The effects of fluctuations in the nutrient supply on the expression of five members of the AGL17 clade of MADS-box genes in rice

Abstract

The ANR1 MADS-box gene in Arabidopsis is a key gene involved in regulating lateral root development in response to the external nitrate supply. There are five ANR1-like genes in Oryza sativa, OsMADS23, OsMADS25, OsMADS27, OsMADS57 and OsMADS61, all of which belong to the AGL17 clade. Here we have investigated the responsiveness of these genes to fluctuations in nitrogen (N), phosphorus (P) and sulfur (S) mineral nutrient supply. The MADS-box genes have been shown to have a range of responses to the nutrient supply. The expression of OsMADS61 was transiently induced by N deprivation but was not affected by re-supply with various N sources. The expression of OsMADS25 and OsMADS27 was induced by re-supplying with NO3(-) and NH4NO3, but downregulated by NH4(+). The expression of OsMADS57 was significantly downregulated by N starvation and upregulated by 3 h NO3(-) re-supply. OsMADS23 was the only gene that showed no response to either N starvation nor NO3(-) re-supply. OsMADS57 was the only gene not regulated by P fluctuation whereas the expression of OsMADS23, OsMADS25 and OsMADS27 was downregulated by P starvation and P re-supply. In contrast, all five ANR1-related genes were significantly upregulated by S starvation. Our results also indicated that there were interactions among nitrate, sulphate and phosphate transporters in rice.

Figure 1. Effect of N deprivation and nitrate resupply on the expression of five ANR1 related genes in rice roots.

Ten-day old rice seedlings grown in complete nutrient solutions were transferred to modified nutrient solutions during which 2.88 mM KNO₃ was the sole nitrogen source for 4 days. Transcript abundance was assayed by qPCR and was expressed relative to the abundance in roots of plants of the same age grown under continuous N (CK). The OsNAR2.1 gene, a known nitrate-regulated gene, was included for comparison. Treatments: CK: continuous KNO₃; −N4h: starved of N for 3 d and resupplied with KCl for 4 h; +N4h: resupplied with KNO₃ for 4 h; −N6h: starved of N for 3 d and resupplied with KCl for 6 h; +N6h: resupplied with KNO₃ for 6 h. The mRNA of OsActin was used as the reference. A Student’s t-test was calculated at the probability of either 5% (*, p<0.05) or (**, P<0.01).

Figure 2. Effect of different N sources on the expression of five ANR1-related genes in rice roots.

Rice seedlings were grown in liquid culture for 14 days with 2.88 mM KNO₃ as the sole nitrogen source and then N starved for 3 d. CK, continuous N; −N, starved for 3 d; +KNO₃, resupplied with 2.88 mM nitrate; +NH₄Cl resupplied with 2.88 mM NH₄ ⁺; and +Gln, resupplied with 2.88 mM glutamine; +NH₄NO₃, resupplied with 1.44 mM NH₄NO₃. The value of related genes were normalized to its CK control respectively. The mRNA of OsActin was used as the reference. Error bars represent _{SE. LSD} values were calculated at the probability of either 5% (*, p<0.05) or (**, P<0.01).

Figure 3. Effect of deprivation and re-supply of phosphate (P) and sulfate (S) on the expression of five ANR1-related genes in rice.

Two-week old rice seedlings grown hydroponically in complete nutrient solution were deprived of P or S or were maintained on complete nutrient supply for 3 d. In the light period on the day of the experiment, one set of the P-starved and S-starved plants were re-supplied with 0.32 mM H₂PO₄ ⁻ and 2.1 mM SO₄ ²⁻ respectively. Roots were harvested 3 h later from controls: continuous nutrient supply (CK); P-deprived (−P); P-resupply (+P); S-deprived (−S); S-resupply (+S). Total RNA was extracted from roots and qPCR reactions were performed in triplicate for each RNA sample. The mRNA of OsActin was used as the reference. The value of related genes were normalized to its CK control respectively. A Student’s t-test was calculated at the probability of either 5% (*, p<0.05) or (**, P<0.01).

Figure 4. Effect of N-deprivation and re-supply on expression of OsNRT2.1, OsNAR2.1, OsIPS1 and OsSULTR1;1 in rice roots.

Rice seedlings were grown hydroponically in a growth cabinet. Nitrogen treatments were as described in Fig. 1. CK: continuous KNO₃; −N4h: starved of N for 3 d and resupplied with KCl for 4 h; +N4h: resupplied with KNO₃ for 4 h; −N6h: starved of N for 3 d and resupplied with KCl for 6 h; +N6h: resupplied with KNO₃ for 6 h. Total RNA was extracted from roots and qPCR reactions were performed in triplicate for each RNA sample. The mRNA of OsActin was used as the reference. A Student’s t-test was calculated at the probability of either 5% (*, p<0.05) or (**, P<0.01).

Figure 5. Effect of P-deprivation and re-supply on expression of OsNRT2.1, OsNAR2.1, OsIPS1 and OsSULTR1;1 in rice roots.

Rice seedlings were grown hydroponically in a growth cabinet. Phosphorous treatments were as described in Fig. 3. C: continuous complete nutrient supply; −P: starved of P; +P: resupplied with H₂PO₄ ⁻ for 3 h. Total RNA was extracted from roots and qPCR reactions were performed in triplicate for each RNA sample. The mRNA of OsActin was used as the reference. A Student’s t-test was calculated at the probability of either 5% (*, p<0.05) or (**, P<0.01).

Figure 6. Effect of S-deprivation and re-supply on expression of OsNRT2.1, OsNAR2.1, OsIPS1 and OsSULTR1;1 in rice roots.

Rice seedlings were grown hydroponically in a growth cabinet. Sulfur treatments were as described in Fig. 3. C: continuous complete nutrient supply; −S: starved of S; +S: resupplied with SO₄ ²⁻ for 3 h. Total RNA was extracted from roots and qPCR reactions were performed in triplicate for each RNA sample. The mRNA of OsActin was used as the reference. A Student’s t-test was calculated at the probability of either 5% (*, p<0.05) or (**, P<0.01).