Molecular cloning and characterization of a novel 1-aminocyclopropane-1-carboxylate oxidase gene involved in ripening of banana fruits

One novel banana fruit ripening related 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene quite different from ACC oxidase genes from other species was cloned. In contrast to other studies, the polypeptide encoded by this gene, named Mh-ACO1, lacks the putative leucine zipper motif which is conserved in all known ACC oxidases including the other previously reported banana ACC oxidase, Mh-ACO2. The locus consists of two nearly identical paralogous ACC oxidase genes arranged in opposite orientation and separated by a 3.1-kb intergenic region. The has only two introns, at positions identical to , which comprises a coding region interrupted by three introns. The predicted amino acid sequence of Mh-ACO1 shares less than 50% identity to those of ACC oxidase from other climacteric fruits, while that of Mh-ACO2 shows more than 65% homology. When expressed in Saccharomyces cerevisiae -encoded protein possessed the enzyme activity for ethylene conversion. The levels of mRNA corresponding to both and increased during fruit ripening and were induced by exogenous ethylene. We conclude that both and contribute to increased ethylene production in fruits and these two genes are differentially expressed in fruits and other organs in banana.     

乙烯催熟徐香猕猴桃的响应面试验及工艺优化研究

为探究外源乙烯催熟徐香猕猴桃的过程中乙烯缓释剂用量、处理时间及贮藏温度对品质的影响,确定可靠的徐香猕猴桃催熟工艺参数,本研究以徐香猕猴桃为原料,乙烯缓释剂用量、处理时间及贮藏温度为影响因素,果实硬度、可溶性固形物、Vc含量、可溶性蛋白含量、总酚含量及感官评分等为响应值进行响应面试验,采用主成分分析法提取特征指标,通过响应面法分析并建立特征指标的二次回归模型并优化参数;利用遗传算法对特征指标进行多目标优化并与响应面优化结果进行比较。结果表明,硬度及总酚含量在主成分分析结果中累计贡献率为63.861%,可作为反映催熟后猕猴桃品质的特征指标;响应面分析得到的优化工艺参数为:乙烯缓释剂用量3.34 g·10kg^-1、处理时间27.72 h、贮藏温度4.21℃;遗传算法多目标优化得到的工艺参数为:乙烯缓释剂用量3.36 g·10 kg^-1、处理时间27.68 h、贮藏温度4.38℃,2种方法的优化结果基本一致。综上,优化工艺可行且硬度、总酚结合Vc含量能够较好地评价乙烯催熟徐香猕猴桃的综合品质。乙烯催熟徐香猕猴桃的最佳工艺参数为:乙烯缓释剂用量3 g·10kg^-1、 处理温度25℃、处理时间28 h、贮藏温度4℃。经后期验证,使用该参数催熟徐香猕猴桃,其后熟时间缩短至5 d左右,较未处理组的后熟时间大大缩短。该优化工艺参数可为满足猕猴桃鲜果销售市场需求,保持果实后熟品质提供一定的参考依据。     

Influence of harvest date within the season and cold storage on cherimoya fruit ripening

Cherimoya ripening with and without prior storage at 8 degrees C was studied in fruit harvested on early- (EH), mid- (MH), and late-season (LH) dates. Most of the differences in the ripening behavior were observed between EH cherimoyas and fruit from the other two harvest dates. During ripening of nonstored fruit, the increases in ethylene production and respiration rates and in soluble sugars and organic acids contents were faster in EH than in the other fruits (which ripened 1 day later). These differences could be due to variations in the physiological stage at harvest as a result of the different heat units (degree/month) accumulated in the last month of fruit development. During ripening of cold-stored fruit the differences in the time to the onset of the increase in ethylene production and in the accumulation of malic and citric acid were minimized, especially after longer storage times, and the relationship of harvest date with the increases in respiration rate was lost. Glucose and fructose accumulation was reduced by prior cold storage, especially after longer storage duration and in MH and LH fruit, but sucrose hydrolysis was almost complete, as in nonstored fruit. It is suggested that glucose and fructose accumulation is more sensitive to low temperature than sucrose metabolism and that this differential sensitivity is more marked in MH and LH cherimoyas. The time to ripen was inversely related to prior cold storage duration and was dependent on harvest date: the later the harvest date, the longer storage time it took to shorten subsequent ripening.     

