山葡萄籽中原花青素的提取

研究了提取溶剂、温度、时间、料液比4个因素对山葡萄籽中原花青素得率的影响.确定最佳提取工艺以70%丙酮为提取剂,在60℃条件下,提取120 min,料液比为17,原花青素的得率为2.31%     

盐胁迫对山葡萄叶绿素荧光参数及超微结构的影响

采用不同浓度NaCl(0、0.1%、0.3%、0.5%)处理山葡萄品种‘左山二’幼苗,于盐胁迫20d时测定叶绿素荧光参数,盐胁迫25d时对0(CK)和0.3%NaCl溶液处理的植株进行叶片超微结构观察,以探讨山葡萄品种的耐盐机理。结果表明:(1)中度(0.3%)或重度(0.5%)盐胁迫下,山葡萄叶初始荧光(F0)显著升高,PSⅡ原初光化学效率(Fv/Fm)、PSⅡ潜在活性(Fv/F0)、光化学淬灭系数(qP)、实际光化学效率(ΦPSⅡ)、非光化学淬灭系数(NPQ)和电子传递效率(ETR)显著降低,表明叶片PSⅡ反应中心破坏,PSⅡ受体侧电子传递受害,通过热耗散散失过剩激发能的能力减弱。(2)中度盐胁迫条件下,叶绿体超显微结构较对照发生明显变化,其外形明显皱缩变短呈不规则球形,大的淀粉粒消失,质壁分离明显,基粒和基质片层界限模糊不清,被膜破损或解体,叶绿素内部质体小球增多,表明其形态结构已受到严重损伤。(3)盐胁迫处理下,山葡萄线粒体嵴排列紊乱,线粒体膜结构模糊不清或溶解,但未见明显的破裂现象,说明线粒体受害程度较叶绿体小,线粒体较叶绿体对NaCl相对不敏感。(4)盐处理下细胞核形态较对照发生改变,核膜溶解或消失,但未完全崩解,说明盐胁迫对细胞核造成了一定损伤。     

Photosynthesis and activity of photosystem II in response to drought stress in Amur Grape (Vitis amurensis Rupr.)

The Amur Grape (Vitis amurensis Rupr.) cultivars ??shuangFeng?? and ??ZuoShanyi?? were grown in shelter greenhouse under natural sunlight and subjected to drought. Sap flow rate, net photosynthetic rate (P _N), and chlorophyll (Chl) fluorescence were measured on Amur Grape leaves subjected to different drought treatments. Significant decreases in P _N were associated with increasing intercellular CO₂ concentration (C _i), suggesting that the reduction in P _N was caused by nonstomatal limitation. Analysis of OJIP transients according to the JIP-test protocol revealed that specific (per PSII reaction center) energy fluxes for light absorption, excitation energy trapping and electron transport have significantly changed. The appearance of a pronounced K-step and J-step in polyphasic rise of fluorescence transient suggested the oxygen-evolving complex and electron transport were inhibited. Drought stress has relatively little effect on the parameter maximal quantum yield of PSII photochemistry (F_v/F_m), but the performance index (PIABS) is more sensitive in different drought treatment. There are cultivar differences in the response of PSII activity to drought, the photosynthetic apparatus of ??ZuoShanyi?? cultivar is more resistant to drought than that of ??ShuangFeng??, and JIP-test could be a useful indicator for evaluation and selection to drought tolerance.     

