Jasmonic Acid Effect on Cucumis sativus L. Growth Is Related to Inhibition of Plasma Membrane Proton Pump and the Uptake and Assimilation of Nitrates.

When plants are exposed to environmental stress, their growth is inhibited. Under such conditions, controlled inhibition of growth is beneficial for plant survival. Jasmonic acid (JA) is a well-known phytohormone that limits plant growth, which has been confirmed in several species. However, its role in cucumber seedlings has not yet been comprehensively investigated. For this reason, we aimed to determine the involvement of JA in the regulation of proteins crucial for growth including plasma membrane proton pump (PM H+-ATPase), PM nitrate transporters, and nitrate reductase (NR). Treatment of cucumber seedlings with JA not only limited their growth but also increased the H2O2 content in their roots. The main sources of ROS generated for signalling purposes are PM NADPH oxidase (RBOH) and superoxide dismutase (SOD). Exposure of seedlings to JA induced the expression of some CsRBOH and SOD encoding genes, suggesting that ROS signalling can be activated by JA. As a consequence of JA exposure, the activity of all analysed proteins was inhibited and the expression of their genes was modified. The results indicate that reduction of PM H+-ATPase activity and the related decrease in nitrate uptake and assimilation are responsible for the root growth retardation of JA-treated plants.

Exogenous Putrescine Modulates Nitrate Reductase-Dependent NO Production in Cucumber Seedlings Subjected to Salt Stress.

Polyamines (PAs) are small aliphatic compounds that participate in the plant response to abiotic stresses. They also participate in nitric oxide (NO) production in plants; however, their role in this process remains unknown. Therefore, the study aimed to investigate the role of putrescine (Put) in NO production in the roots of cucumber seedlings subjected to salt stress (120 mM NaCl) for 1 and 24 h. In salinity, exogenous Put can regulate NO levels by managing NO biosynthesis pathways in a time-dependent manner. In cucumber roots exposed to 1 h of salinity, exogenous Put reduced NO level by decreasing nitrate reductase (NR)-dependent NO production and reduced nitric oxide synthase-like (NOS-like) activity. In contrast, during a 24 h salinity exposure, Put treatment boosted NO levels, counteracting the inhibitory effect of salinity on the NR and plasma membrane nitrate reductase (PM-NR) activity in cucumber roots. The role of endogenous Put in salt-induced NO generation was confirmed using Put biosynthesis inhibitors. Furthermore, the application of Put can modulate the NR activity at the genetic and post-translational levels. After 1 h of salt stress, exogenous Put upregulated CsNR1 and CsNR2 expression and downregulated CsNR3 expression. Put also decreased the NR activation state, indicating a reduction in the level of active dephosphorylated NR (dpNR) in the total enzyme pool. Conversely, in the roots of plants subjected to 24 h of salinity, exogenous Put enhanced the NR activation state, indicating an enhancement of the dpNR form in the total NR pool. These changes were accompanied by a modification of endogenous PA content. Application of exogenous Put led to an increase in the amount of Put in the roots and reduced endogenous spermine (Spm) content in cucumber roots under 24 h salinity. The regulatory role of exogenous Put on NO biosynthesis pathways may link with plant mechanisms of response to salt stress.

Interactions of Polyamines and Phytohormones in Plant Response to Abiotic Stress.

Numerous environmental conditions negatively affect plant production. Abiotic stresses, such as salinity, drought, temperature, and heavy metals, cause damage at the physiological, biochemical, and molecular level, and limit plant growth, development, and survival. Studies have indicated that small amine compounds, polyamines (PAs), play a key role in plant tolerance to various abiotic stresses. Pharmacological and molecular studies, as well as research using genetic and transgenic approaches, have revealed the favorable effects of PAs on growth, ion homeostasis, water maintenance, photosynthesis, reactive oxygen species (ROS) accumulation, and antioxidant systems in many plant species under abiotic stress. PAs display a multitrack action: regulating the expression of stress response genes and the activity of ion channels; improving the stability of membranes, DNA, and other biomolecules; and interacting with signaling molecules and plant hormones. In recent years the number of reports indicating crosstalk between PAs and phytohormones in plant response to abiotic stresses has increased. Interestingly, some plant hormones, previously known as plant growth regulators, can also participate in plant response to abiotic stresses. Therefore, the main goal of this review is to summarize the most significant results that represent the interactions between PAs and plant hormones, such as abscisic acid, brassinosteroids, ethylene, jasmonates, and gibberellins, in plants under abiotic stress. The future perspectives for research focusing on the crosstalk between PAs and plant hormones were also discussed.

Water and Nutrient Recovery for Cucumber Hydroponic Cultivation in Simultaneous Biological Treatment of Urine and Grey Water.

