Nitrate Accumulation and Expression Patterns of Genes Involved in Nitrate Transport and Assimilation in Spinach

Abstract

Excessive accumulation of nitrate in spinach is not only harmful to human beings, but also limits the efficiency of nitrogen usage. However, the underlying mechanism of nitrate accumulation in plants remains unclear. This study analyzed the physiological and molecular characteristics of nitrate uptake and assimilation in the spinach varieties with high or low nitrate accumulation. Our results showed that the variety of spinach with a high nitrate content (So18) had higher nitrate uptake compared to the variety with a low nitrate content (So10). However, the nitrate reductase activities of both varieties were similar, which suggests that the differential capacity to uptake and transport nitrate may account for the differences in nitrate accumulation. The quantitative PCR analysis showed that there was a higher level of expression of spinach nitrate transporter (SoNRT) genes in So18 compared to those in So10. Based on the function of Arabidopsis homologs AtNRTs, the role of spinach SoNRTs in nitrate accumulation is discussed. It is concluded that further work focusing on the expression of SoNRTs (especially for SoNRT1.4, SoNRT1.5 and SoNRT1.3) may help us to elucidate the molecular mechanism of nitrate accumulation in spinach.

Keywords: NRT; Spinacia oleracea; nitrate accumulation; nitrate reductase.

Figure 1

Fresh weights (A) and total nitrogen contents (B) of two spinach varieties (S10 and S18) after 12 h of nitrate treatment. L-So10, the leaf blades of S10; L-So18, the leaf blades of So18; P-So10, the petioles of So10; P-So18, the petioles of So18; R-So10, the roots of So10; and R-So18, the roots of So18. Different letters in columns indicate significant difference between the mean values at p < 0.05 by the LSD test (n = 3). Values are provided as mean ± SD.

Figure 2

Nitrate contents in tissues of two spinach varieties under low (0.5 mmol·L⁻¹) and high (15 mmol·L⁻¹) nitrate treatments. LN-So18, So18 treated with low nitrate concentration; LN-So10, So10 treated with low nitrate concentration; HN-So18, So18 treated with high nitrate concentration; and HN-So10, So10 treated with high nitrate concentration.

Figure 3

¹⁵NO₃⁻-N uptake rate (A) and ¹⁵NO₃⁻-N excess (net NO₃⁻-N uptake amount) (B) of two spinach varieties under low (0.5 mmol·L⁻¹) and high (15 mmol·L⁻¹) nitrate treatments. Values are provided as mean ± SD, n = 3. LN-So18, So18 treated with low nitrate concentration; LN-So10, So10 treated with low nitrate concentration; HN-So18, So18 treated with high nitrate concentration; and HN-So10, So10 treated with high nitrate concentration.

Figure 4

The activities of nitrate reductase (NR) (A) and glutamine synthetase (GS) (B) in shoots of two spinach varieties under low (0.5 mmol·L⁻¹) and high nitrate (15 mmol·L⁻¹) treatments. Values are represented as mean ± SD, n = 3. LN-So18, So18 treated with low nitrate concentration; LN-So10, So10 treated with low nitrate concentration; HN-So18, So18 treated with high nitrate concentration; and HN-So10, So10 treated with high nitrate concentration.

Figure 5

Expression profiles of NRTs genes in tissues of two spinach varieties under two nitrate concentration treatments. S10, the spinach So10 variety; S18, the spinach So18 variety; B, leaf blade; P, petiole; R, root; L, low nitrate (0.5 mmol·L⁻¹) treatment; H, high nitrate (15 mmol·L⁻¹) treatment; 1, 1 h; 7, 7 h; and 12, 12 h.

Figure 6

Expression analysis of genes encoding nitrate transporters in leaf blades, petioles and roots of two spinach varieties under two concentrations (0.5 and 15 mmol·L⁻¹) of nitrate treatments by qRT-PCR. All values represent the averages of three replicates (see Supplementary Table S1 for details). LN-So18, So18 treated with low nitrate concentration; LN-So10, So10 treated with low nitrate concentration; HN-So18, So18 treated with high nitrate concentration; and HN-So10, So10 treated with high nitrate concentration.

Figure 7

Expression analysis of nitrogen assimilation genes in leaf blades, petioles and roots of two spinach varieties under two concentrations (0.5 and 15 mmol·L⁻¹) of nitrate treatments by qRT-PCR. All values represent the averages of three replicates (see Supplementary Table S1 for details). LN-So18, So18 treated with low nitrate concentration; LN-So10, So10 treated with low nitrate concentration; HN-So18, So18 treated with high nitrate concentration; and HN-So10, So10 treated with high nitrate concentration.