Prokaryotic expression, polyclonal antibody preparation of the stilbene synthase gene from grape berry and its different expression in fruit development and under heat acclimation

Stilbene synthase (STS, EC 2.3.1.95) leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. STS expression and regulation are important. Temperature is one of the main external factors affecting phytoalexin accumulation in plant tissues, the effect of temperature on resveratrol synthesis and stilbene synthase expression in grape berries has not been reported before. Here we cloned the full-length sts cDNA with 1179 bp from grape berry via PCR, and then introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and XhoI restriction sites. With the isopropyl-β-d-thiogalactoside (IPTG) induced, the pET-sts was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His–Tag was purified by Ni-NTA His · Bind Resin and then used as the antigen to immunize a New Zealand rabbit. Furthermore, the antiserum was precipitated by 50% saturated ammonium sulfate and DEAE-Sephadex A-50 chromatography to obtain the immunoglobulin G (IgG) fraction. These results provide a substantial basis for the further studies of the STS in grape berry as well as in other species of plants. The sts expression in fruit development and in response to heat acclimation was then assayed. The results indicated STS was regulated in fruits depending on the developmental stage and significantly accumulation of STS mRNA and synthesis of new STS protein during the early of heat acclimation, this work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.     

Pre-anthesis CPPU Treatment Modifies Quality and Susceptibility to Post-harvest Berry Cracking of <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. ‘Thompson Seedless’

Plant growth regulators (PGRs), especially gibberellic acid (GA), are often used in table grape (Vitis vinifera) production to increase berry size. However, overuse of this PGR (for example, more than 200 g·ha^?1·season^?1 of GA) may result in problems with berry condition at the post-harvest stage. A synthetic cytokinin, florchlorfenuron (CPPU), could be considered an alternative to GA. Thus, our objective was to study the effects of pre-anthesis CPPU treatment on berry quality and susceptibility to post-harvest cracking of table grapes. CPPU applied to ‘Thompson Seedless’ inflorescences 48 or 36 days prior to anthesis increased ovary size, number of cells in the ovary pericarp at bloom and berry size at harvest. It also increased the amount of soluble solids and decreased titratable acidity of the fruit, thus allowing an earlier harvest. Finally, if GA was applied during berry growth, CPPU at pre-anthesis improved berry potential tolerance to post-harvest cracking and spoilage. This work demonstrates that application of CPPU at the pre-anthesis stage modifies grape development, improving berry size and potential post-harvest fruit performance facilitating also an early harvest.     

Involvement of phospholipase D in the low temperature acclimation-induced thermotolerance in grape berry

Both phospholipase D (PLD, EC 3.1.4.4) and salicylic acid (SA) play important roles in response to external stimulation and activating defense system in plants. However, roles of the two signals in plants during the development of thermotolerance induced by low temperature acclimation remain unclear. In the experiment presented in the paper, grape berries (Vitis vinifera L. cv. Chardonnay) were pretreated at 8 °C for 3 h and then transferred to 45 °C for heat stress. Compared with the control without low temperature pretreatment, membrane permeability and malondialdehyde (MDA) contents were reduced and the expression of HSP73 increased in the low temperature-pretreated berries under heat stress. During low temperature acclimation, PLD, SA and HSP73 could be activated. Meanwhile, the expression of HSP73 and the accumulation of free SA induced by low temperature can be inhibited by PLD activity inhibitor. All these results suggest that the activation of PLD is an early response to low temperature, and it is involved in the accumulation of free SA and the development of thermotolerance induced by low temperature acclimation.     

