A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): Cloning,characterization and relation to postharvest chilling stress resistance

A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.     

采前低温诱发菠萝果实黑心病

采前低温可诱发菠萝果实黑心病,发病率变化范围为60%~100%。低于21℃的采前低温影响是诱发菠萝黑心病的重要因子之一。在华南菠萝生产区,不同品种对黑心病敏感性的表现不同。因此,培育和栽培抗黑心病的菠萝新品种,将是一个控制黑心病腐烂的重要措施。     

采后菠萝黑心病诱导因素的研究

本文叙述了外源赤霉素ＧＡ３、低温条件对采后菠萝果实黑心病发展的影响．说明外源赤霉素ＧＡ３和低温条件都是诱导菠萝黑心病的重要因子。菠萝黑心病是一种生理失调症。实验中,未发现化学杀菌剂对此种病症显现出防治效果,而采后贮前４０℃高温处理２４小时．则可以减少和延迟此病的发生,因此．后者是一种可以应用于商品菠萝果实黑心病防治的好方法。     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA(3) treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.     

低温与GA_3诱导菠萝黑心病的生理机制

菠萝黑心病是PPO催化氧化酚类物质形成褐色产物所致。低温或GA_3处理提高了PPO活性及其底物——儿茶酚、绿原酸和咖啡酸的含量,也导致了PAL活性增加;低温还使乙烯释放率增大。这些变化均有利于黑心病的发生和发展。     

Inhibitory effects of fruit juices on cytochrome P450 2C9 activity in vitro

There is limited information on the effect of fruits on human cytochrome P450 (CYP) 2C9 activity. The objective of this study was to determine the effect of fruit juice on CYP2C9-mediated drug metabolism. Nine citrus fruits and eight tropical fruits were chosen. We investigated effects of the fruits on diclofenac 4'-hydroxylation and tolbutamide hydroxylation by human liver microsomes. Among the fruits, pineapple juice showed potent inhibition of CYP2C9 activity. The addition of 25 microl (5.0% v/v) of pineapple juice resulted in almost complete inhibition. Next we examined the inhibitory effect of bromelain, a cysteine protease in pineapple. Bromelain also strongly inhibited CYP2C9 activity. In addition, E-64, a cysteine protease inhibitor, almost entirely blocked inhibition by pineapple juice and bromelain. Thus we found that pineapple juice was a potent inhibitor of CYP2C9, and that the inhibitory effect might be due to the bromelain contained in pineapple.     

菠萝中蛋白酶及其他组份在品种间的差异分析

以湛江地区6个主要菠萝品种巴厘、卡因、台农17号、台农16号、粤脆、香水为对象,研究菠萝的组分在品种间的差异及其与菠萝蛋白酶的产率和酶活力之间的相关性,结果显示,不同的菠萝品种间,各组分的含量以及蛋白酶的得率及酶活有显著差异。菠萝果皮和果肉的不同生长部位的蛋白含量、酶活力及酶得率有显著差异。酶得率与蛋白及纤维素含量呈显著正相关;酶活力与糖酸比、得汁率、纤维素含量及Zn含量呈显著负相关,蛋白含量、纤维素含量、糖酸比、得汁率是衡量菠萝产酶品质的重要指标。     

Resonance assignments and secondary structure of a phytocystatin from Ananas comosus

A cDNA encoding a cysteine protease inhibitor, cystatin was cloned from pineapple (Ananas comosus L.) stem. This clone was constructed into an expression vector and expressed in E. coli and purified to homogeneous. The recombinant pineapple cystatins (AcCYS) showed effectively inhibitory activity toward cysteine proteases including papain, bromelain, and cathepsin B. In order to unravel its inhibitory action from structural point of view, multidimensional heteronuclear NMR techniques were used to characterize the structure of AcCYS. The full ¹H, ¹⁵N, and ¹³C resonance assignments of AcCYS were determined. The secondary structure of AcCYS was identified by using the assigned chemical shift of ¹Hα, ¹³Cα, ¹³Cβ, and ¹³CO through the consensus chemical shift index (CSI). The results of CSI analysis suggest 5 β–strands (residues 45–47, 84–91, 94–104, 106–117, and 123–130) and one α–helix (residues 55–73).     

