Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

Ripening Physiology of Fruit from Transgenic Tomato (Lycopersicon esculentum) Plants with Reduced Ethylene Synthesis

The physiological effects of reduced ethylene synthesis in a transgenic tomato (Lycopersicon esculentum) line expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme have been examined. Fruit from the transgenic line 5673 ripen significantly slower than control fruit when removed from the vine early in ripening. In contrast, fruit that remain attached to the plants ripen much more rapidly, exhibiting little delay relative to the control. Ethylene determinations on attached fruit revealed that there was significantly more internal ethylene in attached than detached fruit. The higher ethylene content can fully account for the observed faster on-the-vine ripening. All of the data are consistent with a catalytic role for ethylene in promoting many, although not all, aspects of fruit ripening. Biochemical analyses of transgenic fruit indicated no significant differences from controls in the levels of ACC oxidase or polygalacturonase. Because transgenic fruit are significantly firmer than controls, this last result indicates that other enzymes may have a significant role in fruit softening.     

挥发性香气物质和乙烯生产在"湖景蜜露"桃果实发育过程中的变化

以"湖景蜜露"水蜜桃(Prunus persica L.)为试材,检测了果实从未成熟到成熟发育过程中乙烯生成、呼吸速率及挥发性香气性物质的变化;同时对果实大小、果皮色泽、果肉硬度、可溶性固形物、可滴定酸进行了测定;对与果实乙烯产生密切相关的1-氨基环丙烷-1-羧酸(ACC)含量、ACC合成酶活性、ACC氧化酶活性也进行了测定.结果表明,随果实成熟度的增加,果实大小、果皮L*值、可溶性固形物含量增加,而果实硬度、果皮h°值、可滴定酸含量减少.在未成熟的果实中,C6的醛类(反式-2-己烯醛)和醇类(顺式-3-己烯醇)是主要的成分;乙烯生成量很低;呼吸速率较高.到跃变阶段C6～C12的内酯类物质明显增加,尤其是γ和δ-内酯类成为果实主要的香气挥发性物质.推测果实乙烯、呼吸作用等基本的生理变化可能调节着内酯类物质的生成.在乙烯跃变上升时果肉中ACC氧化酶的活性下降,ACC含量和ACC合成酶活力的变化与乙烯生成量变化的趋势一致.根据以上结果可以认为桃果实主要的香气挥发性物质的形成与乙烯、呼吸跃变的开始密切相关.香气物质形成速率动态变化可能是桃果实发育过程中成熟度的另一个生理学指标.     

苹果果实糖积累特性与品质形成的关系

以'富士'和'国光'苹果为研究对象,对其果实发育过程中糖含量及其代谢关键酶活性的变化进行测定分析,以揭示糖分积累代谢特性对果实品质形成的影响.结果表明:(1)'富士'和'国光'均为己糖积累型果实, '富士'果实以积累果糖最多,果糖/葡萄糖(F/G)值为1.56,而'国光'以积累葡萄糖最多,F/G值仅为0.68;蔗糖在两品种中含量和所占比例均很低,在近成熟期'富士'高于'国光'.(2)'富士'果实蔗糖磷酸合成酶(SPS)和蔗糖合成酶(SS)活性均随果实糖的累积量增加而显著升高,酸性转化酶(AI)活性也渐趋升高,而中性转化酶(NI)活性波动不大,且其糖累积与AI和SPS活性相关性最大,而与NI相关性不大,SS的作用主要表现在发育后期;在 '国光'果实糖积累过程中SPS起主导作用,SS和NI的作用主要表现在发育前期,而AI的作用不大.(3)'富士'和'国光'果实淀粉含量变化趋势相同,在淀粉积累高峰之后,'富士'果实淀粉降解速度更快,其淀粉含量迅速下降且低于'国光',此时其相应淀粉酶活性也高于'国光'.研究发现,'富士'和'国光'果实糖积累和淀粉代谢均存在显著差异,从而直接或间接地影响着果实糖代谢过程,进而导致果实品质的显著差异.     

The Tomato E8 Gene Influences Ethylene Biosynthesis in Fruit but Not in Flowers

We investigated the function of the tomato (Lycopersicon esculentum) E8 gene. Previous experiments in which antisense suppression of E8 was used suggested that the E8 protein has a negative effect on ethylene evolution in fruit. E8 is expressed in flowers as well as in fruit, and its expression is high in anthers. We introduced a cauliflower mosaic virus 35S-E8 gene into tomato plants and obtained plants with overexpression of E8 and plants in which E8 expression was suppressed due to co-suppression. Overexpression of E8 in unripe fruit did not affect the level of ethylene evolution during fruit ripening; however, reduction of E8 protein by cosuppression did lead to elevated levels during ripening. Levels for ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ACC oxidase mRNA were increased approximately 7-fold in fruit of plants with reduced E8 protein. Levels of ACC synthase 2 mRNA were increased 2.5-fold, and ACC synthase 4 mRNA was not affected. Reduction of E8 protein in anthers did not affect the accumulation of ACC or of mRNAs encoding enzymes involved in ethylene biosynthesis. Our results suggest that the product of the E8 reaction participates in feedback regulation of ethylene biosynthesis during fruit ripening.     

