Simultaneous transgenic suppression of LePG and LeExp1 influences rheological properties of juice and concentrates from a processing tomato variety

Processing tomato lines suppressed in the accumulation of ripening-related polygalacturonase or expansin were generated by introduction of transgenes to silence expression of the LePG and LeExp1 genes, respectively. The rheological properties of juice and juice reconstituted from paste produced from lines suppressed in one of these genes, or in both, were compared with azygous controls. When assayed by measuring Bostwick consistency, paste produced from either suppressed LePG or suppressed LeExp1 lines and diluted to 5 degrees Brix was approximately 18% more viscous than that produced from controls. Simultaneous suppression of LePG and LeExp1 produced a small additional increase in viscosity of 4%. Rheometric flow analysis at 5 or 10 degrees Brix also showed substantial increases in the consistency index due to suppression of either LePG or LeExp1 alone, and a small additional increase when both genes were suppressed in the same transgenic line. Measurements by laser diffraction and [1H]NMR showed that suppression of LePG or LeExp1 accumulation altered the size distribution of insoluble particles and modified their surface properties. The data are consistent with suppression of LePG increasing serum viscosity, and suppression of either LePG or LeExp1 altering the properties of the insoluble particles and improving some aspect of particle-particle or particle-serum interaction, or both. However, relative to that caused by suppression of either gene alone, the additional increase in viscosity caused by simultaneous suppression of LePG and LeExp1 together was slight.     

Transgenic overexpression of expansin influences particle size distribution and improves viscosity of tomato juice and paste

Suppression of the expression of a ripening-related expansin gene, LeExp1, in tomato enhanced fruit firmness and overexpression of LeExp1 resulted in increased fruit softening. Because of the incompletely understood relationship between fresh fruit texture and the consistency of processed products, we examined the effects of LeExp1 overexpression on the processing characteristics of tomato fruit. As determined by Bostwick consistency and by controlled strain rheometry, juices and pastes prepared from transgenic tomatoes with suppressed LeExp1 expression had a higher viscosity than preparations from control fruits. However, the viscosity of juice and paste prepared from fruit overexpressing LeExp1 was significantly greater than products from controls or lines with reduced LeExp1. Bostwick consistency increased by 9% (juice) and 6% (paste) in lines with suppressed LeExp1 expression but increased by 27.5% (juice) and 19.5% (paste) in lines overexpressing LeExp1, relative to controls. Determined by laser diffraction, the particles in juice and paste prepared from transgenic fruits with reduced LeExp1 expression were smaller, and preparations from fruits overexpressing LeExp1 had a size distribution indicating more large particles. Analysis of cell wall polysaccharides size indicated that LeExp1 overexpression enhanced depolymerization of water soluble pectins as well as tightly bound matrix glycans. LeExp1 overexpression may allow increased cell wall hydration, resulting in expanded particle size and increased viscosity of products. Because either LeExp1 suppression or overexpression leads to improved consistency, the interactions that contribute to optimal product rheological properties are complex.     

Impact of UV-C irradiation on the cell wall-degrading enzymes during ripening of tomato (Lycopersicon esculentum L.) fruit

The effect of a hormic dose of UV-C (254 nm) on changes in fruit firmness and cell wall-degrading enzyme (CWDE) activity was determined using tomato fruit. Throughout the storage period, a decrease in firmness was jointly observed with an increase of the CWDE (polygalacturonase, pectin methyl esterase, cellulase, xylanase, beta-D-galactosidase, and protease) activity for all treatments, suggesting the involvement of these enzymes in the ripening process. However, the enhancement in the activity of the CWDE was significantly less in fruit subjected to the hormic dose of UV-C. This reduction may explain why irradiated fruit were firmer than control and consequently may explain how UV-C could delay the ripening and senescence process. We suggest that the CWDE are one of the targets of the UV-C, and by this action, irradiation contributed to a delay of the cell wall degradation and consequently retarded softening of the tomato fruit tissues.     