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。     

Changes in polyamines and ethylene during the development and ripening of eggplant fruits (Solanum melongena)

The levels of free polyamines in the endocarpium of eggplants (Solanum melongena) cv. Black Nite were studied during fruit growth and ripening. Other parameters such as weight and volume variation, respiratory intensity, ethylene production, and sugar content were determined. The polyamines found were putrescine and spermidine, with a higher amount of the former. No spermine, agmatine, or cadaverine were found during the development and ripening period of eggplant. At the beginning of fruit development, the levels of putrescine and spermidine observed were 1.67 nmol/g of fresh tissue. Putrescine levels peaked at 17.4 nmol/g of fresh tissue on the ninth day after petal fall, decreasing later to the initial levels. No significant changes in spermidine were observed during the growth period of the fruit. Beginning 9 days after petal fall, there was a rapid increase in fruit weight and volume, which coincided with the maximum content of free polyamines. At the same growth stage, total sugar content was maximum. Ethylene production decreased rapidly from 14.23 to 1.5 microL/kg.h and remained low during the whole growth period.     

Involvement of negative feedback regulation in wound-induced ethylene synthesis in 'Saijo' persimmon

Wounding is one of the most effective stress signals to induce ethylene synthesis in persimmon (Diospyros kaki Thunb.). We found that wound-induced ethylene biosynthesis is subjected to negative feedback regulation in mature 'Saijo' persimmon fruit since ethylene production was enhanced by 1-methylcyclopropene (1-MCP) (an inhibitor of ethylene perception) pretreatment, which was approximately 1.8 fold of that in control tissues (without 1-MCP pretreatment). Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and DK-ACS2 gene expression were substantially increased by 1-MCP pretreatment after 12 h, which resulted in much higher ACC content in 1-MCP pretreated tissues than that in a control after 24 h. These results indicated that wound-induced DK-ACS2 gene expression was negatively regulated by ethylene in mature persimmon fruit. However, 1-MCP pretreatment had no effect on DK-ACO1 gene expression, suggesting the independence of wound-induced DK-ACO1 on ethylene. Out of accord with DK-ACO1 gene expression, ACC oxidase activity was enhanced 48 h after wounding in 1-MCP pretreated tissues, reaching a peak 1.5-fold higher than that in control tissues at 60 h.     

Postharvest polyamine application alleviates chilling injury and affects apricot storage ability

Fruit of two apricot cultivars 'Bagheri' and 'Asgarabadi' were treated with putrescine (Put) or spermidine (Spd) at 1 mM and then were stored at 1 °C for 21 days. Fruit were sampled weekly and stored 2 days at 20 °C for shelf-life study. The treatments reduced ethylene production and maintained the firmness and color of the fruit. Peroxidase (POX), catalase (CAT), superoxide dismutase (SOD), and polyphenol oxidase (PPO) activities and total phenol (TP) concentrations were measured during storage. Both cultivars showed chilling injury (CI) incidence, and the severity in control fruit was higher than either Put or Spd treatments. CI incidence in Spd-treated fruit was lower than that of Put-treated fruit. Polyamine (PA) treatment generally increased antioxidant enzyme activity of fruit during storage. PA treatments may help maintain the quality of apricot fruit during storage by inhibiting ripening and decreasing CI incidence.     

Relationships between respiration, ethylene, and aroma production in ripening banana

Mature green bananas were treated with the ethylene antagonist 1-methylcyclopropene (1-MCP) at intervals during the 24 h period after initiation of ripening with propylene. Following 1-MCP treatment, the fruits were ripened in either air or propylene while ethylene, carbon dioxide, and volatile production and composition were monitored at regular intervals. The application of 1-MCP significantly delayed and suppressed the onset and magnitude of fruit respiration and volatile production. The 1-MCP treatments also caused a quantitative change in the composition of the aroma volatiles, resulting in a substantial increase in the concentration of alcohols and a decrease in their related esters. The results showed that ethylene has a continuing role in integrating many of the biochemical processes that take place during the ripening of bananas.     

Development of apple superficial scald, soft scald, core flush, and greasiness is reduced by MCP.

