Exogenous priming of chitosan induces upregulation of phytohormones and resistance against cucumber powdery mildew disease is correlated with localized biosynthesis of defense enzymes.

In recent years, exploration of biopolymer-based materials to avoid hazardous chemicals in agriculture has gained enormous importance for sustainable crop improvement. In the present study, chitosan a biopolymer derived from crab-shell was used in different concentrations as priming agent to cucumber seeds and were evaluated for its effect to enhance plant growth parameters as well as its ability to induce resistance against powdery mildew disease. Among the treatments, seeds-primed with 2.5 mg/mL exhibited early seedling germination of 90% and vigour of 2665 and also remarkably enhanced the cucumber growth parameters which might be fairly attributed to the stimulation of phytohormones content in primed plants over the controls. More importantly, under greenhouse conditions a significant induced disease protection of 66.6% against powdery mildew disease was noticed in chitosan-pretreated plants at 2.5 mg/mL. The induced resistant plants also showed a significant deposition of lignin, callose and H2O2. Notably, polyphenol oxidase, phenylalanine ammonia-lyase, peroxidase and glucanase defense-responsive enzymes were upregulated in chitosan-primed plants. Considered together, these results determine that the susceptible cucumber cultivar elicits immunity after perception of priming with chitosan to upregulate phytohormones and synthesize defense-responsive enzymes, thereby induce resistance against powdery mildew disease and strengthen the growth-promotion of cucumber plants.

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.

Ectopic expression of CsTGase enhances salt tolerance by regulating polyamine biosynthesis, antioxidant activities and Na+/K+ homeostasis in transgenic tobacco.

Transglutaminases (TGases), mediators of the transamidation of specific proteins by polyamines (PA), play critical roles in PA metabolism in animals, but their functions and regulatory mechanisms are largely unknown in plants. In this study, we demonstrated that TGase from cucumber played a protective role in the regulation of PA metabolism under salt stress. The expression of TGase was induced by salt stress in cucumber. Ectopic overexpression of cucumber TGase in tobacco conferred enhanced tolerance to salt stress based on both external symptoms and membrane integrity. Overexpression lines maintained high levels of PAs under salt stress, suggesting that PAs played a vital role in TGase-induced salt tolerance. In contrast, the levels of Na+ content in the wild-type (WT) plants increased, while they decreased in the overexpression plants. The expression levels of several genes related to ion exchange enhanced, and the Na+/K+ ratio decreased by increased TGase activity under salt stress. The activities of the proton-pump ATPase (H+-ATPase), vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (PPase) were higher in the overexpression lines than in WT plants under salt stress. Moreover, the malondialdehyde (MDA) and H2O2 contents were significantly lower in the overexpression lines than in WT plants, accompanied by increased antioxidant enzyme activity. Taken together, these findings demonstrate that TGase plays protective roles in response to salt stress, which may promote plant survival by regulating PA metabolism and the Na+/K+ balance under salt stress.

2-Hydroxymelatonin mitigates cadmium stress in cucumis sativus seedlings: Modulation of antioxidant enzymes and polyamines.

Cadmium level is continuously increasing in agricultural soils mainly due to anthropogenic activities. Cadmium is one of the most phytotoxic metals in the soils. The present study investigates the possible role of 2-hydroxymelatonin (2-OHMT) in assuagement of Cd-toxicity in cucumber (Cucumis sativus L.) plants. 2-OHMT is an important metabolite produced through interaction of melatonin with oxygenated compounds. Cadmium stress decreased the activity of antioxidant enzymes and polyamines. However, exogenously applied 2-OHMT enhanced plant growth attributes including photosynthetic rate, intercellular CO2 concentration, stomatal conductance and transpiration rate in treated plants. In addition, 2-OHMT induced enhancement of the activity of PAs biosynthesizing enzymes (putrescine, spermidine and spermine) in conjunction with reduction in activity of polyamine oxidase (PAO). 2-OHMT mitigated Cd stress through up-regulation in expression of stress related CS-ERS gene along with the amplified activity of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in treated seedlings. The improved activity of antioxidant scavengers played central role in reduction of hydrogen peroxide (H2O2), electrolyte leakage (EL) and malondialdehyde (MDA) in plants under Cd stress. Recent findings also advocate the positive correlation between PAs and ethylene, as both possess common precursor. The current study reveals that priming seeds with 2-OHMT reduces Cd-toxicity and makes it possible to cultivate cucumber in Cd-contaminated areas. Future experiments will perhaps help in elucidation of 2-OHMT intervened stress mitigation procedure in C. sativus crop. Furthermore, research with reference to potential of 2-OHMT for stress alleviation in other horticultural and agronomic crops will assist in enhancement of crop productivity.