乙烯受体基因LeETR1和LeETR4的克隆及在番茄果实中的表达

摘要：为研究野生型番茄（Lycopersicon esculentum Mill. cv . Lichun）和转反义ACS 番茄果实中乙烯受体基因LeETR1和LeETR4的表达与乙烯的关系，实验用RT-PCR方法扩增了乙烯受体基因LeETR1和LeETR4片段，用两片段作探针进行Northern 杂交。结果表明：两受体基因的表达在番茄果实成熟进程中变化不明显，LeETR4在同一时期的果实外果皮中表达水平低于其在辐射壁和中柱的表达。转反义ACS番茄果实中LeETR1和LeETR4的表达水平显著低于野生型番茄果实，外源乙烯处理转反义ACS番茄果实，促进两个受体基因的表达。可见果实中LeETR1和LeETR4的表达受乙烯调控的影响。     

Compositional changes in 'Bartlett' pear ( Pyrus communis L.) cell wall polysaccharides as affected by sunlight conditions

Preharvest conditions can have a great impact on fruit quality attributes and postharvest responses. Firmness is an important quality attribute in pear, and excessive softening increases susceptibility to bruising and decay, thus limiting fruit postharvest life. Textural characteristics of fruits are determined at least in part by cell wall structure and disassembly. Few studies have analyzed the influence of fruit preharvest environment in softening, cell wall composition, and degradation. In the current work 'Bartlett' pears grown either facing the sun (S) or in the shade (H) were harvested and stored for 13 days at 20 °C. An evaluation of fruit soluble solids, acidity, color, starch degradation, firmness, cell wall yield, pectin and matrix glycan solubilization, depolymerization, and monosaccharide composition was carried out. Sun-exposed pears showed more advanced color development and similar levels of starch degradation, sugars, and acids than shaded fruit. Sunlight-grown pears were at harvest firmer than shade-grown pears. Both fruit groups softened during storage at 20 °C, but even after ripening, sun-exposed pears remained firmer. Sunlight exposure did not have a great impact on pectin molecular weight. Instead, at harvest a higher proportion of water-solubilized uronic acids and alkali-solubilized neutral sugars and a larger mean molecular size of tightly bound glycans was found in sun-exposed pears. During ripening cell wall catabolism took place in both sun- and shade-grown pears, but pectin solubilization was clearly delayed in sun-exposed fruit. This was associated with decreased removal of RG I-arabinan side chains rather than with reduced depolymerization.     

外源5-氨基乙酰丙酸对番茄成熟过程中果实品质和矿质元素含量的影响

5-氨基乙酰丙酸(ALA)为高等植物中叶绿素等卟啉类化合物的生物合成前体,在促进作物生长、增强抗逆性及提高产量方面发挥着重要作用。为探究ALA对番茄果实成熟过程中品质和矿质元素含量的影响,本研究以原味1号品种为试验材料,向绿熟期番茄果实表面涂抹不同浓度(0、50、100、200、300 mg·L^-1)ALA溶液,筛选出促进设施栽培番茄成熟及品质形成的最佳ALA施用浓度,并进一步研究其对设施番茄果实成熟过程中硬度、可溶性固形物以及矿质元素含量的影响。结果表明,外源ALA通过降低果实硬度促进了果实的成熟,并通过提高番茄果实中可溶性固形物含量提升了果实品质。另外,在坐果后40 d,200 mg·L^-1ALA处理的K、Ca和Fe元素含量分别比对照提高了21.82%、56.25%和12.86%,但P、Mg和Cu元素含量分别比对照降低了9.92%、21.74%和25.00%。相关性分析结果表明,可溶性固形物含量与K含量呈正相关关系,与Cu含量呈负相关关系;果实硬度与K含量呈负相关关系。成熟期番茄果实中P含量与Cu含量呈正相关,与Mn、K和Ca含量呈负相关...     