1-Methylcyclopropene (MCP) was used to evaluate the role of ethylene in development of apple (Malus x domestica Borkh.) physiological disorders during storage. Granny Smith, Red Chief Delicious, and Fuji apple fruit were treated with MCP at a concentration of 1 microL L(-)(1) for 12 h at 20 degrees C. For all varieties stored at 0 degrees C, ethylene production and respiration rates were reduced for several months following MCP treatment, and firmness and titratable acidity of treated fruit were higher compared to controls. Apples treated with MCP did not develop superficial scald or peel greasiness through 6 months storage plus ripening at 20 degrees C for 7 days. Core flush was not observed in MCP-treated fruit until 6 months after treatment when the incidence was still lower compared to control fruit. MCP delayed the rise in production of alpha-farnesene and reduced accumulation of its oxidation products.     

Impacts of ionizing radiation on volatile production by ripening gala apple fruit

Apple (Malus x domestica Borkh., cv. Gala) fruit treated with 0.5 microL x L(-1) 1-methylcyclopropene (MCP) or air (non-MCP) for 12 h at 20 degrees C were exposed to gamma radiation at doses of 0, 0.44, 0.88, or 1.32 kGy at 23 degrees C and then stored at 20 degrees C. Production of volatile compounds was measured on the day of irradiation and 1, 3, 7, 14, and 21 days after irradiation. Both MCP treatment and irradiation inhibited ethylene production. MCP treatment reduced production of all volatile esters and alcohols detected, whereas irradiation inhibited production of most, but not all, esters and some alcohols by non-MCP-treated fruit. The inhibition of volatile production following irradiation increased with dose. Production of methyl and propyl esters was inhibited more than that of other esters following irradiation or MCP treatment. The impact of irradiation on production of esters and alcohols by MCP-treated fruit was minimal. Non-MCP-treated fruit irradiated at 0.44 kGy produced the most esters during the 21-day period at 20 degrees C following irradiation, and the ester production rate in these fruit was comparable to that of the nonirradiated fruit 21 days after irradiation. Fruit treated with doses higher than 0.44 kGy did not recover their ability to produce volatile compounds. These results indicate both MCP and ionizing radiation inhibit production of many aroma compounds produced by ripening apple fruit.     

Papaya fruit ripening: response to ethylene and 1-methylcyclopropene (1-MCP)

Ripening affects the quality and nutritional contents of fleshy fruits, and papayas are climacteric fruits very susceptible to postharvest losses due to the fast softening caused by ethylene. This paper reports the changes in respiration, ethylene production, and pulp color and firmness, along with the contents of soluble sugars and major carotenoids, during ripening of 'Golden' papaya, an important Brazilian cultivar that has been exported to North American and European markets. The results obtained for nontreated and ethylene- or 1-MCP-treated papaya suggest that 1-MCP can decrease the quality of treated fruit and that even the use of ethylene for triggering or inducing homogeneous ripening can result in lower quality when compared to that of fruit allowed to ripe naturally.     

Loss of ripening capacity of pawpaw fruit with extended cold storage

The fruit ripening traits of pawpaw [ Asimina triloba (L.) Dunal] were examined after harvest and after cold storage at -2, 2, 4, and 6 degrees C for up to 12 weeks. Generally, fruits stored at 2-4 degrees C for 4 weeks ripened normally, but those stored at -2 degrees C did not ripen normally, those stored at 6 degrees C were overripe, and by 6-8 weeks those stored at 2-4 degrees C had a lower respiration rate and ethylene production, lower firmness, and lower pH than fruit cold-stored for 4 weeks or less. These changes, and the occasional development of brown discoloration in the pulp once the fruits were moved back to room temperature, were evidence of chilling injury by 6 weeks. After harvest and through 4 weeks of cold storage, the main volatile compounds produced by fruit were methyl and ethyl octanoates and hexanoates. Volatile production significantly increased >5-fold in fruit ripened for 72 h after harvest or after removal from up to 4 weeks of cold storage. Fruit cold-stored for 6 weeks or more produced fewer total volatiles and esters but increased levels of such off-flavor compounds as ethyl acetate, ethyl propionate, and hexanoic and decanoic acids. Alcohol acyltransferase (AAT) activity declined in cold-stored fruit but was not correlated with either total volatile production or total ester production. Alcohol dehydrogenase activity did not change during ripening after harvest or cold storage. Lipoxygenase activity was highest just after harvest or after 2 weeks of cold storage, but was low by 4 weeks. Thus, ripening of pawpaw fruit seems to be limited to 4 weeks at 2-4 degrees C with loss of ability to continue ripening and chilling injury symptoms evident at colder temperatures and after longer periods of cold storage.     

