Changes in phosphatase activity during germination and in early phases of cucumber seedlings growth

In cotyledons and roots of Cucumis sativus seedlings, the presence of phosphatases was proved at two optimal pH values: 6.4 and 5.0. The phosphatases are characterized by low substrate specificity. Among the analysed substrates the following were hydrolyzed particularly preferentialy while fissuring: pnitrophenylophosphate (pNPP), adenosinotriphosphate (ATP), pyrophosphate, β-naphthylophosphate and in case of the cotyledon extracts also calcium phytinate. Mo, Zn, Cu and Co present in an incubation mixture inhibited this activity. Analysis of the phosphatase activity in cotyledons and roots of cucumber seedlings showed that in early phases of cucumber growth (germination and the first week of growth in ligth), the activity remained at a relatively high level and had a few peaks connected with the vegetable growth phase whereas the frequency of their accurance was connected with the analysed organ. It was stated that changes of the phosphatase activity were visibly correlated to changes of the phosphorus content in the period under study. In roots similar correlations were not observed

NO₃⁻/H⁺ antiport in the tonoplast of cucumber root cells is stimulated by nitrate supply: evidence for a reversible nitrate-induced phosphorylation of vacuolar NO₃⁻/H⁺ antiport.

Studies in the last few years have shed light on the process of nitrate accumulation within plant cells, achieving molecular identification and partial characterization of the genes and proteins involved in this process. However, contrary to the plasma membrane-localized nitrate transport activities, the kinetics of active nitrate influx into the vacuole and its adaptation to external nitrate availability remain poorly understood. In this work, we have investigated the activity and regulation of the tonoplast-localized H(+)/NO₃(-) antiport in cucumber roots in response to N starvation and NO₃(-) induction. The time course of nitrate availability strongly influenced H(+)/NO₃(-) antiport activity at the tonoplast of root cells. However, under N starvation active nitrate accumulation within the vacuole still occurred. Hence, either a constitutive H(+)-coupled transport system specific for nitrate operates at the tonoplast, or nitrate uses another transport protein of broader specificity to different anions to enter the vacuole via a proton-dependent process. H(+)/NO₃(-) antiport in cucumber was significantly stimulated in NO₃(-)-induced plants that were supplied with nitrate for 24 hours following 6-day-long N starvation. The cytosolic fraction isolated from the roots of NO₃(-)-induced plants significantly stimulated H(+)/NO₃(-) antiport in tonoplast membranes isolated from cucumbers growing on nitrate. The stimulatory effect of the cytosolic fraction was completely abolished by EGTA and the protein kinase inhibitor staurosporine and slightly enhanced by the phosphatase inhibitors okadaic acid and cantharidin. Hence, we conclude that stimulation of H(+)/NO₃(-) antiport at the tonoplast of cucumber roots in response to nitrate provision may occur through the phosphorylation of a membrane antiporter involving Ca-dependent, staurosporine-sensitive protein kinase

Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

Soluble nitrate reductase from cucumber roots (Cucumis sativus L.) was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR) from cotyledon roots and leaves

CsCLCs expression in roots of cucumber grown under different nitrate supply.

<p>The level of <i>CsCLCa</i>, <i>CsCLc</i> and <i>CsCLCg</i> transcript was analyzed by Real-time PCR (A) and PCR (B) using reversed transcribed total RNA isolated from plant roots. For the real-time PCR assay, total RNA was prepared from NO₃⁻-grown (grey bars), NO₃⁻-induced (dark bars) and N-deprived (white bars) plants. The cDNA synthesized from RNA isolated from NO₃⁻-grown plants was also used for the PCR assay (B). The gene encoding clathrin adaptor complex subunit (CACS) was used as internal control. Values are the means ±SE (<i>n</i> = 5–6 measurements from 3 independent experiments). Asterisks indicate a significant difference (<i>P</i><0.05) between the expression of <i>CsCLCs</i> under different availability of nitrate in nutrient solution (A). Presented picture (B) is representative for the results obtained in three to four independent experiments.</p