铜胺氧化酶基因PpCuAO4在桃成熟中的功能鉴定

为明确铜胺氧化酶(CuAO)在桃果实成熟过程中的作用,分别采用1.0、5.0和10.0 mmol·L^-1的CuAO抑制剂氨基胍(AG)对黄水蜜桃果实进行喷施处理,并于桃果实成熟后测定果实硬度。结果表明,5 mmol·L^-1 AG处理组桃果实的硬度保持效果最好。进一步采用5.0 mmol·L^-1 AG处理黄水蜜桃果实,并测定果实成熟相关生理指标。结果表明,AG处理下桃果实在采后7 d内果实硬度均显著高于对照水平,乙烯释放量和呼吸强度均显著低于对照水平,表明抑制CuAO介导的多胺分解可显著延缓桃果实成熟。AG处理显著抑制乙烯合成、生长素转运和细胞壁降解相关基因PpACO1、PpACS、PpPIN1、PpGH3.3、PpPG和PpPME1的表达水平。为进一步明确CuAO在桃成熟中的功能,利用病毒诱导的基因沉默(VIGS)技术沉默腐胺(Put)分解关键基因PpCuAO4。结果显示,转基因桃果实PpCuAO4表达水平仅为对照的18%,Put含量和果实硬度显著高于对照水平,而乙烯释放量和呼吸强度均显著低于对照水平。上述结果表明,PpCuAO4介导的Put分解可以促进桃果实成熟。本研究为进一步深入解析多胺(polyamine)调控桃果实成熟的机制提供了重要的理论依据。     

Temporal sequence of cell wall disassembly events in developing fruits. 1. Analysis of raspberry (Rubus idaeus)

Raspberry fruits were harvested at five developmental stages, from green to red ripe, and the changes in cell wall composition, pectin and hemicellulose solubilization, and depolymerization were analyzed. Fruit softening at intermediate stages of ripening was associated with increased pectin solubilization, which occurred without depolymerization. Arabinose was found to be the most abundant noncellulosic neutral sugar in the cell wall and showed dramatic solubilization late in ripening. No changes in pectin molecular size were observed even at the 100% red stage. Subsequently, as fruit became fully ripe a dramatic depolymerization occurred. In contrast, the hemicellulosic fractions showed no significant changes in content or polymer size during ripening. The paper discusses the sequence of events leading to cell wall disassembly in raspberry fruit.     

Temporal sequence of cell wall disassembly events in developing fruits. 2. Analysis of blueberry (Vaccinium species)

Softening and pathogen susceptibility are the major factors limiting the marketing of blueberries as fresh fruits, and these traits are associated with fruit cell wall structure. However, few studies that characterize wall modifications occurring during development and ripening have been reported for this fruit. In this study the ripening-associated modifications of blueberry fruit cell walls (composition, pectin and hemicellulose solubilization, and depolymerization) at five stages of ripeness have been analyzed. Xylose was found to be the most abundant noncellulosic neutral sugar associated with fruit walls, and the observed high Xyl/Glc ratio suggested that xylans, which are usually a minor hemicellulosic fruit wall component, are abundant in blueberry. The pectic matrix showed increased solubilization at early and intermediate stages of ripening, but no changes were detected in late ripening. Furthermore, little reduction in pectin polymer size occurred during blueberry ripening. In contrast, hemicellulose levels decreased as ripening progressed, and a clear depolymerization of these components was observed. A model for cell wall degradation in this fruit is discussed.     

