Efficient acquisition of iron confers greater tolerance to saline-alkaline stress in rice (Oryza sativa L.)

Abstract

To elucidate the mechanisms underlying tolerance to saline-alkaline stress in two rice genotypes, Dongdao-4 and Jigeng-88, we exposed them to medium supplemented with 10 mM Na₂CO₃ and 40 mM NaCl (pH 8.5). Dongdao-4 plants displayed higher biomass, chlorophyll content, and photosynthetic rates, and a larger root system than Jigeng-88 under saline-alkaline conditions. Dongdao-4 had a higher shoot Na⁺/K⁺ ratio than Jigeng-88 under both control and saline-alkaline conditions. Dongdao-4 exhibited stronger rhizospheric acidification than Jigeng-88 under saline-alkaline conditions, resulting from greater up-regulation of H⁺-ATPases at the transcriptional level. Moreover, Fe concentrations in shoots and roots of Dongdao-4 were higher than those in Jigeng-88, and a higher rate of phytosiderophore exudation was detected in Dongdao-4 versus Jigeng-88 under saline-alkaline conditions. The Fe-deficiency-responsive genes OsIRO2, OsIRT1, OsNAS1, OsNAS2, OsYSL2, and OsYSL15 were more strongly up-regulated in Dongdao-4 than Jigeng-88 plants in saline-alkaline medium, implying greater tolerance of Dongdao-4 plants to Fe deficiency. To test this hypothesis, we compared the effects of Fe deficiency on the two genotypes, and found that Dongdao-4 was more tolerant to Fe deficiency. Exposure to Fe-deficient medium led to greater rhizospheric acidification and phytosiderophore exudation in Dongdao-4 than Jigeng-88 plants. Expression levels of OsIRO2, OsIRT1, OsNAS1, OsNAS2, OsYSL2, and OsYSL15 were higher in Dongdao-4 than Jigeng-88 plants under Fe-deficient conditions. These results demonstrate that a highly efficient Fe acquisition system together with a large root system may underpin the greater tolerance of Dongdao-4 plants to saline-alkaline stress.

Keywords: Oryza sativa; Dongdao-4; Jigeng-88; iron acquisition; iron deficiency; rice; saline-alkaline stress..

Fig. 1.

Effects of saline-alkaline stress on Dongdao-4 and Jigeng-88 seedlings. (A) Growth performance. (B) Photographs of the leaves corresponding to the seedlings in panel (A). (C) Plant height. (D) Dry shoot biomass. Three-week-old rice seedlings grown in normal culture solution were transferred to culture solution supplemented with 40 mM Na⁺ (10 mM Na₂CO₃ and 20 mM NaCl) for 1 day, and then exposed to 60 mM Na⁺ (10 mM Na₂CO₃ and 40 mM NaCl) for 1 week. Bars=10 cm. Data are means±SE (n≥4). Means with different letters are significantly different (P<0.05) within the same treatment. Asterisks indicate significant differences between control and saline-alkaline stress of the same genotype, as determined by Student’s t-test (*P<0.05, **P<0.01, ***P<0.001).

Fig. 2.

(A) Foliar chlorophyll concentration and (B) photosynthetic rates of Dongdao-4 and Jigeng-88 plants grown in normal and saline-alkaline stress conditions. Treatments and statistical analysis were as described in Fig. 1.

Fig. 3.

Effects of saline-alkaline stress on (A, D) K⁺ concentration, (B, E) Na⁺ concentration, and (C, F) Na⁺/K⁺ ratio of shoots and roots in Dongdao-4 and Jigeng-88 seedlings grown in normal and saline-alkaline stress conditions. Treatments and statistical analysis were as described in Fig. 1.

Fig. 4.

Fe concentrations of (B) shoots and (B) roots of Dongdao-4 and Jigeng-88 seedlings grown in normal and saline-alkaline stress conditions. Treatments and statistical analysis were as described in Fig. 1.

Fig. 5.

Effects of saline-alkaline stress on the root system architecture of Dongdao-4 and Jigeng-88 plants. (A) Root phenotypes of the two cultivars under normal conditions. (B) Root phenotypes of the two cultivars under saline-alkaline stress. (C) Adventitious root number. (D) Root surface area. (E) Total root length. (F) Dry root biomass. Treatments and statistical analysis were as described in Fig. 1.

Fig. 6.

