Leucine and spermidine enhance shoot differentiation in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Enhanced numbers of multiple shoots were induced from shoot tip explants of cucumber. The effects of amino acids (leucine, isoleucine, methionine, threonine, and tryptophan) and polyamines (spermidine, spermine, and putrescine) along with benzyladenine (BA) on multiple shoot induction were investigated. A Murashige and Skoog (MS) medium containing a combination of BA (4.44 μM), leucine (88 μM), and spermidine (68 μM) induced the maximum number of shoots (36.6 shoots per explant) compared to BA (4.44 μM) alone or BA (4.44 μM) with leucine (88 μM). The regenerated shoots were elongated on the same medium. Elongated shoots were transferred to the MS medium fortified with BA (4.44 μM), leucine (88 μM), and putrescine (62 μM) for root induction. Rooted plants were hardened and successfully established in soil with a 90% survival rate.     

<Emphasis Type="Italic">p</Emphasis>-Chlorophenoxyisobutyric acid impairs auxin response for gravity-regulated peg formation in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) seedlings

Cucumber (Cucumis sativus L.) seedlings form a specialized protuberance, the peg, on the transition zone between the hypocotyl and the root. When cucumber seeds germinate in a horizontal position, the seedlings develop a peg on the lower side of the transition zone. To verify the role of auxin action in peg formation, we examined the effect of the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB), on peg formation and mRNA accumulation of auxin-regulated genes. Application of PCIB to cucumber seedlings inhibited peg formation. The application of indole-3-acetic acid (IAA) competed with PCIB and induced peg formation. Furthermore, application of PCIB decreased auxin-inducible CsIAA1 mRNA and increased auxin-repressible CsGRP1 mRNA in the lower side of the transition zone. The differential accumulation of CsIAA1 and CsGRP1 mRNAs in the transition zone of cucumber seedlings grown in a horizontal position was smaller in the PCIB-treated seedlings. These results demonstrate that endogenous auxin redistributes and induces the differential expression of auxin-regulated genes, and ultimately results in the suppression or induction of peg formation in the gravistimulated transition zone of cucumber seedlings.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Molecular cloning and structural analysis of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) phosphoenolpyruvate carboxykinase (<Emphasis Type="Italic">CsPCK</Emphasis>) gene

Genomic sequence of the ATP-dependent phosphoeno/pyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing to ascertain the structure of theCsPCK gene. Analysis of a selected positive clone (λcscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns, spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5′-noncoding region of cucumberPCK cDNA, whereas Exon 2 comprises 12 nucleotides of the S′-noncoding region with an N-terminal PEPCK coding sequence. All the exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of theCsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for bothArabidopsis Chromosome 4 (Atg4)PCK and the rice PCX genes, which contain 13 and 12 exons, respectively. Two additionalArabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively) of the PEPCK peptide. TheCsPCK gene promoter has conserved plant-specific as-acting elements within 2 kb of the 5’ flanking region. Several common cis-acting elements of the isocitrate lyase (icl) and malate synthase(ms) gene promoters, identified in theCsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements are discussed here.     

Proanthocyanidins accelerate the germination of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seeds

Proanthocyanidins (PAs) are the end products of the flavonoid biosynthetic pathway in many seeds, but their biological function is rarely unknown during seed germination. In the present study, we observed that PAs pretreatment accelerated cucumber seeds germination with maximum efficiency at 0.15% by measuring germination percentage and radical length. Using inhibitors of abscisic acid (ABA), gibberellins (GA) and alternative oxidase (AOX) and H₂O₂ scavenger pretreatment and gene expression analysis, we found that the accelerated effect of 0.15% PAs on seed germination was due to the decreased ABA biogenesis and enhanced GA production. ROS are induced by PAs pretreatment. Then, the enhanced ROS contributed to GA and ethylene accumulation and ABA decrease in seeds. Moreover, the improvement of GA was involved in the further induction of antioxidant enzymes activities. Therefore, our findings uncover a novel role of PAs in seed germination and clarify the relationships between ROS, ABA, GA and ethylene during seed germination.     