低温打蜡对贮藏菠萝黑心病控制的作用

研究了常温、低温、低温加打蜡 3种贮藏条件下菠萝果实硬度、可溶性固形物 (TSS)、可溶性糖醛酸、多聚半乳糖醛酸酶 (PG)和多酚氧化酶 (PPO)等因素的变化 ,及它们对黑心病发生及发展情况的影响。结果表明 ,低温加打蜡可以延缓果肉硬度、可溶性固形物和可溶性糖醛酸含量的下降 ,降低 PG和 PPO的活性 ,降低黑心病的发病率 ,较好的保持果实品质     

瓜尔豆不同品种抗霜冻性与酯酶同工酶的关系分析

对瓜尔豆10个品种酯酶（EST）同工酶分析及抗霜冻性测试表明,酯酶同工酶带数及酶活性在品种间有明显差异．抗霜冻性强的品种同工酶带数多、活性高,其中品种G1抗霜冻性最强,且具有1条特异EST同工酶带。     

Polyamine Changes and Chilling Injury in Cold-stored Loquat Fruits

Free polyamine levels (spermine (SPM), spermidine (SPD), and putrescine (PUT)) were determined using thin-layer chromatography and fluorometric method in loquat (Eriobotrya japonica Lindl. cv. Dahongpao) fruits stored at 1℃ and 12℃ and in postharvest SPM treated fruits stored at 1℃ respectively to investigate the relationship between changes in polyamines and chilling injury. In the loquat fruits stored at 1℃, SPM level decreased gradually in the first two weeks, then increased sharply and reached a peak value after three weeks, thereafter it decreased rapidly. SPD level decreased steadily during the first three weeks and increased significantly afterwards. PUT level evolved in a similar way as the SPM level did except that it increased slowly in the first two weeks. The fruit showed symptom of chilling injury manifested as flesh leatheriness after three weeks. However, no significant increase and decrease of these three polyamines was detected during storage at the nonchilling temperature (12℃). The SPM-treated fruits maintained high levels of SPM and SPD and remained low level of PUT during storage at 1℃, and no symptom of chilling injury was observed. These results suggested that the increase in SPM level in response to chilling exposure might serve as a defense mechanism against chilling injury while the accumulation of PUT could be a cause of the stress-induced injury and the increase in SPD level could be a consequence of this kind of stress.     

冷胁迫诱导、咖啡因抑制的水稻根新基因片段的分离及表达分析

利用差异显示（Differential display）及H.Y-Yellow琼脂糖凝胶分离等方法,从冷胁迫和甘露醇共同处理的冷敏感水稻（Wase toitsu）根的总RNA中,分离出冷胁迫诱导的cDNA片段,经过测序和同源比较,发现它是一尚未报道的新基因片段。这一基因的表达对温度具有敏感的反应特性,4℃时表达水平明显增加,在正常温度（25℃）下,其表达水平又可快速降低。当幼苗根用0.5mol/L甘露醇预处理2h后,转入冷胁迫,其低温诱导的表达水平增加更为明显。在甘露醇预处理之前加入咖啡因处理根1h,其表达水平明显降低,几乎与正常温度下的对照相同。据此推测,这一基因可能参与了冷胁迫或水分胁迫诱导的耐冷过程中咖啡因敏感信号的传导。      

采后荔枝果实冷害过程中多胺含量的变化

以桂味荔枝果实为材料,研究冷害及外源亚精胺(Spd)处理对果实膜透性和内源多胺含量变化的影响。结果表明,在0℃下贮藏时果实发生冷害过程中,荔枝果皮中腐胺(Put)、Spd、精胺(Spm)含量在14d后明显增加,膜透性快速增大,21d时果皮出现明显的冷害褐变,Put进一步积累,而Spm含量下降,Spd保持较高水平。非冷害温度(3℃)下贮藏时,果皮多胺含量变化相对较小。0℃下果肉的多胺含量和变化幅度低于3℃果实,并延迟7d衰老。外源Spd处理明显提高果实内源多胺含量的同时,延缓了果皮相对膜透性增加,减轻了冷害。这表明果皮中Put的积累可能是荔枝果实冷害的结果,冷藏初期Spm含量的上升可能是果实对冷害的防卫反应。