Water and nutrient deficiencies in soil are becoming a serious threat to crop production. Therefore, usable water and nutrient recovery from wastewater, such as urine and grey water, should be considered. In this work, we showed the possibility of using grey water and urine after processing in an aerobic reactor with activated sludge in which the nitrification process takes place. The resulting liquid (nitrified urine and grey water, NUG) contains three potential factors that can adversely affect plant growth in a hydroponic system: anionic surfactants, nutrient deficits, and salinity. After dilution and supplementation with small amounts of macro- and micro-elements, NUG was suitable for cucumber cultivation. Plant growth on this modified medium (enriched nitrified urine and grey water, NUGE) was similar to that of plants cultivated on Hoagland solution (HS) and reference commercial fertilizer (RCF). The modified medium (NUGE) contained a significant amount of sodium (Na) ions. Therefore, typical effects of salt stress were observed in cucumber plants, including reduced chlorophyll levels, slightly weaker photosynthesis parameters, increased H2O2 levels, lipid peroxidation, ascorbate peroxidase (APX) activity, and proline content in the leaves. In addition, reduced protein levels were observed in plants treated with recycled medium. At the same time, lower nitrate content in tissues was found, which may have resulted from their intensive use by nitrate reductase (NR), the activity of which significantly increased. Although cucumber is a glycophyte, it grew very well in this recycled medium. Interestingly, salt stress and possibly anionic surfactants promoted flower formation, which in turn could positively affect plant yield.

Urine and grey water based liquid fertilizer - Production and the response of plants.

Plant cultivation is a key aspect of future long-distance space missions, and the creation of an efficient food system will not be possible without it. The production of fertilizer in space is based on the recovery of water and nutrients from wastewater, such as urine and grey water. In this study, the fertilizer production process was conducted in an aerobic, activated sludge reactor, where nitrification and the process of carbon removal take place. Treated streams have three potential factors that could affect the plants growth in a hydroponic system (anionic surfactants, nutrients deficiencies, high salinity). The effect of these factors was examined for two hydroponic configurations. Their influence on lettuce yield, quality parameters and stress response were investigated and compared to the control cultivation. The results showed that the main cause of a decrease (up to 24%) in the yield productivity of plants grown on nitrified urine and grey water is oxidative stress originated from a deficiency of elements, not from used anionic surfactant. Enrichment with nutrients resulted in the restoration of proper protein synthesis and an increase in the activity of antioxidant enzymes, which was positively reflected in the qualitative and quantitative parameters of the enriched cultivation (fresh leaves mass equal to 103% of the control). Results also show that Sodium Methyl Cocoyl Taurate (SMCT) surfactant itself after biological treatment used in plant cultivation has no negative effects reflected in lettuce yield or quality.

Role of Plasma Membrane NADPH Oxidase in Response to Salt Stress in Cucumber Seedlings.

Plasma membrane NADPH oxidases (RBOHs, EC 1.6.3.1) are known as the main ROS generators involved in plant adaptation to stress conditions. In the present work, regulation of NADPH oxidase was analyzed in cucumber (Cucumis sativus L. var. Krak) seedlings exposed to salinity. RBOH activity and gene expression, as well as H2O2 content, were determined in the roots of plants treated with 50 or 100 mM NaCl for 1 h, and 50 mM NaCl for 1 or 6 days. It was found that enzyme activity increased in parallel with an enhancement in the H2O2 level in roots exposed to 100 mM NaCl for 1 h, and to 50 mM NaCl for 1 day. The expression of some CsRboh genes was induced by salt. Moreover, an increase in the activity of G6PDH, providing the substrate for the NADPH oxidase, was observed. In seedlings subjected to salinity for a longer time, antioxidant enzymes-including superoxide dismutase, catalase, and ascorbate peroxidase-were activated, participating in maintaining a steady-state H2O2 content in the root cells. In conclusion, NADPH oxidase and endogenous H2O2 up-regulation seem to be early events in cucumber response to salinity.

Involvement of Diamine Oxidase in Modification of Plasma Membrane Proton Pump Activity in Cucumis sativus L. Seedlings under Cadmium Stress.