Isolation and characterization of S-Adenosylmethionine synthase gene from cucumber and responsive to abiotic stress.

S-adenosylmethionine synthetase (SAMS) catalyzes methionine and ATP to generate S-adenosyl-L-methionine (SAM). In plants, accumulating SAMS genes have been characterized and the majority of them are reported to participate in development and stress response. In this study, two putative SAMS genes (CsSAMS1 and CsSAMS2) were identified in cucumber (Cucumis Sativus L.). They displayed 95% similarity and had a high identity with their homologous of Arabidopsis thaliana and Nicotiana tabacum. The qRT-PCR test showed that CsSAMS1 was predominantly expressed in stem, male flower, and young fruit, whereas CsSAMS2 was preferentially accumulated in stem and female flower. And they displayed differential expression profiles under stimuli, including NaCl, ABA, SA, MeJA, drought and low temperature. To elucidate the function of cucumber SAMS, the full-length CDS of CsSAMS1 was cloned, and prokaryotic expression system and transgenic materials were constructed. Expressing CsSAMS1 in Escherichia coli BL21 (DE3) improved the growth of the engineered strain under salt stress. Overexpression of CsSAMS1 significantly increased MDA content, H2O2 content, and POD activity in transgenic lines under non-stress condition. Under salt stress, however, the MDA content of transgenic lines was lower than that of the wild type, the H2O2 content remained high, the polyamine and ACC synthesis in transgenic lines exhibited a CsSAMS1-expressed dependent way. Taken together, our results suggested that both CsSAMS1 and CsSAMS2 were involved in plant development and stress response, and a proper increase of expression level of CsSAMS1 in plants is benificial to improving salt tolerance.

Hydrogen gas promotes the adventitious rooting in cucumber under cadmium stress.

Hydrogen gas (H2) plays an important role in plant development and stress responses. Here, cucumber (Cucumis sativus L.) explants were used to investigate the roles of H2 in adventitious root development under cadmium (Cd) stress and its physiological mechanism. The results showed that hydrogen-rich water (HRW) promoted adventitious rooting under Cd stress and 50% HRW obtained the maximal biological response. Compared with Cd treatment, HRW + Cd treatment significantly reduced the content of malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide radical (O2-), thiobarbituric acid reactive substances (TBARS), ascorbic acid (AsA) and reduced glutathione (GSH), as well as relative electrical conductivity (REC), lipoxygenase (LOX) activity, AsA/docosahexaenoic acid (DHA) ratio, and GSH/oxidized glutathione (GSSG) ratio, while increasing DHA and GSSG content. HRW + Cd treatment also significantly increased in the activity and related gene expression of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). Additionally, HRW + Cd treatment increased the contents of osmotic adjustment substances, as well as the activities of peroxidase (POD) and polyphenol oxidase (PPO), while significantly decreasing indoleacetic acid oxidase (IAAO) activity. In summary, H2 could induce adventitious rooting under Cd stress by decreasing the oxidative damage, increasing osmotic adjustment substance content and regulating rooting-related enzyme activity.

Overexpression of Transglutaminase from Cucumber in Tobacco Increases Salt Tolerance through Regulation of Photosynthesis.

Transglutaminase (TGase) is a regulator of posttranslational modification of protein that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms of TGase-mediated salt tolerance remain largely unknown. Here, we found that the transcription of cucumber TGase (CsTGase) was induced in response to light and during leaf development, and the CsTGase protein was expressed in the chloroplast and the cell wall. The overexpression of the CsTGase gene effectively ameliorated salt-induced photoinhibition in tobacco plants, increased the levels of chloroplast polyamines (PAs) and enhanced the abundance of D1 and D2 proteins. TGase also induced the expression of photosynthesis related genes and remodeling of thylakoids under normal conditions. However, salt stress treatment reduced the photosynthesis rate, PSII and PSI related genes expression, D1 and D2 proteins in wild-type (WT) plants, while these effects were alleviated in CsTGase overexpression plants. Taken together, our results indicate that TGase-dependent PA signaling protects the proteins of thylakoids, which plays a critical role in plant response to salt stress. Thus, overexpression of TGase may be an effective strategy for enhancing resistance to salt stress of salt-sensitive crops in agricultural production.

Acetaminophen detoxification in cucumber plants via induction of glutathione S-transferases.

Many pharmaceutical and personal care products (PPCPs) enter agroecosystems during reuse of treated wastewater and biosolids, presenting potential impacts on plant development. Here, acetaminophen, one of the most-used pharmaceuticals, was used to explore roles of glutathione (GSH) conjugation in its biotransformation in crop plants. Acetaminophen was taken up by plants, and conjugated quickly with GSH. After exposure to 5 mg L-1 acetaminophen for 144 h, GSH-acetaminophen conjugates were 15.2 ±â€¯1.3 nmol g-1 and 1.2 ±â€¯0.1 nmol g-1 in cucumber roots and leaves, respectively. Glutathione-acetaminophen was also observed in common bean, alfalfa, tomato, and wheat. Inhibition of cytochrome P450 decreased GSH conjugation. Moreover, the GSH conjugate was found to further convert to cysteine and N-acetylcysteine conjugates. Glutathione S-transferase activity was significantly elevated after exposure to acetaminophen, while levels of GSH decreased by 55.4% in roots after 48 h, followed by a gradual recovery thereafter. Enzymes involved in GSH synthesis, regeneration and transport were consistently induced to maintain the GSH homeostasis. Therefore, GST-mediated conjugation likely played a crucial role in minimizing phytotoxicity of acetaminophen and other PPCPs in plants.

The functions of cucumber sucrose phosphate synthases 4 (CsSPS4) in carbon metabolism and transport in sucrose- and stachyose-transporting plants.

Sucrose phosphate synthases (SPSs) are rate-limiting sucrose synthesis enzymes present in photosynthetic and non-photosynthetic tissues. The cucumber genome contains three SPSs that can be grouped into families A, B, and C. CsSPS1 and CsSPS2 are highly expressed in flowers and mature leaves, while the expression level of CsSPS4 increased gradually after leaf unfolding in our study and reached its peak after 20 days. In CsSPS4-overexpression tobacco plants, sucrose content and sucrose/starch ratio were increased significantly and resulted in improved leaf yield. By contrast, in CsSPS4-overexpression (CsSPS4-OE) cucumber lines, contents of sucrose and starch were unchanged, and raffinose was increased in transgenic cucumber leaves. The expression of cucumber raffinose family oligosaccharide (RFO)-synthesis-related genes increased obviously in cucumber CsSPS4-OE plants, and the sucrose, raffinose, and stachyose contents increased significantly in the petioles of CsSPS4-OE lines. In CsSPS4-antisense (CsSPS4-A) cucumber lines, decreases occurred in mRNA expression, enzyme activity, sucrose content, sucrose/starch ratio, and stachyose transport, but the RFO-synthesis-related genes were nearly unchanged. Together, these results suggest that overexpression of CsSPS4 can lead to carbon metabolism prioritizing sugar transport in cucumber, and suppression of CsSPS4 likely promotes carbon metabolism to accumulate starch, showing a more complicated carbon distribution model than in transgenic tobacco plants.

