The CsDof1.8-CsLIPOXYGENASE09 module regulates C9 aroma production in cucumber.

Nine-carbon aldehydes and their relative alcohols (C9 aromas) are the main aroma compounds of cucumber (Cucumis sativus L.) fruits and provide a unique cucumber-like note. However, the key regulators of C9 aroma accumulation in cucumber fruit are poorly characterized. Based on C9 aroma dynamic analysis and transcriptome analysis during fruit development of two different cucumber inbred lines, Q16 and Q24, Lipoxygenase09 (CsLOX09) was identified as a candidate gene for C9 aroma accumulation. Additionally, Q24 with higher CsLOX09 expression accumulated more C9 aromas than Q16. To verify the function of CsLOX09, Cslox09 homozygote knockout lines were created. C9 aroma content decreased by 80.79% to 99.16% in these mutants compared to the wild type. To further explore the reasons for the difference in CsLOX09 expression between Q16 and Q24 fruits, a co-expression network was constructed by integrating the C9 aroma-associated metabolism and transcriptomic data. Eighteen candidate transcription factors were highly correlated with the expression of CsLOX09. DNA binding with One Finger 1.8 (CsDof1.8) was confirmed to bind directly to the A/TAAAG motif of the CsLOX09 promoter through dual-luciferase, yeast one-hybrid, chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. Furthermore, C9 aroma content and CsLOX09 expression were significantly increased in the CsDof1.8 overexpression lines. Overall, these data elucidate the metabolic regulation of C9 aromas in cucumber and provide a foundation for facilitating the regulation of flavor in cucumber breeding.

Cell wall invertase 3 plays critical roles in providing sugars during pollination and fertilization in cucumber.

In plants, pollen-pistil interactions during pollination and fertilization mediate pollen hydration and germination, pollen tube growth, and seed set and development. Cell wall invertases (CWINs) help provide the carbohydrates for pollen development; however, their roles in pollination and fertilization have not been well established. In cucumber (Cucumis sativus), CsCWIN3 showed the highest expression in flowers, and we further examined CsCWIN3 for functions during pollination to seed set. Both CsCWIN3 transcript and CsCWIN3 protein exhibited similar expression patterns in the sepals, petals, stamen filaments, anther tapetum, and pollen of male flowers, as well as in the stigma, style, transmitting tract, and ovule funiculus of female flowers. Notably, repression of CsCWIN3 in cucumber did not affect the formation of parthenocarpic fruit but resulted in an arrested growth of stigma integuments in female flowers and a partially delayed dehiscence of anthers with decreased pollen viability in male flowers. Consequently, the pollen tube grew poorly in the gynoecia after pollination. In addition, CsCWIN3-RNA interference plants also showed affected seed development. Considering that sugar transporters could function in cucumber fecundity, we highlight the role of CsCWIN3 and a potential close collaboration between CWIN and sugar transporters in these processes. Overall, we used molecular and physiological analyses to determine the CsCWIN3-mediated metabolism during pollen formation, pollen tube growth, and plant fecundity. CsCWIN3 has essential roles from pollination and fertilization to seed set but not parthenocarpic fruit development in cucumber.

Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus).

BACKGROUND: Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is a significant threat to cucumber (Cucumis sativus) production in many regions. Previous studies have reported the genetic mapping of loci related to ToLCNDV resistance, but no resistance genes have been identified. RESULTS: We conducted map-based cloning of the ToLCNDV resistance gene in cucumber accession No.44. Agroinfiltration and graft-inoculation analyses confirmed the resistance of No.44 to ToLCNDV isolates from the Mediterranean and Asian countries. Initial mapping involving two rounds of phenotyping with two independent F2 populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44 consistently detected major quantitative trait loci (QTLs) on chromosomes 1 and 2 that confer resistance to ToLCNDV. Fine-mapping of Cy-1, the dominant QTL on chromosome 1, using F3 populations narrowed the candidate region to a 209-kb genomic segment harboring 24 predicted genes. Among these genes, DFDGD-class RNA-dependent RNA polymerase (CsRDR3), an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. The CsRDR3 sequence of No.44 contained multiple amino acid substitutions; the promoter region of CsRDR3 in No.44 had a large deletion; and the CsRDR3 transcript levels were greater in No.44 than in SHF. Virus-induced gene silencing (VIGS) of CsRDR3 using two chromosome segment substitution lines harboring chromosome 1 segments derived from No.44 compromised resistance to ToLCNDV. CONCLUSIONS: Forward and reverse genetic approaches identified CsRDR3, which encodes a DFDGD-class RNA-dependent RNA polymerase, as the gene responsible for ToLCNDV resistance at the major QTL Cy-1 on chromosome 1 in cucumber. Marker-assisted breeding of ToLCNDV resistance in cucumber will be expedited by using No.44 and the DNA markers developed in this study.

