A novel plastid localization of chalcone synthase in developing grape berry

Chalcone synthase (CHS, E.C. 2.3.1.74) is an entrance enzyme of flavonoid metabolism and a critical point to regulation of biosynthesis of different flavonoid compounds that directly contribute to color and monthfeel of grape and wine. In the present experiment, subcellular localization of CHS in developing grape berry was performed via immunogold electron microscopy technique. The result showed that CHS was localized in rough endoplasmic reticulum (ER) and cytoplasm of the skin cells, while few gold particles representing CHS were found on the cell wall. Besides, two novel localized sites of CHS were observed within a cell of developing grape berry, one being the plastid-distributed throughout developmental stages and the other being vacuole-distributed at late developmental stage. It is speculated that these novel localized patterns may relate to abundant and multi-branch flavonoid metabolism in grape berry. This work will provide new insight for the regulation of different branch pathways leading to diverse flavonoid compounds.     

Pollination: A key event controlling the expression of genes related to phytohormone biosynthesis during grapevine berry formation

Berry formation is the process of ovary conversion into a functional fruit, and is characterized by abrupt changes in the content of several phytohormones, associated with pollination and fertilization. Much effort has been made in order to improve our understanding of berry development, particularly from veraison to post-harvest time. However, the period of berry formation has been poorly investigated, despite its importance. Phytohormones are involved in the control of fruit formation; hence it is important to understand the regulation of their content at this stage. Grapevine is an excellent fleshy-fruit plant model since its fruits have particularities that differentiate them from those of commonly studied organisms. For instance, berries are prepared to cope with stress by producing several antioxidants and they are non-climacteric fruits. Also its genome is fully sequenced, which allows to identify genes involved in developmental processes. In grapevine, no link has been established between pollination and phytohormone biosynthesis, until recently. Here we highlight relevant findings regarding pollination effect on gene expression related to phytohormone biosynthesis, and present results showing how quickly this effect is achieved.     

Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharomyces pombe

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the S. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.     

Tailored biosynthesis of gibberellin plant hormones in yeast

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway.We then built a GA₄-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA₄. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA₇ (trace amounts) and GA₃ (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA₃-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes.ClassificationBiological Sciences, Applied Biological Sciences.     

Influence of maceration temperature in red wine vinification on extraction of phenolics from berry skins and seeds of grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.     

Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.     

叶片黄化对葡萄生理和果实品质的影响

为了探究叶片黄化对葡萄生理和果实生长的伤害机制,试验以生长在河南省荥阳市蔡寨村表现黄化症状的4年生‘阳光玫瑰’成龄树为试材,通过测定并分析黄化植株与正常植株的茎叶生长量、叶绿素含量、光合特性、叶绿素荧光参数、叶片解剖结构以及果实品质和产量的变化特征,考察叶片黄化后对葡萄生长量、光合指标、叶绿素荧光特性以及果实品质的影响,以期为葡萄开展黄化矫正栽培提供参考依据。研究表明,(1)黄化植株叶片的叶绿素含量、叶片大小、叶片重量、新梢长度、枝条粗度均显著降低;(2)黄化植株叶片的净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(G_s)显著下降,而胞间CO₂浓度(C_i)显著上升;(3)黄化植株叶片的叶绿素荧光参数初始荧光(F_o)、光下最小荧光产量(F_o′)、最大荧光产量(F_m)、相对电子传递速率(ETR)、实际光合量子产量Y(Ⅱ)均比正常植株显著降低;(4)黄化植株叶片厚度、栅栏组织厚度和栅海比比正常植株显著升高,上表皮厚度和...     

A genetic analysis of seed and berry weight in grapevine.

Fruit size and seedlessness are highly relevant traits in many fruit crop species, and both are primary targets of breeding programs for table grapes. In this work we performed a quantitative genetic analysis of size and seedlessness in an F1 segregating population derived from the cross between a classical seeded (Vitis vinifera L. 'Dominga') and a newly bred seedless ('Autumn Seedless') cultivar. Fruit size was scored as berry weight (BW), and for seedlessness we considered both seed fresh weight (SFW) and the number of seeds and seed traces (SN) per berry. Quantitative trait loci (QTL) analysis of BW detected 3 QTLs affecting this trait and accounting for up to 67% of the total phenotypic variance. QTL analysis for seedlessness detected 3 QTLs affecting SN (explaining up to 35% of total variance) and 6 affecting SFW (explaining up to 90% of total variance). Among them, a major effect QTL explained almost half of the phenotypic variation for SFW. Comparative analysis of QTLs for these traits reduced the number of grapevine genomic regions involved, one of them being a major effect QTL for seedlessness. Association analyses showed that microsatellite locus VMC7F2, closely linked to this QTL, is a useful marker for selection of seedlessnes.     

Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.     

Effect of pollination and fertilization on the expression of genes related to floral transition, hormone synthesis and berry development in grapevine

In the present work, the effect of assisted fertilization on anatomical, morphological and gene expression changes occurring in carpels and during early stages of berry development in Vitis vinifera were studied. Inflorescences were emasculated before capfall, immediately manually pollinated (EP) and fruit development was compared to emasculated but non-pollinated (ENP) and self-pollinated inflorescences (NESP). The diameter of berries derived from pollinated flowers (EP and NESP) was significantly higher than from non-pollinated flowers (ENP) at 21 days after emasculation/pollination (DAE), and a rapid increase in the size of the inner mesocarp, together with the presence of an embryo-like structure, were observed. The expression of gibberellin oxidases (GA20ox and GA2ox), anthranilate synthase (related to auxin synthesis) and cytokinin synthase coding genes was studied to assess the relationship between hormone synthesis and early berry development, while flower patterning genes were analyzed to describe floral transition. Significant expression changes were found for hormone-related genes, suggesting that their expression at early stages of berry development (13 DAE) is related to cell division and differentiation of mesocarp tissue at a later stage (21 DAE). Expression of hormone-related genes also correlates with the expression of VvHB13, a gene related to mesocarp expansion, and with an increased repression of floral patterning genes (PISTILLATA and TM6), which may contribute to prevent floral transition inhibiting fruit growth before fertilization takes place.     

An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

Background  

The response regulators represent the elements of bacterial two-component system and have been characterized from dicot plants like Arabidopsis but little information is available on the monocots, including the cereal crops. The aim of this study was to characterize type-A response regulator genes from rice, and to investigate their expression in various organs as well as in response to different hormones, including cytokinin, and environmental stimuli.     

An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

Background  

Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents.