Involvement of nitric oxide in water stress-induced responses of cucumber roots

The effect of water deficit on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) seedling roots using bio-imaging with an NO-selective fluorophor, diaminofluorescein-2-diacetate (DAF-2DA). Roots subjected to mild (5 and 10 h) water deficit showed slightly enhanced NO synthesis in cells of root tips and in the surrounding elongation zone. However, severe (17 h) stress resulted in an intensive NO production localized mainly in and above the elongation zone. Water stress-induced NO generation was blocked by a specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as well as nitrate reductase (NR) and partially by nitric oxide synthase (NOS-like) inhibitors.A pharmacological approach was used in order to verify the capacity of NO to induce adaptive responses of cucumber roots to water deficit. A positive correlation was found between NO donor (SNP 100 μM and GSNO 100 μM) pretreatment and plant hydration status, measured as relative water content (RWC) during progressive dehydration. At an early stage (5 h) of stress duration NO caused a periodical increase in lipoxygenase (LOX) activity, correlated with time-dependent enhancement of lipid peroxidation. Beginning from 10 h up to severe stress (17 h) exogenous NO was able to diminish LOX activity and alleviate water deficit-induced membrane permeability and lipid peroxidation, measured as TBARS content and visualised by histochemical staining in situ. Observed changes via NO were accompanied by a significant reduction of proline level, suggesting that the accumulation of this osmolyte might not be essential in water stress tolerance. Obtained results clearly indicate that NO augmentation is likely to help the plant at the initial stage of tissue dehydration to trigger efficient mechanisms, mitigating severe water deficit effects in roots of cucumber seedlings.     

Interaction Between Polyamine and Nitric Oxide Signaling in Adaptive Responses to Drought in Cucumber

The effect of polyamines (PAs) on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) primary leaves using bio-imaging with an NO-selective fluorophor, DAF-2DA. Seedlings pretreated with PAs and subjected to water deficit showed early (after 5 h) and transient NO production. The amplitude of the response depended on the form of the applied polyamine. Spermine (1.0 mM) and spermidine (1.0 mM) induced higher NO-dependent fluorescence compared with putrescine (1.0 mM) and the control. The NO production was blocked by tungstate, an inhibitor of nitrate reductase, and partially by an inhibitor of nitric oxide synthase (NOS-like) enzymes. NO donor administration preceding drought had no effect on endogenous PA levels but was positively correlated with an alleviation of water deficit-induced membrane permeability and lipid peroxidation. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a membrane-permeable NO scavenger, markedly reversed the NO donor effects. Similarly, pretreating seedlings with PAs resulted in lower ion leakage from the membrane and modified lipid peroxidation. The results indicate that NO may act downstream of PAs in cucumber seedlings under water stress.     

黄瓜抗黑星病相关基因的差异表达分析

以抗黑星病黄瓜材料HX1为试材,接种黑星病菌（Cladosporium cucumerinum）2h、8h、20h、32h和72h的叶片作为试验方（Tester）,相应的未接种叶片作为对照方（Driver）,利用SSH技术,构建了黑星病菌侵染初期的正向和反向cDNA-SSH文库。用巢式引物PCR检测插入片段,获得了200个阳性克隆,通过测序,除去重复序列,共得到105个Unique ESTs。与非冗余蛋白数据库进行BLASTx比对,结果显示,17条ESTs未找到同源序列,88条非重复序列和已知基因的同源性较高,占全部ESTs序列的83.8%,其中86条ESTs与非冗余蛋白数据库已知功能的蛋白具有高度的相似性。结合高密度点阵膜杂交差异筛选,阳性率为75.0%。经初步分析这些序列的功能,差异表达的ESTs功能涉及能量和基础代谢、信号转导、蛋白和核酸代谢、光合作用及逆境中特异表达的基因等方面。为研究黄瓜抗黑星病基因提供了依据。     

Hydrogen peroxide involvement in formation and development of adventitious roots in cucumber

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems. The present study demonstrates that H₂O₂ was generated in seedling explants after the primary roots were removed, and it mediates the auxin response prior to adventitious root formation in cucumber (Cucumis sativus L. Ganfeng 8). When compared with the controls, treatment of cucumber seedling explants after primary roots removal with either 20–40 mM H₂O₂ or 10 μM IAA significantly increased the number of adventitious roots, and treatment with 10–50 mM H₂O₂ significantly increased the fresh weight of adventitious roots. The effects of H₂O₂ on promoting the formation and growth of adventitious roots were eliminated by 2 mM ascorbic acid, 100 U CAT or 1 μM DPI, and the effects of IAA were eliminated by 4 mM ascorbic acid, 100 U CAT or 5 μM DPI. Treatment with either 4 mM ascorbic acid or 1–5 μM DPI inhibited the formation and growth of adventitious roots, and these inhibitory effects were partly reversed by exogenous H₂O₂.Furthermore, a higher concentration of endogenous H₂O₂ was detected in seedling explants 3 h after the primary roots were removed. However, in 10 μM DPI-treated seedling explants, the concentration of endogenous H₂O₂ was markedly reduced by DPI. Results obtained suggest that H₂O₂ may function as a signaling molecule, involved in the formation and development of adventitious roots in cucumber.     

百合病毒的DNA芯片检测技术研究

根据已知的黄瓜花叶病毒,百合无症病毒、百合斑驳病毒基因核苷酸序列,设计引物和探针,制备寡核苷酸芯片。用Cy3标记核苷酸引物,不对称RT-PCR扩增产物与芯片上的寡核苷酸探针杂交,荧光扫描仪检测并分析信号。研究制备的基因芯片能够检测侵染百合的3种重要病毒核酸的特异性荧光信号,该项技术具有特异、灵敏、快速的优点。     

Ultrastructure of Necrotic Lesions in Nicotiana glutinosa Leaves Locally Infected with a Variant of Cucumber mosaic virus

CMV(Y/GM2)tr is a variant of Cucumber mosaic virus strain Y [CMV(Y)] which infects Nicotiana species, including N. glutinosa, to induce necrotic local lesions (NLLs) in inoculated leaves, although all other CMV strains including CMV(Y) systemically infect Nicotiana species. To investigate the morphological features of this unique host response in N. glutinosa leaves infected with CMV(Y/GM2)tr, the ultrastructure of cells surrounding completely collapsed NLLs in virus‐inoculated N. glutinosa leaves was compared with that of normal cells of mock‐inoculated N. glutinosa leaves. The changes, which have been reported in other several virus–host plant systems showing the hypersensitive response (HR), were frequently observed in cells surrounding the NLLs. Furthermore, clumping of the nuclear matrix within the nuclei, which is a feature of programmed cell death, also occurred in these cells. These results indicated that the HR‐like host response occurred at the fine structural level in the cells of N. glutinosa plants infected with CMV(Y/GM2)tr.     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000