钙和ABA在番茄果实乙烯生物合成中的相互作用

研究了钙和ABA对不同成熟阶段和不同成熟模式番茄(Lycopersicon esculentum cv．Lichun)果实乙烯生成的影响，分析两者在乙烯生物合成系统中的相互作用。普通番茄绿熟果实组织圆片在用100μmol／L ABA和100mmol/L钙处理后，乙烯生成均很快增加。当用这两种试剂同时处理番茄果实圆片时，乙烯生成增加量明显比单一处理增加量大，表明两者在对绿熟番茄果实乙烯生成的影响中有协同作用关系。钙离子鳌合剂EGTA(乙二醇双乙胺醚-N，N’-四乙酸)对乙烯生成有抑制作用，用ABA和EGTA同时处理番茄圆片时，乙烯的生成量比EGTA单一处理的生成量要高。当用这些试剂处理未成熟番茄果实则产生不同的效果，ABA明显抑制乙烯生成，而钙却依然促进乙烯的生物合成，ABA和钙同时处理果实时，乙烯生成量介于二者单一处理之间，表明ABA和钙在未成熟番茄果实乙烯生成中表现出相互抑制作用。在转反义ACS番茄中，ABA和钙抑制未成熟果实的乙烯生成，而都促进了绿熟果实的乙烯生成。这表明ABA和钙在番茄果实不同成熟阶段和不同成熟模式的乙烯生物合成过程中有着不同的相互作用关系。     

Effect of jasmonates on ethylene biosynthesis and aroma volatile emission in Japanese apricot infected by a pathogen (Colletotrichum gloeosporioides)

The effects of the application of the jasmonic acid derivative n-propyl dihydrojasmonate (PDJ) on ethylene biosynthesis, volatile compounds, and endogenous jasmonic acid (JA) and methyl jasmonate (MeJA) were examined in Japanese apricot (Prunus mume Sieb.) infected by a pathogen (Colletotrichum gloeosporioides). The fruit were dipped into 0.4 mM PDJ solution before inoculation with the pathogen and stored at 25 °C for 6 days. The inoculation induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene, JA, and MeJA. In contrast, PDJ application reduced the endogenous JA, MeJA, and ethylene production and expression of the ACC oxidase gene (PmACO1) caused by the pathogen infection. The lesion diameter with C. gloeosporioides decreased upon PDJ application. The alcohol, ester, ketone, and lactone concentrations and alcohol acyltransferase (AAT) activity increased in the pathogen-infected fruit, but were decreased by PDJ application. These results suggest that PDJ application might influence ethylene production through PmACO1 and that aroma volatile emissions affected by pathogen infection can be correlated with the ethylene production, which is mediated by the levels of jasmonates.     

Influence of 1-methylcyclopropene on ripening,storage life,and volatile production by d'Anjou cv. pear fruit

d'Anjou cv. pear fruit (Pyrus communis L.) exposed at harvest to 0, 0.42, 4.2, or 42 micromol m(-)(3) 1-methylcyclopropene (1-MCP) for 12 h at 20 degrees C were stored at 1 degrees C for up to 8 months. After storage, half of the fruit was continuously exposed to ethylene (0.45 or 4-18 mmol m(-)(3)) for 7 days at 20 degrees C. All fruit treated with 1-MCP had lower respiration and ethylene production compared to untreated controls. Fruit quality changes were delayed following 1-MCP treatment, as was development of superficial scald and peel yellowing. The duration of 1-MCP-induced responses was dependent on 1-MCP treatment concentration. When 1-MCP-treated fruit began to ripen, softening and production of volatile compounds proceeded similar to that of untreated fruit. Post-storage ethylene exposure did not consistently stimulate ripening of fruit previously treated with 1-MCP. Efficacy of ethylene treatment depended on 1-MCP concentration and storage duration.     

Antioxidants in raspberry: on-line analysis links antioxidant activity to a diversity of individual metabolites