Cucumber (Cucumis sativus L.) is a crop plant being the third most-produced vegetable developed as a new model plant. Heavy metal pollution is a serious global problem that affects crop production. An industrial activity has led to high emissions of Cd into the environment. Plants realize adaptive strategies to diminish the toxic effects of Cd. They can remove excess toxic ions of heavy metals from the cytoplasm to the outside of cells using the metal/proton antiport. The proton gradient needed for the action of the antiporter is generated by the plasma membrane (PM) H+-ATPase (EC 3.6.3.14). We have shown that treatment of cucumber plants with Cd stimulated the diamine oxidase (DAO, EC 1.4.3.6) activity in roots. Under cadmium stress, the PM H+-ATPase activity also increased in cucumber seedlings. The stimulating effect of Cd on the PM H+-ATPase activity and expression of three genes encoding this enzyme (CsHA2, CsHA4, CsHA8) was reduced by aminoguanidine (AG, a DAO inhibitor). Moreover, we have observed that H2O2 produced by DAO promotes the formation of NO in the roots of seedlings. The results presented in this work showed that DAO may be an element of the signal transduction pathway, leading to enhanced PM H+-ATPase activity under cadmium stress.

Interaction between the signaling molecules hydrogen sulfide and hydrogen peroxide and their role in vacuolar H+ -ATPase regulation in cadmium-stressed cucumber roots.

Vacuolar H+ -ATPase (V-ATPase; EC 3.6.3.14) is the main enzyme responsible for generating a proton gradient across the tonoplast. Under cadmium (Cd) stress conditions, V-ATPase activity is inhibited. In the present work, hydrogen sulfide (H2 S) and hydrogen peroxide (H2 O2 ) cross-talk was analyzed in cucumber (Cucumis sativus L.) seedlings exposed to Cd to explain the role of both signaling molecules in the control of V-ATPase. V-ATPase activity and gene expression as well as H2 S and H2 O2 content and endogenous production were determined in roots of plants treated with 100 µM CdCl2 and different inhibitors or scavengers. It was found that H2 S donor improved photosynthetic parameters in Cd-stressed cucumber seedlings. Cd-induced stimulation of H2 S level was correlated with the increased activities of the H2 S-generating desulfhydrases. Increased H2 O2 and lowered H2 S contents in roots were able to reduce V-ATPase activities similar to Cd. H2 O2 and H2 S-induced modulations in V-ATPase activities were not closely related to the transcript level of encoding genes, suggesting posttranslational modifications of enzyme protein. On the other hand, exogenous H2 O2 raised H2 S content in root tissues independently from the desulfhydrase activity. Although treatment of control plants with H2 S significantly stimulated NADPH oxidase activity and gene expression, H2 S did not affect H2 O2 accumulation in roots exposed to Cd. The results suggest the existence of two pathways of H2 S generation in Cd-stressed cucumber roots. One involves desulfhydrase activity, as was previously demonstrated in different plant species. The other, the desulfhydrase-independent pathway induced by H2 O2 /NADPH oxidase, may protect V-ATPase from inhibition by Cd.

Involvement of NR and PM-NR in NO biosynthesis in cucumber plants subjected to salt stress.

Nitrate reductase (NR) mainly reduces nitrate to nitrite. However, in certain conditions it can reduce nitrite to NO. In plants, a plasma membrane-associated form of NR (PM-NR) is present. It produces NO2- for nitrite NO/reductase (Ni-NOR), which can release NO into the apoplastic space. The effect of 50 mM NaCl on NO formation and the involvement of NR in NO biosynthesis were studied in cucumber seedling roots under salt stress. In salt-stressed roots, the amount of NO was higher than in control. The application of tungstate abolished the increase of NO level in stressed roots, indicating that NR was responsible for NO biosynthesis under the test conditions. The involvement of other molybdoenzymes was excluded using specific inhibitors. Furthermore, higher cNR and PM-NR activities were observed in NaCl-treated roots. The increase in NR activity was due to the stimulation of CsNR genes expression and posttranslational modifications, such as enzyme dephosphorylation. This was confirmed by Western blot analysis. Moreover, the increase of nitrite tissue level in short-term stressed roots and the nitrite/nitrate ratio, with a simultaneous decrease of nitrite reductase (NiR) activity, in both short- and long-term stressed roots, could promote the production of NO by NR in roots under salt stress.

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.

Response of nitrate reductase activity and NIA genes expression in roots of Arabidopsis hxk1 mutant treated with selected carbon and nitrogen metabolites.

In plants sugar sensing and signal transduction involves pathways dependent or independent on HXK1 as a glucose sensor. Research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. The effect of selected carbon and nitrogen metabolites on nitrate reductase (NR) activity in Arabidopsis thaliana wild type (WT) and hxk1 mutant roots was studied. Exogenously supplied sugar, sucrose (Suc) and organic acid, 2-oxoglutarate (2-OG) led to an increase in the total and actual activity of NR. It was due to both the increase in expression of NIA genes and NR activation state. The stimulatory effect of Suc and 2-OG on nitrate reduction was less pronounced in hxk1 mutant roots with T-DNA insertion in the AtHXK1 gene encoding hexokinase1 (HXK1) and characterized by reduced hexokinase activity and root level of G6P and F6P. On the other hand, it was shown that exogenous glucose did not mimic Suc-mediated NR activation in Arabidopsis roots. Taken together, this data suggest that the Suc signaling pathway might be independent from hexose's sensor dependent mechanism.