Effect of root applied 24-epibrassinolide on carbohydrate status and fermentative enzyme activities in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) seedlings under hypoxia

The effects of 24-epibrassinolide (EBR) added to nutrient solution on growth of cucumber (Cucumis sativus L.) under root-zone hypoxia were investigated. Cucumber seedlings were hydroponically grown for 8 days in normoxic and hypoxic nutrient solutions with and without addition of EBR at 1 μg l⁻¹. EBR exerted little influence on plant performance in the normoxic nutrient solution, while the chemical alleviated root-zone hypoxia-induced inhibition of root and shoot growth and net photosynthetic rate (Pn). EBR added to hypoxic nutrient solution caused an increase in the concentration of fructose, sucrose, and total soluble sugars in the roots but not in the leaves. Root-zone hypoxia enhanced the activities of lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and pyruvate decarboxylase in the roots. Interestingly, EBR further enhanced ADH activity but lowered LDH activity in hypoxic roots. These results suggest that EBR added to hypoxic nutrient solution may stimulate the photosynthate allocation down to roots and the shift from lactate fermentation to alcohol fermentation in hypoxic roots, resulting in the increase in ATP production through glycolysis and the avoidance of cytosolic acidosis and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

Fine mapping of a male sterility gene <Emphasis Type="Italic">ms</Emphasis>-<Emphasis Type="Italic">3</Emphasis> in a novel cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) mutant

Key message

The cucumber male sterility gene ms - 3 was fine mapped in a 76 kb region harboring an MMD1 -like gene Csa3M006660 that may be responsible for the male sterile in cucumber.

Abstract

A cucumber (Cucumis sativus L.) male sterile mutant (ms-3) in an advanced-generation inbred line was identified, and genetic analysis revealed that the male sterility trait was controlled by a recessive nuclear gene, ms-3, which was stably inherited. Histological studies suggested that the main cause of the male sterility was defective microsporogenesis, resulting in no tetrad or microspores being formed. Bulked segregant analysis (BSA) and genotyping of an F₂ population of 2553 individuals were employed used to fine map ms-3, which was delimited to a 76 Kb region. In this region, a single non-synonymous SNP was found in the Csa3M006660 gene locus, which was predicted to result in an amino acid change. Quantitative RT-PCR analysis of Csa3M006660 was consistent with the fact that it plays a role in the early development of cucumber pollen. The protein encoded by Csa3M006660 is predicted to be homeodomain (PHD) finger protein, and the high degree of sequence conservation with homologs from a range of plant species further suggested the importance of the ms-3 non-synonymous mutation. The data presented here provide support for Csa3M006660 as the most likely candidate gene for Ms-3.

     

Identification and transcriptional analysis of dehydrin gene family in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Dehydrins (DHNs) are a group II late embryogenesis abundant (LEA) proteins that play essential roles in plant growth, development and responses to diverse environmental stimuli. Here, four DHNs in cucumber genome were identified using bioinformatics-based methods according to the highly conserved K-, Y- and S-segments, including 1 Y_nK_n-type, 2 Y_nSK_n-type, and 1 SK_n-type DHNs. All of them are intrinsically disordered proteins (IDPs) and possess a large number of disorder-promoting amino acids. Secondary structure prediction revealed that each of them is composed of high proportion of alpha helix and random coil. Gene structure and phylogenetic analyses with DHNs from cucumber and several other species revealed that some closely related DHN genes had similar gene structures. A number of cis-elements involved in stress responses and phytohormones were found in each CsDHN promoter. The tissue expression profiles suggested that the CsDHN genes have overlapping, but different expression patterns. qRT-PCR results showed that three selected CsDHN genes could respond to heat, cold, osmotic and salt stresses, as well as to signaling molecules such as H₂O₂ and ABA. These results lay a solid foundation for future functional investigation of the cucumber dehydrin gene family in tissue development and stress responses in plants.     

Molecular characterization and isolation of the <Emphasis Type="Italic">F/f</Emphasis> gene for femaleness in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

The biological processes leading to sex expression in plants are of tremendous practical significance for fruit production of many agricultural and horticultural crops. Sex-expression studies in cucumber showed that the different sex types are determined by three major genes: M/m, F/f and A/a. The M/m gene in the dominant condition suppresses stamina development and thus leads to female flowers. The F/f gene in the dominant condition shifts the monoecious sex pattern downwards and promotes femaleness by causing a higher level of ethylene in the plant. To investigate the molecular character of the gene F/f, we used nearly isogenic gynoecious (MMFF) and monoecious (MMff) lines (NIL) produced by our own backcross programme. Our investigations confirmed the result of other groups that an additional genomic ACC synthase (key enzyme of ethylene biosynthesis) sequence (CsACS1G) should exist in gynoecious genotypes. A linkage was also verified between the F/f locus and the CsACS1G sequence with our plant material. After the exploration of different Southern hybridization patterns originating from different CsACS1 probes, a restriction map of the CsACS1 locus was constructed. By using this restriction map, the duplication of the CsACS1 gene and following mutation of the CsACS1G gene could be explained. The promoter regions of the genes CsACS1G and CsACS1 were amplified in a splinkerette PCR and sequenced. An exclusive amplification of the new isolated sequence (CsACS1G) in gynoecious (MMFF) and sub-gynoecious (MMFf) genotypes confirmed that the isolated gene is the dominant F allele.     

A mutant in the <Emphasis Type="Italic">CsDET2</Emphasis> gene leads to a systemic brassinosteriod deficiency and <Emphasis Type="Italic">super compact </Emphasis>phenotype in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Key message

A novel dwarf cucumber mutant, scp-2, displays a typical BR biosynthesis-deficient phenotype, which is due to a mutation in CsDET2 for a steroid 5-alpha-reductase.

Abstract

Brassinosteroids (BRs) are a group of plant hormones that play important roles in the development of plant architecture, and extreme dwarfism is a typical outcome of BR-deficiency. Most cucumber (Cucumis sativus L.) varieties have an indeterminate growth habit, and dwarfism may have its value in manipulation of plant architecture and improve production in certain production systems. In this study, we identified a spontaneous dwarf mutant, super compact-2 (scp-2), that also has dark green, wrinkle leaves. Genetic analyses indicated that scp-2 was different from two previously reported dwarf mutants: compact (cp) and super compact-1 (scp-1). Map-based cloning revealed that the mutant phenotype was due to two single nucleotide polymorphism and a single-base insertion in the CsDET2 gene that resulted in a missense mutation in a conserved amino acid and thus a truncated protein lacking the conserved catalytic domains in the predicted steroid 5α-reductase protein. Measurement of endogenous hormone levels indicated a reduced level of brassinolide (BL, a bioactive BR) in scp-2, and the mutant phenotype could be partially rescued by the application of epibrassinolide (EBR). In addition, scp-2 mutant seedlings exhibited dark-grown de-etiolation, and defects in cell elongation and vascular development. These data support that scp-2 is a BR biosynthesis-deficient mutant, and that the CsDET2 gene plays a key role in BR biosynthesis in cucumber. We also described the systemic BR responses and discussed the specific BR-related phenotypes in cucumber plants.

     

Identification and functional characterization of <Emphasis Type="Italic">APRR2</Emphasis> controlling green immature fruit color in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Nitrogen (N) is a macronutrient essential for plant growth and development. Meanwhile, grafting is a method used to alleviate stress tolerance of various biotic and abiotic factors. This study aims to investigate how pumpkin grafting (PG) improves N use efficiency of watermelon. A commercial watermelon cultivar “Zaojia 8424” [Citrullus lanatus (Thunb.) Matsum. and Nakai.] was self-grafted and then grafted onto pumpkin (Cucurbita maxima?×C. moschata) rootstock cv. Qingyan Zhenmu No. 1. The grafted plants were exposed to two levels of N (9 and 0.2 mM) under hydroponic conditions. The grafted plants were harvested at days 11 and 22 after low N (0.2 mM) treatment. PG improved the N use efficiency of watermelon scion through the vigorous root system of pumpkin rootstock that enhanced the uptake and accumulation of N, P, K, Ca, Mg, B, and Mn in watermelon. Gene expressions of nitrate reductase (Cla002787, Cla002791, and Cla023145) and nitrite reductase (Cla013062) genes were increased, promoting N assimilation. Mesophyll thickness and SPAD index (relative chlorophyll measurement) were also improved. Furthermore, pumpkin rootstock also enhanced the supply of zeatine riboside (ZR) and isopentenyl adenosine (iPA) in the leaves, promoting shoot growth. All these lead to improved plant growth and nitrogen use efficiency of pumpkin rootstock-grafted watermelon plants.     

