ABA-responsive AREB1/ABI3-1/ABI5 cascade regulates IAA oxidase gene SlDAO2 to inhibit hypocotyl elongation in tomato.

Hypocotyl elongation is dramatically influenced by environmental factors and phytohormones. Indole-3-acetic acid (IAA) plays a prominent role in hypocotyl elongation, whereas abscisic acid (ABA) is regarded as an inhibitor through repressing IAA synthesis and signalling. However, the regulatory role of ABA in local IAA deactivation remains largely uncharacterized. In this study, we confirmed the antagonistic interplay of ABA and IAA during the hypocotyl elongation of tomato (Solanum lycopersicum) seedlings. We identified an IAA oxidase enzyme DIOXYGENASE FOR AUXIN OXIDATION2 (SlDAO2), and its expression was induced by both external and internal ABA signals in tomato hypocotyls. Moreover, the overexpression of SlDAO2 led to a reduced sensitivity to IAA, and the knockout of SlDAO2 alleviated the inhibitory effect of ABA on hypocotyl elongation. Furthermore, an ABA-responsive regulatory SlAREB1/SlABI3-1/SlABI5 cascade was identified to act upstream of SlDAO2 and to precisely control its expression. SlAREB1 directly bound to the ABRE present in the SlDAO2 promoter to activate SlDAO2 expression, and SlABI3-1 enhanced while SlABI5 inhibited the activation ability of SlAREB1 by directly interacting with SlAREB1. Our findings revealed that ABA might induce local IAA oxidation and deactivation via SlDAO2 to modulate IAA homoeostasis and thereby repress hypocotyl elongation in tomato.

Salicylic acid regulates adventitious root formation via competitive inhibition of the auxin conjugation enzyme CsGH3.5 in cucumber hypocotyls.

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 (Kcat/Km) 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.

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings.

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.

Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus).

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.

Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.

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.

Dynamic changes in bacterial communities in the recirculating nutrient solution of cucumber plug seedlings cultivated in an ebb-and-flow subirrigation system.

Ebb-and-flow subirrigation systems are highly efficient, water-saving and environmentally friendly. However, one concern with these recirculating systems is the possible transmission of plant pathogens. Here, through 16S rRNA-targeted Illumina sequencing, the bacterial dynamics in a recirculating nutrient solution were characterized for cucumber plug seedlings cultivated in an ebb-and-flow system in summer and winter. Both the bacterial number and diversity in the nutrient solution increased immediately after the first irrigation cycle; then, these values were gradually stable with recirculating irrigation. In summer and winter, different bacterial compositions and changing patterns were observed. In summer, the predominant genera in the nutrient solution included Comamonas, Pseudomonas, Acinetobacter, Reyranella, Sphingobium, Bradyrhizobium, Sphingomonas, and Acidovorax. Of those genera, during recirculating irrigation, the relative abundance of Bradyrhizobium gradually decreased, whereas those of Pseudomonas, Reyranella, Sphingobium, Sphingomonas, and Acidovorax gradually increased. In winter, the bacterial communities were mainly composed of Nevskia, Bosea, Sphingobium, Acidovorax, Pseudomonas, and Hydrocarboniphaga. Of those genera, the relative abundance of Bosea, Sphingobium, and Acidovorax showed an increasing trend, whereas those of Nevskia and Hydrocarboniphaga decreased overall. Furthermore, in both summer and winter, no plant pathogenic bacteria on cucumber could be detected; however, some potentially beneficial bacteria, including Comamonas testosteroni, Acinetobacter baumannii, Pseudomonas aeruginosa, P. koreensis and Sphingobium yanoikuyae, colonized the nutrient solution and exhibited increased relative abundances during irrigation. The colonization of these bacteria might facilitate the plant growth promotion. Inoculation of the microbes from the effluent nutrient solution also promoted the growth of cucumber seedlings, but did not lead to any disease. The present data elucidate the bacterial dynamics in a cucumber cultivation ebb-and-flow system and provide useful information for biological control during cucumber seedling production.

Bacterial communities in the rhizosphere, phyllosphere and endosphere of tomato plants.

Plants harbor diverse bacterial communities, which play crucial roles in plant health and growth, in their rhizosphere, phyllosphere and endosphere. Tomato is an important model for studying plant-microbe interactions, but comparison of its associated bacterial community is still lacking. In this study, using Illumina sequencing of 16S rRNA amplicons, we characterized and compared the bacterial size and community from rootzone soil as well as the rhizosphere, phyllosphere and endosphere of roots, stems, leaves, fruits and seeds of tomato plants that were grown in greenhouse conditions. Habitat (soil, phyllospheric, and endophytic) structured the community. The bacterial communities from the soil-type samples (rootzone soil and rhizosphere) showed the highest richness and diversity. The lowest bacterial diversity occurred in the phyllospheric samples, while the lowest richness occurred in the endosphere. Among the endophytic samples, both bacterial diversity and richness varied in different tissues, with the highest values in roots. The most abundant phyla in the tomato-associated community was Proteobacteria, with the exception of the seeds and jelly, where both Proteobacteria and Firmicutes were dominant. At the genus level, the sequences of Pseudomonas and Acinetobacter were prevalent in the rhizosphere, and in the phyllosphere, more than 97% of the sequences were assigned to Acinetobacter. For the endophytes, Acinetobacter, Enterobacter, and Pseudomonas were the abundant genera in the roots, stems and leaves. In the fruits, the bacterial endophytes varied in different compartments, with Enterobacter being enriched in the pericarp and seeds, Acinetobacter in the placenta, and Weissella in the jelly. The present data provide a comprehensive description of the tomato-associated bacterial community and will be useful for better understanding plant-microbe interactions and selecting suitable bacterial taxa for tomato production.