CRISPR/Cas9靶向敲除番茄SNAC4/9突变体的构建、鉴定及分析

NAC转录因子参与植物生长发育、果实色素积累、细胞壁形态形成和植株衰老等过程。为系统研究SNAC4(SlNAC48,基因ID:101247735)和SNAC9(SlNAC19,基因ID:101248665)在番茄成熟衰老进程中的精准功能,本试验设计SNAC4/9敲除靶点,通过重叠聚合酶链式反应（overlapping PCR）法构建单引导RNA(sgRNA)表达盒,采用金门分子克隆（golden gate）方法将单个或多个sgRNA表达盒组装至pYLCRISPR/Cas9载体。PCR和测序结果表明,SNAC4/9敲除载体构建成功,通过农杆菌转化法将编码Cas9蛋白和sgRNA的基因导入Micro-Tom番茄细胞,对阳性苗进行靶点和脱靶位点检测,结果表明番茄成功突变且未脱靶,通过鉴定T1代突变体进一步证明,CRISPR/Cas9基因编辑番茄的靶点在代际之间可以稳定遗传。与野生型相比,SNAC4/9敲除果实色素积累减少,成熟延迟,表明SNAC4/9在番茄果实成熟中发挥重要作用。本研究为进一步探究SNAC4/9调控番茄果实色素代谢和胞壁代谢机制提供了遗传材料和试验基础。     

Identification and quantification of stilbenes in fruits of transgenic tomato plants (Lycopersicon esculentum Mill.) by reversed phase HPLC with photodiode array and mass spectrometry detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection was employed to study the accumulation of stilbenes and other naturally occurring polyphenol intermediates of flavonoid pathway in tomato fruits of plants genetically modified to synthesize resveratrol. The transgenic tomato fruits were obtained by overexpression of a grapevine gene encoding the enzyme stilbene synthase in tomato plants (Lycopersicon esculentum Mill.). Stilbenes and flavonoids, either glycosylated or free, were simultaneosly identified by electrospray interface (ESI)-MS in negative ionization mode and were quantified by PDA detection at the wavelength corresponding to their maximum absorbance. The two detectors were coupled online with an HPLC system utilizing a narrow-bore C18 reversed-phase column, which was eluted by a multistep gradient of increasing concentration of acetonitrile in water containing 0.5% (v/v) formic acid. The results of these analysis revealed that the genetic modification of the tomato plants originated different levels of accumulation of four stilbenes (i.e., trans- and cis-piceid and trans- and cis-resveratrol) in their fruit depending on the stages of ripening. Either at immature or at mature stages of ripening the stilbenes were preferentially accumulated in the fruit peel as the glycosylated form. The highest amount of trans-piceid and trans-resveratrol were found in the peel of fruits harvested at mature stage of ripening. The variations in the levels of rutin, naringenin, and chlorogenic acid found in the samples extracted from the fruits of transgenic tomato plants, in comparison to that determined in the control lines, seemed to be related to the genetic transformation, whose effect on the flavonoid biosynthetic pathway needs to be elucidated by additional studies.     

Effect of dips in a 1-methylcyclopropene-generating solution on 'Harrow Sun' plums stored under different temperature regimes

The effect of postharvest dips in a 1-methylcyclopropene-generating solution of the formulation AFxRD-038 (Rohm & Haas) on plum fruit (Prunus salicina Lindell cv. 'Harrow Sun') quality and ripening during storage was determined. Fruit weight loss, tissue firmness, soluble solids content (SSC), titratable acidity (TA), ethylene production, respiration, and the activities of the cell wall modifying enzymes polygalacturonase (PG), 1,4-beta-D-glucanase/glucosidase (EGase), beta-galactosidase (beta-gal), and pectin methylesterase (PME) were measured. Fruit reddening, anthocyanin content, and phenylalanine ammonia-lyase (PAL) activity were also analyzed. The 1-MCP-treated fruit showed reduced ethylene production and respiration rate and delayed softening, which was associated with the reduction in the activity of PG, EGase, and beta-gal. The immersion in 1-MCP-generating solutions also decreased weight and acidity loss without modifying the fruit SSC. The immersion treatment was particularly effective in the fruit stored at 5 degrees C, keeping higher overall quality, maintaining lower levels of anthocyanins and PAL activity, and preventing flesh reddening. The present data show that beneficial effects in delaying plum fruit ripening and controlling chilling injury can be obtained by dipping the fruits in a solution of this novel 1-MCP-generating formulation.     

