CmERF1 acts as a positive regulator of fruits and leaves growth in melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an important horticultural and economic crop. ETHYLENE RESPONSE FACTOR1 (ERF1) plays an important role in regulating plant development, and the resistance to multiple biotic and abiotic stresses. In this study, developmental biology, molecular biology and biochemical assays were performed to explore the biological function of CmERF1 in melon. Abundant transcripts of CmERF1 were found in ovary at green-yellow bud (GYB) and rapid enlargement (ORE) stages. In CmERF1 promoter, the cis-regulatory elements for indoleacetic acid (IAA), methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), gibberellic acid (GA), light and low temperature responses were found. CmERF1 could be significantly induced by ethylene, IAA, MeJA, SA, ABA, and respond to continuous light and low temperature stresses in melon. Ectopic expression of CmERF1 increased the length of siliqua and carpopodium, and expanded the size of leaves in Arabidopsis. Knockdown of CmERF1 led to smaller ovary at anthesis, mature fruit and leaves in melon. In CmERF1-RNAi #2 plants, 75 genes were differently expressed compared with control, and the promoter regions of 28 differential expression genes (DEGs) contained the GCC-box (AGCCGCC) or DRE (A/GCCGAC) cis-acting elements of CmERF1. A homolog of cell division cycle protein 48 (CmCDC48) was proved to be the direct target of CmERF1 by the yeast one-hybrid assay and dual-luciferase (LUC) reporter (DLR) system. These results indicated that CmERF1 was able to promote the growth of fruits and leaves, and involved in multiple hormones and environmental signaling pathways in melon.

MicroRNA Profiling Revealed the Mechanism of Enhanced Cold Resistance by Grafting in Melon (Cucumis melo L.).

Grafting is widely used to improve the resistance to abiotic stresses in cucurbit plants, but the effect and molecular mechanism of grafting on cold stress are still unknown in melon. In this study, phenotypic characteristics, physiological indexes, small-RNA sequencing and expression analyses were performed on grafted plants with pumpkin rootstock (PG) and self-grafted plants (SG) to explore the mechanism of changed cold tolerance by grafting in melon. Compared with SG plants, the cold tolerance was obviously enhanced, the malondialdehyde (MDA) content was significantly decreased and the activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; peroxidase, POD) were significantly increased in PG plants. Depend on differentially expressed miRNA (DEM) identification and expression pattern analyses, cme-miR156b, cme-miR156f and chr07_30026 were thought to play a key role in enhancing low-temperature resistance resulting from grafting. Subsequently, 24, 37 and 17 target genes of cme-miR156b, cme-miR156f and chr07_30026 were respectively predicted, and 21 target genes were co-regulated by cme-miR156b and cme-miR156f. Among these 57 unique target genes, the putative promoter of 13 target genes contained the low-temperature responsive (LTR) cis-acting element. The results of qRT-PCR indicated that six target genes (MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732) displayed the opposite expression pattern to their corresponding miRNAs. Furthermore, MELO3C002370, MELO3C016713 and MELO3C012858 were significantly downregulated in cold-resistant cultivars and upregulated in cold-sensitive varieties after cold stimulus, and they acted as the key negative regulators of low-temperature response in melon. This study revealed three key miRNAs and three putative target genes involved in the cold tolerance of melon and provided a molecular basis underlying how grafting improved the low-temperature resistance of melon plants.

The effects of different rootstocks on aroma components, activities and genes expression of aroma-related enzymes in oriental melon fruit.

Grafting is widely applied in the cultivation of melon. In this study, 'Qinmi No.1' (Cucumis melo L.(QG)) and 'Ribenxuesong' (Cucurbita maxima Duch. (RG)) were used as rootstocks for 'Qingxin Yangjiaocui' (Cucumis melo L.). The results showed that grafting with muskmelon rootstocks had no significant effect on fruit aroma, but grafting with pumpkin rootstocks significantly reduced the odor intensity and odor preference scores of melon fruits. Compared with the fruits from self-grafted plants (SG), four new aromatic volatiles with a sweet smell were detected, the alcohol dehydrogenase (ADH) activity was significantly decreased at 30 DAP, but unaffected at 42 DAP in QG fruits. There was no difference for alcohol acetyltransferase (AAT) activity between QG and SG fruits. The expression level of CmADH2 was significantly higher at 30 DAP and 42 DAP, but CmAAT2 was significantly lower at 42 DAP in QG fruits compared with SG fruits. In RG fruits, the main aroma compounds including butanoic acid ethyl ester, 2-methyl-2-butene-1-al, and 2-methylheptan-1-al were absent, while the volatile compounds with unpleasant odor characteristics including trans, cis-2,6-nonadien-1-ol, (E,E)-2,4-heptadienal, octanoic acid, and styrene were detected. Compared with SG fruits, 1-nonanol and 1-heptanol with green odor characteristics were significantly increased, but eucalyptol and farnesene with fruity aroma characteristics were significantly decreased in RG fruits. The ADH activity of RG fruits was significantly lower than that of SG fruits at 30 DAP and the AAT activity was significantly lower than that of SG fruits at 42 DAP. In addition, the expression levels of CmADH and CmAAT homologs in RG fruits were significantly lower than those in SG or QG fruits. These results show that grafting with pumpkin rootstocks affected the main aroma components, reduced ADH and AAT activities, and down-regulated the expression levels of CmADHs and CmAATs in the melon fruits. This study reveals the mechanism of different rootstocks on melon fruit aroma quality, and lays a theoretical foundation for the selection of rootstocks in melon production. Future studies using overexpression or CRISPR/CAS system to obtain stable transgenic lines of genes encoding key aromatic volatiles, would be promising to effectively improve the flavor quality of melon.