Genome-wide identification of Brassinosteroid insensitive 1-associated receptor kinase 1 genes and expression analysis in response to pathogen infection in cucumber (Cucumis sativus L.).

BACKGROUND: BAK1 (Brassinosteroid insensitive 1-associated receptor kinase 1) plays an important role in disease resistance in plants. However, the function of BAK1 family in cucumber and the decisive genes for disease-resistance remain elusive. RESULTS: Here, we identified 27 CsBAK1s in cucumber, and classified them into five subgroups based on phylogenetic analysis and gene structure. CsBAK1s in the same subgroup shared the similar motifs, but different gene structures. Cis-elements analysis revealed that CsBAK1s might respond to various stress and growth regulation. Three segmentally duplicated pairwise genes were identified in cucumber. In addition, Ka/Ks analysis indicated that CsBAK1s were under positive selection during evolution. Tissue expression profile showed that most CsBAK1s in Subgroup II and IV showed constitutive expression, members in other subgroups showed tissue-specific expression. To further explore whether CsBAK1s were involved in the resistance to pathogens, the expression patterns of CsBAK1s to five pathogens (gummy stem blight, powdery mildew, downy mildew, grey mildew, and fusarium wilt) reveled that different CsBAK1s had specific roles in different pathogen infections. The expression of CsBAK1-14 was induced/repressed significantly by five pathogens, CsBAK1-14 might play an important role in disease resistance in cucumber. CONCLUSIONS: 27 BAK1 genes were identified in cucumber from a full perspective, which have important functions in pathogen infection. Our study provided a theoretical basis to further clarify the function of BAK1s to disease resistance in cucumber.

Gain-of-function of the 1-aminocyclopropane-1-carboxylate synthase gene ACS1G induces female flower development in cucumber gynoecy.

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.

A mutation in CsGME encoding GDP-mannose 3,5-epimerase results in little and wrinkled leaf in cucumber.

KEY MESSAGE: Map-based cloning revealed that a mutation in a highly conserved amino acid of the CsGME gene encoding GDP-mannose 3,5-epimerase, causes the phenotype of little and wrinkled leaves in cucumbers. Leaf size is a critical determinant of plant architecture in cucumbers, yet only a few genes associated with this trait have been mapped or cloned. Here, we identified and characterized a mutant with little and wrinkled leaves, named lwl-1. Genetic analysis revealed that the phenotype of the lwl-1 was controlled by a single recessive gene. Through map-based cloning, the lwl-1 locus was narrowed down to a 12.22-kb region exclusively containing one fully annotated gene CsGME (CsaV3_2G004170). CsGME encodes GDP-mannose 3,5-epimerase, which is involved in the synthesis of ascorbic acid (ASA) and one of the components of pectin, RG-II. Whole-length sequencing of the 12.22 kb DNA fragment revealed the presence of only a non-synonymous mutation located in the sixth exon of CsGME in lwl-1, resulting in an amino acid alteration from Pro363 to Leu363. This mutation was unique among 118 inbred lines from cucumber natural populations. CsGME expression significantly reduced in various organs of lwl-1, accompanied by a significant decrease in ASA and pectin content in leaves. Both CsGME and Csgme proteins were localized to the cytoplasm. The mutant phenotype exhibited partial recovery after the application of exogenous boric acid. Silencing CsGME in cucumber through VIGS confirmed its role as the causal gene for lwl-1. Transcriptome profiling revealed that CsGME greatly affected the expression of genes related to the cell division process and cell plate formation. This study represents the first report to characterize and clone the CsGME in cucumber, indicating its crucial role in regulating leaf size and development.

