果实成熟的基因调控

果实的成熟过程是由一系列生理生化变化过程组成,这些变化过程受到外界环境条件、植物激素和基因的调控。随着近年来有关果实成熟衰老的基因的分离、定性及反义基因技术在控制果实成熟上的成功应用,对揭示果实成熟衰老的分子机理起到了重要作用。本文就近年来果实成熟基因调控研究进展作一简要评述 。     

果实成熟的基因调控

果实的成熟过程是由一系列生理生化学变化过程组成,这些变化过程受到外界环境条件、植激素和基因的调控。随着近年来有关果实成熟衰老的基因的分离,定性及反义基因技术在控制果实成熟上的成功应用,对揭示果实成熟衰老的分子机理起到了重要作用。本文就近来果实成熟基因调控研究进展作一简要评述。     

果实成熟衰老过程中蛋白质组学研究进展

蛋白质组学已开始应用于果实成熟衰老研究,以明确蛋白差异表达与成熟衰老的关系和深入揭示果实成熟衰老过程的分子机制。本文综述了蛋白质组学在果实成熟衰老研究中的重要性、果实样品蛋白的提取制备方法,重点介绍了蛋白质组学在果实成熟衰老机制、果实抗病性机制、冷害机制以及采后处理对果实成熟调控研究中的应用,分析了蛋白质组学在果实成熟衰老研究中存在的不足,提出了今后研究的方向。     

乙烯受体在果实成熟及花衰老中的研究进展

乙烯受体是乙烯信号转导网络的第一个转导元件,通过调控受体基因的表达,可以调节植物对乙烯的敏感性,以调控果实的成熟及花衰老进程的响应。随着人们对乙烯受体研究的深入,乙烯受体突变体及受体抑制剂在采后果实和切花保鲜上的应用已受到广泛关注。就近年来关于乙烯受体的相关研究进展进行综述,重点介绍了乙烯受体的分子调控机制及乙烯受体在果实成熟和花衰老中的应用,并对今后乙烯受体的研究方向作了展望,以期为进一步研究提供参考。     

果实的成熟及调控

果实的成熟及调控朱广廉（北京大学生物学系１００８７１）果实（特别是肉质果实）的成熟通常是指其可食部分达到良好的食用品质,可以认为果实的成熟是果实发育的终点和走向衰老的前奏。成熟后的肉质果实易变软、腐烂、难于贮藏。全世界每年因保存不当而变质、腐烂所造成...     

番茄果实成熟过程中钙调素含量变化及其与乙烯生成的关系

应用酶联免疫吸附法(ELISA)测定番茄(Lycopersicon esculentum Mill大红品种)果实成熟过程中钙调素(CaM)含量的变化。果实开始成熟(发白期),CaM含量随着呼吸跃变上升,成熟时(粉红期)达到最大,过熟衰老时则下降。果实内部乙烯浓度、ACC含量及其合成酶活性也随跃变而增加,随过熟衰老而降低。GaM含量在果实不同部位中的分布有明显差异,跃变上升期以子房腔组织含量最高,并由中心向外逐渐降低,外周果皮含量最低。此时用外源乙烯催熟处理促进各部位CaM增加。成熟衰老时子房腔组织首先衰老,CaM含量大为降低,但在中柱和果皮中却高于跃变上升期。外源乙烯促进衰老使CaM下降。Ca~(2+)促进番茄圆片CaM含量增高和乙烯产生,CaM抑制剂CPZ,TFP在降低CaM含量的同时也抑制乙烯的产生。     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

采后黄花梨(Pyrus pyrifolia Nakai.)果实中丙二烯氧合酶的生理功能

以不同成熟时期黄花梨果实为材料 ,研究果实采后成熟衰老进程中丙二烯氧合酶 (AOS)与几个成熟衰老相关因子的关系 ,探讨AOS的生理功能。结果表明 :2 0℃下不同成熟时期果实成熟衰老进程中的AOS活性变化均为峰形曲线 ,活性峰值出现在采后 10～ 12d ,先于乙烯跃变峰 2～ 4d ;果实成熟衰老各种相关因子的变化峰值出现的先后顺序依次是 :脂氧合酶(LOX)、自由基 (O- ·2 )、AOS、ACC (1 氨基环丙烷 1 羧酸 )合成酶、ACC、ACC氧化酶 ,最后为乙烯跃变峰的出现。 1℃下贮藏果实的AOS活性、乙烯合成和其他成熟衰老相关酶活性均受到强烈抑制 ,ACC和O- ·2 含量也较低 ,果实衰老进程被显著延缓。推测AOS是乙烯合成的上游调控因子之一。     