Effects of ultraviolet-B irradiation on the cuticular wax of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) cotyledons

Cucumber seedlings were grown under three doses of supplemental ultraviolet-B (UV-B) irradiation to examine the effects on the surface structure of the cotyledons. Medium and high doses of irradiation induced glazing (formation of translucent, glossy layers) on the adaxial surfaces of cotyledons, especially those exposed to a high dose of UV-B. Observation with a scanning electron microscope revealed that the adaxial surfaces of cotyledons exposed to a medium dose of UV-B and controls became rough in appearance, but unevenness of the surface was not apparent in cotyledons irradiated with a high dose of UV-B. UV-B irradiation affected the types and amounts of alkanes and primary alcohols, the main components of cucumber cuticular wax. Based on cotyledon area, the amounts of these components were significantly higher in cotyledons irradiated with a medium dose of UV-B than in controls. This effect could be a consequence of small cotyledon area and constant wax production in the cotyledons irradiated with a medium dose of UV-B. The distribution patterns of homologs within the alkane and primary alcohol fractions shifted during growth to longer alkyl chain length in the control cotyledons. UV-B irradiation repressed these changes, suggesting that UV-B acts on cuticular wax biosynthetic pathways.     

Salicylic acid and phenolic compounds under cadmium stress in cucumber plants (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

In this pot experiment, cucumbers (Cucumis sativus L.) were grown in a model soil contaminated by three different concentrations of cadmium (40, 160, and 320 mg.kg^?1) with different accompanied anions (Cl^?, SO₄ ^2?). In all variants, the most Cd (90 %) was accumulated in the roots, but higher content in the case of Cl^?. The distribution of Cd in various cucumber organs was as follows: root > stem > leaf > fruits. However, in variants with higher doses of Cd with SO₄ ^2?, the ratio was changed as follows: root > leaf > stem > fruits. In all variants, least of Cd (max. 1 %) was found in fruits. Variants with the highest Cd doses were significantly different by comparison with all other variants, but higher content was in the case of Cl^? anion. Stimulation effect on the biomass production and growth of aerial parts and roots of plants in all variants with Cd was observed. Toxicity symptoms, mainly in the presence of leaf chlorosis and yellowing, were more visible in the variants with Cl^?, in comparison with SO₄ ^2?. The amounts of phenol compounds in leaves rose almost in all variants. Only the variants with higher Cd content with SO₄ ^2? showed slight reduction. One possible explanation of reduced content may be their bounding on Cd. The content of salicylic acid was reduced in all variants with Cd treatment. However, it is difficult to conclude their role in plant defence responses to heavy metal, because their actual defence mechanism is still unclear. However, from these results, we can suggest that the accompanying anion and the form in which Cd exists may have an impact on the involvement of various antioxidant systems.     

Long-term suspension cultures of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) with high embryogenic potential

Cucumber (Cucumis sativus L.) cytokinin-independent embryogenic cell suspension cultures were derived and maintained for more than 3.5 years without losing the embryogenic potential. The preparation and the characteristics of the cucumber embryogenic cell suspension possess many similarities to that of carrot. The cultures were induced from hypocotyl explants of in vitro grown cucumber plants in liquid MS media containing 2,4-dichlorophenoxyacetic acid as the sole growth regulator during 6 weeks and they contained a heterogeneous array of several different types of single cells and cell clusters (PEMs). The established cell suspensions were subcultured in 1-week interval, while the inoculation density was optimized to 2.0 × 10⁵ cells ml⁻¹ using cell viability as a marker. Somatic embryos were obtained after the transfer of the proembryogenic masses to a hormone-free semisolid MS medium with a frequency of 388 ± 57 somatic embryos per 1 ml of packed cell volume of the established cucumber embryogenic culture within 7 days. The frequency of normal somatic embryos with two cotyledons was found to be 78%. Such embryos possessed the potential of spontaneous maturation and the embryo conversion rates were 87%. The yield of normally growing plants was much higher compared with that previously described for cucumber systems. Somatic embryo-derived plants were successfully transferred to the greenhouse where they flowered and fruited.