Edible coatings influence fruit ripening, quality, and aroma biosynthesis in mango fruit

The effects of different edible coatings on mango fruit ripening and ripe fruit quality parameters including color, firmness, soluble solids concentrations, total acidity, ascorbic acid, total carotenoids, fatty acids, and aroma volatiles were investigated. Hard mature green mango (Mangifera indica L. cv. Kensigton Pride) fruits were coated with aqueous mango carnauba (1:1 v/v), Semperfresh (0.6%), Aloe vera gel (1:1, v/v), or A. vera gel (100%). Untreated fruit served as the control. Following the coating, fruits were allowed to dry at room temperature and packed in soft-board trays to ripen at 21+/-1 degrees C and 55.2+/-11.1% relative humidity until the eating soft stage. Mango carnauba was effective in retarding fruit ripening, retaining fruit firmness, and improving fruit quality attributes including levels of fatty acids and aroma volatiles. Semperfresh and A. vera gel (1:1 or 100%) slightly delayed fruit ripening but reduced fruit aroma volatile development. A. vera gel coating did not exceed the commercial mango carnauba and Semperfresh in retarding fruit ripening and improving aroma volatile biosynthesis.     

Effects of Fe deficiency chlorosis on yield and fruit quality in peach (Prunus persica L. Batsch)

The effects of iron (Fe) deficiency on fruit yield and quality were measured in two peach cultivars, Carson (yellow-skin fruit) and Babygold (red-skin fruit). In both cultivars, Fe deficiency caused major decreases in fruit fresh weight per tree and number of fruits per tree. Fruits from Fe-deficient peach trees had a smaller size, resulting in a large decrease in the percentage of commercially acceptable fruits, whereas fruit firmness was unaffected. In cv. Babygold, Fe deficiency greatly decreased the red color of the fruit skin. Part of these results was likely associated with a delay in fruit ripening. When fruits with similar appearance were compared, taking into account fruit size, color, and firmness, Fe deficiency generally led to higher concentrations of organic anions (especially succinate and quinate), vitamin C, and phenolic compounds and to lower total sugar/total organic acid ratios. This could lead to decreased fruit eating quality and to a slight improvement in fruit nutritional value.     

Effects of nitrogen application on flavor compounds of cherry tomato fruits

A pot experiment was conducted to determine the effects of N application on volatile compounds, taste compounds, and firmness of fresh tomato fruits. Each pot was filled with 8 kg of clean sand. The experiment consisted of six nitrogen (N)‐application rates with 0, 2.25, 4.50, 9.00, 18.00, and 36.00 mmol N L^–1 in the nutrient solution. Volatile compounds, soluble sugars, soluble solids, titratable acidity, and firmness of fresh tomato fruits were measured. The results show that increasing N application increased the concentrations of 1‐penten‐3‐one, hexanal, cis‐3‐hexenal, 2‐methyl‐4‐pentenal, trans‐2‐hexenal, 6‐methyl‐5‐hepten‐2‐one, titratable acidity, soluble sugars, and soluble solids. By contrast, increasing N supply decreased the concentration of phenylacetaldehyde and first increased and then decreased the concentrations of 2E–4E‐hexadienal and the firmness of fresh tomato fruits. Close relationships between the concentration of various volatile compounds, titratable acidity, soluble sugars, and soluble solids were found. However, concentrations of these flavor compounds were very poorly correlated with fruit firmness. Based on contributions of these compounds to tomato flavor, we assume that moderate high N supply improves tomato flavor, whereas excessive N supply can deteriorate it.     

Technique for rapid, small-scale analysis of vitamin C levels in fruit and application to a tomato mutant collection

We present a technique for easy, rapid analysis of both total and reduced forms of vitamin C in fruits using microplates and a plate reader. This technique has been compared with a spectrofluorometric technique classically used for assaying vitamin C in fresh tomato. We have applied these methods to a population of 118 tomato mutant lines and controls in search of variability for this trait. Six lines, identified as having high vitamin C levels, and four lines having low vitamin C levels have been chosen for further study. The vitamin C levels have been compared with sugar concentration, dry matter content, fruit weight, titratable acidity, and firmness. The correlations that often exist in tomato varieties between sugar and vitamin C content (positive correlation) or fruit weight and vitamin C content (negative correlation) can be uncoupled in the lines selected for further analysis.     

Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion

Although light is recognized as one of the main factors influencing fruit carotenogenesis, the specific role of UV-B radiation has been poorly investigated. The present work is addressed to assess the molecular events underlying carotenoid accumulation in presence or absence of ultraviolet-B (UV-B) light in tomato fruits of wild-type and high pigment-1 (hp-1), a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression analyses indicated that in wild-type fruits UV-B radiation mainly negatively affects the carotenoid biosynthetic genes encoding enzymes downstream of lycopene both in flesh and peel, suggesting that the down-regulation of genes CrtL-b and CrtL-e and the subsequent accumulation of lycopene during tomato ripening are determined at least in part by UV-B light. In contrast to wild-type, UV-B depletion did not greatly affect carotenoid accumulation in hp-1 and generally determined minor differences in gene expression between control and UV-B-depleted conditions.     

Influence of 1-methylcyclopropene and storage atmosphere on changes in volatile compounds and fruit quality of conference pears

Conference pears (Pyrus communis L.) were treated with 25 and 50 nL L(-1) 1-methylcyclopropene (1-MCP) at -0.5 degrees C for 24 h, then stored for up to 22 weeks in air (NA) and controlled atmosphere (CA). After 7 and 14 weeks of storage, fruits were retreated with 1-MCP. After 7, 14, and 22 weeks of storage, fruits were kept for up to 7 days at 20 degrees C in air for poststorage ripening. The effects of 1-MCP treatment declined with duration of storage in both storage atmospheres, indicating that retreatments had little additional effects on subsequent ripening. Ethylene production was lower and firmness was higher in 50 nL L(-1) fruits, while the 25 nL L(-1) dose was not very different from the control. Development of superficial scald was not prevented by 1-MCP treatments, but the severity of the symptoms was influenced. The 1-MCP effects were perceivable on texture (juiciness) and flavor. Control fruit and 25 nL L(-1) fruit reached their best sensory quality after 14 weeks of storage, while 50 nL L(-1) fruit reached the same sensory quality later, keeping a fresh flavor when the quality of control fruit declined and became watery or grainy. The fresh flavor in 50 nL L(-1) fruit was probably due to the presence below the odor detection threshold concentrations of the volatile compounds responsible for the "ripe pear" aroma, mainly of butanol and ethyl butanoate. CA prolonged or enhanced the effects of 1-MCP; 1-MCP cannot substitute for CA but can reinforce the CA effects.     

番茄果实成熟衰老相关蛋白酶鉴定

为进一步研究蛋白酶与成熟衰老的关系,试验以‘918’番茄(Lycopersicum esculentum Mill.cv.918)果实为材料,采用改进的含明胶的十二烷基硫酸钠聚丙烯酰胺凝胶电泳方法对番茄果实中的蛋白酶进行检测,结果发现番茄果实中有3个蛋白酶,命名为SP-1、SP-2和SP-3,其活性伴随着果实后熟衰老发生变化。乙烯处理可提高番茄果实蛋白酶SP-2的活性,使SP-1和SP-3活性被提前检出,而1-甲基环丙烯处理对蛋白酶起相反作用。番茄果实蛋白酶受果实后熟衰老和乙烯的双重调控,是衰老相关蛋白酶。该文为进一步研究蛋白酶与果蔬衰老的关系提供参考。