Figure legends
Figure 1. Enrichment for plasma membrane (PM) proteins. A-Western blot analysis of the sub-cellular fractions obtained from the roots of ‘Topas’ plants grown under nitrogen (N)-sufficient conditions. The compartment-specific marker proteins were: H+-ATPase (PM marker), VDAC (mitochondrial), BiP (ER), MDAR (cytosolic), V-ATPase (vacuolar). Abbreviations are used as follows, C- crude extract, S- soluble fraction, M- microsomal fraction, LP- lower phase, PMe- plasma membrane fraction.
Figure 2. Heat map representation of proteins with differential abundance (p-value < 0.05, fold change > 1.5) in the two potato cultivars contrasting in their nitrogen (N) deficiency response, grown under either sufficient or deficient N conditions. According to the K-means algorithm, the proteins formed seven clusters. Relative abundance has been color-coded following Z-score normalization. Each column represents one treatment. Abbreviation used: HN- N sufficient treatment, LN- N deficient treatment. The aquaporin proteins PIP1;1 and PIP1;3 are marked with an asterisk each.
Figure 3. Effect on nitrogen (N) deficiency on theStPIP1;1 and StPIP1;3 transcript abundances in roots of the potato cultivars ‘Lambada’ and ‘Topas’. The expression of genes was quantified using qRT-PCR. The expression of genes was measured using qRT-PCR. Bars (black: N-sufficient, white: N-deficient conditions) labeled by a different letter indicate statistically significant differences (p < 0.05), error bars represent ±SD.
Figure 4. The ammonium transport ability of PIP1;1 and PIP1;3 studied in yeast by a growth complementation assay. The Δmep1-3mutant was co-transformed with the combination of empty vector (EV) and indicated PIP cDNA. Cultures spotted on the medium containing indicated amount of ammonium or 0.2% proline (positive control) as nitrogen sources. The growth of the transformants was recorded after 8-10 days of incubation at 30 °C. Displayed image represents a group of images assembled from different growth conditions.
Figure 5. Effect on nitrogen (N) deficiency on theAtPIP1;1 and AtPIP1;3 transcript abundances in the roots of in-vitro grown Arabidopsis WT plants. The expression of genes was quantified using qRT-PCR. Bars represents N-sufficient (black) and N deficient (white) treatment. The statistical significance in transcript abundance induced by N deficiency is indicated by asterisks (Student’s t-test ,***: p < 0.001), error bars represent ±SD.
Figure 6 . Effect of nitrogen (N) deficiency on growth ofArabidopsis thaliana pip mutants. Plants were grown in the presence of either 60 mM (sufficient) or 30 mM (deficient) NH4NO3. Leaves fresh weight was determined after 5 weeks of cultivation. Bars represents N-sufficient (black) and N deficient (white) treatment. The data present the mean of 8 plants per treatment for 3 independent growth experiments. Letters above the histogram columns indicate significant differences in mean values, as predicted by a two way ANOVA (p-value < 0.05), error bars represent ±SD.
Figure 7 . The response of Arabidopsis WT and pipmutants to nitrogen deficiency. A- Nitrogen (N) and carbon (C) content of the leaves. B- Leaf phenylpropanoid and anthocyanin contents. Bars represents N-sufficient (black) and N deficient (white) treatment. The data present the mean of 8 plants per treatment for 3 independent growth experiments. Letters above the histogram columns indicate significant differences in mean values, as predicted by a two way ANOVA (p-value < 0.05), error bars represent ±SD.