The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.AFRC Research Group in Plant Gene Regulation     

Silver ions inhibit the ethylene-stimulated production of ripening-related mRNAs in tomato

Abstract. Silver ions effectively inhibited both the initiation and the continuation of tomato ( Lyeopersicon esculentum Mill) ripening. Studies of protein synthesis in vivo showed that application of 2 mol m⁻³ silver thiosulphate to mature green fruit prevented the appearance of several novel proteins associated with ripening, including the softening enzyme polygalacturonase. However, total protein synthesis, as judged by the incorporation of [³⁵S] methionine into proteins, continued unabated after silver treatment. Ripening was also arrested when silver was supplied after ripening had begun. The accumulation of several ripening-related mRNAs, including that for polygalacturonase, was studied by translation in vitro and using cDNA clones as hybridization probes. Silver was shown to prevent the appearance of polygalaturonase mRNA when supplied to mature green fruit and to cause a rapid reduction in the concentration of mRNA for polygalacturonase and other ripening-related proteins when supplied after ripening had begun. It is proposed that silver exerts its effects due to interaction with the ethylene perception mechanism. The results suggest that perception of ethylene is vital not only for the initiation of ripening but also for the continued expression of genes required for ripening.     

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.     

Inhibition of expression of tomato-ripening genes at high temperature

Abstract. Ripening tomato fruits incubated at 35°C fail to achieve normal pigmentation, soften little and show a marked decline in ethylene evolution. Labelling studies in vivo indicate that protein synthesis continues throughout incubation at 35°C although the spectrum of labelled proteins is different to that observed at 25°C. Translation of mRNAs in vitro shows traces of several 'heat-shock' mRNAs at 35°C and the loss of several others normally found in fruit ripened at 25°C. Using ripening-related cDNA clones as hybridization probes the expression of 12 ripening-related genes was followed during incubation at 25°C and 35°C. In general, there was a marked decline in the amounts of these mRNAs following incubation of ripening fruit at 35°C. In particular, mRNA homologous to pTOM 6, a cDNA clone coding for polygalacturonase, a major cell wall degrading enzyme, showed a rapid decline following incubation at 35°C and after 72-h at elevated temperature was undetectable. There was no recovery of expression during 120 h at 35°C and the application of exogenous ethylene did not overcome the inhibition of ripening or lead to the renewed accumulation of polygalacturonase mRNA. It is proposed that the failure to soften normally at elevated temperature is due, in part, to the suppression of polygalacturonase mRNA and that the inhibition of other facets of ripening at 35°C is due to the inhibition or reduced expression of other, as yet unidentified, ripening-related genes.     

Molecular biology of fruit ripening and its manipulation with antisense genes

Considerable progress in tomato molecular biology has been made over the past five years. At least 19 different mRNAs which increase in amount during tomato fruit ripening have been cloned and genes for enzymes involved in cell wall degradation (polygalacturonase and pectinesterase) and ethylene synthesis (ACC synthase) have been identified by conventional procedures. Transgenic plants have been used to identify regions of DNA flanking fruit-specific, ripening-related and ethylene-regulated genes and trans-acting factors which bind to these promoters have also been identified.Antisense genes expressed in transgenic plants have proved to be highly effective for inhibiting the specific expression of ripening-related genes. These experiments have changed our understanding of how softening occurs in tomato fruit. Antisense techniques have also been used to identify genes encoding enzymes for carotenoid biosynthesis (phytoene synthase) and ethylene biosynthesis (the ethylene-forming enzyme). The altered characteristics of fruit transformed with specific antisense genes, such as retarded ripening and resistance to splitting, may prove to be of value to fruit growers, processors and ultimately the consumer.     

Identification of an ETR1-homologue from mango fruit expressing during fruit ripening and wounding

We have isolated a mango (Mangifera indica L.) cDNA homologue of the ethylene receptor gene ETR-1, referred to as METR1, which codes for a polypeptide of 802 amino acids with a predicted molecular weight of 89 kDa. The amino acid sequence is highly homologous (over 80 percnt;) to ETRs from other fruits. Genomic Southern blot analysis indicates that two or more ETR homologues exist in mango. RNA blot analysis revealed that the level of METR1 mRNA in the mesocarp increased during fruit ripening. In addition, it was found that the METR1 mRNA increases transiently during wounding of the tissue. This is the first report of an ETR homologue showing an induction during fruit ripening and wounding.     