乙烯对番茄成熟过程中果皮细胞核超微结构的影响

应用冰冻蚀刻和透射电镜观察了番茄成熟过程中果皮细胞核染色质的变化。成熟过程开始启动时(发白期),异染色质不断减少、分散,直至转色成熟。果实成熟衰老时细胞核形态畸变,染色质结构瓦解。经外源乙烯处理后果实成熟加速,异染色质减少,核孔数量增加,NBD可消除乙烯的作用。     

大豆microRNA基因GmMIR160A负调控植物叶片衰老进程

叶片衰老是受内外多种因子影响的遗传发育进程。生长素、细胞分裂素和乙烯等多种植物激素是调控叶片衰老的重要内部因子,它们通过长或短距离运输形成叶片组织内特定的区域分布和浓度梯度,从而直接或间接参与植物叶片衰老过程。分子遗传学表明,细胞分裂素和乙烯分别是叶片衰老的抑制子和正调节子,而生长素如何参与叶片衰老的分子机制目前还不清晰。植物体内成熟小分子RNA由小RNA基因转录并通过特定酶加工形成的21~23bp的双链RNA分子。这些小分子通过不完全配对方式抑制其靶基因转录和/或表达,参与植物生长发育多个过程,然而这类小RNA分子如何调控植物叶片衰老发育过程目前则还鲜有报告。大豆是重要的油料作物,具有典型的单次结实性衰老特征。研究大豆叶片衰老具有重要的科学意义和深远的应用价值。该文采用实时荧光定量PCR(qPCR)技术分析大豆(Glycine max)micro RNA基因GmMIR160A的表达模式,发现大豆第一复叶中GmMIR160A表达受外源生长素和黑暗处理的诱导,暗示该基因是生长素快速响应的叶片衰老相关基因。为进一步探究GmMIR160A在大豆叶片发育中的功能,构建了肾上腺皮质激素(Glucocorticoid,GR)类似物地塞米松(Dexamethasone,DEX)诱导表达GmMIR160A双元表达载体并通过农杆菌介导的子叶节方法转化野生型大豆。通过抗性筛选和基因组PCR鉴定并结合表型分析,共获得了4株诱导表达的稳定遗传转基因植株(株系OX-3、OX-5、OX-7和OX-8)。GmMIR160A过表达植株根、茎、叶、花和果实在形态学上与野生型相比无显著差异,但叶片的叶绿素含量增加、最大光量子效率(Fv/Fm)增强。进一步分子分析发现,转基因大豆叶片中GmARFs和衰老标记基因(GmCYSP1)表达明显下降,表明大豆Gma-miR160通过抑制靶基因GmARFs的表达来负调控植物叶片的衰老进程。该文揭示了生长素通过小分子RNA调控叶片发育一条新途径,为研究植物激素调控植物叶片衰老提供了新的思路。     

Effects of Abscisic Acid and Benzyladenine on Fruits of Normal and rin Mutant Tomatoes

Since ethylene application did not induce ripening in detached fruits of the nonripening mutant rin we initiated studies to determine possible involvement of other hormones. We proposed that the lack of ripening in mutant rin tomato fruit may result from a lack of abscisic acid or from excessive endogenous levels of cytokiuin. Application of abscisic acid (3 x 10(-5)m and 10(-3)m) to detached fruits of a normal strain (Lycopersicon esculentum Mill. cv. ;Rutgers') reduced the time to initiate ripening by about 50%. This acceleration of the onset of ripening appeared not to be due to an increased rate of ethylene production. Abscisic acid did not alter respiration or ethylene production or induce ripening in rin fruit. Ripening in Rutgers fruit was not influenced by treatment with 6-benzyladenine (4.44 x 10(-6)m, 4.44 x 10(-5)m or 1.8 x 10(-4)m). Fruits of the mutant rin showed no response to exogenous BA. However, senescence rates of leaf disks of both Rutgers and rin were significantly inhibited by as little as 10(-7)m exogenous benzyladenine. The results are discussed in relation to previous studies of the physiology of rin fruits and it is concluded that endogenous levels of ABA and cytokinins do not account for the lack of ripening in rin fruit.     

Pectin esterase gene family in strawberry fruit: study of FaPE1, a ripening-specific isoform

Pectin esterases (PE, EC 3.1.1.11) catalyse the demethylation of pectin. As a result of its activity, structural interactions among cell wall components during cell wall turnover and loosening are affected. In plants, PEs are typically encoded by a gene family. This family has been studied in strawberry (Fragaria x ananassa Duch.) in order to investigate the role of distinct PE genes during fruit ripening and senescence. By a combination of a PCR-based library screening and RT-PCR four different strawberry PE cDNAs, termed FaPE1 to FaPE4, have been isolated. Differential expression of each FaPE gene in various organs and during fruit development was revealed by northern blot. FaPE1 is specifically expressed in fruit, showing an increasing expression during the ripening process up to a maximum in the turning stage. Concerning hormone regulation, auxin treatment increased FaPE1 mRNA levels in green fruit, whereas exogenous ethylene decreased FaPE1 mRNA levels in ripe and senescing fruits. It is proposed that this repression of FaPE1 expression could be involved in textural changes occurring during fruit senescence.     