Transcriptional regulation of the V-ATPase subunit c and V-PPase isoforms in Cucumis sativus under heavy metal stress.

Two electrogenic proton pumps, vacuolar H(+) transporting ATPase (V-ATPase, EC 3.6.3.14) and vacuolar H(+) transporting inorganic pyrophosphatase (V-PPase, EC 3.6.1.1), co-exist in the vacuolar membrane of plant cells. In this work, all CsVHA and CsVHP genes encoding V-ATPase and V-PPase, respectively, were identified in the cucumber genome. Among them, three CsVHA-c genes for V-ATPase subunit c and two CsVHP1 genes for type I V-PPase were analyzed in detail. Individual isogenes were differentially regulated in plant tissues and during plant development as well as under changing environmental conditions. CsVHA-c1 and CsVHA-c2 showed similar tissue-specific expression patterns with the highest levels in stamens and old leaves. CsVHP1;1 was predominantly expressed in roots and female flowers. In contrast, both CsVHA-c3 and CsVHP1;2 remained in a rather constant ratio in all examined cucumber organs. Under heavy metal stress, the transcript amount of CsVHA-c1 and CsVHP1;1 showed a pronounced stress-dependent increase after copper and nickel treatment. CsVHA-c3 was upregulated by nickel only whereas CsVHA-c2 was induced by all metals with the most visible effect of copper. Additionally, CsVHP1;2 showed a tendency to be upregulated by copper and zinc. We propose that CsVHA-c1, CsVHA-c2 and CsVHP1;1 are essential elements of mechanisms involved in adaptation of cucumber plants to copper toxicity.

Regulation of nitrate reduction in Arabidopsis WT and hxk1 mutant under C and N metabolites.

As in plants sugar sensing and signal transduction involve pathways dependent or independent on hexokinase 1 (HXK1) as a glucose sensor, research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. An Arabidopsis mutant with T-DNA insertion in the AtHXK1 gene and defects in glucose signaling (hxk1) was used to determine nitrate reductase (NR) activity, NIA genes expression in leaves after 8-h treatment with sugars (glucose and sucrose), organic acids [2-oxoglutarate (2OG)] and amino acids (glutamine and glutamate). Sugars, especially sucrose, caused induction of NR actual activity accompanied by an increase of the NR activation state, indicating the posttranslational nature of the modifications. Those modifications were observed in wild-type (WT) and hxk1 leaves, suggesting that regulation of NR activity by sugars does not involve HXK1 as a glucose sensor. Moreover, sugars enhanced expression of NIA genes. However, a higher level of NIA transcripts did not lead to an increase of total NR activity in sugar-treated plants. This may suggest that posttranslational modification of NR is fundamental regulatory mechanisms controlling NR activity in response to C metabolites. Treatment of plants with 2-OG also modified NR through the posttranslational modifications. Elevation of actual NR activity and the enzyme activation state in WT and hxk1 leaves was observed. Amino acids caused a decrease of NIA gene expression and NR activities in WT and hxk1 leaves indicating that mutation in the hexokinase-dependent glucose signaling pathway did not interrupt the amino acid feedback regulation of NR.

Effect of short-term salinity on the nitrate reductase activity in cucumber roots.

In short-term experiments, the effect of high salinity on cucumber (Cucumis sativus) nitrate reductase activity was studied. The 60-min exposure of cucumber roots to 200 mM NaCl resulted in significant increase of the actual NR activity (measured in the presence of Mg²+), whereas the total enzyme activity (measured with EDTA) was not affected. NaCl-induced stimulation of the actual NR activity was rapidly reversed upon transfer of roots to salt-free solution. The increase in actual activity was completely prevented by microcystin-LR and cantharidin, protein phosphatases inhibitors. In addition, a significant decrease in ATP level was also observed in roots incubated with NaCl. These data suggest that the reversible protein phosphorylation is involved in the induction of NR activity during the first hour of salt stress. The effect of short-term salinity on the expression of genes encoding for nitrate reductase in cucumber roots was also studied. 200 mM NaCl diminished the increase in CsNR1 expression observed in control roots. During the same time period, the expression of CsNR2 was not affected, whereas the expression of CsNR3 decreased significantly after 1h incubation of the excised roots in both, control and salt-containing nutrient solutions. Incubation of roots in the presence of iso-osmotic concentration of PEG had no effect on both, NR activity and expression. This indicates that only the ionic component of salt stress was involved in the salt-induced modifications of nitrate reductase activity.