The effect of soluble fractions (A) and soluble fraction isolated from NO₃

<p>⁻-<b>induced roots, protein kinase and phosphatase inhibitors or EGTA (B) on proton-coupled nitrate transport in tonoplast membranes obtained from NO₃</b>⁻-<b>grown plant roots.</b> Cytosolic soluble fraction (supernatant 120 000 g, 50 µl) alone or with other compounds was added to the reaction media containing 0.25 M sucrose, 1 mM DTT, 10 µM acridine orange and tonoplast membranes (50 µg of protein). After 5-min long incubation, 10 mM KNO₃ was introduced into the membranes to initiate proton efflux from the vesicles observed as the acridine orange absorbance increase. At first, the rate of H⁺-coupled nitrate antiport was measured in the presence of KNO₃ (light grey bars) and cytosolic fractions isolated from NO₃⁻-grown (dark grey bars), NO₃⁻-induced (dark bars) or N-deprived (white bars) plants (A). In further experiments, H⁺/NO₃⁻ activity was also determined in the presence of KNO₃, cytosolic fraction obtained from NO₃⁻-induced plants and phosphatase inhibitors (black bars) or kinase inhibitor (white bars) or EGTA (striped bars) (B). Protein kinase inhibitor, staurosporine and phosphatase inhibitors, okadaic acid (OA) and cantharidin were used at 5 µM and 2 µM concentration, respectively, whereas EGTA was applied to the media at final 5 mM concentration. In control assays, equal amounts of water or DMSO was used instead of cytosolic fraction/EGTA or inhibitors, respectively (light grey bars). Values are the means ±SE (<i>n</i> = 5–6 measurements from 4–6 independent tonoplast preparations). Asterisks indicate a significant difference (<i>P</i><0.05) between H⁺-coupled NO₃⁻ transport in tonoplast isolated from different plants.</p

The properties of H⁺-coupled transport of sulphate and chloride in tonoplast vesicles isolated from the roots of cucumbers grown under different nitrate supply.

<p>The time-course of acridine orange absorbance change after the addition of different concentration of K₂SO₄ (A) or KCl (C) to the reaction media containing ΔpH-energized tonoplast vesicles obtained from NO₃⁻-grown (grey bars), NO₃⁻-induced (dark bars) or N-deprived (white bars) plants. The effect of sulphate (B) or chloride (D) concentration on the acridine orange absorbance ΔpH-energized, tonoplast obtained from NO₃⁻-grown (squares), NO₃⁻-induced (triangles), or N-deprived (circles) plants. Values are the means ±SE (<i>n</i> = 5–6 measurements from 4–6 independent tonoplast preparations). Asterisks indicate a significant difference (<i>P</i><0.05) between H⁺-coupled SO₄²⁻ and Cl⁻ transport in tonoplast isolated from different plants. The Km and R² values were calculated using GraphPrism Software. The −1/Km values are indicated by red arrows. V represents the ΔA₄₉₅×min⁻¹×mg⁻¹ protein.</p

The activity of proton-coupled nitrate transport in the tonoplast membranes isolated from cucumbers grown under different nitrate supply.

<p>50 µg of tonoplast membrane protein was incubated with 20 mM Tris-Mes 0,25 M sucrose, 1 mM DTT and acridine orange until a stable baseline was reached (3–5 min). The increase in acridine orange absorbance in membranes isolated from N-deprived (A), NO₃⁻-grown (B) and NO₃⁻-induced (C) plants was initiated by the addition of different concentrations of KNO₃ into the reaction media (indicated by the arrows) and monitored during the following 3 min. Presented values are representative for the results obtained in three to four independent experiments with each experiment done in triplicate. D. The rate of nitrate accumulation in tonoplast vesicles isolated from N-deprived (white bars), NO₃⁻-grown (light grey bars) and NO₃⁻-induced (dark bars) plants determined by HPLC. The reaction mixture for HPLC assay was deprived of pH-sensitive probe. Values are the means ±SE (<i>n</i> = 5–6 measurements from 4–6 independent tonoplast preparations). Asterisks indicate a significant difference (<i>P</i><0.05) between proton-coupled nitrate transport activities in tonoplast membranes isolated from different plants.</p