Key words: Dongdao-4, iron acquisition, iron deficiency, Jigeng-88,Oryza sativa, rice, saline-alkaline stress.

Plant materials, growth conditions, and stress treatments

Two rice genotypes,O.sativa L. ssp.Japonica cv. Dongdao-4 and Jigeng-88, were used in this study. Seeds were germinated in tap water at 37 °C for 2 days and transferred on to moist tissue paper for 2 days at 30 °C in the dark. The germinated seedlings were then transferred to nutrient solution containing 1.425 mM NH₄NO₃, 0.42 mM NaH₂PO₄, 0.510 mM K₂SO₄, 0.998 mM CaCl₂, 1.643 mM MgSO₄, 0.168 mM Na₂SiO₃, 0.125 mM Fe-EDTA, 0.019 mM H₃BO₃, 0.009 mM MnCl₂, 0.155 mM CuSO₄, 0.152 mM ZnSO₄, and 0.075 mM Na₂MoO₄. This hydroponic experiment was carried out in a growth chamber maintained at 30 °C/22 °C (day/night) with a 14-h photoperiod and relative humidity maintained at approximately 70%.

For analysis of tolerance to saline-alkaline stress, Dongdao-4 and Jigeng-88 plants were grown in the culture solution for 3 weeks. Then, half of the plants 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. The remaining plants were kept in the original culture solution as a control. The solution was refreshed on a daily basis. The pH of the control hydroponic solution was adjusted to 6.0, while the solution used for saline-alkaline treatment was adjusted to pH 8.5.

To evaluate the effects of Fe deficiency on the two rice genotypes, Dongdao-4 and Jigeng-88 seedlings were cultured in the control hydroponic solution for 2 weeks and then transferred to a hydroponic solution supplemented with 0.01 μM Fe-EDTA for 2 weeks.

Measurements of plant growth

Shoots and roots of the rice seedlings were harvested and oven-dried at 75 °C for 2 days until they reached constant mass. To analyze root morphological parameters, roots were scanned with an Epson digital scanner (Expression 10000XL, Epson Inc.) and scans were analyzed with WinRHIZO/WinFOLIA software (Regent Instruments Inc.).

Determination of metal content

Shoots and roots of rice seedlings were harvested and dried as described above. They were ground into fine powder and digested with 6 mL nitric acid and 2 mL perhydrol using a microwave system (MARS, CEM). Total metal content was determined by inductively coupled plasma mass spectrometry (ICAP6300; Thermo Scientific, Waltham, MA, USA).

Measurements of chlorophyll concentration

To determine chlorophyll concentration in Dongdao-4 and Jigeng-88 plants, newly formed leaves were harvested, weighed, and extracted with aqueous ethanol (95% v/v). The absorbance (A) of the supernatant was recorded at wavelengths of 663 and 645 nm. Total chlorophyll concentration was calculated as 8.02A₆₆₃+20.21A₆₄₅, and was expressed as mg chlorophyll g^–1 fresh weight. Total chlorophyll concentration values (SPAD values) were also determined on the fully expanded youngest leaves of seedlings with a portable chlorophyll meter (SPAD-502, Minolta Sensing).

Measurements of photosynthetic characteristics

Photosynthetic rates of rice seedlings were measured between 8.30 and 11.30 h with a LI-6400 XT portable photosynthesis system equipped with a LED leaf cuvette (Li-Cor, Lincoln, NE, USA). Artificial illumination was applied to the leaves in the chamber from a red–blue 6400-02B LED light source attached to the sensor head giving continuous light (1000 μmol m^–2 s^–1 photosynthetic photon flux density); ambient CO₂ concentration was ~500 μmol CO₂ mol^–1. At least 15 individual Dongdao-4 and Jigeng-88 plants in each stress treatment were selected for measuring photosynthetic rates.

Measurement of external solution pH

To measure root-induced pH changes, pH in the growth medium was monitored continuously starting at 9:00 h, using a pH-sensitive microelectrode (pH-HJ90B, Shanghai). In the saline-alkaline stress experiment, the solution was refreshed on a daily basis. The pH of the control hydroponic solution was adjusted to 5.9, while the solution used for saline-alkaline stress treatment was adjusted to pH 8.5. In the Fe-deficient stress experiment, the pH of the medium was adjusted to 5.9 and the solution was renewed every 4 days.