A comprehensive Study of Juglone's Effect on Polyphenol Oxidase in Cucumber: In Vitro Experiments and Computational Docking and Dynamics Insights.

This study explores the impact of juglone on cucumber (Cucumis sativus cv. Beith Alpha), scrutinizing its effects on seed germination, growth, and the polyphenol oxidase (PPO) enzyme's activity and gene expression. Employing concentrations ranging from 0.01 to 0.5 mM, we found juglone's effects to be concentration-dependent. At lower concentrations (0.01 and 0.1 mM), juglone promoted root and shoot growth along with germination, whereas higher concentrations (0.25 and 0.5 mM) exerted inhibitory effects, delineating a threshold for its allelopathic influence. Notably, PPO activity surged, especially at 0.5 mM in roots, hinting at oxidative stress involvement. Real-time PCR unveiled that juglone modulates PPO gene expression in cotyledons, peaking at 0.1 mM and diminishing at elevated levels. Correlation analyses elucidated a positive link between juglone-induced root growth and cotyledon PPO gene expression but a negative correlation with heightened root enzyme activity. Additionally, germination percentage inversely correlated with root PPO activity, while PPO activities positively associated with dopa and catechol substrates in both roots and cotyledons. Molecular docking studies revealed juglone's selective interactions with PPO's B chain, suggesting regulatory impacts. Protein interaction assessments highlighted juglone's influence on amino acid metabolism, and molecular dynamics indicated juglone's stronger, more stable binding to PPO, inferring potential alterations in enzyme function and stability. Conclusively, our findings elucidate juglone's dose-dependent physiological and biochemical shifts in cucumber plants, offering insights into its role in plant growth, stress response, and metabolic modulation.

The synthesis of triacylglycerol by diacylglycerol acyltransferases (CsDGAT1A and CsDGAT2D) is essential for tolerance of cucumber's resistance to low-temperature stress.

KEY MESSAGE: CsDGAT1A and CsDGAT2D play a positive regulatory role in cucumber's response to low-temperature stress and positively regulate the synthesis of triacylglycerol (TAG). Triacylglycerol (TAG), a highly abundant and significant organic compound in plants, plays crucial roles in plant growth, development, and stress responses. The final acetylation step of TAG synthesis is catalyzed by diacylglycerol acyltransferases (DGATs). However, the involvement of DGATs in cucumber's low-temperature stress response remains unexplored. This study focused on two DGAT genes, CsDGAT1A and CsDGAT2D, investigating their function in enhancing cucumber's low-temperature stress tolerance. Our results revealed that both proteins were the members of the diacylglycerol acyltransferase family and were predominantly localized in the endoplasmic reticulum. Functional analysis demonstrated that transient silencing of CsDGAT1A and CsDGAT2D significantly compromised cucumber's low-temperature stress tolerance, whereas transient overexpression enhanced it. Furthermore, the TAG content quantification indicated that CsDGAT1A and CsDGAT2D promoted TAG accumulation. In conclusion, this study elucidates the lipid metabolism mechanism in cucumber's low-temperature stress response and offers valuable insights for the cultivation of cold-tolerant cucumber plants.

Genome-Wide Characterization of Fructose 1,6-Bisphosphate Aldolase Genes and Expression Profile Reveals Their Regulatory Role in Abiotic Stress in Cucumber.