Expression patterns of ABCE model genes during flower development of melon (Cucumis melo L.).

In production, most cultivars of melon are andromonoecious and characterized by carrying both male and bisexual flowers on the same plant. In this study, four A-class genes (CmAP1a, CmAP1b, CmAP2a and CmAP2b), two B-class genes (CmAP3 and CmPI), two C-class genes (CmAGa and CmAGb) and four E-class genes (CmSEP1,2,3,4) were identified in melon. However, no D-class gene of melon was identified. The conserved domains of ABCE function proteins showed relatively high similarity between Arabidopsis and melon. The expression patterns of ABCE homeotic genes in different flower buds of melon suggested that transcripts of CmAP1a, CmPI and CmSEP1 in bisexual buds were significantly lower than that in male flower buds, while the expression levels of CmAGa, CmAGb and CmSEP4 in bisexual flower buds were significantly higher than that in male flower buds. There was no significant difference in expression levels of other ABCE model genes between male buds and bisexual buds. Subsequently, qRT-PCR was performed in different floral organs of bisexual flowers in melon. For A class genes, CmAP1a and CmAP1b showed the highest accumulation in sepals than petals, stamens and pistil, while CmAP2a and CmAP2b revealed the highest expression in pistil than other three floral organs. For B class genes, CmAP3 and CmPI were highly accumulated in petals and stamens though CmAP3 also showed abundant accumulation in pistil. For C class genes, the expression levels of CmAGa and CmAGb were higher in stamens and pistil than that in sepals and petals. For E class genes, CmSEP1 showed higher expression level in sepals and petals than stamens and pistil. CmSEP2, CmSEP3 and CmSEP4 showed the highest accumulation in pistil than other floral organs. These results provided a theoretical basis for studying the function of ABCE homeotic genes in floral organs development of melon.

Distinct metabolic profiling is correlated with bisexual flowers formation resulting from exogenous ethephon induction in melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an agronomically important vegetable. Most cultivars of melon are andromonoecious and bisexual flowers only emerged from the leaf axil of lateral branches. However, the regulatory mechanism contributing to the occurrence of bisexual flowers were still obscure. In this study, ethephon was applied in two common cultivars of melon. In control without ethephon treatment, no bisexual flower was made in the main stem. However, 6.56 ± 1.42 and 6.63 ± 0.55 bisexual flowers were respectively induced in main stem of 'Yangjiaocui-QX' and 'Lvbao' after ethephon treatment, and induced bisexual flowers distributed in 12-20 nodes of main stem. During the formation of bisexual flowers, 41 metabolites were significantly up-regulated and 98 metabolites were significantly down-regulated. According to the KEGG enrichment analysis of 139 different metabolites, a total of 30 pathways were mapped and KEGG terms of "Phenylalanine, tyrosine and tryptophan biosynthesis", "Phenylalanine metabolism" and "Flavone and flavonol biosynthesis" were significantly enriched. In three significantly enriched KEGG terms, shikimic acid, L-tryptophan, L-phenylalanine, and kaempferol were significantly up-regulated while L-tyrosine, 4-hydroxycinnami acid and luteolin were significantly down-regulated in ET compared to CK. Different metabolites were also classified depend on major class features and 14 classes were acquired. The results of metabonomics and endogenous hormone identification indicated that ethylene could enhance the concentration of salicylic acid, methyl jasmonate, ABA and IAA. This study provided an important theoretical foundation for inducing bisexual flowers in main stem and breeding new varieties of melon in future.

Transcriptome profiling reveals the occurrence mechanism of bisexual flowers in melon (Cucumis melo L.).