Aminoethoxyvinylglycine Effects on Late-Season Apple Fruit Maturation

Aminoethoxyvinylglycine (AVG) inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, and thus blocks ethylene synthesis. Preharvest foliar application of AVG to apple (Malus domestica Borkh.) fruit retards several key events of maturation including climacteric ethylene production, starch conversion to sugars, fruit softening, and abscission zone development. Although the impact of AVG on apple fruit maturation is well known, the biochemical basis of these effects is not well understood. The effects of AVG application on Redchief Delicious apple fruit maturation were studied. AVG applied four weeks prior to harvest significantly reduced internal ethylene levels, amylose degradation, and accumulation of sucrose, glucose, and sorbitol. Because AVG application coincidentally inhibited starch degradation and the increase in internal ethylene, we investigated the enzymatic basis of starch mobilization in apple fruit. Amylase activity was somewhat reduced in AVG-treated fruit. Amylase activity was less in AVG-treated fruit during the early stages of starch mobilization. Starch phosphorylase activity increased dramatically during the later stages of starch mobilization, but was not affected by AVG treatment. Soluble starch synthase activity was also unaffected by AVG treatment and remained constant throughout the eight-week harvest period. Moreover, AVG did not affect the levels of amylopectin, fructose, malate, ascorbate, citrate, or anthocyanin. These results suggest that apple fruit ripening has both ethylene-dependent and -independent processes occurring simultaneously.     

1-Aminocyclopropane-1-Carboxylic Acid Levels in Ripening Apple Fruits

1-Aminocyclopropane-1-carboxylic acid (ACC) in amino acid fractionsof apple fruits was assayed by chemical conversion to ethylene.The specificity of the assay was checked with other amino acids;homocysteine was the only naturally occurring compound foundto yield significant amounts of ethylene in the assay. Analysisof the thiol content of apples showed that homocysteine couldnot be a significant source of interference. Interference froman uncharacterized component of amino acid fractions was lessthan 20% of the ACC level in unripe fruit and insignificantin ripe fruit. Liquid chromatographic assay gave results inclose agreement with the standard assay. Higher apparent ACClevels were measured in unfractionated apple juice than in thestandard assay. Both of these methods and the liquid chromatographicassay were used on a number of apple samples during ripening.All three methods showed that ACC increased 30–40 foldwhereas ethylene production increased by a factor of 10⁴. Inindividual apples the ACC level increased about one day laterthan ethylene production. Key words: Apple fruit, 1-Aminocyclopropane-1-carboxylic acid, Analytical methods, Ethylene     

Effect of methylglyoxal bis-(guanylhydrazone) on polyamine and ethylene biosynthesis of apple fruit after harvest

Polyamine and ethylene both play important roles in fruit ripening, whose biosynthetic pathways share a common substrate, S-adenosylmethionine (SAM). To unravel the interrelationship between polyamine and ethylene, their metabolism and expression of relevant genes were investigated in apple fruit (Malus domestica Borkh.) treated with methylglyoxal bis-(guanylhydrazone) (MGBG). The MGBG-treated fruit had higher ethylene production until 16 days after treatment (DAT) with preceding accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) than control fruit and then decreased to nearly the same level as control. Ethylene promotion at the early stage by MGBG was accompanied by increased expression of apple ACC synthase (Md-ACS1) and ACC oxidase (MdACO). The expression of apple SAM synthase (MdSAMS) in MGBG-treated fruit was slightly higher than that in control. On the other hand, significant changes in free polyamine titers were observed at some stages, but the changes did not show consistent trends. Based on these observations, possible relationship between polyamine and ethylene pathways was discussed.     

Effect of low oxygen and high carbon dioxide on the levels of ethylene and 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

The association of the level of ACC and the ethylene concentration in ripening apple fruit (Malus sylvestris Mill, var. Ben Davis) was studied. Preclimacteric apple contained small amounts of ACC and ethylene. With the onset of the climacteric and a concomitant decrease in flesh firmness, the level of ACC and ethylene concentration both increased markedly. During the postclimacteric period, ethylene concentration started to decline, but the level of ACC continued to increase. Ethylene production and loss of flesh firmness of fruits during ripening were greatly suppressed by treatments with low O₂ (O₂ 1–3%, CO₂ O%) or high CO₂ (CO₂ 20–30%, O₂ 15–20%) at the preclimacteric stage. However, after 4 weeks an accumulation of ACC was observed in treated fruits when control fruit was at the postclimacteric stage. Treatment of fruit with either low O₂ or high CO₂ at the climacteric stage resulted in a decrease of ethylene production. However, the ACC level in fruit treated with low O₂ was much higher than both control and high CO₂ treated fruit; it appears that low O₂ inhibits only the conversion of ACC to ethylene, resulting in an accumulation of ACC. Since CO₂ inhibits ethylene production but does not result in an accumulation of ACC, it appears that high CO₂ inhibits both the conversion of ACC to ethylene and the formation of ACC.     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.     