Visualization and measurement of ferric reductase activity

Intact rice roots were thoroughly washed with saturated CaSO₄ (pH 5.9) and then spread on an agar sheet containing 0.75% (w/v) agar, 0.1 mM Fe³⁺-EDTA, 0.01 M MES buffer (pH 5.9), and 0.3 mM ferrozine. The incubation for visualization of ferric reductase was conducted in a growth chamber under light. After 1 d incubation, ferric reductase-induced color changes along the roots become visible; a purple color indicated that Fe³⁺ was reduced to Fe²⁺ by ferric reductase and chelated by ferrozine.

For measuring ferric reductase activity, roots of Dongdao-4 and Jigeng-88 plants were immersed in saturated CaSO₄ solution. After washing three times with deionized water, the roots were transferred to 20 mL of nutrient solution containing 0.1 mM Fe³⁺-EDTA, 10 mM MES buffer (pH 5.9), and 0.3 mM ferrozine for 1 h incubation in a growth chamber under light. The absorbance of the solution at 562 nm was measured. Ferric reductase activity was calculated based on the absorbance and expressed as μmol Fe²⁺ g^–1 FW root h^–1.

Determination of root phytosiderophore release

Roots of Dongdao-4 and Jigeng-88 plants were rinsed thoroughly with deionized water after immersion in saturated CaSO₄ solution, then placed in 40 mL of aerated deionized water for 2 h, to collect the root exudates at 12.00 h. Phytosiderophores were indirectly quantified by calculating the amount of Fe³⁺ mobilized from Fe hydroxide, following protocols described byInalet al. (2007). In brief, an aliquot of 0.5 mL 1mM FeCl₃ was added to 9 mL of the root exudates and the mixture was incubated for 1 h with shaking to produce Fe³⁺-phytosiderophore compounds. An aliquot of 1 mL 0.5 M sodium acetate (pH 7.0) was added to the mixture, which was shaken for another 15 min to precipitate the remaining Fe³⁺. The solution was then filtered. The filtrate was acidified by adding 0.2 mL 1.5 M H₂SO₄ together with 0.5 mL 1.15 M hydroxylammonium chloride and then incubated for 20 min at 55 °C, in order to convert Fe³⁺-phytosiderophore to Fe²⁺-phytosiderophore. An aliquot of 90 μL 0.03 M ferrozine was added to the filtrate to chelate the reduced Fe-phytosiderophore complex. The absorption of the filtrate at 562 nm was recorded and the phytosiderophore release rates were calculated as Fe equivalents.

RNA isolation and real-time RT-PCR

Total RNA of leaves and roots was isolated using RNAiso reagent (Takara) and reverse transcribed into first-strand cDNA with a PrimeScript^® RT Reagent kit (Takara). Real-time PCR was performed in an optical 96-well plate with an Applied Biosystems Stepone TM Real-Time PCR system. Each reaction contained 7.5 μl of 2× SYBR Green Master Mix reagent, 0.5 μl of cDNA samples, and 0.6 μl of 10 μM gene-specific primers in a final volume of 15 μL. The thermal cycle used was as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The following primers were used:OsIRO2, 5ʹ-CTCCCATCGTTTCGGCTACCT-3ʹ and 5ʹ-GCTGGGCACTCCTCGTTGATC-3ʹ;OsIRT1, 5ʹ-CGTC TTCTTCTTCTCCACCACGAC-3ʹ and 5ʹ-GCAGCTGATGATCG AGTCTGACC-3ʹ;OsNAS1, 5ʹ-GTCTAACAGCCGGACGATCGA AAGG-3ʹ and 5’-TTTCTCACTGTCATACACAGATGGC-3ʹ;OsNAS2, 5ʹ-TGAGTGCGTGCATAGTAATCCTGGC-3ʹ and 5ʹ-CA GACGGTGACAAACACCTCTTGC-3ʹ (Inoueet al. 2003);OsYSL15, 5ʹ-ACTGGTACCCTGCAAACATAC-3ʹ and 5ʹ-GCAAT GATGCTTAGCAAGAAG-3ʹ; Os12g0638700, 5ʹ-CTTGCCTCT GCTGTTTACCT-3ʹ and 5ʹ-GCTTCACAACCGATTCTACAT-3ʹ; Os03g0100800, 5ʹ-GCTGTTGCCTATCAGGAAGT-3ʹ and 5ʹ-TCA AAGAGTGGGAGAAGACC-3ʹ; Os03g0689300, 5ʹ-AAACGATG CTCCAGCCCTAA-3ʹ and 5ʹ-AATGGCGGGAAATCAAACT C-3ʹ; andactin, 5ʹ-ACCACAGGTATTGTGTTGGACTC-3ʹ and 5ʹ-AGAGCATATCCT TCATAGATGGG-3ʹ. Amplification ofactin (GenBank accession no.AB047313) was used as an internal control. The relative expression level was analyzed by the comparative Ct method.