[Effects of exogenous 2,4-epibrassinolide on the growth and redox balance of cucumber seedlings under NaHCO3 stress]. / å¤–æºè¡¨æ²¹èœç´ å† é ¯å¯¹NaHCO3胁迫下黄瓜幼苗生长及氧化还原平衡的影响.

The effects of 0.2 µmol·L-1 exogenous 2,4-epibrassinolide (EBR) on the growth and reactive oxygen species metabolism of cucumber seedlings ('Jinyan 4' cucumber) under salt-alkaline stress (30 mmol·L-1 NaHCO3) were examined by hydroponics method. The results showed that NaHCO3 stress significantly induced production of O2-· and accumulation of H2O2 in leaves and roots, resulting in the increases of MDA content and electrolyte leakage. Under NaHCO3 stress, activities of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, dehydroascorbatereductase, monodehydrodroascorbate reductase, and glutathione reductase as well as contents of ascorbic acid and glutathione were firstly increased and then decreased with progress of stress time. Exogenous EBR application significantly increased the activities of antioxidant enzymes and contents of antioxidants as well as the ratio of AsA/DHA (dehydroascorbic acid) and GSH/GSSG (L-glutathione oxidized) in leaves and roots of cucumber under NaHCO3 stress. Such changes improved the redox hemostasis in plants, reduced the level of reactive oxygen species, and alleviated the membrane lipid peroxidation. Together, they increased the alkaline tolerance of cucumber seedlings.

Sequenced ascorbate-proline-glutathione seed treatment elevates cadmium tolerance in cucumber transplants.

During its life cycle, plant has to cope with a number of abiotic stresses including cadmium stress. Cadmium (Cd) is highly toxic to plant and greatly influences its growth and entire metabolism. Antioxidants have to enable plant to beat such stresses. Therefore, effects of ascorbate (AsA), proline (Pro) and glutathione (GSH) applied, as seed soaking solutions, singly or in a sequence on cucumber transplant growth, physio-biochemical attributes and antioxidant defense system activity were investigated under 2 mM Cd stress. Adding Cd to transplants in irrigation water reduced photosynthetic efficiency, and nutrient (K+ and Ca2+) contents, while increased the activity of defense systems (non-enzymatic and enzymatic antioxidants) and Cd2+ contents in roots and leaves. Exogenous AsA, Pro and GSH applied singly or in a sequence improved transplant growth (e.g., shoot length, leaf area, shoot fresh weight and shoot dry weight), photosynthetic efficiency (i.e., SPAD chlorophyll, Fv/Fm and PI), transplant health (i.e., increased leaf MSI and RWC, and decreased root and leaf Cd2+ contents), antioxidant defense systems activity (enzymatic; superoxide dismutase, catalase, glutathione reductase and ascorbate peroxidase, and non-enzymatic; Pro, AsA and GSH antioxidants) and nutrient (K+ and Ca2+) contents. These positive results were obtained under irrigation with or without Cd, AsA. Sequenced AsA-Pro-GSH was the best treatment of which this study recommends to use, followed by GSH treatment, for growing cucumber transplants under Cd stress.

Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses.

Plasma membrane (PM) H+-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H+-ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H+-ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H+-ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H+-ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H+-ATPase and NADPH oxidase are key factors.

A pathogenic long noncoding RNA redesigns the epigenetic landscape of the infected cells by subverting host Histone Deacetylase 6 activity.

