cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Characterization of tomato endo-β-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection

The tomato (Lycopersicon esculentum Mill.) endo--1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.This revised version was published online in August 2004 with corrections to Fig. 1 and Fig. 5.     

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     

Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits

We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a λgtll expression library constructed from MG pericarp poly(A)⁺ RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits.     

Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato

Differential screening of a cDNA bank constructed from ripe tomato fruit mRNA allowed the isolation of cDNA clone 2A11 which is entirely fruit-specific, is expressed at steadily increasing levels from anthesis to breaker, and accounts for approximately 1% of the messenger RNA in mature tomato fruit. A genomic clone corresponding to the 2A11 cDNA was isolated from a tomato genomic library. Sequence comparison of the cDNA clone with the genomic clone shows they are identical over the shared region with the genomic clone possessing a single large intron near the 5 end of the message.The open reading frame of 2A11 would encode a sulfur-rich polypeptide 96 amino acids in length. The identity of the putative protein is unknown. In situ hybridization shows that the 2A11 message is found throughout the pericarp cells in a tomato fruit. In contrast, in situ hybridization of early ripening stages with a polygalacturonase probe shows higher mRNA levels in cells of the outer pericarp and cells surrounding the vascular regions of the pericarp.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

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.     

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product -aminobutyric acid (GABA) in fruit, are discussed.     

Developmental regulation of phospholipase D in tomato fruits

The catabolism of phospholipids initiated by phospholipase D (PLD, EC 3.1.4.4) is an inherent feature of developmental processes that include fruit growth and ripening. In cherry tomatoes (Lycopersicon esculentum Mill.), soluble and membrane-associated PLD activities increased during fruit development, which peaked at the mature green and orange stages. The increase in PLD activity was associated with a similar increase in the intensity of a 92 kDa band as demonstrated by western blot analysis. A full-length cDNA having 2430 bp and encoding a putative polypeptide with 809 amino acids, was isolated using tomato RNA, RT-PCR and 5' and 3' rapid amplification of cloned ends (RACE). Analysis of the primary and secondary structures showed the presence of the C2 domain, the PLD domain and several other features characteristic of PLD alpha. Microtom tomato plants transformed with antisense PLD alpha cDNA, were similar to untransformed plants and showed normal fruit set and development. The ethylene climacteric was delayed by over 7 d in the antisense PLD fruits, indicative of a slower ripening process. The leaves and unripened fruits of antisense PLD microtom plants possessed lowered PLD activity and PLD protein, as demonstrated by western blotting. However, during ripening, PLD activity in the transgenic fruits was maintained at a higher level than that in the untransformed control. Immunolocalization of PLD in microtom tomato fruits revealed the cytosol-membrane translocation of PLD during fruit development. The ripe fruits of antisense PLD celebrity plants possessed lowered PLD expression and activity and showed increased firmness and red colour. These results suggest that the expression of antisense PLD cDNA could be variable in different tomato varieties. The potential role of PLD in ethylene signal transduction events is discussed.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

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。     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

Changes in the protein and activity levels of the plastid NADH–plastoquinone–oxidoreductase complex during fruit development

Plastids contain an NADH dehydrogenase complex (Ndh complex) homologous to the mitochondrial complex I (EC 1.6.5.3). In this work, we have analysed the changes in the Ndh complex during ripening of pepper (Capsicum annum L., cv. Maor) and tomato (Lycopersicon esculentum Mill., cv. Marglobe) fruits. The Ndh complex was mainly present in the outer pericarp of tomato fruits, whereas it was evenly distributed in the pericarp of pepper. In both kinds of fruit we observed a decrease in the total amount of Ndh complex from the green to the red stage of development. This decrease corresponds to parallel decreases in the content and activity of the complex in plastids during the transition from chloroplasts to chromoplasts. Levels of plastidial quinol peroxidase activity were also higher during the first stages of tomato fruit development than during the latter stages of ripening. However, when referred to total plastid protein, the amount and activity of the Ndh complex in chloroplasts isolated from green fruits was higher than in chloroplasts isolated from leaves. These results strongly suggest that function of the Ndh complex, probably related to a plastidial electron transport chain, can be important during the first stages of fruit development.