The fructose-1,6-bisphosphate aldolase (FBA) gene family exists in higher plants, with the genes of this family playing significant roles in plant growth and development, as well as response to abiotic stresses. However, systematic reports on the FBA gene family and its functions in cucumber are lacking. In this study, we identified five cucumber FBA genes, named CsFBA1-5, that are distributed randomly across chromosomes. Phylogenetic analyses involving these cucumber FBAs, alongside eight Arabidopsis FBA proteins and eight tomato FBA proteins, were conducted to assess their homology. The CsFBAs were grouped into two clades. We also analyzed the physicochemical properties, motif composition, and gene structure of the cucumber FBAs. This analysis highlighted differences in the physicochemical properties and revealed highly conserved domains within the CsFBA family. Additionally, to explore the evolutionary relationships of the CsFBA family further, we constructed comparative syntenic maps with Arabidopsis and tomato, which showed high homology but only one segmental duplication event within the cucumber genome. Expression profiles indicated that the CsFBA gene family is responsive to various abiotic stresses, including low temperature, heat, and salt. Taken together, the results of this study provide a theoretical foundation for understanding the evolution of and future research into the functional characterization of cucumber FBA genes during plant growth and development.

A mutation in CsHY2 encoding a phytochromobilin (PΦB) synthase leads to an elongated hypocotyl 1(elh1) phenotype in cucumber (Cucumis sativus L.).

KEY MESSAGE: The elongated hypocotyl1 (elh1) mutant in cucumber is due to a mutation in CsHY2, which is a homolog of the Arabidopsis HY2 encoding the phytochromobilin (PΦB) synthase for phytochrome biosynthesis Hypocotyl length is a critical determinant in establishing high quality seedlings for successful cucumber production, but knowledge on the molecular regulation of hypocotyl growth in cucumber is very limited. Here, we reported identification and characterization of a cucumber elongated hypocotyl 1 (elh1) mutant. We found that the longer hypocotyl in elh1 was due to longitudinal growth of hypocotyl cells. With fine mapping, the elh1 locus was delimited to a 20.9-kb region containing three annotated genes; only one polymorphism was identified in this region between two parental lines, which was a non-synonymous SNP (G28153633A) in the third exon of CsHY2 (CsGy1G030000) that encodes a phytochromobilin (PΦB) synthase. Uniqueness of the mutant allele at CsHY2 was verified in natural cucumber populations. Ectopic expression of CsHY2 in Arabidopsis hy2-1 long-hypocotyl mutant led to reduced hypocotyl length. The PΦB protein was targeted to the chloroplast. The expression levels of CsHY2 and five phytochrome genes CsPHYA1, CsPHYA2, CsPHYB, CsPHYC and CsPHYE were all significantly down-regulated while several cell elongation related genes were up-regulated in elh1 mutant compared to wild-type cucumber, which are correlated with dynamic hypocotyl elongation in the mutant. RNA-seq analysis in the WT and mutant revealed differentially expressed genes involved in porphyrin and chlorophyll metabolisms, cell elongation and plant hormone signal transduction pathways. This is the first report to characterize and clone the CsHY2 gene in cucumber. This work reveals the important of CsHY2 in regulating hypocotyl length and extends our understanding of the roles of CsHY2 in cucumber.

Identification of a putative candidate gene encoding 7-dehydrocholesterol reductase involved in brassinosteroids biosynthesis for compact plant architecture in Cucumber (Cucumis sativus L.).

KEY MESSAGE: By the strategy of bulked segregant analysis sequencing combined with genetic mapping, CsDWF5, which encodes 7 dehydrocholesterol reductase that involved in brassinosteroids biosynthesis, was identified as the candidate gene for cpa. Dwarf architecture is one of the most important breeding goals in crops. The biosynthesis and signal transduction of brassinosteroids (BRs) have a great impact on plant growth and development including plant architecture. Here, we identified a compact plant architecture (cpa) mutant from an EMS-induced cucumber population. cpa displayed the extremely dwarf phenotype with shortened internode and petiole, darkened and wrinkled leaf. Genetic analysis revealed that cpa was caused by a single recessive gene. By the strategy of bulked segregant analysis sequencing combined with genetic mapping, CsDWF5, encoding a 7-dehydrocholesterol reductase that involved in sterol biosynthesis, was identified as the candidate gene for cpa. One single nucleotide mutation (G→A) in splicing site causing 3-bp insertion (TAG) was found in the first base of the sixth intron of CsDWF5 in cpa, which furtherly resulted in the frameshift mutation and got a premature stop codon. The expression of CsDWF5 gene was significantly down regulated in different tissues of the cpa mutant compared with that in wild type. The phenotype of cpa could be partially recovered by exogenous BR treatment. Transcriptome analysis identified 1096 genes that exhibited differential expression between the cpa mutant and wild type. KEGG enrichment analysis indicated that differentially expressed genes were significantly enriched in BR biosynthesis and plant-pathogen interaction pathways. These results provide perspectives on the molecular mechanisms underlying the dwarfing phenotype in cucumber.

