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Main Conclusion: Exogenous SA treatment at appropriate concentrations promotes adventitious root formation in cucumber hypocotyls, via competitive inhibiting the IAA-Asp synthetase activity of CsGH3.5, and increasing the local free IAA level. Adventitious root formation is critical for the cutting propagation of horticultural plants. Indole-3-acetic acid (IAA) has been shown to play a central role in regulating this process, while for salicylic acid (SA), its exact effects and regulatory mechanism have not been elucidated. In this study, we showed that exogenous SA treatment at the concentrations of both 50 and 100 µM promoted adventitious root formation at the base of the hypocotyl of cucumber seedlings. At these concentrations, SA could induce the expression of CYCLIN and Cyclin-dependent Kinase (CDK) genes during adventitious rooting. IAA was shown to be involved in SA-induced adventitious root formation in cucumber hypocotyls. Exposure to exogenous SA led to a slight increase in the free IAA content, and pre-treatment with the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) almost completely abolished the inducible effects of SA on adventitious root number. SA-induced IAA accumulation was also associated with the enhanced expression of Gretchen Hagen3.5 (CsGH3.5). The in vitro enzymatic assay indicated that CsGH3.5 has both IAA- and SA-amido synthetase activity and prefers aspartate (Asp) as the amino acid conjugate. The Asp concentration dictated the functional activity of CsGH3.5 on IAA. Both affinity and catalytic efficiency (K cat /K m ) increased when the Asp concentration increased from 0.3 to 1 mM. In contrast, CsGH3.5 showed equal catalytic efficiency for SA at low and high Asp concentrations. Furthermore, SA functioned as a competitive inhibitor of the IAA-Asp synthetase activity of CsGH3.5. During adventitious formation, SA application indeed repressed the IAA-Asp levels in the rooting zone. These data show that SA plays an inducible role in adventitious root formation in cucumber through competitive inhibition of the auxin conjugation enzyme CsGH3.5. SA reduces the IAA conjugate levels, thereby increasing the local free IAA level and ultimately enhancing adventitious root formation.

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H2O2) accumulation. The application of exogenous SA and the chemical scavenger of H2O2 reduces the excess H2O2 and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H2O2 and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H2O2 levels.

Phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway, plays a critical role in plant growth, development, and adaptation. PAL enzymes are encoded by a gene family in plants. Here, we report a genome-wide search for PAL genes in watermelon. A total of 12 PAL genes, designated ClPAL1-12, are identified . Nine are arranged in tandem in two duplication blocks located on chromosomes 4 and 7, and the other three ClPAL genes are distributed as single copies on chromosomes 2, 3, and 8. Both the cDNA and protein sequences of ClPALs share an overall high identity with each other. A phylogenetic analysis places 11 of the ClPALs into a separate cucurbit subclade, whereas ClPAL2, which belongs to neither monocots nor dicots, may serve as an ancestral PAL in plants. In the cucurbit subclade, seven ClPALs form homologous pairs with their counterparts from cucumber. Expression profiling reveals that 11 of the ClPAL genes are expressed and show preferential expression in the stems and male and female flowers. Six of the 12 ClPALs are moderately or strongly expressed in the fruits, particularly in the pulp, suggesting the potential roles of PAL in the development of fruit color and flavor. A promoter motif analysis of the ClPAL genes implies redundant but distinctive cis-regulatory structures for stress responsiveness. Finally, duplication events during the evolution and expansion of the ClPAL gene family are discussed, and the relationships between the ClPAL genes and their cucumber orthologs are estimated.

Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway, and therefore plays a key role in both plant development and stress defense. In many plants, PAL is encoded by a multi-gene family, and each member is differentially regulated in response to environmental stimuli. In the present study, we report that PAL in cucumber (Cucumis sativus L.) is encoded for by a family of seven genes (designated as CsPAL1-7). All seven CsPALs are arranged in tandem in two duplication blocks, which are located on chromosomes 4 and 6, respectively. The cDNA and protein sequences of the CsPALs share an overall high identity to each other. Homology modeling reveals similarities in their protein structures, besides several slight differences, implying the different activities in conversion of phenylalanine. Phylogenic analysis places CsPAL1-7 in a separate cluster rather than clustering with other plant PALs. Analyses of expression profiles in different cucumber tissues or in response to various stress or plant hormone treatments indicate that CsPAL1-7 play redundant, but divergent roles in cucumber development and stress response. This is consistent with our finding that CsPALs possess overlapping but different cis-elements in their promoter regions. Finally, several duplication events are discussed to explain the evolution of the cucumber PAL genes.