The presence of antioxidant compounds can be considered as a quality parameter for edible fruit. In this paper, we studied the antioxidant compounds in raspberry (Rubus idaeus) fruits by high-performance liquid chromatography (HPLC) coupled to an on-line postcolumn antioxidant detection system. Both developmental and genetic factors were assessed by comparing fruits from a single cultivar of different ripening stages and by comparing ripe fruits of 14 raspberry cultivars, respectively. The HPLC-separated antioxidant compounds were identified using HPLC-photodiode array coupled to mass spectrometry (quadrupole time-of-flight tandem mass spectrometry), using a reference lock mass for determining accurate masses. The dominant antioxidants could be classified as anthocyanins, ellagitannins, and proanthocyanidin-like tannins. During fruit ripening, some anthocyanins were newly produced, while others, like cyanidin-3-glucoside, were already present early in fruit development. The level of tannins, both ellagitannins and proanthocyanidin-like tannins, was reduced strongly during fruit ripening. Among the 14 cultivars, major differences (>20-fold) were observed in the levels of pelagonidin type anthocyanins and some proanthocyanidin type tannins. The content of ellagitannins varied approximately 3-fold. The findings presented here suggest that the content of individual health-promoting compounds varies significantly in raspberry, due to both developmental and genetic factors. This information will assist in the future development and identification of raspberry lines with enhanced health-promoting properties.     

1-methylcyclopropene increases storability and shelf life in climacteric and nonclimacteric plums

The effect of 1-methylcyclopropene (1-MCP) at three different doses (0.25, 0.50, and 0.75 microL L(-1)) on the ripening processes of a climacteric, cv. Santa Rosa, and a suppressed climacteric type, cv. Golden Japan, plum was studied. For both cultivars, positive effects were observed in terms of inhibition of ethylene production and delays of the physical, chemical, and biochemical changes associated with ripening. 1-MCP-treated plums were firmer with lower weight loss, reduced degrees Brix/titratable acidity ratios, and lower color changes during cold storage and subsequent shelf life at 20 degrees C than controls. For most factors, the effectiveness of 1-MCP was dose-dependent in Santa Rosa but dose-independent for Golden Japan.     

Benzylglucosinolate, benzylisothiocyanate, and myrosinase activity in papaya fruit during development and ripening

Papaya is a climacteric fruit that has high amounts of benzylglucosinolates (BG) and benzylisothiocyanates (BITC), but information regarding levels of BG or BITC during fruit development and ripening is limited. Because BG and BITC are compounds of importance from both a nutritional and a crop yield standpoint, the aim of this work was to access data on the distribution and changes of BG and BITC levels during fruit development and ripening. BG and BITC levels were quantified in peel, pulp, and seeds of papaya fruit. Volatile BITC was also verified in the internal cavity of the fruit during ripening. The influence of the ethylene in BG and BITC levels and mirosinase activity was tested by exposing mature green fruits to ethylene and 1-methylcyclopropene (1-MCP). The highest BG levels were detected in seeds, followed by the peel and pulp being decreased in all tissues during fruit development. Similarly, the levels of BITC were much higher in the seeds than the peel and pulp. The levels of BG for control and ethylene-treated fruit were very similar, increasing in the pulp and peel during late ripening but not changing significantly in seeds. On the other hand, fruit exposed to 1-MCP showed a decrease in BG amount in the pulp and accumulation in seed. The treatments did not result in clear differences regarding the amount of BITC in the pulp and peel of the fruit. According to the results, ethylene does not have a clear effect on BITC accumulation in ripening papaya fruit. The fact that BG levels in the pulp did not decrease during ripening, regardless of the treatment employed, and that papaya is consumed mainly as fresh fruit, speaks in favor of this fruit as a good dietary source for glucosinolate and isothiocyanates.     

低温贮藏对猕猴桃果实成熟软化相关基因表达影响

为探究低温对果实采后成熟软化与淀粉降解的影响,以红阳猕猴桃果实为试验材料,研究在低温贮藏期间,猕猴桃果实硬度,可溶性固形物、乙烯、淀粉含量以及淀粉酶相关基因的变化。结果表明,低温贮藏能显著抑制猕猴桃果实采后成熟软化,延缓果实淀粉的降解和可溶性固形物含量的增加,维持贮藏期间果实较高的硬度。低温贮藏抑制乙烯合成关键基因AcACO1和AcACS1的表达,抑制乙烯合成。同时,低温贮藏显著抑制淀粉降解相关基因AcAMY1、AcBAM1/3、AcISA3、AcLDA1和AcDPE1的表达。在低温贮藏后期,淀粉酶相关基因AcAMY1、AcBAM1/3、AcLDA1和AcDPE1的表达与乙烯释放速率有关。综上,低温贮藏延缓猕猴桃采后成熟软化进程与淀粉降解密切相关,可能主要通过抑制乙烯合成,从而影响贮藏后期淀粉降解速率,最终延缓果实软化进程。本研究结果为猕猴桃采后低温贮藏提供了理论依据。