Membrane-associated protein kinase activities in the developing mesocarp of grape berry

Fruit development is a process involving various signals and gene expression. Protein phosphorylation catalyzed by protein kinases is known to play a key role in eukaryotic cell signalling and so may be involved in the regulation of fruit development. Using the method of exogenous substrate phosphorylation, we characterised the calcium-dependent and calmodulin-independent protein kinase (CDPK) activity and the myelin basic protein (MBP)-phosphoralating activity that could be due to a mitogen-activated protein kinase (MAPK)-like activity in the developing mesocarp of grape berry. The CDPK activity was shown to be predominantly localised in the plasma membrane, while the MAPK-like activity was predominantly associated with endomembranes. The assays of bivalent cation requirement showed that Mn2+ could to a certain extent replace Mg2+ in the incubation system for the protein kinase activities. Both CDPK and MAPK-like activities were resistant to heat treatment. The activities of the two enzymes were fruit developmental stage-specific with the highest activities of both enzymes in the lag growth phase before the ripening stage, suggesting strongly the important roles of the detected CDPK and MAPK-like activities in the fruit development.     

Combined Mass Mapping and Biochemical Characterization of Grape β-Glycosidase-enriched Extract

A beta-glucosidase enzyme activity was enriched from skins of ripe grape berry by cell wall fractionation, hydrophobic interaction and cation-exchange chromatographies. This enriched enzyme extract contained several beta-glycosidase activities hydrolyzing a wide range of synthetic and natural monoglycosides and diglycosides, as well as a beta-fructosidase activity. The enzyme extract was further characterized by two-dimensional gel electrophoresis coupled to peptide mass fingerprinting of eight spots using MALDI-TOF mass spectrometry. No beta-glucosidase but a beta-fructosidase associated to the relevant spot at 66 kDa/pI 5.1 was identified. Taken together all results issued from the biochemical characterization, the substrate specificity and the mass spectrometry-based identification of this enriched enzyme extract, we propose that this protein could be a specific beta-fructosidase isoform associated with a broad spectrum of beta-glycosidase activities in grape berry skin and involved in cell wall modifications which occur during the ripening-induced thickness of the grape.     

Characterization of microsomal and mitochondrial phospholipase D activities and cloning of a phospholipase D alpha cDNA from strawberry fruits.

Phospholipase D alpha (PLD, EC 3.1.4.4)) is a key enzyme involved in membrane deterioration that occurs during fruit ripening and senescence. The biochemical and molecular characteristics of PLD was studied in strawberry (Fragaria ananassa Duch) fruits, which are non-climacteric fruits. PLD activity was primarily associated with the mitochondrial and microsomal fractions and showed increased activity during development. Optimal pH levels of activity were observed at 5.5 and 6.5 for mitochondrial PLD and at 5 and 7 for microsomal PLD. Calcium enhanced microsomal PLD activity at 1-40 microM levels. PLD activity followed Michaelis-Menten kinetics. Lineweaver-Burk analysis gave Km values in the range of 114 and 277 microM using dipalmitoylphosphatidylcholine (DPPC) as substrate for mitochondrial and microsomal PLD, respectively. The Vmax value for the microsomal PLD was nearly 12-fold higher than that of mitochondrial PLD. A 2874 bp full-length cDNA for PLD alpha was amplified from strawberry fruit mRNA using RT-PCR and 5'- and 3'-RACE encoding an 810 amino acid-polypeptide. The predicted strawberry PLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The strawberry PLD alpha showed a high degree of similarity to other PLD alphas from plants. The implications of PLD regulation during ripening of fruits are discussed.     

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.     

葡萄贮藏过程中落粒与离区酶活性变化及植物生长调节物质的关系

研究了无核白葡萄(Vitis vinifera L.)采后贮藏过程中离区纤维素酶、果胶酯酶(Pectinesterase,PE)、多聚半乳糖醛酸酶(polygalacturonase,PG)、脂氧合酶(lipoxygenase,LOX)和过氧化物酶(peroxidase,POD)活性的变化与落粒的关系及植物生长调节物质对其的影响。结果表明,葡萄在贮藏过程中,伴随浆果落粒的增加,离区纤维素酶、PG、LOX、POD活性升高,PE活性下降。离区纤维素酶、PG、LOX等酶的活性与葡萄落粒程度之间呈显著正相关。外源ABA和CEPA处理能增强离区纤维素酶、PG、LOX活性,促进落粒;GA3,IAA处理则能抑制离区纤维素酶、PG、LOX活性,减轻落粒。ABA对落粒的促进效应及GA3对纤维素酶活性和落粒的抑制效应尤为明显,表明GA3与ABA比值在葡萄采后落粒过程中起重要的作用。     

Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of Δ1-Pyrroline-5-Carboxylate Synthetase mRNA or Protein

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 μmol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera Δ¹-pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steady-state levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.     