Characterization of the Fatty Acid Desaturase Genes in Cucumber: Structure, Phylogeny, and Expression Patterns.

Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber.

Quantitative Proteomic Profiling of Early and Late Responses to Salicylic Acid in Cucumber Leaves.

Salicylic acid (SA) is an important phytohormone that plays vital regulatory roles in plant growth, development, and stress responses. However, studies on the molecular mechanism of SA, especially during the early SA responses, are lagging behind. In this study, we initiated a comprehensive isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the early and late SA-responsive proteins in leaves of cucumber (Cucumis sativus L.) seedlings. Upon SA application through the roots, endogenous SA accumulated in cucumber leaves. By assaying the changes in marker gene expression and photosynthetic rate, we collected samples at 12 h and 72 h post treatment (hpt) to profile the early and late SA responsiveness, respectively. The iTRAQ assay followed by tandem mass spectrometry revealed 135 differentially expressed proteins (DEPs) at 12 hpt and 301 DEPs at 72 hpt. The functional categories for these SA-responsive proteins included in a variety of biochemical processes, including photosynthesis, redox homeostasis, carbohydrate and energy metabolism, lipid metabolism, transport, protein folding and modification, proteolysis, cell wall organization, and the secondary phenylpropanoid pathway. Conclusively, based on the abundant changes of these DEPs, together with their putative functions, we proposed a possible SA-responsive protein network. It appears that SA could elicit reactive oxygen species (ROS) production via enhancing the photosynthetic electron transferring, and then confer some growth-promoting and stress-priming effects on cells during the late phase, including enhanced photosynthesis and ROS scavenging, altered carbon metabolic flux for the biosynthesis of amino acids and nucleotides, and cell wall reorganization. Overall, the present iTRAQ assay provides higher proteome coverage and deepened our understanding of the molecular basis of SA-responses.

[Roles of phenylalanine ammonia-lyase in low temperature tolerance in cucumber seedlings].

To reveal the roles of phenylalanine ammonia-lyase (PAL) in low temperature tolerance in cucumber seedlings, a specific PAL inhibitor (AOPP) was sprayed to the seedlings, and then the stress tolerance was determined. The results suggested that both gene expression and enzymatic activity of PAL in cucumber leaves were induced under low temperature. The seedlings pretreated with AOPP showed lower PAL activity and less accumulation of phenolics and flavonoids. Low temperature caused damages in cucumber seedlings, and pretreatment of AOPP aggravated these damages. Compared to the control, the seedlings pretreated with AOPP showed significantly higher relative electrolyte leakage and MDA production, lower maximum photochemical efficiency of PSII (Fv/Fm) but higher photo-chemical quenching coefficient Y(NO), and reduced expression of low temperature-responsive genes (PR1-la, COR47, P5CS and HSP70). In cucumber seedlings, low temperature stress induced the accumulation of H2O2, increased the contents of ascobate (AsA) but decreased the contents of dehydroascobate (DHA), and thus reduced the value of AsA: DHA. In the AOPP-pretreated seedlings, the activities of antioxidant enzymes (CAT and APX) were significantly repressed, and excess H2O2 accumulated. The value of AsA: DHA was also lower than the control. Furthermore, co-application of H2O2 scavenger alleviated the low temperature-induced damages in the AOPP-pretreated seedlings, while coapplication of a CAT inhibitor made the seedlings more sensitive to low temperature stress. These results indicated that under low temperature stress, the enhanced activities of PAL could increase the biosynthesis of phenylpropanoid compounds and activate the cellular antioxidant enzymes, which could scavenge the excess ROS and maintain the cellular redox status, and thereby reduce the photo- and oxidative damages caused by low temperature stress.

[Effects of exogenous salicylic acid on membrane lipid peroxidation and photosynthetic characteristics of Cucumis sativus seedlings under drought stress].

To approach the related mechanisms of exogenous salicylic acid (SA) in improving plant drought-resistance, this paper studied the effects of applying exogenous SA to the rhizosphere on the plant growth, membrane lipid peroxidation, proline accumulation, water use efficiency, net photosynthetic rate (Pn), and chlorophyll fluorescence parameters of cucumber (Cucumis sativus) seedlings under drought stresses (60% and 50% of saturated water capacity). Applying SA relieved the inhibitory effects of drought stress on plant growth, Pn, and water use efficiency, decreased membrane lipid peroxidation, and promoted proline accumulation. Meanwhile, the SA decreased the decrements of the maximum photochemical efficiency of PS II, actual photochemical efficiency of PS II, potential activity of PS II, effective photochemical efficiency of PS II, and photochemical quenching coefficient under drought stress significantly, and limited the increase of non-photochemical quenching coefficient. All the results suggested that applying exogenous SA could alleviate the oxidation damage of cell membrane resulted from the drought-caused membrane lipid peroxidation, improve the Pn by increasing PS II activity to benefit water utilization, enhance the regulation capability of osmosis to decrease water loss and increase water use efficiency, and thereby, improve the plant drought-resistance.

[Roles of spermidine in induced resistance of cucumber seedlings to Botrytis cinerea Pers].

Under the conditions of root substrate injection and foliar spray of different concentration spermidine, this paper measured the disease index, defense enzyme activities, and contents of disease resistance-related substances in cucumber (Cucumis sativus L.) seedlings infected by Botrytis cinerea Pers., aimed to investigate the roles of spermidine in induced resistance of cucumber seedlings to B. cinerea. The results showed that after treated with spermidine, the disease index decreased by 25.4%, phenylalanine ammonia lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO) activities increased by 48.4%, 635.9% and 38.9%, respectively, and total polyphenol, flavonoid and lignin contents increased significantly, compared with the control. All of these suggested that spermidine could induce the disease resistance of cucumber seedlings, and its optimum concentration was 150 mg L(-1).