Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway

Purpose of work  

Melons have short shelf-lives due to fruit ripening caused by ethylene production. The 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene is essential for ethylene biosynthesis. As fruit ripening in other fruit crops can be deterred by down-regulation of ACC oxidase expression, we have carried out similar work to improve fruit quality and shelf-life of the melon Cucumis melo.     

Ethylene regulation of fruit ripening: Molecular aspects

Progress in ethylene regulating fruit ripening concerning itsperception and signal transduction and expression of ACC synthaseand ACC oxidase genes is reviewed. ACC synthase and ACC oxidasehave been characterized and their genes cloned from various fruittissues. Both ACC synthase and ACC oxidase are encoded bymultigene families, and their activities are associated withfruit ripening. In climacteric fruit, the transition toautocatalytic ethylene production appears to be due to a seriesof events in which ACC sythase and ACC oxidase genes have beenexpressed developmentally. Differential expression of ACCsynthase and ACC oxidase gene family members is probably involvedin such a transition that ultimately controls the onset of fruitripening.In comparison to ACC synthase and ACC oxidase, less is knownabout ethylene perception and signal transduction because of thedifficulties in isolating and purifying ethylene receptors orethylene-binding proteins using biochemical methods. However, theidentification of the Nr tomato ripening mutant as anethylene receptor, the applications of new potent anti-ethylenecompounds and the generation of transgenic fruits with reducedethylene production have provided evidence that ethylenereceptors regulate a defined set of genes which are expressedduring fruit ripening. The properties and functions of ethylenereceptors, such as ETR1, are being elucidated.Application of molecular genetics, in combination withbiochemical approaches, will enable us to better understand theindividual steps leading from ethylene perception and signaltransduction and expression of ACC synthase and ACC oxidase genefamily member to the physiological responses.     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Expression of ACC synthase is enhanced earlier than that of ACC oxidase during fruit ripening of mume (Prunus mume)

Mume (Japanese apricot: Prunus mume Sieb. et Zucc.) is a climacteric fruit that produces large amounts of ethylene as it ripens. Ripening is accompanied by marked increases in the activities of two ethylene-biosynthetic enzymes, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. To study the molecular aspects of ripening of mume, we isolated cDNA clones for proteins that we considered likely to be involved in the biosynthesis and perception of ethylene during ripening, namely, ACC synthase, ACC oxidase and the ethylene receptor. Northern blotting analysis revealed the markedly increased expression of ACC synthase prior to that of ACC oxidase and the increase in ethylene production during ripening. Overall, the levels of the mRNAs for the genes corresponded closely to the levels of activity of the ethylene-biosynthetic enzymes. Exposure of mature green mume fruit to ethylene for 12 h induced strong expression of ACC synthase, as well as of ACC oxidase. Wounding of the pericarp of mume fruit induced the expression of ACC synthase but not of ACC oxidase. The rate of ethylene production increased only slightly after wounding. These results suggest that expression of the genes for ACC synthase and ACC oxidase must be activated sequentially for maximum production of ethylene during ripening of mume fruit and that several mechanisms regulate the expression of ethylene-biosynthetic genes during ripening.     

The never ripe mutation blocks ethylene perception in tomato.

Seedlings of tomato fruit ripening mutants were screened for their ability to respond to ethylene. Ethylene induced the triple response in etiolated hypocotyls of all tomato ripening mutants tested except for one, Never ripe (Nr). Our results indicated that the lack of ripening in this mutant is caused by ethylene insensitivity. Segregation analysis indicated that Nr-associated ethylene insensitivity is a single codominant trait and is pleiotropic, blocking senescence and abscission of flowers and the epinastic response of petioles. In normal tomato flowers, petal abscission and senescence occur 4 to 5 days after the flower opens and precede fruit expansion. If fertilization does not occur, pedicel abscission occurs 5 to 8 days after petal senescence. If unfertilized, Nr flowers remained attached to the plant indefinitely, and petals remained viable and turgid more than four times longer than their normal counterparts. Fruit development in Nr plants was not preceded by petal senescence; petals and anthers remained attached until they were physically displaced by the expanding ovary. Analysis of engineered 1-aminocyclopropane-1-carboxylate (ACC) synthase-overexpressing plants indicated that they are phenotypic opposites of Nr plants. Constitutive expression of ACC synthase in tomato plants resulted in high rates of ethylene production by many tissues of the plant and induced petiole epinasty and premature senescence and abscission of flowers, usually before anthesis. There were no obvious effects on senescence in leaves of ACC synthase overexpressers, suggesting that although ethylene may be important, it is not sufficient to cause tomato leaf senescence; other signals are clearly involved.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

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.     

果实成熟过程相关调控基因研究进展

果实成熟过程中,多聚半乳糖醛酸酶（ＰＧ）参与果胶的分解,从而在果实软化中起作用,新近发现,果实软化过程中,协同展蛋白具有一定的作用：ＡＣＣ合成酶（ＡＣＳ）、ＡＣＣ氧化酶（ＡＣＯ）和ＡＣＣ脱氨酶与乙烯合成直接有关,ＡＣＳ是乙烯形成的关键酶,由多基因家族编码,各个基因协同表达,每一基因都有自己的转录特性,新近不断发现果实中ＡＣＳ基因家族中的新成员;ＡＣＯ是一种与膜结合的酶,这种酶具有结构上的立体专一性     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening

Ethylene is required for climacteric fruit ripening. Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development. LeHB-1, a previously uncharacterized tomato homeobox protein, was shown by gel retardation assay to interact with the promoter of LeACO1 , an ACC oxidase gene expressed during ripening. Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening. Conversely, ectopic overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on injected plants triggered altered floral organ morphology, including production of multiple flowers within one sepal whorl, fusion of sepals and petals, and conversion of sepals into carpel-like structures that grew into fruits and ripened. Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.     

Manipulation of ethylene biosynthesis

The plant hormone ethylene is involved in many plant processes ranging from seed germination to leaf and flower senescence and fruit ripening. Ethylene is synthesized from methionine, via S-adenosyl-L-methionine (SAM) and 1-amino-cyclopropane-1-carboxylic acid (ACC). The key ethylene biosynthetic enzymes are ACC synthase (ACS) and ACC oxidase (ACO). Manipulation of ethylene biosynthesis by chemicals and gene technology is discussed. Biotechnological modification of ethylene synthesis is a promising method to prevent spoilage of agricultural and horticultural products.     

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.     

The Role of Ethylene in the Shedding of Red Raspberry Fruit

The production of ethylene by red raspberry (Rubus idaeus L.cv. Glen Clova) fruit increased climacterically during development.The concentration of ethylene within green fruit was low butincreased substantially as fruit abscission and ripening commenced.The receptacle contained higher concentrations than the drupeletsat all stages measured. In the mature ripening fruit the ethyleneconcentrations were found to be physiologically significant,and would accelerate the abscission of large green non-abscisingfruit if supplied as a fumigant. The addition of ethylene toripe fruit did not accelerate abscission, probably because saturatinglevels occurred naturally within these fruit. Reduction of ethylenesynthesis rates using the inhibitor of ethylene production aminoethoxyvinylglycine(AVG) reduced the rate of abscission zone weakening which occursin detached large green fruit. The rate of ethylene productionwas found to be dependent on the supply of the precursor l-aminocyclopropane-l-carboxylicacid (ACC). This only accumulated to any extent in those ripefruit with high rates of ethylene production. Rubus idaeus, raspberry, abscission, fruit ripening, ethylene, aminocyclopropane-l-carboxylic acid