Seasonal pattern of oviposition by the North American grape berry moth (Lepidoptera: Tortricidae)

The seasonal patterns of oviposition by the North American grape berry moth, Paralobesia viteana (Clemens) (Lepidoptera: Tortricidae) were monitored in juice grape (Vitis labrusca) vineyards in southwest Michigan. Egg deposition was recorded throughout the growing season at two vineyards in 2006, and at four vineyards from 2007 to 2009. In each vineyard, a random sample of 100 grape clusters was visually inspected twice‐weekly and the number of newly laid eggs was counted. We found that oviposition was continuous but variable throughout the season. Egg deposition started in early June coinciding with early grape bloom, continued at low level until mid‐ to late July, intensified in August close to veraison, and ended in September often before harvest. There were no consistent periods without oviposition that would indicate distinct generations. To determine the contribution of moth immigration into the vineyard to the pattern of oviposition, six grape plants located at the edge of a vineyard next to woods were covered with field cages and stocked with infested fruit. Oviposition and berry infestation were followed weekly on covered and exposed plants. Although higher numbers of eggs and infested berries were found on fruit of exposed vines than enclosed vines, egg deposition and berry infestation followed the same pattern in both treatments. This result indicates that the seasonal pattern of egg deposition is not dependent on immigration of grape berry moth of wild grape origin. The pattern of oviposition by grape berry moth described here contributes to the difficulty of controlling this pest using conventional insecticides with short residual activity.     

Localization of stilbene synthase in Vitis vinifera L. during berry development

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.     

Characterization of Protein Kinases in Apple and Grape During Fruit Development

The protein kinases were identified at different developmental stages of apple (Malus domestica L. Borth. cv. Red Starking) and grape (Vitis vinifera L. × V.lubrusca L. cv. Kyoho) fruit, and these protein kinases were found to be greatly activated by Ca2+. This demonstrated that calcium-dependent protein kinase plays an important role in apple and grape fruits. The results also showed that calmodulin (CaM) had no stimulation effect, but the CaM antagonists, such as calmidazolium, W7 and trifluoroacetic acid (TFA), had some inhibitory effects on the activities of this kind of protein kinase in both fruits, indicating that the detected protein kinases may be a kind of calmodulin-like domain protein kinase or calcium-dependent protein kinase (CDPK). There were distinct differences between apple and grape fruits, both in the characteristics and in the activity evolution patterns of the protein kinases during fruit development. The activity of calcium-dependent protein kinase was little changed in the three stages of apple fruit development, while in grape fruit the activity of this protein kinase was much higher in stage Ⅱ than those in stage Ⅰ and stage Ⅲ. The calcium-dependent protein kinase in apple fruits was strongly activated by Mn2+, and it was sensitive to heat treatment, but in grape fruits it was neither stimulated by Mn2+ nor sensitive to high temperature. Besides, phosphatidyl-serine (PS) had little effect on the activities of calcium-dependent protein kinase in grape fruits, but had stimulative effects in apple fruits. It was thus proposed that there might be a kind of calcium- and phosphatide-activated protein kinase, i.e., PKC, in apple fruits. The differences among the kinds, characteristics and the activity evolution patterns of protein kinases during apple and grape fruit development, suggest that the protein kinases may be involved in the process of fruit development.     

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.     

Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development

The phenylpropanoid pathway yields a variety of phenolics that are closely associated with fruit qualities in addition to structural and defense-related functions. However, very little has been reported concerning its metabolism in fruit. This experiment was designed to assess changes of eleven phenolic acids in grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) and explore both the activities and amounts of three key enzymes--phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL)--catalyzing the biosynthesis of these compounds during berry development. Finally, the subcellular localizations of the enzymes within berry tissues were also investigated using immuno-gold electron microscopic technique. The results indicated that the contents of gallic, protocatechuic, gentisic and caffeic acid all changed drastically during berry development, while other compounds containing p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric, ferulic and sinapic acid varied only slightly. Activities of PAL, C4H and 4CL showed similar pattern changes with two accumulated peaks throughout berry development. In addition, their activities all showed a highly positive correlation with the total contents of phenolic acids, whereas the immunoblotting analysis showed that changes in enzyme activities were independent of the enzyme amounts. Results from the subcellular-localization study revealed that PAL was mainly present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, C4H was found primarily in the chloroplast (plastid) and nucleus and 4CL predominantly in the secondarily thickened walls and the parenchyma cells of mesocarp vascular tissue.     

Geographic variation in diapause induction: the grape berry moth (Lepidoptera: Tortricidae)

Diapause in insects occurs in response to environmental cues, such as changes in photoperiod, and it is a major adaptation by which insects synchronize their activity with biotic resources and environmental constraints. For multivoltine agricultural insect pests, diapause initiation is an important consideration in management decisions, particularly toward the end of the growing season. The grape berry moth, Paralobesia viteana (Clemens), is the main insect pest affecting viticulture, and this insect responds to postsummer solstice photoperiods to initiate diapause. Because the range of grape berry moth extends from southern Canada to the southern United States, different populations are exposed to different photoperiodic regimes. We quantified the diapause response in grape berry moth populations from Arkansas, Michigan, New York, Pennsylvania, Texas, and Virginia, and observed latitudinal variation in diapause initiation. Populations from Michigan, New York, and Pennsylvania responded significantly different than those from Arkansas, Texas, and Virginia. We also observed, as a consequence of our experiments, that the timing of our laboratory studies influenced grape berry moth's response to photoperiod, ceteris paribus. Experiments that were conducted when grape berry moth would be naturally in diapause resulted in a significant higher proportion of diapausing pupae at photoperiods (i.e., >15 h) that generally do not induce diapause, suggesting that attention should be paid to the timing of behavioral and physiological experiments on insects. This relationship between photoperiod and diapause induction in grape berry moth across geographic regions will provide applicable knowledge to improve pest management decisions.     

日灼对酿酒葡萄‘霞多丽’果实品质与解剖结构的影响

以酿酒葡萄品种‘霞多丽’为试验材料,采用石蜡切片与CFDA荧光染色观察日灼果皮细胞结构与细胞活性变化,同时测定日灼发生后葡萄果实品质及相关生理指标变化,以揭示日灼对果实品质与细胞结构的影响。结果表明:(1)随着葡萄日灼病加重,果实表皮颜色由浅黄色逐渐加深,后期甚至出现细胞坏死。(2)果实发生日灼后,果实硬度与含水量下降,细胞壁含量增加,果皮从外向内第1~3层细胞明显变小,细胞壁增厚。(3)随着日灼病加重,果皮细胞破裂,且破裂数量增加,细胞活性也随之下降,果皮保护功能逐渐丧失,果肉细胞逐渐失水导致了果实皱缩;重度日灼果实周缘维管束木质部导管受到果皮细胞失水断裂的影响,出现断裂变形。(4)在葡萄果实日灼发生过程中,受到高温与强光照胁迫影响,同时伴随着水分散失增加,果实可溶性固形物含量和总糖含量增加,但有机酸含量降低,糖酸比随之增加;以上各品质指标值的大小与果实水分含量密切相关。研究发现,日灼引起了葡萄果实结构变化与生理代谢的紊乱,随着日灼程度加重,果皮细胞逐渐死亡,果实内水分大量散失,果实糖含量增加,严重影响了葡萄果实的外观和内在品质。