Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with K _ms 10–20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit. Daniel Manríquez and Islam El-Sharkawy contributed equally to the work. Accession numbers for Cm-ADH1 (ABC02081), and Cm-ADH2 (ABC02082).     

苹果果实香气产生过程中氨基酸和脂肪酸含量及一些相关酶活性的变化

研究了苹果果实成熟期间香气和乙烯的产生动态,以及游离氨基酸、游离脂肪酸含量和脂氧合酶(LOX)、醇-酰基转移酶(AAT)活性的变化.结果表明,果实香气物质是随着乙烯释放的增加而产生和增加的.在此过程中,异亮氨酸大量积累.游离脂肪酸在果实香气很少时呈增加趋势;随着香气产生的增多而迅速下降;乙烯高峰过后又有增加.脂氧合酶活性随着果实成熟而提高,其活性在乙烯释放达到高峰时达到最大值,之后迅速下降.醇-酰基转移酶活性在果实开始产生香气时迅速增加,之后保持较高活性.     

Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening.

Two genes (CM-AAT1 and CM-AAT2) with strong sequence homology (87% identity at the protein level) putatively involved in the formation of aroma volatile esters have been isolated from Charentais melon fruit. They belong to a large and highly divergent family of multifunctional plant acyl-transferases and show at most 21% identity to the only other fruit acyl-transferase characterized so far in strawberry. RT-PCR studies indicated that both genes were specifically expressed in fruit at increasing rates in the early and mid phases of ripening. Expression was severely reduced in ethylene-suppressed antisense ACC oxidase (AS) fruit and in wild-type (WT) fruit treated with the ethylene antagonist 1-MCP. Cloning of the two genes in yeast revealed that the CM-AAT1 protein exhibited alcohol acyl-transferase activity while no such activity could be detected for CM-AAT2 despite the strong homology between the two sequences. CM-AAT1 was capable of producing esters from a wide range of combinations of alcohols and acyl-CoAs. The higher the carbon chain of aliphatic alcohols, the higher the activity. Branched alcohols were esterified at differential rates depending on the position of the methyl group and the nature of the acyl donor. Phenyl and benzoyl alcohols were also good substrates, but activity varied with the position and size of the aromatic residue. The cis/trans configuration influenced activity either positively (2-hexenol) or negatively (3-hexenol). Because ripening melons evolve the whole range of esters generated by the recombinant CM-AAT1 protein, we conclude that CM-AAT1 plays a major role in aroma volatiles formation in the melon.     

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.     

甜瓜乙烯应答因子基因在果实发育成熟过程中的表达特性

根据已报道的甜瓜CMe-ERF1和CMe-ERF2基因cDNA序列设计合成特异性引物,应用RT-PCR技术从甜瓜品种‘河套蜜瓜’成熟果实中克隆得到CMe-ERF1和CMe-ERF2基因cDNA全长编码区序列,分别为498bp和822bp.序列比对分析表明,得到的cDNA序列与已报道的Andes甜瓜相应基因的cDNA序列完全一致.果实不同发育时期实时定量PCR检测结果表明,CMe-ERF1、CMe-ERF2基因表达与甜瓜果实成熟及乙烯生成量显著相关,表明该基因可能对果实成熟起重要作用.     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.     

Functional allelic diversity of the apple alcohol acyl-transferase gene <Emphasis Type="Italic">MdAAT1</Emphasis> associated with fruit ester volatile contents in apple cultivars

Flavour is an important key factor of apple (Malus × domestica Borkh.) fruit quality, and its improvement is an important but complex breeding goal. Acetate esters are quantitatively the most important volatile compounds in apple fruit, and only a few of them dominate the typical aroma of a cultivar. Alcohol acyl-transferase (AAT) is a key enzyme involved in the last step of ester biosynthesis. The aim of this study was to target single nucleotide polymorphisms (SNPs) in an AAT candidate gene genetically associated with ester quantitative trait loci (QTL), to enable functional marker development for marker-assisted apple breeding programs. The AAT gene inventory of apple was characterized by in-silico mining of the assembled Golden Delicious genome, and 17 putative AAT genes in total were defined. MdAAT1 located on chromosome 2 was selected as the main candidate gene associated with QTL for different acetate esters, and its allelic diversity was assessed by direct amplicon sequencing in a collection of 102 apple cultivars characterized for ester volatile profiles. Sequencing a 468 bp nucleotide sequence of the MdAAT1 coding region resulted in the detection of four SNPs. In total, 18 different SNP haplotypes/heterozygous patterns were generated from the four SNPs identified within the apple collection. Association analyses resulted in highly significant associations of both individual SNPs and distinct haplotypes with the content of four acetate esters, including hexyl acetate, butyl acetate and 2-methyl-butyl acetate. About a third (31) of the 102 apple cultivars possessed the specific MdAAT1 haplotype H1 (C-A-C-A) and were characterized by strongly decreased ester concentrations. The contrasting H8 haplotype (T-G-T-G) was found in 28 varieties but was associated with normal to elevated ester concentrations. The observed association suggests a putative causal functional relationship between MdAAT1 and production of key apple esters.     