Statistical analysis

All data were analyzed by ANOVA using SAS statistical software. Significant differences were evaluated using Student’st-test.

Effects of saline-alkaline stress on growth

To compare the tolerance of Dongdao-4 and Jigeng-88 plants to saline-alkaline stress, 3-week-old seedlings of the two rice genotypes were exposed to growth solution supplemented with 60 mM Na⁺ and at pH 8.5 for 1 week. As shown inFig. 1A, no differences in the growth of the two genotypes were detected when they were grown in the control solution. Upon exposure of the two genotypes to saline-alkaline medium, foliar chlorosis was observed in Jigeng-88, but not Dongdao-4 (Fig. 1B). In addition, the saline-alkaline stress treatment led to suppression of shoot and root growth in both genotypes; the magnitude of reduction in shoot and root growth of Dongdao-4 plants was significantly less than that in Jigeng-88 plants, such that plant height and dry shoot biomass of Dongdao-4 seedlings were significantly higher than those of Jigeng-88 seedlings (Fig. 1C,D).

To explore the physiological mechanism by which Dongdao-4 plants exhibited greater growth than Jigeng-88 plants under saline-alkaline conditions, we compared the effects of saline-alkaline stress on foliar chlorophyll concentration and photosynthetic rates (Pn) of the two genotypes. Both chlorophyll concentration and Pn of the two genotypes were comparable under control conditions (Fig. 2A,B). Exposure to saline-alkaline medium led to a significant reduction in foliar chlorophyll concentration in Jigeng-88 seedlings, while Dongdao-4 plants maintained a relatively constant foliar chlorophyll concentration when challenged by saline-alkaline stress, leading to a significant higher chlorophyll concentration in Dongdao-4 plants (Fig. 2A). Similarly, there were no differences in Pn in the two genotypes under control conditions (Fig. 2B). There were significant reductions in Pn in both genotypes after exposure to saline-alkaline stress, but the magnitude of the reduction in Pn was greater in Jigeng-88 than in Dongdao-4, leading to a significantly higher Pn in Dongdao-4 (Fig. 2B). These results suggest that Dongdao-4 seedlings are more tolerant to saline-alkaline stress than Jigeng-88 seedlings.

Effect of saline-alkaline stress on Na⁺ and K⁺ concentrations

Given that rice plants suffering from saline-alkaline stress have to cope with excess toxic Na⁺ in the growth substrate, we compared the effects of saline-alkaline stress on Na⁺ and K⁺ concentrations as well as Na⁺/K⁺ ratios in shoots and roots of the two genotypes. Shoots of Dongdao-4 plants showed a lower K⁺ concentration and higher Na⁺ concentration than Jigeng-88 plants under control, non-saline-alkaline conditions (Fig. 3A,B). Exposure to saline-alkaline stress led to a significant increase in Na⁺ concentration and reduction in K⁺ concentration in shoots of both Dongdao-4 and Jigeng-88 plants (Fig. 3A,B). These changes resulted in a higher K⁺ concentration in shoots of Jigeng-88 than in shoots of Dongdao-4, whereas no significant differences in Na⁺ concentration in shoots between the two genotypes were found under saline-alkaline conditions (Fig. 3A,B). The greater increase in Na⁺ concentration and decrease in K⁺ concentration in shoots of Dongdao-4 led to a higher Na⁺/K⁺ ratio in Dongdao-4 than in Jigeng-88 plants (Fig. 3C). Under control conditions, a higher K⁺ concentration in roots of Jigeng-88 than in roots of Dongdao-4 was detected, while Na⁺ concentrations in roots of both genotypes were comparable (Fig. 3D,E). There were significant increases in root Na⁺ concentrations and reductions in root K⁺ concentrations of the two genotypes upon exposure to saline-alkaline medium, leading to comparable K⁺ and Na⁺ concentrations in the roots of the two genotypes (Fig. 3D,E). A higher Na⁺/K⁺ ratio in roots of Dongdao-4 than Jigeng-88 plants grown in control medium was found (Fig. 3F). The increases in root Na⁺ concentration and decreases in K⁺ concentration associated with saline-alkaline treatment led to significant increases of Na⁺/K⁺ ratio in the roots of both genotypes; the Na⁺/K⁺ ratio of the two genotypes did not differ significantly under saline-alkaline stress (Fig. 3F).