果实多聚半乳糖醛酸酶分子生物学研究进展

多聚半乳糖醛酸酶（PG酶）是一种在植物细胞壁降解中起重要的作用的酶。作者介绍了PG酶在果实成熟软化中的作用。概述了PG酶基因及其表达调控,评述了乙烯对PG酶合成的影响。     

Reevaluation of the changes in polygalacturonases in tomatoes during ripening

A procedure was developed for the differential extraction of polygalacturonases (PG) I and II from tomatoes (Lycopersicon esculentum Mill.). Extraction of pericarp tissue from ripe fruit at conventional conditions of 1.0 M NaCl and pH 6.0 yielded nearly equal amounts of the two enzymes. However, most of the PG activity could be extracted also with water at pH 1.6, and the water extract contained only PG II. Subsequent extraction of the pellet with 1.0 M NaCl at pH 6.0 and 10.0 yielded some PG I and high levels of PG converter, the protein in tomatoes that reacts with PG II to form PG I. Application of this procedure to tomatoes at different stages of ripening showed that PG II appeared as ripening began and then increased during ripening. Much lower levels of PG I than of PG II were extracted at all stages of ripeness. The PG converter was present in unripe fruit and increased during ripening. The results demonstrate that PG I is formed when PG II and PG converter are solubilized simultaneously and that PG II is the only endogenous PG in tomatoes.Abbreviation PG polygalacturonase     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

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     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

Foliar application of aminoethoxyvinylglycine (AVG) delays fruit ripening and reduces pre-harvest fruit drop and ethylene production of bagged “Kogetsu” apples

Covering apple fruits with double layer waterproof bags to enhance fruit quality and evenness of blush colour is typical on many cultivars in Korea and Japan. Aminoethoxyvinylglycine (AVG) applied to unbagged apple fruits at 3–4 weeks before commercial harvest reduces ethylene production in the fruit, delays fruit ripening and reduces pre-harvest fruit drop. Spray application of AVG to trees of bagged apples should have no effect on apple ripening as there is␣no direct contact with the fruit and the translocation of AVG in apple trees is regarded as negligible. However, preliminary experiments suggested that AVG applied to trees of bagged apples reduced pre-harvest fruit drop in “Kotgetsu” apples. This study investigated the effect of spray treatments of 125 ppm of AVG on fruit drop, fruit ripening (firmness, starch conversion and soluble solids) and ethylene production to whole trees with bagged or unbagged “Kogetsu” fruit, as well as sprays of only the bagged or unbagged fruit on trees on two orchards. AVG applied to whole trees with unbagged apples reduced fruit drop from an average of 58.9% to 10.4%, delayed starch conversion and decreased ethylene production. AVG applied to whole trees with bagged fruit was equally effective in reducing pre-harvest drop, delaying fruit ripening and reducing ethylene production. Application of AVG to unbagged fruit only was nearly as effective as application to whole trees with unbagged fruit but application to bagged fruit only had no effect on fruit ripening or ethylene production. Application of AVG to bagged fruit only did reduce fruit drop to an average of 42.5% but this was not as effective as spraying unbagged fruit only or whole trees with bagged fruit. Possible mechanisms for this effect are discussed.     

Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display

Fruit ripening is a complex developmental process that involves specific changes in gene expression and cellular metabolism. In climateric fruits these events are coordinated by the gaseous hormone ethylene, which is synthesized autocatalytically in the early stages of ripening. Nonclimacteric fruits do not synthesize or respond to ethylene in this manner, yet undergo many of the same physiological and biochemical changes associated with the production of a ripe fruit. To gain insight into the molecular determinants associated with nonclimacteric fruit ripening, we examined mRNA populations in ripening strawberry fruit using polymerase chain reaction (PCR) differential display. Five mRNAs with ripening-enhanced expression were identified using this approach. Three of the mRNAs appear to be fruit-specific, with little or no expression detected in vegetative tissues. Sequence analysis of cDNA clones revealed positive identities for three of the five mRNAs based on homology to known proteins. These results indicate that the differential display technique can be a useful tool to study fruit ripening and other developmental processes in plants at the RNA level.     

