Candidate cucumber genes ranked according to their expression stability as determined by NormFinder.

<p>Stability values are listed from the most stable to the least stable gene.</p>*<p>Samples from two-week-old plants grown under nitrate, ammonia, glutamine or glutamate for 4 or 12 hours.</p>**<p>Samples from 4-week-old plants grown under nitrogen deficiency, 0.5 mM nitrate, 10 mM nitrate, temporary nitrate provision, temporary nitrate starvation or temporary nitrate re-supply.</p

GeNorm based evaluation of candidate gene expression in samples from plants grown in different nitrogen compounds.

<p>Average expression stability values (<i>M</i>) of the remaining candidate cucumber reference genes during stepwise exclusion of the least stable reference gene in roots (a), stems (c), leaves (e) and all cucumbers organs taken together (g). The lowest the M values indicate the most stable expression of candidate cucumber genes. Determination of optimal number of reference genes based on pairwise variation (V) analysis of normalization factors of the candidate reference genes in roots (b), stems (d), leaves (f) and all cucumber organs taken together (h). The V_n/n+1 value was calculated for every comparison between two of the twelve consecutive candidate reference genes. According to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072887#pone.0072887-Vandesompele1" target="_blank">[14]</a>, additional (n+1)^th reference gene should be included into analysis whenever the V_n/n+1 value drops below the 0.15 threshold.</p

BestKeeper based evaluation of reference genes stability in cucumber plants grown in varying NO₃⁻ supply.

<p>The stability values were calculated based on the pairwise correlation between genes and BI (BestKeeper Index). The highest Person coefficient values representing the most stable genes are marked in bold. Genes ranked at the lowest positions by geNorm and NormFinder for each set of analyzed samples were not included (-) in BestKeeper evaluation.</p

Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition

<div><p>In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target <i>CsNRT1.1</i> gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH₃). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EFα proved to be the most suitable for normalization of <i>CsNRT1.1</i> expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress.</p></div

BestKeeper based evaluation of reference genes stability in cucumber plants grown in different nitrogen compounds.

<p>The stability values were calculated based on the pairwise correlation between genes and BI (BestKeeper Index). The highest Person coefficient values representing the most stable genes are marked in bold. Genes ranked at the lowest positions by geNorm and NormFinder for each set of analyzed samples were not included (-) in BestKeeper evaluation.</p

GeNorm based evaluation of candidate gene expression in samples from plants grown in different nitrate supply.

<p>Average expression stability values (<i>M</i>) of the remaining candidate cucumber reference genes during stepwise exclusion of the least stable reference gene in roots (a), stems (c), leaves (e) and all cucumbers organs taken together (g). The lowest the M values indicate the most stable expression of candidate cucumber genes. Determination of optimal number of reference genes based on pairwise variation (V) analysis of normalization factors of the candidate reference genes in roots (b), stems (d), leaves (f) and all cucumber organs taken together (h). The V_n/n+1 value was calculated for every comparison between two of the twelve consecutive candidate reference genes. According to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072887#pone.0072887-Vandesompele1" target="_blank">[14]</a>, additional (n+1)^th reference gene should be included into analysis whenever the V_n/n+1 value drops below the 0.15 threshold.</p

Description of cucumber candidate reference genes based on the comparison with their Arabidopsis orthologs.

<p>Seven of the twelve candidate cucumber reference genes (<i>CACS</i>, <i>TIP41</i>, <i>PDF2</i>, <i>GW881873</i>, <i>YSL8</i>, <i>HEL</i>) have been recently retrieved from the whole cucumber genome sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072887#pone.0072887-Migocka1" target="_blank">[36]</a> using the novel reference genes identified in <i>Arabidopsis</i> as the query sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072887#pone.0072887-Czechowski1" target="_blank">[3]</a>. The commonly used remaining five genes (<i>ACT</i>, <i>TUA</i>, <i>UBI-1</i>, <i>EFα</i>, <i>CYP</i>) were previously available in the Genbank database as partial cDNAs. The full cDNAs and exon/intron organization of all 12 candidate genes were established using BlastN, and FGENESH or FGENESH+.</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 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

V-ATPase activity and quantity in tonoplast vesicles isolated from roots of cucumber plants grown under different nitrate supply.

<p>A–B. The rate of MgATP-dependent, bafilomycin-sensitive proton transport in tonoplast under constant (squares, grey bars) or temporary (triangles, dark bars) NO₃⁻ supply and under N-deprivation (circles, white bars). Figure A is representative for the results obtained in six independent experiments. Figure B presents the average values ± SD of four independent experiments. Asterisks indicate a significant difference (<i>P</i><0.05) between transport activity of V-ATPase under different nitrate supply. C. NO₃⁻-sensitive hydrolytic activity in membranes obtained from NO₃⁻-grown (light grey bars), NO₃⁻-induced (dark grey bars) and N-deprived (white bars) plants. Data are the average values ± SD of three independent experiments. Asterisks indicate a significant difference (<i>P</i><0.05) between V-ATPase activity under different nitrate supply. D. A representative Coomassie blue-stained gel of total tonoplast protein isolated from plants grown under different nitrate regime. 15 µg of tonoplast proteins were separated by SDS-PAGE on a 10% linear acrylamide gel. The positions of PAGE molecular mass markers are shown in kilodaltons on the right of the gel image. E. Immunoblot of V-ATPase subunit a on tonoplast membranes obtained from plants grown under different nitrate regime. Presented picture is representative for the results obtained in three to four independent experiments.</p