Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus)

We have isolated a cDNA for Cm-HMGR, encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in melon (Cucumis melo L. reticulatus; Genbank Accession No. AB021862). Cm-HMGR encodes a polypeptide of 588 amino acids that contains two transmembrane domains and a catalytic domain. Database searches revealed that Cm-HMGR shows homology to HMG1 (63.7%) and HMG2 (70.3%) of tomato, to HMG1 (77.2%) and HMG2 (69.4%) of Arabidopsis thaliana, and to HMGR of tobacco (72.6%). Functional expression in a HMG-CoA reductase-deficient mutant yeast showed that Cm-HMGR products mediate the synthesis of mevalonate. Northern analysis revealed that the level of Cm-HMGR mRNA in the fruit increased after pollination and markedly decreased at the end of fruit enlargement. During ripening, Cm-HMGR mRNA levels increased markedly in the fruit. In parallel with mRNA expression, Cm-HMGR activity increased after pollination, whereas no Cm-HMGR activity was detectable during fruit ripening. Our results suggest that Cm-HMGR is important during early post-pollination development of the fruit in melon.     

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.     

An endo-1,4-β-glucanase expressed at high levels in rapidly expanding tissues

Plant developmental processes involving modifications to cell wall structure, such as cell expansion, organ abscission and fruit ripening, are accompanied by increased enzyme activity and mRNA abundance of endo-1,4--glucanases (EGases). An EGase cDNA clone, Ce14, isolated from tomato (Lycopersicon esculentum) has been shown to be identical to a tomato pistil-predominant EGase cDNA, TPP18. In addition to its previously reported expression during certain stages of early pistil development, Ce14 mRNA was also detected at high levels in the growing zones of etiolated hypocotyls (about 2.5-fold less than in pistils) and in young expanding leaves (about 3.5-fold less than in pistils). The abundance of Ce14 mRNA declined precipitously in older tissues as cells became fully expanded, and was barely detectable in mature vegetative tissues. Ce14 mRNA abundance was also low in abscission zones, and did not increase as abscission progressed. In fruit, Ce14 mRNA was present at low levels during fruit expansion, but was essentially absent during subsequent fruit development and ripening. Treatment of etiolated hypocotyls with ethylene or high concentrations of auxin sufficient to induce rapid lateral cell expansion and hypocotyl swelling also brought about an approximate doubling of Ce14 mRNA abundance, suggesting that Ce14 mRNA accumulation may be promoted directly or indirectly by ethylene. Thus, accumulation of Ce14 mRNA was found to be correlated with rapid cell expansion in pistils, hypocotyls and leaves.     

Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening

Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5-bisphosphate carboxylase decreased steadily during fruit development from 23 ± 8 nmoles per minute per milligram protein at the mature green stage to 13.4 ± 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio of carboxylase to oxygenase activity, which was about 10. Catalase activity reached a maximum during the climacteric, simultaneously with increased ethylene and CO₂ formation. Glycolate oxidase activity decreased during early stages of development and was barely detectable at the climacteric. Hydroxypyruvate reductase, associated with serine formation by the glycerate pathway, increased in specific activity during early stages of tomato fruit ripening. In the fruit of the rin tomato mutant, which does not ripen normally, none of these changes in enzyme activity occurred.     

Detection of expansin proteins and activity during tomato fruit ontogeny

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.     

A fruit-specific phosphoenolpyruvate carboxylase is related to rapid growth of tomato fruit

Malic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement of phospho enolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in organic acid accumulation and tomato fruit development was investigated. Two PEPCases, named LYCes;Ppc1 and LYCes;Ppc2 and mapped to chromosomes 12 and 7, respectively, were shown to be differentially expressed during tomato fruit development. LYCes;Ppc1 mRNA was present in all fruit tissues and in all other plant organs examined. In contrast, LYCes;Ppc2 was strongly and specifically expressed in fruit from the end of cell division to ripening. No LYCes;Ppc2 expression was detected by northern blot in other plant tissues. In fruit, the increase in LYCes;Ppc2 mRNA was closely followed by an increase in fruit PEPCase protein and activity, and was coincident with the increased accumulation of malate and citrate during the initial period of rapid growth rate, from 8 to 20 days post anthesis. Localization of LYCes;Ppc2 mRNA in young tomato fruit by in situ hybridization revealed that LYCes;Ppc2 is preferentially expressed in large cells of the pericarp and in enlarging cells of the gel surrounding the seeds. Examination of the kinetic and regulatory properties of the PEPCases of growing and ripening fruit further showed that PEPCase in growing fruit is less sensitive to low pH and malate inhibition, indicating a high phosphorylation state and/or the presence of a PEPCase isoform with these characteristics. Taken together, these results indicate that in developing tomato fruit PEPCase is probably important in permitting the synthesis of organic acids to provide the turgor pressure necessary for cell expansion.     

Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase

Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-specific isoform of phytoene synthase) in wild-type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.     

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.     

Endo-beta-mannanase activity increases in the skin and outer pericarp of tomato fruits during ripening

Activity of endo-beta-mannanase increases during ripening of tomato (Lycopersicon esculentum Mill.) fruit of the cultivar Trust. beta-Mannoside mannohydrolase is also present during ripening, but its pattern of activity is different from that of endo-beta-mannanase. The increase in endo-beta-mannanase activity is greatest in the skin, and less in the outer and inner pericarp regions. This enzyme is probably bound to the walls of the outermost cell layers of the fruit during ripening, and it requires a high-salt buffer for effective extraction. The enzyme protein, as detected immunologically on Western blots, is present during the early stages of ripening, before any enzyme activity is detectable. The mRNA for the enzyme is also present at these stages; endo-beta-mannanase may be produced and sequestered in a mature-sized inactive form during early ripening. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-beta-mannanase activity, but a cause-and-effect relationship between the enzyme and ripening is unlikely because some cultivars which ripen normally do not exhibit any endo-beta-mannanase activity in the fruit.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

The early light-inducible protein (ELIP) gene is expressed during the chloroplast-to-chromoplast transition in ripening tomato fruit

Chloroplast-to-chromoplast transitions during fruit ripening require massive transformation of the plastid internal membrane structure as the photosynthetic apparatus is disassembled. Early Light-Inducible Proteins (ELIPs) are known to accumulate in chloroplasts during thylakoid biogenesis and under stressful conditions. To determine if ELIP may also play a role in thylakoid disassembly during the chloroplast-to-chromoplast transition, ELIP mRNA expression was measured in tomato, Lycopersicon esculentum Mill. cv. Rutgers. An EST clone was identified in the Tomato Genome Project/Solanaceae Genomics Network database that has high sequence similarity with the amino acid sequence of Arabidopsis ELIP1 and ELIP2. It has complete identity in the two conserved regions of the protein. Genomic Southern blots indicate that the gene is a single copy in tomato. The genomic sequence shows the three-exon structure typical of ELIP sequences from other species. mRNA for this gene is barely detectable on northern blots from etiolated seedlings, but transiently accumulates to high levels 2 h after transfer to the light. Greenhouse-grown tomatoes were used to measure ELIP mRNA accumulation during fruit development and ripening. Tomato ELIP mRNA is detectable in all stages of fruit ripening, but is most abundant in the breaker/turning stage of development. A survey of tomato EST databases revealed that ELIP cDNA is also relatively abundant in developing flowers, which contain yellow chromoplasts. Combined, these results suggest that ELIP may play a newly-recognized role in the chloroplast-to-chromoplast transition process.     

Novel promoters that induce specific transgene expression during the green to ripening stages of tomato fruit development

Fruit-specific promoters have been used as genetic engineering tools for studies on molecular mechanism of fruit development and advance in fruit quality and additional value by increasing functional component. Especially fruit-ripening specific promoters have been well utilized and studied in tomato; however, few studies have reported the development of promoters that act at fruit developing stages such as immature green and mature green periods. In this study, we report novel promoters for gene expression during the green to ripening stages of tomato fruit development. Genes specifically expressed at tomato fruit were selected using microarray data. Subsequent to confirmation of the expression of the selected 12 genes, upstream DNA fragments of the genes LA22CD07, Les.3122.2.A1_a_at and LesAffx.6852.1.S1_at which specifically expressed at fruit were isolated from tomato genomic DNA as promoter regions. Isolated promoter regions were fused with the GUS gene and the resultant constructs were introduced into tomato by agrobacterium-mediated transformation for evaluation of promoter activity in tomato fruit. The two promoters of LA22CD07, and LesAffx.6852.1.S1_at showed strong activity in the fruit, weak activity in the flower and undetectable activity in other tissues. Unlike well-known fruit-ripening specific promoters, such as the E8 promoter, these promoters exhibited strong activity in green fruit in addition to red-ripening fruit, indicating that the promoters are suitable for transgene expression during green to ripening stages of tomato fruit development. KEY MESSAGE: Novel fruit-specific promoters have been identified and are suitable for transgene expression during green to ripening stages of tomato fruit development.     

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     

钙对不同成熟期番茄果实的PG活性及其合成的影响

本文研究了钙处理不同成熟期番茄果实对果壁组织中钙含量与转化、多聚半乳糖醛酸酶(PG)活性与 PG 合成的影响。结果表明,钙处理绿熟期的番茄果实可使总钙和可溶性钙含量明显增加,并较多转化为结合钙;后期处理,进入和转化的钙都减少。同样,钙处理愈早,对果实 PG 活性的抑制愈强,绿熟期处理可完全抑制 PG 活性。凝胶电泳结合钌红染色,证明绿熟期果实无 PG,PG 是在果实成熟过程中新合成的。钙处理愈早,对 PG 合成的抑制愈强,绿熟期钙处理可完全抑制 PG 合成。     

Effect of the Calcium on Polygalacturonase (PG) Activity and Synthesis at Different Ripening Stages of Tomato Fruits

It has been reported that PG is a key enzyme related to the tomato fruit ripening and that the application of calcium can dramatically decrease the PG activity and delay the ripening of fruits. In this paper the effects of calcium treament at various ripening stages on the transformation of absorbed calcium, PG activity and PG synthesis in tomato fruits were studicd. According to the analysis of calcium by atomic absorption spectroscopy, it was shown that the soluble and total calcium contents in pericarp of fruits treated with calcium at mature-green stage were increased significantly, and that more soluble calcium was transformed into bound calcium. Both the absorption and transformation of calcium decreased in fruits treated with calcium at later stage of ripening. The inhibition of calcium on PG activity was most effective by treatment at mature-green stage, but less effective at later stage of ripening. One reason for the decrease of calcium inhibition was probably due to the decline of calcium absorption as fruit ripening. The polyacrylamide gel electrophoresis of PG showed that PG with a molecular weight of 46.7 kD was absent in mature-green fruits, and PG synthesis occurred only at the later stage of ripening. It seems that the earlier the treatment was done the more effective of the calcium inhibition of PG synthesis. Based on the above results, it was concluded that the PG plays a major role in ripening and senescence of tomato fruits, and both PG synthesis and its activity were inhibited by calcium. In order to delay the ripening and senescence of tomato fruits, the treatment with calcium should be done at mature-green stage.     

Protein synthesis in relation to ripening of pome fruits

Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. ¹⁴C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases during the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment with cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis.     

Regulation of isoprenoid/cholesterol biosynthesis in cells from mevalonate kinase-deficient patients

Mevalonic aciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS) are two inherited disorders both caused by depressed mevalonate kinase (MK) activity. MK is the first enzyme to follow the highly regulated 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), which catalyzes the rate-limiting step in the isoprenoid/cholesterol biosynthesis pathway. In fibroblasts of MA patients, but not of HIDS patients, HMGR activity is elevated under normal growth conditions. This activity is down-regulated when cells are supplemented with the isoprenoid precursors geraniol, farnesol, and geranylgeraniol, and a mixture of 25-hydroxycholesterol and cholesterol. This indicates that the regulation of the pathway in these cells is not disturbed. The elevated HMGR activity is probably due to a shortage of non-sterol isoprenoid end products, as indicated by normal HMGR mRNA levels in MA fibroblasts. Furthermore, the HMGR activity in MA cells was more sensitive to geranylgeraniol suppression and less sensitive to sterol suppression than the HMGR activity in low density lipoprotein receptor-deficient cells. HMGR activity in MA cells was down-regulated also by addition of its product mevalonate to the culture medium. Thus, it appears that the elevation of mevalonate levels, which are high in MA patients and moderate in HIDS patients, allows the cells to compensate for the depressed MK activity. Indeed, the isoprenylation of Ras and RhoA protein appeared normal in HIDS and MA fibroblasts under normal conditions but showed increased sensitivity toward inhibition of HMGR by simvastatin. Our results indicate that MK-deficient cells maintain the flux through the isoprenoid/cholesterol biosynthesis pathway by elevating intracellular mevalonate levels.