Viroids - ancient plant-pathogenic long noncoding RNAs - have developed a singular evolutionary strategy based on reprogramming specific phases of host-metabolism to ensure that their infection cycle can be completed in infected cells. However, the molecular aspects governing this transregulatory phenomenon remain elusive. Here, we use immunoprecipitation assays and bisulfite sequencing of rDNA to shown that, in infected cucumber and Nicotiana benthamina plants, Hop stunt viroid (HSVd) recruits and functionally subverts Histone Deacetylase 6 (HDA6) to promote host-epigenetic alterations that trigger the transcriptional alterations observed during viroid pathogenesis. This notion is supported by the demonstration that, during infection, the HSVd-HDA6 complex occurs in vivo and that endogenous HDA6 expression is increased in HSVd-infected cells. Moreover, transient overexpression of recombinant HDA6 reverts the hypomethylation status of rDNA observed in HSVd-infected plants and reduces viroid accumulation. We hypothesize that the host-transcriptional alterations induced as a consequence of viroid-mediated HDA6 recruitment favor spurious recognition of HSVd-RNA as an RNA Pol II template, thereby improving viroid replication. Our results constitute the first description of a physical and functional interaction between a pathogenic RNA and a component of the host RNA silencing mechanism, providing novel evidence of the potential of these pathogenic lncRNAs to physically redesign the host-cell environment and reprogram their regulatory mechanisms.

Nitric oxide is required for hydrogen gas-induced adventitious root formation in cucumber.

Hydrogen gas (H2) is involved in plant development and stress responses. Cucumber explants were used to study whether nitric oxide (NO) is involved in H2-induced adventitious root development. The results revealed that 50% and 100% hydrogen-rich water (HRW) apparently promoted the development of adventitious root in cucumber. While, the responses of HRW-induced adventitious rooting were blocked by a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), NO synthase (NOS) enzyme inhibitor N(G)-nitro-l-arginine methylester hydrochloride (l-NAME) and nitrate reductase (NR) inhibitor NaN3. HRW also increased NO content and NOS and NR activity both in a dose- and time-dependent fashion. Moreover, molecular evidence showed that HRW up-regulated NR genes expression in explants. The results indicate the importance of NOS and NR enzymes, which might be responsible for NO production in explants during H2-induced root organogenesis. Additionally, peroxidase (POD) and indoleacetic acid oxidase (IAAO) activity was significantly decreased in the explants treated with HRW, while HRW treatment significantly increased polyphenol oxidase (PPO) activity. In addition, cPTIO, l-NAME and NaN3 inhibited the actions of HRW on the activity of these enzymes. Together, NO may be involved in H2-induced adventitious rooting, and NO may be acting downstream in plant H2 signaling cascade.

[Effects of calcium and ABA on photosynthesis and related enzymes activities in cucumber seedlings under drought stress]. / é’™ä¸Žè„±è½é ¸å¯¹å¹²æ—±èƒè¿«ä¸‹é»„ç“œå¹¼è‹—å ‰åˆåŠç›¸å ³é ¶æ´»æ€§çš„å½±å“.

To investigate the effect of calcium and ABA on photosynthesis and the activities of antioxidant enzymes in cucumber seedlings under drought stress, the cucumber was used as the expe-riment materials, normal nutrient solution culture was considered as the control, and PEG-6000 application in the nutrient solution simulated the drought stress. There were five different treatments which were spraying water, ABA, CaCl2+ABA, LaCl3(calcium channel inhibitor)+ABA and EGTA (calcium ion chelating agent)+ABA under drought stress. The results showed that drought stress inhibited the growth of cucumber seedlings, and reduced the activities of antioxidant enzymes, nitrate reductase, net photosynthetic rate and fluorescence parameters of the cucumber seedlings leaves. The application of ABA reduced the inhibition of activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), photosynthesis (Pn, gs) and the fluorescence parameters (Fv'/Fm', qP and ETR), and decreased the damage of drought stress on plant. Spraying CaCl2+ABAsignificantly promoted the positive effect of ABA, while EGTA+ABA and LaCl3 +ABA didn't show the promoting effect.

The functions of a cucumber phospholipase D alpha gene (CsPLDα) in growth and tolerance to hyperosmotic stress.

Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. In this research, a qRT-PCR analysis indicated that the expression of a cucumber phospholipase D alpha gene (CsPLDα) was induced by salt and drought stresses in the roots and leaves. To further study the roles of CsPLDα in regulating plant tolerance to salt, polyethylene glycol (PEG) and abscisic acid (ABA) stresses, transgenic tobacco plants constitutively overexpressing CsPLDα were produced. A qRT-PCR analysis showed that the CsPLDα transcript levels were high in transgenic tobacco lines, whereas no expression was found in wild type (WT) tobacco, indicating that CsPLDα was successfully transferred into the tobacco genome and overexpressed. Under normal conditions for 30 d, seeds of transgenic lines germinated neatly, and the seedlings were robust and bigger than WT plants. When treated with different concentrations of NaCl, PEG and ABA, germination rates and seedling sizes of the transgenic lines were significantly greater than WT. In addition, the germination times for transgenic lines were also remarkably shorter. Further studies indicated that transgenic lines had longer primary roots and more biomass accumulation than WT plants. The water loss in transgenic lines was also much lower than in WT. These findings suggest that the CsPLDα overexpression positively regulates plant tolerance to hyperosmotic stresses, and that CsPLDα is involved in the ABA regulation of stomatal closure and the alleviation of ABA inhibition on seed germination and seedling growth.

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2.

Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu(+) (Km ∼1 or 0.5 µM, respectively) and similar affinity to Ag(+) (Km ∼2.5 µM). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu(+) transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.

Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress.

The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxidase activity in cucumber roots. Plants were treated with cadmium for 1, 3 or 6 days. Some of the plants after 3-day exposure to cadmium were transferred to a medium without the heavy metal for the next 3 days. Treatment of plants with cadmium for 6 days stimulated the activity of NADPH oxidase. The highest stimulation of O2(•-) production by NADPH oxidase was observed in post-stressed plants, which was correlated with the stimulation of activity of PM H(+)-ATPase in the same conditions. In order to examine the effects of cadmium stresses on the expression level of genes encoding NADPH oxidase, putative cucumber homologs encoding RBOH proteins were selected and a real-time PCR assay was performed. NADPH is a substrate for oxidase; thus alterations in the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-isocitrate dehydrogenase and NADP-malic enzyme under cadmium stress were studied. The activity of NADPH dehydrogenases was increased under cadmium stress. The results indicate that PM NADPH oxidase could be involved in plants' response to cadmium stress by affecting the activity of PM H(+)-ATPase, and NADPH-generating enzymes could play important roles in this process.

Molecular and biochemical properties of two P1B2-ATPases, CsHMA3 and CsHMA4, from cucumber.

P1B-ATPases (heavy metal ATPases, HMAs) constitute a multigenic subfamily of P-ATPases involved in the transport of monovalent and divalent heavy metals in plant cells. Here, we present the organization of genes encoding the HMA family in the cucumber genome and report the function and biochemical properties of two cucumber proteins homologous to the HMA2-4-like plant HMAs. Eight genes encoding putative P1B -ATPases were identified in the cucumber genome. Among them, CsHMA3 was predominantly expressed in roots and up-regulated by Pb, Zn and Cd excess, whereas the CsHMA4 transcript was most abundant in roots and flowers of cucumber plants, and elevated under Pb and Zn excess. Expression of CsHMA3 in Saccharomyces cerevisiae enhanced yeast tolerance to Cd and Pb, whereas CsHMA4 conferred increased resistance of yeast cells to Cd and Zn. Immunostaining with specific antibodies raised against cucumber proteins revealed tonoplast localization of CsHMA3 and plasma membrane localization of CsHMA4 in cucumber root cells. Kinetic studies of CsHMA3 and CsHMA4 in yeast membranes indicated differing heavy metal cation affinities of these two proteins. Altogether, the results suggest an important role of CsHMA3 and CsHMA4 in Cd and Pb detoxification and Zn homeostasis in cucumber cells.