Differential effects of abscisic acid and ethylene on the fruit maturation of <Emphasis Type="Italic">Litchi chinensis</Emphasis> Sonn.

Two litchi cultivars, a well-coloured ‘Nuomici’ and a poorly coloured ‘Feizixiao’, were used to investigate changes in endogenous abscisic acid (ABA) concentration and ethylene production during fruit maturation and to test the effects of exogenous growth regulators on litchi fruit maturation. Abscisic acid concentration in both the aril and pericarp increased with fruit maturation. Transfusion of ABA into the fruit 3 weeks before harvest accelerated, whereas transfusion of 6-benzyl aminopurine (6-BA) retarded sugar accumulation and pigmentation. The effect of 6-BA was assumed to link with the resultant decrease in ABA. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) concentration and ACC oxidase (ACO) activities in the aril remained relatively constant during sugar accumulation. Transfusion of aminooxyacetic acid (AOA) significantly decreased ACC concentration but had no effect on sugar accumulation in the aril. These results suggested that endogenous ABA, but not ethylene, was critical for the sugar accumulation. However, the roles of ABA and ethylene in pericarp pigmentation were rather complicated. Application of exogenous ABA promoted anthocyanin synthesis significantly, but had very little effect on chlorophyll degradation. Ethylene production in litchi fruit decreased with development, but a transient increase of endogenous ethylene production was detected just around the colour break in ‘Nuomici’. Enhanced ACO activity in the pericarp was detected during pigmentation. Ethrel at 400 mg l⁻¹ showed no effect on pericarp coloration, but accelerated chlorophyll degradation and anthocyanin synthesis at a much higher concentration (800 mg l⁻¹). Fruit dipped in ABA solution alone yielded no effect on chlorophyll degradation, but the combined use of ABA and Ethrel at 400 mg l⁻¹ enhanced both chlorophyll degradation and anthocyanin synthesis. These results indicated the possible synergistic action of ethylene and ABA during litchi fruit colouration. ABA is suggested to play a more crucial role in anthocyanin synthesis, while ethylene is more important in chlorophyll degradation. ABA can increase the sensitivity of pericarp tissue to ethylene.     

Availability of assimilates and formation of aroma compounds in apples as affected by the fruit/leaf ratio

Availability of assimilates in apple trees ( Malus domestica cv. Jonagored) was affected by removing young fruits to obtain 3 ranges of fruit/leaf ratios with average values of 130, 268 and 381 fruits per kg leaf dry matter. Fruit analyses were carried out at fruit harvest and 4 times during a 3-week ripening period. The analyses included detection of volatile aroma components from the juice by headspace gas chromatography. At a low fruit/leaf ratio, higher concentrations of total dry matter, soluble solid and titrateable acids were found. The flesh was also firmer, and ethylene development proceeded at a lower rate and reached a lower maximum value. Aroma compounds consisted of ca 20% esters, 73% alcohols and 6% C-6 aldehydes. The production of butylacetate and hexylacetate, which were the dominating esters, peaked during the ripening period and was most pronounced at the lowest fruit/leaf ratios. At the last sampling date this was also the case for butanol, which was the dominating alcohol. Other esters and alcohols behaved similarly, while C-6 aldehydes showed no significant differences in the fruit/leaf ratio. We suggest that the greater availability of assimilates when internal competition is relieved at a low fruit/leaf ratio causes increased accumulation of fatty acid aroma precursors and aroma compounds as well as of sugars, acids and other compounds in the fruits.     

1-MCP对‘粉红女士’苹果冷藏期间品质变化和香气形成的影响

以'粉红女士'苹果为试验材料,研究了1 μL/L 1-MCP(1-甲基环丙烯)对苹果冷藏期间乙烯释放速率、呼吸速率、果实硬度以及香气成分和相对含量的影响.结果表明,1-MCP处理可显著抑制'粉红女士'苹果冷藏期间呼吸作用和乙烯释放,有效延缓果实硬度的下降.冷藏期内'粉红女士'苹果香气物质主要有醇类、醛类、酯类、烯类、酸类和烷烃类等,并以酯类香气为主(占46.15%);1-MCP能显著减少果实贮藏期间酯类、醇类和烷烃类香气成分种类和相对含量,处理果中酯类和醇类香气成分种类比同期对照分别减少了50%和78%,主要香气成分丁酸己酯在处理和对照果实的相对含量分别为1.12%～1.73%和1.87%～5.18%.可见,1-MCP处理对'粉红女士'苹果具有良好保鲜效果,也显著地抑制了贮藏期间香气的形成.     

