Carbon starvation reduces carbohydrate and anthocyanin accumulation in red-fleshed fruit via trehalose 6-phosphate and MYB27

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel-coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red-fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6-phosphate. RNA-Seq analysis showed down-regulation of both gene-encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6-phosphate synthase gene TPS1.1a was down-regulated, whereas putative regulatory TPS7 and TPS11 were strongly up-regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up-regulated, whereas MYB27 was also up-regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6-phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits.     

Dry matter accumulation in citrus fruit is not limited by transport capacity of the pedicel

The vascularization of the pedicel in Marisol clementine (Citrus clementina Hort. ex Tanaka) has been characterized in relation to fruit growth. Phloem and xylem formation occurred during the first half of the period of fruit growth. Phloem cross-sectional area reached its maximum value by the end of fruitlet abscission, 78 d after anthesis (DAA), shortly after the rate of accumulation of dry matter in fruitlets reached its maximum value. Secondary xylem formation occurred until day 93, well after the end of fruitlet abscission. At fruit maturity, xylem accounted for 42-46 % of the cross-section of the pedicel. Vessels differentiated in this late-formed xylem. Formation of phloem and early xylem was directly related to fruitlet size (and growth rate). Differences in the rate of formation of conductive tissues in the pedicel of the developing fruitlets followed rather than preceded the differences in growth rate. Specific mass transfer (SMT) in the phloem was highest in the fastest growing fruitlets, and peaked during the late stages of fruitlet abscission (72-78 DAA) and during the main period of fruit growth (107-121 DAA). Application of a synthetic auxin to developing fruits, either at the end of flowering (2,4-D) or by day 64 after flowering (2,4-DP), increased the growth rate of the fruit and fruit size at maturity (8-13 % increase in fruit diameter at maturity). These auxin applications also enhanced the formation of conductive tissues in the pedicel, with a specific effect on phloem formation. Applying auxin at flowering resulted in a reduction in the phloem SMT by days 72-78, whereas auxin application on day 64 increased this parameter. Despite this difference in behaviour, which resulted from the different time-course of the growth response of the fruit to auxin applications, these applications increased fruit size to a similar extent. Severing 37 % of the phloem of the pedicel during the main period of fruit growth resulted in an increase in the specific mass transfer in the phloem but had no influence on fruit growth. These observations demonstrate that the transport capacity in the phloem of the pedicel does not limit fruit growth and, within the limits of our experiments, an increase in demand by the fruit appeared to be matched by an increase in SMT. The dependence of late xylem formation (after the period of fruitlet abscission) on fruitlet growth was demonstrated in Salustiana orange [Citrus sinensis (L.) Osbeck] by means of controlling fruit growth through the manipulation of leaf area. Fruit growth at this time was more closely related to leaf area than to carbohydrate levels, suggesting that it may be limited by current photosynthesis.     

Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis

Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO, and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.     

Pollination and Resource Constraints on Fruit Set and Fruit Size ofPersoonia rigida(Proteaceae)

Flower and immature fruit abscission can be caused by proximatefactors preventing the development of all flowers into maturefruits. Two potential limiting factors, pollen supply and carbohydrateavailability, were assessed as relative constraints on accumulationof fruit dry matter inPersoonia rigida(Proteaceae). Naturalpollen transfer was highly efficient, and no evidence of self-incompatibilitywas found. Levels of fruit set (67.4% following open-pollinationand 50.4–58.8% following hand-pollinations) may be thehighest recorded for a species of Proteaceae with hermaphroditicflowers. Fruit abscission occurred principally in a single phasefrom 4–10 weeks post-anthesis, with a peak during theeighth week post-anthesis, and this early abscission was theresult of fertilization failure in some flowers. Manipulationof carbohydrate availability by girdling and/or defoliationof branches provided evidence that carbohydrate supply affectedfruit set only when supply was very low. Fruit abscission dueto very low carbohydrate availability on defoliated brancheswas evident during the early abscission phase but was more pronouncedduring the second half of the fruit development period (>17weeks post-anthesis), coinciding with the period of greatestdry matter demand by the fruits. Minimal fruit abscission occurredduring this later period on leaf-bearing branches. Increasesin carbohydrate supply had no effect on fruit set, but fruitsize was highly sensitive to carbohydrate availability. Subtendingand adjacent leaves were identified as major contributors tothe carbohydrate supply of fruits, although some carbohydratecould be obtained from beyond the fruiting branch. Despite thehigh levels of natural fruit set, both pollen supply and resourceavailability were indicated as potential constraints on accumulationof fruit dry matter inP. rigida.Copyright 1999 Annals of BotanyCompany. Persoonia rigidaR.Br., Proteaceae, pollination, carbohydrates, fruit set, fruit size, abscission, girdling.     