Mapping and identification of CsSh5.1, a gene encoding a xyloglucan galactosyltransferase required for hypocotyl elongation in cucumber (Cucumis sativus L.).

KEY MESSAGE: CsSh5.1, which controls hypocotyl elongation under high temperature conditions in cucumber, was mapped to a 57.1 kb region on chromosome 5 containing a candidate gene encoding a xyloglucan galactosyltransferase. Hypocotyl growth is a vital process in seedling establishment. Hypocotyl elongation after germination relies more on longitudinal cell elongation than cell division. Cell elongation is largely determined by the extensibility of the cell wall. Here, we identified a spontaneous mutant in cucumber (Cucumis sativus L.), sh5.1, which exhibits a temperature-insensitive short hypocotyl phenotype. Genetic analysis showed that the phenotype of sh5.1 was controlled by a recessive nuclear gene. CsSh5.1 was mapped to a 57.1 kb interval on chromosome 5, containing eight predicted genes. Sequencing analysis revealed that the Csa5G171710 is the candidate gene of CsSh5.1, which was further confirmed via co-segregation analysis and genomic DNA sequencing in natural cucumber variations. The result indicated that hypocotyl elongation might be controlled by this gene. CsSh5.1 encodes a xyloglucan galactosyltransferase that specifically adds galactose to xyloglucan and forms galactosylated xyloglucans, which determine the strength and extensibility of the cell walls. CsSh5.1 expression in wild-type (WT) hypocotyl was significantly higher than that in sh5.1 hypocotyl under high temperature, suggesting its important role in hypocotyl cell elongation under high temperature. The identification of CsSh5.1 is helpful for elucidating the function of xyloglucan galactosyltransferase in cell wall expansion and understanding the mechanism of hypocotyl elongation in cucumber.

CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits.

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after ß-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Genome-Wide Identification, Diversification, and Expression Analysis of Lectin Receptor-Like Kinase (LecRLK) Gene Family in Cucumber under Biotic Stress.

Members of the lectin receptor-like kinase (LecRLKs) family play a vital role in innate plant immunity. Few members of the LecRLKs family have been characterized in rice and Arabidopsis, respectively. However, little literature is available about LecRLKs and their role against fungal infection in cucumber. In this study, 60 putative cucumber LecRLK (CsLecRLK) proteins were identified using genome-wide analysis and further characterized into L-type LecRLKs (24) and G-type LecRLKs (36) based on domain composition and phylogenetic analysis. These proteins were allocated to seven cucumber chromosomes and found to be involved in the expansion of the CsLecRLK gene family. Subcellular localization of CsaLecRLK9 and CsaLecRLK12 showed green fluorescence signals in the plasma membrane of leaves. The transcriptional profiling of CsLecRLK genes showed that L-type LecRLKs exhibited functional redundancy as compared to G-type LecRLKs. The qRT-PCR results indicated that both L- and G-type LecRLKs showed significant response against plant growth-promoting fungi (PGPF-Trichoderma harzianum Rifai), powdery mildew pathogen (PPM-Golovinomyces orontii (Castagne) V.P. Heluta), and combined (PGPF+PPM) treatments. The findings of this study contribute to a better understanding of the role of cucumber CsLecRLK genes in response to PGPF, PPM, and PGPF+PPM treatments and lay the basis for the characterization of this important functional gene family.

A Vacuolar Invertase CsVI2 Regulates Sucrose Metabolism and Increases Drought Tolerance in Cucumis sativus L.