Evidences for Chlorogenic Acid — A Major Endogenous Polyphenol Involved in Regulation of Ripening and Senescence of Apple Fruit

To learn how the endogenous polyphenols may play a role in fruit ripening and senescence, apple pulp discs were used as a model to study the influences of chlorogenic acid (CHA, a major polyphenol in apple pulp) on fruit ripening and senescence. Apple (‘Golden Delicious’) pulp discs prepared from pre-climacteric fruit were treated with 50 mg L^-1 CHA and incubated in flasks with 10 mM MES buffer (pH 6.0, 11% sorbitol). Compared to the control samples, treatment with CHA significantly reduced ethylene production and respiration rate, and enhanced levels of firmness and soluble solids content of the pulp discs during incubation at 25°C. These results suggested that CHA could retard senescence of the apple pulp discs. Proteomics analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry (MALDI-TOF/TOF) revealed that the expressions of several key proteins correlated to fruit ripening and senescence were affected by the treatment with CHA. Further study showed that treating the pulp discs with CHA remarkably reduced levels of lipoxygenase, β-galactosidase, NADP-malic enzyme, and enzymatic activities of lipoxygenase and UDP-glucose pyrophosphorylase, all of which are known as promoters of fruit ripening and senescence. These results could provide new insights into the functions of endogenous phenolic compounds in fruit ripening and senescence.     

Molecular and genetic regulation of fruit ripening

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During this maturation process, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organisms. In addition, advanced or over-ripening and senescence, especially through tissue softening and eventual decay, render fruit susceptible to invasion by opportunistic pathogens. While ripening and senescence are often used interchangeably, the specific metabolic activities of each would suggest that ripening is a distinct process of fleshy fruits that precedes and may predispose the fruit to subsequent senescence.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

Cloning of 9-cis-epoxycarotenoid dioxygenase (NCED) gene and the role of ABA on fruit ripening

In order to understand more details about the role of abscisic acid (ABA) in fruit ripening and senescence, six 740 bp cDNAs (LeNCED1, LeNCED2, PpNCED1, VVNCED1, DKNCED1 and CMNCED1) which encode 9-cis-epoxycarotenoid dioxygenase (NCED) as a key enzyme in ABA biosynthesis, were cloned from fruits of tomato, peach, grape, persimmon and melon using an RT-PCR approach. A Blast homology search revealed a similarity of amino acid 85.76% between the NCEDs. A relationship between ABA and ethylene during ripening was also investigated. At the mature green stage, exogenous ABA treatment increased ABA content in flesh, and promoting ethylene synthesis and fruit ripening, while treatment with nordihydroguaiaretic acid (NDGA), inhibited them, delayed fruit ripening and softening. However, ABA inhibited the ethylene synthesis obviously while NDGA promoted them when treated the immature fruit with these chemicals. At the breaker, NDGA treatment cannot block ABA accumulation and ethylene synthesis. Based on the results obtained in this study, it was concluded that ABA plays different role in ethylene synthesis system in different stages of tomato fruit ripening.Key words: tomato, NCED gene, ABA, ethylene, fruit ripening, peach, grape, persimmon, melon     

Calcium in plant senescence and fruit ripening

Abstract. Calcium has long been associated with regulation of the ripening process of fruit and post-harvest storage life. Specifically, maintenance of relatively high calcium concentrations in fruit tissue results in a slower rate of ripening, as seen in lower respiration rates, reduced ethylene production, and slower softening of fruit flesh. There are also some specific fruit disorders such as bitter pit, which can be prevented if sufficient calcium is present. Senescence of other plant tissues such as leaves and flowers has also been shown to be retarded by the application of calcium.
Work leading to the above information is reviewed and discussed in the context of what is currently known of cellular regulation of calcium in plants. The major sites for the action of calcium in senescence and ripening are suggested to be in membrane structure and function, and in cell wall structure. Although high external concentrations of calcium are an advantage in reducing the rate of senescence or ripening, it is emphasized that normal cell function requires the maintenance of low concentrations of free calcium in the cell cytosol. It is suggested that one possible feature of senescence is a breakdown in such cellular regulation.