Developmental changes in antioxidant metabolites, enzymes, and pigments in fruit exocarp of four tomato (Lycopersicon esculentum Mill.) genotypes: β-carotene, high pigment-1, ripening inhibitor, and ‘Rutgers’

In surface cell layers of fleshy fruit, antioxidants must limit photooxidative reactions that generate reactive oxygen species (ROS) in high light. Our objective was to measure changes in the concentrations of antioxidant metabolites and pigments, and the activities of enzymes of the Mehler-peroxidase, ascorbate-glutathione cycle in fruit exocarp tissue under non-stress conditions of the following fruit-specific tomato (Lycopersicon esculentum Mill.=Solanum lycopersicum) mutants and their parent: (1) beta-carotene (B), (2) high pigment (hp-1), (3) ripening inhibitor (rin), and (4) the nearly isogenic wild-type 'Rutgers'. Developmental variables included days after anthesis (DAA) and fruit surface color. The highest total ascorbic acid (AsA) concentration was in the exocarp of immature green fruit of hp-1, being 32% higher than 'Rutgers'. The hp-1 mutant also had the highest chlorophyll and total carotenoid concentrations, comprised mostly of lycopene in red ripe fruit; whereas, beta-carotene comprised 90% of the carotenoids in B. Although enzyme activities varied within genotype, they generally increased with development, then decreased as fruit maturity was reached, being coupled with AsA and glutathione (GSH) concentrations. In all mutants, dark-green (DG) exocarp had more chlorophyll and protein, higher concentrations of reduced AsA and GSH, and usually lower enzyme activities than light-green (LG) exocarp taken from the same fruit.     

Patterns of carbohydrate and fatty acid changes under nitrogen starvation in the microalgae Haematococcus pluvialis and Nannochloropsis sp.

The aim of this research was to study the impact of nitrogen starvation on the production of two major secondary metabolites, fatty acids and carbohydrates, in two microalgae: Nannochloropsis sp. and Haematococcus pluvialis. The major response to nitrogen starvation in both algae occurred within the first 2 days, accompanied by a sharp reduction in chlorophyll content. However, the pattern of the response differed between the two microalgae. In H. pluvialis, the first response to nitrogen starvation was intensive production of carbohydrates, accumulating to up to 63% of dry weight by day 1; on day 2, the total carbohydrate content decreased and was partially degraded, possibly to support fatty acid synthesis. Under these conditions, H. pluvialis accumulated up to 35% total fatty acids in the biomass. In Nannochloropsis sp., the immediate and major response, which was maintained throughout the entire period of exposure to stress, was production of fatty acids, accumulating up to 50% of dry weight, while carbohydrate content in the biomass remained stable at 18%. In addition, we tested the effect of the lipid-synthesis inhibitor sesamol, known to inhibit malic enzyme, on the balance between total fatty acid and carbohydrate contents in H. pluvialis and Nannochloropsis sp. In both cultures, sesamol inhibited fatty acid accumulation, but the carbohydrate content was reduced as well, albeit to a lesser extent. These findings demonstrate the complexity of the stress-response and the potential link between fatty acid and carbohydrate synthesis.     

Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0–1000 V/cm for a selected duration in the range 10–1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.     

Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions

Irreversible photolabeling by [3H]flunitrazepam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS-polyacrylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15-25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.     

大颗粒活化腐植酸肥对苹果土壤团聚体和有机碳的影响

新型大颗粒活化腐植酸肥(LAF)在苹果化肥减量和果实稳产方面的效果显著,探明其对土壤团聚体和有机碳的影响是揭示该新型肥料对苹果土壤结构影响的重要依据。本研究设置4个LAF处理:LAF₁[全量施肥,施肥时期及重量比(下同):萌芽期∶膨果期∶成熟期=3∶4∶3]、LAF₂(全量施肥,萌芽期∶膨果期∶成熟期=2∶3∶5)、LAF₃(减量1/4施肥,萌芽期∶膨果期∶成熟期=2∶3∶5)、LAF₄(减量1/3施肥,萌芽期∶膨果期∶成熟期=2∶3∶5),以不施肥(CK)处理为对照。通过4年盆栽试验,研究苹果土壤团聚体组成、稳定性和有机碳对不同施肥处理的响应。结果表明: 1)与CK相比,LAF各处理显著提高了土壤水稳性大团聚体含量,>2 mm和2~0.25 mm粒径团聚体含量分别提高了53.4%~77.5%和12.3%~17.0%,且提高幅度随施肥量的增加而增大,其中LAF₁处理土壤水稳性大团聚体含量最高。2)LAF各处理在各粒径团聚体含量上差异不显著,其中2~0.25 mm粒径团聚体含量所占的比例最高。3)与CK相比,LAF各处理均显著提高了团聚体平均重量直径(MWD)和几何平均直径(GMD),降低了分形维数(D),其中LAF₁处理的MWD和GMD值最高,对土壤团聚体稳定性提升效果最好。4)除LAF₄外,其他LAF处理土壤有机碳含量均显著高于CK,其中LAF₂处理土壤有机碳含量最高;LAF各处理均增加了土壤各粒径团聚体有机碳含量,LAF₁、LAF₂、LAF₃处理显著提高了>2 mm粒径团聚体有机碳含量,且>2 mm团聚体有机碳对总有机碳的贡献率最大;LAF各处理的水稳性大团聚体有机碳对总有机碳的贡献率均显著高于CK,且贡献率均在66.0%以上,其中LAF₁处理最高。综上,施用LAF在促进苹果土壤水稳性大团聚体形成和稳定性、提高团聚体有机碳含量上应用效果显著,其中全量施用效果最好。施用LAF可作为改善苹果土壤结构和提升土壤肥力的有效措施。     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