Vacuolar invertase (VI) can irreversibly degrade sucrose into glucose and fructose and involve in plants abiotic-stress-tolerance. Cucumber (Cucumis sativus L.) is susceptible to drought stress, especially during the seedling stage. To date, the involvement of VI in drought tolerance in cucumber seedlings is in urgent need of exploration. In the present study, a cucumber vacuolar invertase gene, CsVI2, was isolated and functionally characterized. The results showed that (1) CsVI2 showed vacuolar invertase activity both in vivo and in vitro; (2) the transcript level of CsVI2, along with VI activity, was significantly induced by drought stress. Moreover, the expression of sucrose synthase 3 (CsSUS3) was increased and that of sucrose phosphate synthase 1 (CsSPS1) was decreased after exposure to drought stress, which was followed by an increase in sucrose synthase activity and a decrease in sucrose phosphate synthase activity; (3) CsVI2-overexpressing transformed cucumber seedlings showed enhanced vacuolar invertase activity and drought tolerance and 4) protein-protein interaction modelling indicated that a cucumber invertase inhibitor, CsINVINH3, can interact with CsVI2. In summary, the results indicate that CsVI2 as an invertase can regulate sucrose metabolism and enhance drought stress in cucumber seedlings.

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.

NO is involved in H2-induced adventitious rooting in cucumber by regulating the expression and interaction of plasma membrane H+-ATPase and 14-3-3.

MAIN CONCLUSION: NO was involved in H2-induced adventitious rooting by regulating the protein and gene expressions of PM H+-ATPase and 14-3-3. Simultaneously, the interaction of PM H+-ATPase and 14-3-3 protein was also involved in this process. Hydrogen gas (H2) and nitric oxide (NO) have been shown to be involved in plant growth and development. The results in this study revealed that NO was involved in H2-induced adventitious root formation. Western blot (WB) analysis showed that the protein abundances of plasma membrane H+-ATPase (PM H+-ATPase) and 14-3-3 protein were increased after H2, NO, H2 plus NO treatments, whereas their protein abundances were down regulated when NO scavenger carboxy-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTI O) was added. Moreover, the mRNA abundances of the HA3 and 14-3-3(7) gene as well as the activities of PM H+-ATPase (EC 3.6.1.35) and H+ pump were in full agreement with the changes of protein abundance. Phosphorylation of PM H+-ATPase and the interaction of PM H+-ATPase and 14-3-3 protein were detected by co-immunoprecipitation analysis. H2 and NO significantly up regulated the phosphorylation of PM H+-ATPase and the interaction of PM H+-ATPase and 14-3-3 protein. Conversely, the stimulation of PM H+-ATPase phosphorylation and protein interaction were significantly diminished by cPTIO. Protein interaction activator fusicoccin (FC) and inhibitor adenosine monophosphate (AMP) of PM H+-ATPase and 14-3-3 were used in this study, and the results showed that FC significantly increased the abundances of PM H+-ATPase and 14-3-3, while AMP showed opposite trends. We further proved the critical roles of PM H+-ATPase and 14-3-3 protein interaction in NO-H2-induced adventitious root formation. Taken together, our results suggested that NO might be involved in H2-induced adventitious rooting by regulating the expression and the interaction of PM H+-ATPase and 14-3-3 protein.

Responses of antioxidative enzymes and gene expression in Oryza sativa L and Cucumis sativus L seedlings to microcystins stress.

