Polyamines (PAs) play a vital role in response to abiotic stress of higher plants. However, limited studies examined the interplay between PAs and signal molecules. Therefore, in this study, we tried to elucidate the crosstalk between PAs, abscisic acid (ABA), nitric oxide (NO) and hydrogen peroxide (H2O2) under chilling stress conditions were determined using tomato seedlings (Lycopersicon esculentum Mill.) cv. Moneymaker). The study showed that during chilling stress (4 oC, 0 h, 12 h and 24 h), application of Spd and Spm could elevate NO and H2O2 levels, enhance nitrite reductase (NR), nitric oxide synthase (NOS)-like, and polyamine oxidase (PAO) activities, upregulate LeNR relative expression, but did not influence LeNOS1 expression. While putrescine (Put) treatment had no this obvious impact in tomato leaves. Moreover, H2O2 might act as an upstream signal to stimulate NO production under chilling stress. In recovery period (25/15 oC, 10 h), above-mentioned parameters induced by PAs application were back to the control levels. In turn, seedlings pretreated with sodium nitroprusside (SNP, an NO donor) improved Put and Spd levels through increasing arginine decarboxylase (LeADC, LeADC1), ornithine decarboxylase (LeODC) and Spd synthase (LeSPDS) expressions in tomato leaves throughout treatment period. Put content appeared increases first, and then Spd content began to rise under chilling stress. Exogenously applied NO did not improved S-adenosylmethionine decarboxylase (LeSAMDC) and Spm synthase (LeSPMS) expressions, which led to constant Spm level under chilling stress and recover period. On the other hand, exogenous Put significantly increased ABA content and LeNCED1 transcript level, ABA treatment could alleviate the electrolyte leakage (EL) induced by D-arginine (D-Arg, an inhibitor of Put). Taken together, it is concluded that Spd and Spm enhanced NO production in tomato seedlings in H2O2-dependent way under chilling stress, which through NR and NOS pathways. ABA is involved in Put induced chilling stress tolerance. Furthermore, NO could improve the Put and the Spd contents under chilling stress.

Late blight, caused by Phytophthora infestans, is severely damaging to the global tomato industry. Micro-RNAs (miRNAs) have been widely demonstrated to play vital roles in plant resistance by repressing their target genes. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) method has been continuously improved and extensively applied to edit plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9 in tomato has not been studied yet. We knocked out miR482b and miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 system. Two transgenic plants with silenced miR482b and miR482c simultaneously and one transgenic line with silenced miR482b alone were obtained. Compared with wild-type plants, the disease symptoms of three transgenic plants upon infection were reduced, accompanied by increased expression of their common target nucleotide binding site-leucine-rich repeat genes and decreased levels of reactive oxygen species. Furthermore, silencing miR482b and miR482c simultaneously was more resistant than silencing miR482b alone in tomato. More importantly, we found that knocking out miR482b and miR482c can elicit expression perturbation of other miRNAs, suggesting cross-regulation between miRNAs. Our study demonstrated that editing miR482b and miR482c simultaneously with CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant tomatoes, and cross-regulation between miRNAs may reveal the novel mechanism in tomato–P. infestans interactions.

Drought stress severely impairs plant growth and production. Lipoxygenase (LOX), a master regulator for lipid peroxidation, is critical for direct or indirect response to abiotic stresses. Here, we found that drought stress induced the transcription of CmLOX10 in leaves of oriental melon seedlings. Reverse genetic approaches and physiological analyses revealed that silencing CmLOX10 increased drought susceptibility and stomatal aperture in oriental melon seedlings, and that ectopic overexpression of CmLOX10 in Arabidopsis enhanced drought tolerance and decreased the stomatal aperture. Moreover, the transcription of jasmonic acid (JA)-related genes and JA accumulation were significantly induced in CmLOX10-overexpressed Arabidopsis, which were reversely suppressed in CmLOX10-silenced seedlings during the stage of drought stress. Foliar application of JA further verified that JA enhanced drought tolerance and induced stomatal closure in leaves of melon seedlings. In addition, the feedback regulation of CmLOX10 was induced by JA signaling, and the expression level of CmMYC2 was increased by JA and drought treatment. Yeast one-hybrid analysis showed that CmMYC2 directly bound to the promoter of CmLOX10. In summary, we identified the important roles of CmLOX10 in the regulation of drought tolerance in oriental melon seedlings through JA- mediated stomatal closure and JA signaling-mediated feedback through CmMYC2.

Our previous study has indicated that a long noncoding RNA (lncRNA), lncRNA39026, can be responsive to Phytophthora infestans infection. However, the function and regulation mechanism of lncRNA39026 during tomato resistance to P. infestans are unknown. In this study, an lncRNA39026 sequence was cloned from tomato Zaofen No. 2, and this sequence contained an endogenous target mimicry for miR168a, which might suppress the expression of miR168a. LncRNA39026 was strongly downregulated at 3 h in the tomato plants infected with P. infestans, and its expression level displayed a negative correlation with the expression level of miR168a and a positive correlation with the expression levels of SlAGO1 genes (target gene of miR168a) upon P. infestans infection. Tomato plants in which lncRNA39026 was overexpressed displayed enhanced resistance to P. infestans, decreased level of miR168a, and increased level of SlAGO1, whereas the resistance was impaired, level of miR168a was increased, and level of SlAGO1 was decreased after lncRNA39026 silencing. In addition, lncRNA39026 could also induce the expression of pathogenesis-related (PR) genes, as shown by increased and decreased expression levels of PR genes in tomato plants with overexpressed and silenced lncRNA39026, respectively. The result demonstrated that lncRNA39026 might function to decoy miR168a and affect the expression of PR genes in tomato plants, increasing resistance to disease.

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.