Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development.
Polyamines (PAs) play a vital role in the responses of higher plants to abiotic stresses. However, only a limited number of studies have examined the interplay between PAs and signal molecules. The aim of this study was to elucidate the cross-talk among PAs, abscisic acid (ABA), nitric oxide (NO), and hydrogen peroxide (H2O2) under chilling stress conditions using tomato seedlings [(Lycopersicon esculentum Mill.) cv. Moneymaker]. The study showed that during chilling stress (4°C; 0, 12, and 24 h), the application of spermidine (Spd) and spermine (Spm) elevated NO and H2O2 levels, enhanced nitrite reductase (NR), nitric oxide synthase (NOS)-like, and polyamine oxidase activities, and upregulated LeNR relative expression, but did not influence LeNOS1 expression. In contrast, putrescine (Put) treatment had no obvious impact. During the recovery period (25/15°C, 10 h), the above-mentioned parameters induced by the application of PAs were restored to their control levels. Seedlings pretreated with sodium nitroprusside (SNP, an NO donor) showed elevated Put and Spd levels throughout the treatment period, consistent with increased expression in leaves of genes encoding arginine decarboxylase (LeADC. LeADC1), ornithine decarboxylase (LeODC), and Spd synthase (LeSPDS) expressions in tomato leaves throughout the treatment period. Under chilling stress, the Put content increased first, followed by a rise in the Spd content. Exogenously applied SNP did not increase the expression of genes encoding S-adenosylmethionine decarboxylase (LeSAMDC) and Spm synthase (LeSPMS), consistent with the observation that Spm levels remained constant under chilling stress and during the recovery period. In contrast, exogenous Put significantly increased the ABA content and the 9-cis-epoxycarotenoid dioxygenase (LeNCED1) transcript level. Treatment with ABA could alleviate the electrolyte leakage (EL) induced by D-Arg (an inhibitor of Put). Taken together, it is concluded that, under chilling stress, Spd and Spm enhanced the production of NO in tomato seedlings through an H2O2-dependent mechanism, via the NR and NOS-like pathways. ABA is involved in Put-induced tolerance to chilling stress, and NO could increase the content of Put and Spd under chilling stress.
Lipoxygenases (LOXs) play important role in the synthesis of volatile organic compounds (VOCs), which influence the aroma of fruit. In this study, we elucidate that there is a positive relationship between LOXs activity and VOC production in melon (Cucumis melo), and CmLOX genes are involved in fruit aroma generation in melon. To this end, we tested four aroma types of melon that feature a thin pericarp: two aromatic cultivars of the oriental melons (C. melo var. makuwa Makino), ‘Yu Meiren’ (YMR) and ‘Cui Bao’ (CB); a non-aromatic oriental pickling melon (C. melo var. conomon), ‘Shao Gua’ (SHAO); and a non-aromatic snake melon (C. melo L. var. flexuosus Naud), ‘Cai Gua’ (CAI). A principal component analysis (PCA) revealed that the aromas of SHAO and CAI are similar in nature because their ester contents are lower than those of YMR and CB. Ethyl acetate, benzyl acetate, (E, Z)-2, 6-nonadienal and menthol are four principal volatile compounds that affect the aromatic characteristics of these four types of melons. The LOX activity and total ester content in YMR were the highest among the examined melon varieties. The expression patterns of 18 CmLOX genes were found to vary based on the aromatic nature of the melon. Four of them were highly expressed in YMR. Moreover, we treated the fruit disks of YMR with LOX substrates (linoleic acid and linolenic acid) and LOX inhibitors (n-propyl gallate and nordihydroguariaretic acid). Substrate application promoted LOX activity and induced accumulation of hexanal, (2E)-nonenal and straight-chain esters, such as ethyl acetate. In contrast, LOX inhibitors decreased the levels of these compounds. The effect of CmLOXs in the biosynthesis of esters in melons are discussed.
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