Microcystins (MCs) have become an important global environmental issue, causing oxidative stress, which is an important toxic mechanism for MCs in plants. However, the regulating mechanism of antioxidative enzymes in plants in adapting to MCs stress remains unclear. We studied the dynamic effects of MCs at different concentrations (5, 10, 50 and 100 µg/L) in rice and cucumber seedlings on relative growth rate (RGR), and reactive oxygen species and malondialdehyde (MDA) content, and antioxidative enzyme activities, during a stress period (MCs exposed for 1, 3, 5 and 7 d) and recovery period (7 d). During the stress period, MCs at 5 µg/L inhibited RGR in cucumber and promoted RGR in rice. The contents of superoxide anion (O2·-), hydrogen peroxide (H2O2) and MDA increased and RGR declined in both crops with time and intensity of MCs stress. For cucumber, all these parameters responded earlier to MCs stress, and O2·-, MDA and RGR were more responsive to MCs stress than in rice. Moreover, catalase (CAT) and peroxidase (POD), and the relative expressions of CAT genes increased in both crops at 5-100 µg/L MCs, whereas relative expression of POD genes increased only in cucumber. Diversely, superoxide dismutase (SOD) response to MCs in cucumber leaves was later than for rice. MCs at 100 µg/L decreased the relative expression of SOD genes in cucumber but did not change SOD activity. During the recovery period, all the above indicators in both crops were higher than the control and lower than in the stress period. Conversely, RGR was lower than in the control and higher than in the stress period, except for cucumber which was lower, and MDA content higher than the stress period at 100 µg/L MCs. Overall, these results indicated that cucumber was more sensitive to MCs than rice, and SOD, CAT and POD play an important role in plant response to MCs stress.

A Mutation in CsYL2.1 Encoding a Plastid Isoform of Triose Phosphate Isomerase Leads to Yellow Leaf 2.1 (yl2.1) in Cucumber (Cucumis Sativus L.).

The leaf is an important photosynthetic organ and plays an essential role in the growth and development of plants. Leaf color mutants are ideal materials for studying chlorophyll metabolism, chloroplast development, and photosynthesis. In this study, we identified an EMS-induced mutant, yl2.1, which exhibited yellow cotyledons and true leaves that did not turn green with leaf growth. The yl2.1 locus was controlled by a recessive nuclear gene. The CsYL2.1 was mapped to a 166.7-kb genomic region on chromosome 2, which contains 24 predicted genes. Only one non-synonymous single nucleotide polymorphism (SNP) was found between yl2.1 and wt-WD1 that was located in Exon 7 of Csa2G263900, resulting in an amino acid substitution. CsYL2.1 encodes a plastid isoform of triose phosphate isomerase (pdTPI), which catalyzes the reversible conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) in chloroplasts. CsYL2.1 was highly expressed in the cotyledons and leaves. The mesophyll cells of the yl2.1 leaves contained reduced chlorophyll and abnormal chloroplasts. Correspondingly, the photosynthetic efficiency of the yl2.1 leaves was impaired. Identification of CsYL2.1 is helpful in elucidating the function of ptTPI in the chlorophyll metabolism and chloroplast development and understanding the molecular mechanism of this leaf color variant in cucumber.

Zinc and Copper Enhance Cucumber Tolerance to Fusaric Acid by Mediating Its Distribution and Toxicity and Modifying the Antioxidant System.

Fusaric acid (FA), the fungal toxin produced by Fusarium oxysporum, plays a predominant role in the virulence and symptom development of Fusarium wilt disease. As mineral nutrients can be protective agents against Fusarium wilt, hydroponic experiments employing zinc (Zn) and copper (Cu) followed by FA treatment were conducted in a glasshouse. FA exhibited strong phytotoxicity on cucumber plants, which was reversed by the addition of Zn or Cu. Thus, Zn or Cu dramatically reduced the wilt index, alleviated the leaf or root cell membrane injury and mitigated against the FA inhibition of plant growth and photosynthesis. Cucumber plants grown with Zn exhibited decreased FA transportation to shoots and a 17% increase in toxicity mitigation and showed minimal hydrogen peroxide, lipid peroxidation level with the increased of antioxidant enzymes activity in both roots and leaves. Cucumber grown with additional Cu absorbed less FA but showed more toxicity mitigation at 20% compared to with additional Zn and exhibited decreased hydrogen peroxide level and increased antioxidant enzymes activity. Thus, adding Zn or Cu can decrease the toxicity of the FA by affecting the absorption or transportation of the FA in plants and mitigate toxicity possibly through chelation. Zn and Cu modify the antioxidant system to scavenge hydrogen peroxide for suppressing FA induction of oxidative damage. Our experiments could provide a theoretical basis for the direct application of micro-fertilizer as protective agents in farming.