Indole-3-acetic acid, ethylene, and abscisic acid metabolism in developing muskmelon (Cucumis melo L.) fruit

Hormonal metabolism associated with fruit development in muskmelon was investigated by measuring IAA, ABA, and ACC levels in several tissues at various stages of development. In addition, levels of conjugated IAA and ABA were determined in the same tissues. Ethylene production, which is believed to signal the ripening and senescence of mature fruit, was also measured. Ethylene production was highest in the outer tissue near the rind and gradually declined during maturation, except for a dramatic increase in all fruit tissues at the climacteric. In contrast to ethylene production, ACC levels increased during maturation and remained equal throughout the fruit until the climacteric, when levels in the outer tissues increased nearly 5-fold over levels in the inner tissues. The consistent presence of ACC indicates that ACC oxidase rather than the availability of ACC regulates ethylene production in developing fruits. ABA and ABA esters generally declined during maturation, however an increase in ABA esters associated with the outer mesocarp tissue was observed in fully mature, climacteric fruit. IAA and IAA conjugates were only found in the outer tissue near the rind, and their levels remained low until the fruit was fully mature and entering the climacteric. At that time, increased levels of conjugates were detected. The late burst of hormonal metabolism in the outer mesocarp tissue appeared to signal its degeneration and the deterioration that typically occurs in ripening fruit. The tissue-specific conjugation of IAA and ABA, in addition to the production of climacteric ethylene, may represent part of the signaling mechanism initiating ripening and eventual deterioration of tissues in muskmelon fruits.Abbreviations ABA abscisic acid - ACC 1-aminocylopropane-1-carboxylic acid - DAP days after pollination - IAA indole-3-acetic acid     

Reduced ethylene synthesis by transgenic tomatoes expressing S-adenosylmethionine hydrolase

We have utilized a gene from bacteriophage T3 that encodes the enzyme S-adenosylmethionine hydrolase (SAMase) to generate transgenic tomato plants that produce fruit with a reduced capacity to synthesize ethylene. S-adenosylmethionine (SAM) is the metabolic precursor of 1-aminocyclopropane-1-carboxylic acid, the proximal precursor to ethylene. SAMase catalyzes the conversion of SAM to methylthioadenosine and homoserine. To restrict the presence of SAMase to ripening fruit, the promoter from the tomato E8 gene was used to regulate SAMase gene expression. Transgenic tomato plants containing the 1.1 kb E8 promoter bore fruit that expressed SAMase during the breaker and orange stage of fruit ripening and stopped expression after the fruit fully ripened. Plants containing the 2.3 kb E8 promoter expressed SAMase at higher levels during the post-breaker phases of fruit ripening and had a substantially reduced capacity to synthesize ethylene.     

番茄果实中乙烯与多聚半乳糖醛酸酶的关系

乙烯与多聚半乳糖醛酸酶(PG)都是果实成熟过程中关键的调节因子.一方面,在有乙烯合成缺陷的转反义ACS番茄和乙烯感受缺陷的Nr突变体番茄果实中PG基因表达量都明显下降,PG酶活性明显降低;用外源乙烯(100 μL/L)处理绿熟期番茄果实使PG基因的表达明显增强,而1-甲基环丙烯(1-MCP,1 μL/L)处理转色期番茄果实明显抑制PG基因表达.另一方面,转反义PG基因番茄果实乙烯释放量在授粉后低于其野生型,番茄乙烯受体基因LeETR4和乙烯反应因子LeERF2基因表达量比野生种低.PG降解果胶的产物D-GA(100 mg/L)促进未熟期番茄果实中的乙烯生成和LeETR4、LeERF2基因的表达.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid