Correlation of ASN2 gene expression with ammonium metabolism in Arabidopsis

Abstract

In Arabidopsis, asparagine (Asn) synthetase is encoded by a small gene family (ASN1, ASN2, and ASN3). It has been shown that ASN1 and ASN2 exhibit reciprocal gene expression patterns toward light and metabolites. Moreover, changes in total free Asn levels parallel the expression of ASN1, but not ASN2. In this study, we show that ASN2 expression correlates with ammonium metabolism. We demonstrate that the light induction of ASN2 is ammonium dependent. The addition and removal of ammonium exerted fast and reciprocal effects on the levels of ASN2 mRNA, specifically under light-grown conditions. NaCl and cold stress increased cellular free ammonium and ASN2 mRNA levels in a coordinated manner, suggesting that the effects of stress on ASN2 expression may be mediated via accumulation of ammonium. The correlation between ASN2 and cellular ammonium metabolism was further demonstrated by analysis of ASN2 transgenic plants. When plants were grown on Murashige and Skoog medium containing 50 mm ammonium, ASN2 overexpressors accumulated less endogenous ammonium compared with the wild-type Colombia-0 and ASN2 underexpressors. When plants were subjected to high-light irradiance, ammonium levels built up. Under such conditions, ASN2 underexpressors accumulated more endogenous ammonium than the wild-type Colombia-0 and ASN2 overexpressors. These results support the notion that ASN2 is closely correlated to ammonium metabolism in higher plants.

Figure 1.

Ammonium controls the light induction of ASN2. Twelve-day-old seedlings grown on Murashige and Skoog agar plates under a regular day/light cycle (16 h of light and 8 h of dark) were transferred to new Murashige and Skoog medium containing 20 mm (Lanes 1 & 2) or no (Lanes 3 & 4) ammonium. NaNO₃ was added to ammonium-free medium to maintain an equal molar concentration of nitrogen resources. Plants were subsequently treated under continuous light (L) or continuous dark (D) conditions for 48 h. Total RNA was extracted from the seedlings as described in “Materials and Methods.” An aliquot of 15 μg of total RNA from each line was loaded onto each lane. Northern-blot analysis was performed as described in “Materials and Methods.”

Figure 2.

Ammonium controls the light induction of ASN2. Seedlings were germinated and grown under a regular day/light cycle on Murashige and Skoog agar plates containing no (A) or 20 mm (B) ammonium for 14 d before being transferred to Murashige and Skoog agar plates containing 20 mm (A) and no (B) ammonium respectively, and grown under continuous light. NaNO₃ was added to ammonium-free medium to maintain an equal molar concentration of nitrogen resource. Samples were collected at time 0, 2, 4, 8, 16, 24, and 48 h (lanes 1-7, respectively) after treatments. Total RNA was extracted from the seedlings as described in “Materials and Methods.” An aliquot of 15 μg of total RNA from each line was loaded onto each lane. Northern-blot analysis was performed as described in “Materials and Methods.”

Figure 3.

NaCl and cold stresses increase the levels of ASN2 mRNA. The effects of NaCl (200 mm; A) and cold (10°C; B) on the levels of ASN2 mRNA were tested. Twelve-day-old seedlings grown on Murashige and Skoog agar plates under a regular day/light cycle were transferred to new growth medium (A) or were subjected to new growth temperature (B) for 4 (lane 4), 8 (lane 5), and 16 h (lane 6). Samples of control plants without stress treatments were also collected at same time points (lanes 1-3). Total RNA was extracted from the seedlings as described in “Materials and Methods.” An aliquot of 15 μg of total RNA from each line was loaded onto each lane. Northern-blot analysis was performed as described in “Materials and Methods.”

Figure 4.

Endogenous free ammonium content increases under stress treatments. Samples were harvested as described in Figure 3. Free ammonium content was assayed as described in “Materials and Methods.” Each bar represents an average of five samples. Error bars = ses. The data was analyzed by one-way analysis of variance (ANOVA) followed by lsd test. An asterisk indicates significant difference when compared NaCl-(lanes 2, 5, and 8) and cold-(lanes 3, 6, and 9) treated samples to untreated controls (lanes 1, 4, and 7), with a P value less than 0.05.

Figure 5.

Northern-blot analysis of ASN2 transgenic lines. Twelve-day-old seedlings of the wild-type Col-0 (lanes 1 and 2), the ASN2-underexpressing line 401-A4-c1 (lanes 3 and 4), and the ASN2-overexpressing line 402-A1-b1 (lanes 5 and 6) grown on Murashige and Skoog agar plates under a regular day/light cycle were subsequently treated under continuous light (L) or continuous dark (D) conditions for 48 h. Total RNA was extracted from the seedlings as described in “Materials and Methods.” An aliquot of 15 μg of total RNA from each line was loaded onto each lane. Northern-blot analysis was performed as described in “Materials and Methods.”

Figure 6.

Endogenous free ammonium content decreases in ASN2-overexpressing transgenic lines grown on ammonium-containing medium. Ten-day-old seedlings of the wild-type Col-0 (lanes 1 and 6), ASN2-underexpressing lines 401-A4-c1 (lanes 2 and 7) and 401-A1-a8 (lanes 3 and 6), and ASN2-overexpressing lines 402-A1-b1 (lanes 4 and 9) and 402-IT-13 (lanes 5 and 10) grown under a regular day/light cycle on regular Murashige and Skoog (containing 20 mm ammonium) agar plates were transferred to Murashige and Skoog medium supplemented with 20 (lanes 1-5) or 50 mm (lanes 6-10) exogenous ammonium. Free ammonium content was assayed as described in “Materials and Methods.” Each bar represents an average of five samples. Error bars = ses. The data was analyzed by one-way ANOVA followed by lsd test. An asterisk indicates significant difference with a P value less than 0.05.

Figure 7.

Endogenous free ammonium content increases in ASN2 antisense transgenic lines grown under high-light irradiance. Twelve-day-old seedlings of the wild-type Col-0 (lanes 1 and 2), ASN2-underexpressing lines 401-A4-c1 (lanes 3 and 4) and 401-A1-a8 (lanes 5 and 6), and ASN2-overexpressing lines 402-A1-b1 (lanes 7 and 8) and 402-IT-13 (lanes 9 and 10) grown under a regular day/light cycle at low light irradiance (35 μE) on regular Murashige and Skoog agar plates were exposed to continuous light at low (35 μE; lanes 1, 3, 5, 7, and 9) or high (150 μE; lanes 2, 4, 6, 8, and 10) for 72 h. Free ammonium contents were assayed as described in “Materials and Methods.” Each bar represents an average of five samples. Error bars = ses. The data was analyzed by one-way ANOVA followed by lsd test. An asterisk indicates significant difference with a P value less than 0.05.