绿色巴夫藻受紫外(UV－B)胁迫后的超补偿生长效应

以绿色巴夫藻(Pavlova viridis)为实验材料,设置了18、36、54、65、86和108 J·m^-2 6个UV-B辐射剂量处理组,以无紫外辐射为对照,解除胁迫后,处理组和对照组在相同接种密度和相同条件下培养12 d,测定了生长过程中的吸光值、生物量、叶绿素a、类胡萝卜素、可溶性蛋白质和胞内多糖含量.结果表明,在UV-B胁迫下,绿色巴夫藻细胞生长受到显著抑制,6个处理组细胞的相对增长率比对照下降了16.15%～60.00% (P＜0.05).但在胁迫解除后,各胁迫处理的藻细胞生长指标均显著高于对照(P＜0.05),证明绿色巴夫藻在胁迫后的恢复生长中出现超补偿生长现象.恢复培养第12天,最大吸光值、生物量、叶绿素a、类胡萝卜素、可溶性蛋白质和胞内多糖含量分别比对照提高了22.38%、15.00%、26.15%、23.81%、11.63%和27.58%.藻类中存在超补偿生长特性为微藻生物活性物质的开发提供了有效途径.     

外源ABA和GA3对红肉脐橙果皮主要色素含量变化和果实着色的影响

测定了红肉脐橙(Citrus sinensis Osbeskcv.CaraCara)果实发育期间和果实转色前用不同浓度的外源ABA和GA₃处理后果皮叶绿素和类胡萝卜素的动态含量,并测定了外源ABA和GA₃处理后成熟红肉脐橙果皮色泽的表现。结果表明:红肉脐橙果皮叶绿素含量于9月20日出现最大值,为0.1469mg·g^-1FW;类胡萝卜素含量于12月20日达到最大值,为0.0321mg·g^-1FW;果实转色前,用外源ABA处理后加速了果皮叶绿素的降解,但也抑制了果皮类胡萝卜素的积累,用GA₃处理后延缓了果皮叶绿素的降解,同样抑制了果皮类胡萝卜素的积累,严重阻碍了果皮类胡萝卜素的合成;外源ABA或GA₃处理均不利于果实色泽品质的提高。     

Vegetative and reproductive responses,oil yield and composition from olive trees (Olea europaea) under contrasting water availability during the dry winter‐spring period in central Argentina

In Argentina, the climatic pattern of the olive production areas is characterised by a marked water deficit during winter and spring months. A field experiment was carried out to evaluate the effect of water availability during the pre‐flowering–flowering period on vegetative, reproductive and yield responses of olive trees grown in central Argentina. From the end of autumn to mid‐spring, four irrigation treatments were imposed to olive trees (Olea europaea, cv. Arbequina and Manzanilla) at 0, 25, 50 and 75% estimated crop evapotranspiration (ETc). Also, a control treatment was kept at 100% ETc for the entire year. For the first crop year evaluated, water deficit applied at early June, approximately 4 months prior to bloom, reduced the vegetative shoot growth and delayed the flowering time, resulting in shortening of the fruit maturation period and, ultimately, decreased fructification. Trees irrigated with high (75% of ETc) and full (100% of ETc) winter‐spring water supply presented significantly higher values of flower density, fruit density and final fruit yield which resulted in water productivity (kg fruits mm^?1 of irrigation/ha) enhancements of about 500% (cv. Arbequina) and 330% (cv. Manzanilla) with respect to those obtained from the corresponding unirrigated treatments. Differences between treatments in oil content and composition were primarily attributed to variations in fruit maturity. Differences in fatty acid composition were stronger in cv. Arbequina where a gradual increase in oleic acid content was registered in parallel to the increase in irrigation water supply. From a practical stand point, results obtained from most of the analysed parameters were quite similar for both T75 and T100 treatments. Thus, the possible convenience of irrigation at T75% ETc should be considered since it may warrant profitable olive production while saving a considerably quantity of irrigation water in the olive production area in central Argentina.     

外源ABA和GA3对红肉脐橙果皮主要色素含量变化的影响

测定了红肉脐橙果实发育期间果皮叶绿素和总类胡萝卜素含量的变化动态,并在果实转色前用不同浓度的外源ABA和GA₃处理后测定果皮色素含量的变化动态。结果表明,红肉脐橙果皮叶绿素总量于9月20日出现最大值(0.1469mg/gfw),总类胡萝卜素含量于12月20日达到最大值(0.0321mg/gfw);转色前用ABA处理后加速果皮叶绿素的降解,但抑制果皮类胡萝卜素的积累;转色前用GA₃处理后延缓果皮叶绿素的降解,并同样抑制果皮类胡萝卜素的积累,严重阻碍了果皮类胡萝卜素的合成。