Candidate genes and QTLs for fruit ripening and softening in melon

Different factors affect the quality of melon fruit and among them long shelf life is critical from the consumer’s point of view. In melon, cultivars showing both climacteric and non-climacteric ripening types are found. In this study we have investigated climacteric ripening and fruit softening using a collection of near-isogenic lines (NILs) derived from the non-climacteric melon parental lines PI 161375 (SC) and “Piel de Sapo” (PS). Surprisingly, we found that QTL eth3.5 in NIL SC3-5b induced a climacteric-ripening phenotype with increased respiration and ethylene levels. Data suggest that the non-climacteric phenotypes from PI 161375 and “Piel de Sapo” may be the result of mutations in different genes. Several QTLs for fruit flesh firmness were also detected. Candidate genes putatively involved in ethylene regulation, biosynthesis and perception and cell wall degradation were mapped and some colocations with QTLs were observed. These results may provide additional data towards understanding of non-climacteric ripening in melon.     

Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.     

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     

Characterization of two cDNA clones for mRNAs expressed during ripening of melon (Cucumis melo L.) fruits

In vitro translation of mRNAs and polyacrylamide gel electrophoresis of proteins from melons revealed that several mRNAs increased in amount during ripening, indicating the existence of other ripening genes in addition to those cloned previously. To identify ripening-related genes we have screened a ripe melon cDNA library and isolated two novel cDNA clones (MEL2 and MEL7) encoding unidentified proteins. Southern analysis revealed that MEL2 and MEL7 are encoded by low-copy-number genes. The MEL2 cDNA clone is near full-length, corresponds to a 1600 nucleotide mRNA that accumulates during ripening and encodes a predicted protein rich in hydrophobic amino acids. The MEL7 cDNA clone is full-length, corresponds to a mRNA of 0.7 kb which accumulates during early ripening stages and is also present at low levels in other organs of the melon plant. The MEL7 predicted polypeptide is 17 kDa and shows significant homology with the major latex protein from opium-poppy. Wounding and ethylene treatment of unripe melon fruits 20 days after anthesis showed that MEL2 and MEL7 mRNAs are only induced by ethylene.     

Engineering melon plants with improved fruit shelf life using the TILLING approach

Background

Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening.

Methodology/Principal Findings

To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect.

Conclusions/Significance

We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.     

The Synthesis of Ethylene in Melon Fruit during the Early Stage of Ripening

The levels of mRNA and polypeptide for a 1-aminocyclopropane-1-carboxylate(ACC) oxidase were studied to identify the tissues in whichthe synthesis of ethylene first occurs during the initial stageof ripening. RNA and immunoblot analysis showed that the levelsof the mRNA and polypeptide for ACC oxidase were very low inunripe fruit. They first became detectable in the placentaltissue at the pre-climacteric stage, and then their levels increasedin the mesocarp tissue during the climacteric increase in theproduction of ethylene. Two mRNAs for ACC synthase (transcribedfrom ME-ACS1 and ME-ACS2) were detected in the placental tissueand seeds at the pre-climacteric stage, but only the level ofME-ACS1 mRNA, which has been characterized as the mRNA for awound-inducible ACC synthase, increased in mesocarp, placentaltissues and seeds during ripening. The level of ME-ACS2 mRNAthat was isolated from etiolated seedlings of melon, did notchange markedly during ripening. These results suggest thatthe central region of melon fruit (placental tissue and seeds)plays a major role in the production of ethylene during theearly stage of ripening. ³These three authors made equal contribution to this study.     

The respiratory climacteric is present in Charentais (Cucumis melo cv. Reticulatus F1 Alpha) melons ripened on or off the plant

Ripening of climacteric fruit is accompanied by an increasein respiration and autocatalytic ethylene synthesis. In harvestedmelons, there is variation in the magnitude and duration ofthe respiratory climacteric depending on the cultivar. It hasrecently been reported that, while the ripening-associated increasein ethylene production is present, the respiratory climactericis absent in ripening melon fruit attached to the plant, leadingto the suggestion that climacteric respiration is an artifactof harvest. To address the universality of this phenomenon,ripening behaviour in the melon cultivar Charentais (Cucumismelo cv. Reticulatus F1 Alpha), was investigated and the resultsshow that the respiratory climacteric occurs in fruit ripenedboth on and off the plant. Key words: Cucumis melo, ethylene, respiratory climacteric     

Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4.     

Role of ethylene in the biosynthetic pathway of aliphatic ester aroma volatiles in Charentais Cantaloupe melons.

Compared to other melon types, Cantaloupe Charentais melons are highly aromatic with a major contribution to the aroma being made by aliphatic and branched esters. Using a transgenic line in which the synthesis of the plant hormone ethylene has been considerably lowered by antisense ACC oxidase mRNA (AS), the aliphatic ester pathway steps at which ethylene exerts its regulatory role were found. The data show that the production of aliphatic esters such as hexyl and butyl acetate was blocked in AS fruit and could be reversed by ethylene. Using fruit discs incubated in the presence of various precursors, the steps at which ester formation was inhibited in AS fruit was shown to be the reduction of fatty acids and aldehydes, the last step of acetyl transfer to alcohols being unaffected. However, treating AS fruit with the ethylene antagonist 1-methylcyclopropene resulted in about 50% inhibition of acetyl transfer activity, indicating that this portion of activity was ethylene-dependent and this was supported by the low residual ethylene concentration of AS fruit discs (around 2 microl l(-1)). In conclusion, the reduction of fatty acids and aldehydes appears essentially to be ethylene-dependent, whilst the last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyltransferases.     

A novel tomato F‐box protein,SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F‐BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene‐dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening‐associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening‐related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene‐related alterations, including inhibition of fruit ripening, attenuated triple‐response and delayed petal abscission. Yeast‐two‐hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3‐mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.