Most cultivated melons are andromonoecies in which male flowers arose both in main stem and lateral branches but bisexual flowers only emerged from the leaf axils of lateral branches. However, bisexual flowers emerged in leaf axils of main stem after ethephon treatment. Therefore, the mechanism regulating the occurrence of bisexual flowers were investigated by performing transcriptome analysis in two comparison sets: shoot apex of main stem (MA) versus that of lateral branches (LA), and shoot apex of main stem after ethephon treatment (Eth) versus control (Cont). KEGG results showed that genes involved in "plant hormone signal transduction", "MAPK signaling pathway" and "carbon metabolism" were significantly upregulated both in LA and Eth. Further, details of DEGs involved in ethylene signaling pathway were surveyed and six genes were co-upregulated in two comparison sets. Among these, CmERF1, downstream in ethylene signaling pathway, showed the most significantly difference and expressed higher in bisexual buds than that in male buds. Furthermore, fifteen DEGs were found to contain GCC box or CRT/DRE cis-element for CmERF1 in their putative promoter region, and these DEGs involved in several plant hormones signaling pathway, camalexin synthesis, carbon metabolism and plant pathogen interaction.

Metabolomic analysis of the occurrence of bitter fruits on grafted oriental melon plants.

Grafting has been widely applied to melon (Cucumis melo L.) production to alleviate obstacles of continuous cropping and control soil-borne diseases. However, grafting often leads to a decline of fruit quality. For example, sometimes bitter fruits are produced on grafted plants. However, the underlying physiological mechanism still remains unclear. This study investigated the effects of different rootstocks on the taste of fruits of the Balengcui, an oriental melon cultivar, during summer production. The results showed that all grafted plants with Cucurbita maxima Duch. rootstocks produced bitter fruits, while non-grafted plants and plants grafted onto muskmelon rootstocks produced no bitter fruits. Liquid chromatography-mass spectrometry and metabonomic analysis were performed to investigate the mechanism underlying the occurrence of bitter fruits. Metabolite comparisons of fruits from plants grafted onto Ribenxuesong rootstocks both with non-grafted plants and plants grafted onto muskmelon rootstocks showed that 17 metabolites including phospholipids, cucurbitacins and flavonoids, exhibited changes. The three Cucurbitacins, Cucurbitacin O, Cucurbitacin C, and Cucurbitacin S, increased dramatically. The 10 phospholipids PS(18:1(9Z)/18:2(9Z,12Z)), PS(P-18:0/15:0), PA(18:1(11Z)/18:1(11Z)), PE(16:0/18:0), PS(O-16:0/17:2(9Z,12Z)), PI(16:0/18:2(9Z,12Z)), PA(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PS(P-16:0/17:2(9Z,12Z)), PS(22:0/22:1(11Z)), and PA(17:1(9Z)/0:0)) were significantly decreased, while two PA (16:0/18:2 (9Z, 12Z) and 16:0/18:1 (11Z)), two flavonoids (pelargonidin 3-(6''-malonylglucoside)-5-glucoside and malvidin 3-rutinoside) significantly increased in fruits of plants grafted onto Cucurbita maxima Duch. rootstocks. These metabolites were involved in the glycerophospholipid metabolic pathway, the mevalonate pathway, and the phenylpropanoid pathway. In summary, these results showed that the bitter fruits of grafted Balengcui were caused by Cucurbita maxima Duch. rootstocks. Phospholipids, cucurbitacins, and flavonoids were the key contributors for the occurrence of bitter fruits in Balengcui melon after grafting onto Cucurbita maxima Duch. rootstocks.

[Changes in amino acid and fatty acid contents as well as activity of some related enzymes in apple fruit during aroma production].

Aroma volatiles from apple (Malus domestica Borkh. var. Starkrimson) fruit at different stages of maturity were collected by solid adsorbent-Tenax-GC and determined by thermodesorption and GC-MS. Production of propyl acetate, butyl acetate, ethyl 2-methyl-butanoate and total ester volatiles and changes in concentration of the precursors of aroma biosynthsis--free amino acids and fatty acids and activities of lipoxygenases (LOX) and alcohol acetyltransferase (AAT) in apple fruits during ripening were studied. The results showed that propyl acetate and total esters were very low when the endogenous ethylene formation of the fruit was very low. At the stage of the increase in ethylene production, the rate of formation of propyl acetate and total esters increased. Butyl acetate appeared at the beginning of ethylene rise and increased thereafter. Ethyl 2-methyl-butanoate was produced at the beginning of climacteric stage and then increased sharply (Figs.1). These facts suggest that the aroma production is closely related to ethylene production. Among the 14 free amino acids detected in fruit, isoleucine which is considered to be the biosynthetic precursor of some branched chain esters showed a great increase during fruit ripening while the others decreased or remained stable (Table 1). The accumulation of isoleucine suggested that isoleucine supply in fruit may not limit the biosynthesis of esters with branched chain alkyl groups. Concentrations of free fatty acids such as palmitic, linolenic, oleic, linoleic, stearic acids increased before the increase of aroma production, decreased with the increase of aroma production and showed an increase at postclimacteric stages (Fig.2). LOX activity increased at climacteric stages and declined rapidly thereafter. AAT activity increased sharply at the early stage of fruit maturity when the aroma was very low and remained at a stable high level during fruit ripening (Fig.3) indicating that the AAT activity is not the limiting factor for aroma formation in apple fruit.