binding and response to stress, respectively. Both V-PPase isoforms were expressed at higher levels in the late post-véraison stage of grape berry ripening. Results also showed that the expression of grapevine V-PPase was induced by cold stress.