Effects of saline-alkaline stress on root acidification. (A) Time course of changes in the pH of the growth solution when intact rice roots were incubated for 7 days under control and saline-alkaline stress conditions. The solution was refreshed on a daily basis and its pH was adjusted to 8.5. (B–D) Expression patterns of the plasma membrane H⁺-ATPase genes (B) Os12g0638700, (C) Os03g0100800, and (D) Os03g0689300 in roots of Dongdao-4 and Jigeng-88 plants grown in control and saline-alkaline medium for 7 days. Data are means±SE (n≥3). Means with different letters are significantly different (P<0.05) within the same treatments. Asterisks indicate significant differences between control and saline-alkaline stress treatments of the same genotype, as determined by Student’s t-test (*P<0.05, **P<0.01, ***P<0.001).

Fig. 7.

Effects of saline-alkaline stress on phytosiderophore release from roots of Dongdao-4 and Jigeng-88. Data (means±SE; n≥3) were collected after exposure of the rice plants to saline-alkaline medium for 7 days. Means with different letters are significantly different (P<0.05) within the same treatment. Asterisks indicate significant differences between control and saline-alkaline stress treatments of the same genotype, as determined by Student’s t-test (**P<0.01, ***P<0.001).

Fig. 8.

Effects of saline-alkaline stress on the expression of six Fe-deficiency-related genes in Dongdao-4 and Jigeng-88 plants: (A) OsIRO2, (B) OsIRT1, (C) OsNAS1, (D) OsNAS2, (E) OsYSL15, and (F) OsYSL2. Total RNA was extracted from rice seedlings grown under control and saline-alkaline stress conditions for 1 week. Transcript levels were measured by real-time RT-PCR. Actin was used as an internal control. The transcript levels of OsIRT1 and OsYSL2 were multiplied by 1000. Error bars are calculated based on three biological replicates. Treatments and statistical analysis were as described in Fig. 1.

Fig. 9.

Physiological responses of the Dongdao-4 and Jigeng-88 rice genotypes to different Fe levels. (A) Growth performance. (B) Photographs of leaves corresponding to the seedlings in panel (A). (C) Plant height. (D) Dry shoot biomass. (E) Dry root biomass. (F) Total chlorophyll (SPAD) values. Two-week-old rice seedlings were exposed to Fe-sufficient (+Fe) or Fe-deficient (–Fe) medium for 2 weeks. Bars=10 cm. Data are means±SE (n≥3). Means with different letters are significantly different (P<0.05) within the same treatments. Asterisks indicate significant differences between +Fe and –Fe treatment of the same genotype, as determined by Student’s t-test (*P<0.05, **P<0.01, ** P<0.001).

Fig. 10.

Fe concentration in (A) shoots and (B) roots of Dongdao-4 and Jigeng-88 seedlings grown in Fe-sufficient or Fe-deficient conditions. Treatments and statistical analysis were as described in Fig. 9.

Fig. 11.

Effects of different levels of Fe supplementation on root acidification. (A) Time course of changes in the pH of the growth solution following exposure of plants previously grown in Fe-sufficient (+Fe) medium to Fe-deficient (–Fe) medium for 9 days. The arrow indicates the time when the growth medium was renewed; at the time of medium renewal, the pH was adjusted to 5.9. (B–D) Expression patterns of the plasma membrane H⁺-ATPase genes (B) Os12g0638700, (C) Os03g0100800, and (D) Os03g0689300 in roots of Dongdao-4 and Jigeng-88 plants exposed to +Fe and –Fe medium for 9 days. Treatments and statistical analysis were as described in Fig. 9.

Fig. 12.

Effects of Fe deficiency on the rate of phytosiderophore secretion from roots of Dongdao-4 and Jigeng-88 plants. Data are means±SE (n=4). Treatments and statistical analysis were as described in Fig. 9.

Fig. 13.

Effects of Fe deficiency on the expression of six Fe-deficiency-responsive genes in roots of Dongdao-4 and Jigeng-88 plants: (A) OsIRO2, (B) OsIRT1, (C) OsNAS1, (D) OsNAS2, (E) OsYSL15, and (F) OsYSL2. Total RNA was extracted from rice seedlings grown under Fe-sufficient or Fe-deficient medium for 1week. Transcript levels were measured by real-time RT-PCR. Actin was used as an internal control. Error bars are calculated based on three biological replicates. Treatments and statistical analysis were as described in Fig. 9.