Effect of plant stilbene precursors on the biosynthesis of resveratrol in Vitis amurensis Rupr. Cell cultures

The biosynthesis of resveratrol after the application of a precursor for biosynthesis, i.e., phenylalanine (Phe), has been studied. The application of Phe has been shown to increase significantly the expression of the phenylalanine-ammonia-lyase (PAL) and stilbene synthase (STS) genes and enhance the production of resveratrol by 8.5 times. Data on resveratrol production after the addition of Phe and coumaric acid (CA) were compared with known analogs.     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

Chromosomal variability of ginseng cells transformed with plant oncogene rolC

Chromosome numbers were was studied in ginseng cell line 1c transformed with Agrobacterium rhizogenes strain A4, which carried plasmid pRiA4, and with A. tumefaciens strain GV3101, which carried vector pPCV002-35S rolC. As compared with the nontransformed cell line 1c, tumor cell cultures 1c-A4 and 1c-rolC and the tissues of rolC teratoma (excluding leaves) displayed higher polyploidy and aneuploidy. The 1c-A4 and 1c-rolC hairy-root cultures also had aneuploid and polyploid cells, but the chromosome variation was lower than in tumor cells or the initial culture 1c. Generally, an increase of chromosome variation in cultivated cells was the main effect of the integration of several oncogenes, which were in the A. rhizogenes A4 T-DNA, or of the individual rolC gene in the ginseng genome. Another effect consisted in stabilization of the chromosome number in some differentiated transgenic tissues. Possible reasons for this effect are discussed.     

Effects of the Calmodulin Antagonist W7 on Resveratrol Biosynthesis in Vitis amurensis Rupr.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca²⁺/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.     

The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.     

Phosphinothricin stimulates somatic embryogenesis in grape (Vitis sp. L.)

The effect of phosphinothricin concentration on embryo production from an embryogenic callus of Chancellor (Vitis L. complex interspecific hybrid) was tested. Embryogenic callus was cultured on medium supplemented with nine phosphinothricin concentrations (0, 0.1, 0.5, 1, 1.5, 2, 3, 5, and 10 mg/l). The highest number of embryos per plate was observed at 0.5 mg/l phosphinothricin. The use of phosphinothricin to stimulate embryo production did not affect embryo germination and plantlet formation. Three germination techniques were compared. Embryo dehydration or growth on Transfergelsolidified medium gave higher germination rates than chilling treatments. Most germinated somatic embryos produced secondary embryos from the hypocotyl after a few weeks of culture. Regardless of the germination technique, the plantlet conversion rate was very low.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962) basal medium - NN Nitsch and Nitsch (1969) medium - PPT phosphinothricin     

An NMR microscopic study of grape (Vitis vinifera L.)

Summary Mature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by¹H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T₂-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T₁-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T₁-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T₂-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.Abbrevations BF bright field - FDA fluorescein diacetate - FI field inhomogeneity - FOV field of view - NMR nuclear magnetic resonance - RF radiofrequency - T₁ spin-lattice relaxation time - T₂ spin-spin relaxation time - TE echo time - TMS tetramethylsilane - TR repeat time     

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

Oncogene alterations in in vitro transformed rat tracheal epithelial cells

10 derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 nontumorigenic cell line transformed in vitro by treatment with 7,12-dimethyl-benz[a]anthracene (DMBA), benzo[a]pyrene (BP) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for oncogene alterations. No abnormalities of Ha-ras were seen that were suggestive of amplification, rearrangement or the presence of RFLPs. Analysis of specific-point mutations in Ha-ras using Pst I digestion (codon 12, GGA to GCA) or Ha-ras and Ki-ras using Xba I (codon 61, CAA to CTA) were negative. In one cell line derived by DMBA treatment, changes in the c-myc restriction digest pattern were seen after incubation with Bam HI and Hind III. Northern analysis revealed consistent differences between normal and transformed cells when probed with Ha-ras; c-myc expression was of low intensity, and the expression of Ki-ras could not be detected. Transfection of RTE cell DNAs into NIH/3T3 cells did not result in the appearance of morphologic transformants. The studies suggest that Ha-ras or Ki-ras codon 61 A to T transversions (CAA to CTA) are not associated with the immoral/tumorigenic phenotype in RTE cells transformed by DMBA or TPA, and are in contrast to results reported in some other biological systems.     

Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5′- and 3′-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5′- and 3′-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5′- and 3′- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway.     

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.)

Summary. Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1→3)-β-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth. Correspondence: P. Bowen, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada