Involvement of polyamines in gibberellin-induced development of seedless grape berries

The roles of polyamines (PAs) in the development of seedless grape berries induced by gibberellin (GA₃) was investigated. The development of seedless grape berries was stimulated by the application of putrescine (Put), but not by that of spermidine (Spd) and spermine (Spm), regardless of the presence of GA₃. At harvest, the fresh weight of seedless grape berries treated with 500 ppm Put + 25 ppm GA₃ and 500 ppm Put increased to 111 and 112%, respectively, of the control. Treatment with methylglyoxal-bis (guanyl hydrazone), a potent inhibitor of S-adenosylmethionine decarboxylase that plays a role in Spd and Spm synthesis, did not affect the development of seedless grape berries induced by 100 ppm GA₃. The application of 100 ppm GA₃ significantly increased endogenous free Put levels. Levels of free Spd and Spm were not affected by GA₃. Although the levels of endogenous perchloric acid insoluble bound PAs were higher than those of free PAs, obvious changes in the levels of bound PAs were not observed. These results indicate that free Put is implicated in the development of seedless grape berries induced by GA₃.     

The induction of α-amylase activity by sucrose starvation in suspension-cultured rice cells is regulated by polyamines

When suspension-cultured rice ( Oryza sativa L. cv. Tainan 5) cells were deprived of sucrose, α-amylase (EC 3.2.1.1) activity in the cells and the culture medium increased markedly. The increase in activity of α-amylase caused by sucrose starvation in the cells and the medium was strongly reduced in the presence of exogenously added spermine. Putrescine and spermidine also inhibited, though only slightly, the increase in α-amylase activity caused by sucrose starvation. Preincubation of the enzyme extract or enzyme in the medium with polyamines had no effect on α-amylase activity. Sucrose starvation resulted in lower polyamine levels in rice suspension cells. D-Arginine and α-methylomithine, inhibitors of polyamine biosynthesis, caused reduced levels of polyamines and increased activity of α-amylase in rice suspension cells cultured in the presence of sucrose. Our results indicate that the induction of α-amylase activity by sucrose starvation in rice suspension cells is mediated, at least partly, through the internal level of polyamines.     

Effect of light exposure on linalool biosynthesis and accumulation in grape berries

Linalool is an important compound that contributes to the floral aroma in wines. This study showed the effect of light exposure on linalool accumulation in berries. The grape bunches were covered with films that block the full light spectrum (Shade) and the UV spectrum (UV-block), and a transparent film (Control). The linalool content was significantly higher in juice from Control-covered berries than in juice from Shade- and UV-block-covered berries, and the expression levels of the representative genes in linalool biosynthesis in Shade- and UV-block-covered berries were markedly lower than in Control-covered berries. These findings suggest that exposing berries to light is essential for linalool biosynthesis. To reflect sunlight onto grape clusters, reflective sheets were placed on the ground of a vineyard. The linalool content in berries exposed to sunlight reflected from the reflective sheets was higher than those in the control.     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.     

葡萄细胞悬浮培养生产白藜芦醇

以巨峰葡萄果皮为外植体,在添加2.0 mg/L 6-苄基嘌呤（6-BA）和0.1 mg/L 2,4-二氯苯氧基（2,4-D）的B5培养基上诱导葡萄愈伤组织; 以50 g/L的初始接种量在添加1.0 mg/L 6-BA和0.05 mg/L 2,4-D的B5液体培养基上建立葡萄悬浮培养体系。在25～27 ℃下,摇床振荡暗培养（120～130 r/min）18 d后,葡萄细胞生物量和白藜芦醇含量达到最大值（16.17 g/L、95.69 μg/g干质量）。在培养第12天时,向培养基中添加100 μmol/L茉莉酸甲酯（MeJA）,经过6 d处理,细胞中白藜芦醇含量达235.73 μg/g干质量。     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Changes in the activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase in suspension-cultured cells of Vitis

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) is the first enzyme in the shikimate pathway, which leads to the biosynthesis of the aromatic amino acids. These amino acids are utilized as precursors for the synthesis of some secondary metabolites. The relationship between the accumulation of anthocyanin and the activity of DAHP synthase in suspension cultures of Vitis hybrid (Bailey Alicante A) was investigated. The activity of the plastidic isozyme, designated DS-Mn, was very low throughout the culture of cells. However, the activity of the cytosolic isozyme, designated DS-Co, increased transiently and then decreased after transfer of cells to fresh medium, reaching minimum levels during the logarithmic phase. Thereafter, the activity of DS-Co increased rapidly prior to the accumulation of anthocyanin. When phosphate was removed from the culture medium, growth of cells was limited and rapid accumulation of anthocyanin occurred, coincident with the termination of cell division. The activity of phenylalanine ammonia-lyase continued to increase from day 1 and the activity of DS-Co in phosphate-free culture also was 1.6-fold greater than that in the control culture on day 1, while the activity of DS-Mn was unaffected by this treatment. These results suggest a close correlation between the activity of DS-Co and the biosynthesis of anthocyanin.     

Field evaluation of water transport in grape berries during water deficits

The net flow in vascular and transpirational components of the grape berry water budget was evaluated during water deficits imposed at different stages of fruit development. Diurnal fluctuations in berry diameter were measured on field-grown grapevines ( Vitis vinifera L. cv. Cabernet Sauvignon) by using electronic displacement transducers. Water deficits were imposed by withholding irrigation, and water potentials of mid-shoot leaves, basal stem xylem and clusters were determined with a pressure chamber. The relative net flows through pedicel xylem and phloem and through berry transpiration were estimated pre-veraison and post-veraison. The xylem functioned nearly exclusively in providing net inflow pre-veraison, while the phloem was clearly dominant post-veraison. Accordingly, the amplitude of diurnal contraction was markedly smaller post-veraison than pre-veraison. The amplitude of diurnal contraction increased dramatically with decreasing plant water status pre-veraison, yet exhibited little sensitivity to low vine water status post-veraison. Measurements of the difference in water potential between clusters and source stems did not provide evidence of a gradient that would elicit significant water movement from the cluster to the stem at any time of the day. This was true for both irrigated and non-irrigated vines, although the non-irrigated vines exhibited a smaller gradient favoring inflow throughout much of the day. The gradient for xylem water transport to the cluster was considerably smaller post-veraison than pre-veraison. The results showed that berry transpiration functioned as the primary pathway for water loss both pre- and post-veraison.     

Phenology of flowering and starch accumulation in grape (Vitis vinifera L.) cuttings and vines

BACKGROUND AND AIMS: A reliable protocol for flowering and fruiting in cuttings was developed with the aim of (a) studying inflorescence and flower development in grapevine cuttings and field plants, and (b) assisting haploid plant production. METHODS: Inflorescence and flower development was studied in 'Gewurztraminer' (GW) and 'Pinot Noir' (PN) grape vines and cuttings grown in a glasshouse, along with variations in starch in the flowers. As there is a strong relationship between flower development and starch, the starch content of reproductive structures was estimated. KEY RESULTS: Inflorescence and flower development were similar in the vines and cuttings with consistent differences between the two cultivars. Indeed, the ontogenesis of male and female organs is not synchronous in GW and PN, with both female and male meiosis occurring earlier in PN than in GW. Moreover, changes of starch reserves were similar in the two plant types. CONCLUSIONS: Cuttings have a similar reproductive physiology to vines, and can be used to study grape physiology and to develop haploid plants.     

Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars

Sucrose was found to modulate polyphenol accumulation in Vitis vinifera cell cultures. The production of anthocyanins increased 12-fold after addition of 0.15 m sucrose, while that of stilbenes was only slightly affected. Sucrose did not play a physical role because metabolic sugars were required for the induction of polyphenol accumulation. Indeed, the polyols, mannitol and sorbitol, had no effect on this accumulation. We established a model system to investigate the mechanism of sucrose regulation of polyphenol production without inhibition of grape cell growth. After addition of sucrose to the culture medium, the major sugars accumulated in grape cells were glucose and fructose, reaching 40% of the dry weight. The increase in the level of these hexoses closely coincided with the increase in anthocyanin accumulation in grape cells. Received: 18 August 1997 / Revision received: 6 November 1997 / Accepted: 5 January 1998     

Effect of gibberellic acid and uniconazol on embryo abortion in the stenospermocarpic grape cultivars Emperatriz and Perlon

Hypothesizing that seed abortion in stenospermocarpic grapes (Vitis vinifera L.) is caused by high gibberellin levels in the seed during the first stages of its development, we studied the effect of gibberellic acid GA₃ and uniconazol (a GAs biosynthesis inhibitor) on this phenomenon. In vitro germination was analyzed in the seedless cultivars Emperatriz and Perlon, which were treated with 60 and 120 mg.^-l 1 uniconazol (5 and 15 days before bloom) and 100 mg.^-l 1 GA₃ (5 days after bloom). In addition, endogenous levels of free gibberellins in flowers and seeds of Emperatriz and Perlon were compared with their seedeed progenitor Emperador. Clusters were harvested at bloom and 20 days after bloom for gibberellin analysis and at commercial maturity for in vitro culture of the seeds. Considerable gibberellin activity was found in the three cultivars, but only small differences were detected between the seedless and the seeded genotypes. Exogenous applications of GA₃ had a deleterious effect on seed growth and on in vitro germination. Uniconazol also inhibited in vitro germination, though not affecting the total number of germinating embryos plus those rescued from non-germinating seeds. In conclusion, gibberellins do not appear to be directly involved in seed abortion of the stenospermocarpic cultivars Emperatriz and Perlon, although their participation in a more complex scenario should not be rejected, taking into account that in Perlon germination rates are positively correlated with the number of clusters per plant. Treatments with growth regulators also modified berry number per cluster, berry weight and rachis morphology. Finally, the plant source was a determinant affecting germination rates in vitro.     

Habitat diversification tactic for improving biological control: parasitism of the western grape leafhopper

In a previous study we demonstrated greater abundance of the parasitoid Anagrus epos (Girault) in grape vineyards located downwind of prune trees that function as overwintering habitats. This study examines whether these higher A. epos numbers translated into higher egg parasitism rates of the grape leafhopper, Erythroneura elegantula (Osborn). Paired commercial wine-grape vineyard plots, one with and one without adjacent prune trees, were studied within a complete block design in northern and central California. A. epos was the key mortality factor affecting E. elegantula eggs. Point estimates of A. epos parasitism rates were significantly greater in vineyards associated with prune trees during the first E. elegantula generation in both 1991 and 1992. No consistent differences in parasitism rates were observed during the second or third generations. The results indicated that prune trees enhance early season parasitism rates. Cumulative estimates of egg parasitism across E. elegantula generations demonstrated that enhanced early-season parasitism resulted in a net season-long increase in the degree of mortality imposed by A. epos on E. elegantula eggs. Two factors were found to influence parasitism rates: the abundance of early-season A. epos adults moving into vineyards and the density of E. elegantula eggs in vineyards. Our results indicate that diversification of vineyards using prune trees supports overwintering populations of a specialist parasitoid and thereby alters host-parasitoid interactions to favor enhanced parasitism in vineyards.     

Replacement of Mg2+ by polyamines in the aminoacylation of tRNA from Phaseolus

The ability of putrescine, spermidine and spermine to replace Mg²⁺ ions in the charging reaction of tRNA was estimated for seventeen amino acids. The polyamines promoted only the transfer reaction in the case of Leu, Ile, Val, Tyr and Arg. A synergistic effect was observed when spermine was added to a suboptimal concentration of Mg²⁺ (charging at only 5% of the optimal level). This synergistic effect was not observed for Ala, Asp-NH₂, His, Lys and Ser. Kinetic studies showed a slower aminoacylation rate in those experiments when spermine and Mg⁺² (at 5% of the Mg²⁺ optimal concn) were used together than with Mg²⁺ (at the optimal concn) alone.     