5种新疆苹果资源的果实品质性状比较与评价

选择5种果皮和果肉颜色不同的新疆苹果地方特色资源克孜阿尔玛、卡拉阿尔玛、阿克阿尔玛、伊犁野苹果、柠檬海棠,以栽培品种富士为对照,通过果实外观品质、内在品质、营养品质和香气品质4个方面的指标进行品质精细化评价,并考察了红肉苹果果肉花青苷合成途径相关基因的表达情况,以明确新疆苹果地方资源不同果实品质指标的差异,为新疆苹果地方资源的合理利用提供资料。结果表明:(1)5种特色资源均为中、小果型苹果,其中红肉品种克孜阿尔玛的外观品质优于栽培品种富士,而柠檬海棠外观品质指标均处于最低水平。(2)阿克阿尔玛的糖、酸含量均显著低于富士,柠檬海棠的酸含量高于富士及其他品种。(3)伊犁野苹果的果肉硬度显著高于富士,而红肉资源品种的果肉硬度均低于富士,肉质松软、不耐储运,但其总酚、类黄酮含量最为丰富,抗氧化能力最强,均显著高于栽培品种富士。(4)香气品质中,红皮资源卡拉阿尔玛的挥发性物质种类及含量最为丰富,其含量高于最低的栽培品种富士近2倍,红肉资源的挥发性物质含量最低;红肉资源的挥发性物质以醇类为主,而红皮资源卡拉阿尔玛、伊犁野苹果和柠檬海棠以醛类物质为主。(5)主成分分析显示,各个新疆苹果地方资源营养、香气品质等综合品质表现为:克孜阿尔玛卡拉阿尔玛阿克阿尔玛富士伊犁野苹果柠檬海棠。(6)红肉苹果的果肉花青苷合成途径相关基因表达分析表明,其果肉花青苷积累过程中的关键基因是UFGT。     

Metabolism of 1-Aminocyclopropane-l-Carboxylic Acid during Apple Fruit Development

The metabolism of [U^–14C] 1-aminocyclopropane-1-carboxylicacid (ACC) supplied to whole fruits of apple (Malus domesticaBorkh., cv. Cox's Orange Pippin) was investigated. Radioactiveethylene was recovered in mercuric acetate traps and an acidicmetabolite was formed in proportions which varied little withthe absolute amount of substrate supplied. The amount of ACCusually supplied did not cause immediate, rapid ethylene productionby mature, pre-climacteric fruit but the onset of productionwas earlier than in untreated fruit. The radioactive acidic metabolite was purified by four chromatographicprocedures and activity was coincident with authentic 1-malonylamino)cyclopropane-1-carboxylic acid (MACC). The presence of thiscompound was confirmed by gas chromatography linked to massspectrometry. MACC was a major metabolite of [¹⁴C] ACC supplied to applesthroughout fruit development. The proportion converted to ethylenewas low but increased with endogenous ethylene production inthe final samples. MACC was shown to be a natural constituent of apple fruits andto accumulate to the amol kg^–1 level. Key words: 1-Aminocyclopropane-l-carboxylic acid, Ethylene, 1 (Malonylamino) cyclopropane-1-carboxylic acid, Malus domestica     

Role of ethylene in the biosynthetic pathway of aliphatic ester aroma volatiles in Charentais Cantaloupe melons.

Compared to other melon types, Cantaloupe Charentais melons are highly aromatic with a major contribution to the aroma being made by aliphatic and branched esters. Using a transgenic line in which the synthesis of the plant hormone ethylene has been considerably lowered by antisense ACC oxidase mRNA (AS), the aliphatic ester pathway steps at which ethylene exerts its regulatory role were found. The data show that the production of aliphatic esters such as hexyl and butyl acetate was blocked in AS fruit and could be reversed by ethylene. Using fruit discs incubated in the presence of various precursors, the steps at which ester formation was inhibited in AS fruit was shown to be the reduction of fatty acids and aldehydes, the last step of acetyl transfer to alcohols being unaffected. However, treating AS fruit with the ethylene antagonist 1-methylcyclopropene resulted in about 50% inhibition of acetyl transfer activity, indicating that this portion of activity was ethylene-dependent and this was supported by the low residual ethylene concentration of AS fruit discs (around 2 microl l(-1)). In conclusion, the reduction of fatty acids and aldehydes appears essentially to be ethylene-dependent, whilst the last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyltransferases.     

Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.