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.     

Roles of leaf in regulation of root and shoot growth from single node softwood cuttings of grape (Vitis vinifera)

The role of leaf in regulation of root and shoot growths in single node softwood cuttings of grape (Vitis vinifera) was characterised. Leafy cuttings showed early rooting, vigorous root growth and subsequent shoot development. Defoliation at planting induced early sprouting, but adversely affected rooting and decreased the survival of cuttings irrespective of pre‐planting treatment with 100 μM indole 3‐acetic acid (IAA). Treatment with IAA did not affect the percent rooting of leafy cuttings but increased root and shoot growth. Leaf weight (wt) and leaf area of the cuttings showed a highly significant correlation to root wt of the new plant at 4 wk after planting, while cutting stem + petiole wt was either not or less significantly correlated to root and shoot weights of the subsequent plant. The greater the area or wt of leaf, the better the root and shoot growths, implying that leaf contributed to adventitious root growth. However, retaining the leaf for just 2 days was enough to stimulate rooting in more than 80% of the cuttings, suggesting that leaf tissue could also induce root formation. Root growth increased with the period of leaf retention but leaf removal before 3 wk triggered sprouting leading to high mortality in rooted cuttings. Bringing the leaf closer to the rooting zone by preparing leaf at base (LAB) cuttings delayed rooting and sprouting compared with the standard leaf at top (LAT) cuttings. An inhibitory effect on rooting and sprouting by the exposed upper internode region in LAB cuttings is suggested.     

Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells ( Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H⁺-ATPase activity. Plasma membrane-enriched vesicles were prepared and the H⁺-ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H⁺-ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2m M dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H⁺-ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH.
This rapid irreversible inhibition of the plasma membrane H⁺-ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material.     

Developmental changes in the diurnal water budget of the grape berry exposed to water deficits

The diurnal water budget of developing grape (Vitis vinifera L.) berries was evaluated before and after the onset of fruit ripening (veraison). The diameter of individual berries of potted ‘Zinfandel’ and ‘Cabernet Sauvignon’ grapevines was measured continuously with electronic displacement transducers over 24 h periods under controlled environmental conditions, and leaf water status was determined by the pressure chamber technique. For well-watered vines, daytime contraction was much less during ripening (after veraison) than before ripening. Daytime contraction was reduced by restricting berry or shoot transpiration, with the larger effect being shoot transpiration pre-veraison and berry transpiration post-veraison. The contributions of the pedicel xylem and phloem as well as berry transpiration to the net diurnal water budget of the fruit were estimated by eliminating phloem or phloem and xylem pathways. Berry transpiration was significant and comprised the bulk of water outflow for the berry both before and after veraison. A nearly exclusive role for the xylem in water transport into the berry was evident during pre-veraison development, but the phloem was clearly dominant in the post-veraison water budget. Daytime contraction was very sensitive to plant water status before veraison but was remarkably insensitive to changes in plant water status after veraison. This transition is attributed to an increased phloem inflow and a partial discontinuity in berry xylem during ripening.     

蔗糖和硝酸铵对黄连悬浮培养细胞生长和小檗碱含量的影响

在６、７－Ｖ液体培养基中较为适合黄连细胞生长的蔗糖浓度为．３％,当蔗糖浓度增加到７％时,细胞生长速率开始下降,但小檗碱含量提高到５．２０％．小檗碱产率为４５６．６０ｍｇ／Ｌ（２３天）。基本培养基中随着补加ＮＨ４ＮＯ３浓度的增加,抑制细胞生长愈严重;补加０．８ｇ／ＬＮＨ４ＮＯ３的小檗碱含量比未加的增加约１０％,随着浓度的增加又急剧下降。