Effect of saline-alkaline stress on Fe concentration

The concentration of plant-available Fe in saline-alkaline soils is often low due to immobilization, and this often becomes a limiting factor for crop yield and quality. Therefore, plants that are tolerant to saline-alkaline soils may also be equipped with an efficient Fe acquisition system. To test this hypothesis, we measured shoot Fe concentrations in the two genotypes grown in control and saline-alkaline media. The two genotypes exhibited comparable shoot Fe concentrations when grown in control medium (Fig. 4A). Upon exposure to saline-alkaline medium there was a significant reduction in shoot Fe concentration of Jigeng-88 seedlings, whereas shoot Fe concentration in Dongdao-4 seedlings remained relatively unchanged, leading to a significantly higher shoot Fe concentration in Dongdao-4 plants (Fig. 4A). The two genotypes showed comparable root Fe concentrations when grown in control medium. Exposure to saline-alkaline solution led to similar reductions in root Fe concentrations in the two genotypes, such that no significant difference was found between the genotypes (Fig. 4B).

Effects of saline-alkaline stress on root system architecture

Root system architecture is an important trait responsible for efficient Fe acquisition under Fe-deficient conditions. The observation that Dongdao-4 seedlings had a higher foliar Fe concentration than Jigeng-88 seedlings under saline-alkaline conditions prompted us to evaluate whether the root systems of the two genotypes differed in their response to saline-alkaline treatment. As shown inFig. 5, exposure of the two genotypes to saline-alkaline medium led to significant reductions in their adventitious root number and root surface area; the reduction was greater in Jigeng-88 than in Dongdao-4 plants (Fig. 5C,D), such that Dongdao-4 seedlings exhibited significantly greater adventitious root number and root surface area. The root system of the two genotypes was comparable when grown in the control solution; the total root length and dry root biomass of Dongdao-4 and Jigeng-88 seedlings were comparable under control conditions. Both parameters were markedly reduced in the two genotypes by saline-alkaline treatment, with a greater reduction in Jigeng-88 than in Dongdao-4 plants, leading to a significantly longer total root length and higher dry root biomass in Dongdao-4 plants (Fig. 5E,F). These results suggest that the larger root system of Dongdao-4 seedlings under saline-alkaline stress may facilitate their Fe acquisition, thus providing them with tolerance to Fe deficiency.

Effect of saline-alkaline stress on Fe deficiency-induced acidification of the rhizosphere

Acidification of the rhizosphere can facilitate Fe acquisition by plants. To test whether the two rice genotypes differed in their capacity to acidify the rhizosphere, we compared the effects of saline-alkaline stress on rhizosphere acidification by monitoring changes in medium pH. On exposure to saline-alkaline stress, a significantly greater reduction in growth medium pH was observed for Dongdao-4 plants relative to Jigeng-88 plants (Fig. 6A).

We further investigated the effect of saline-alkaline stress on transcripts of H⁺-ATPases in the two genotypes. No differences in levels of the three transcripts encoding rice H⁺-ATPases (Os12g0638700,Os03g0100800, andOs03g0689300) were detected between Dongdao-4 and Jigang-88 plants grown in control medium (Fig. 6B–D). The expression levels of the three genes were markedly up-regulated by saline-alkaline treatment in Dongdao-4 (Fig. 6B–D). In contrast, expression ofOs12g0638700 andOs03g0689300 in Jigeng-88 was suppressed by the saline-alkaline treatment, leading to significantly higher expression levels for these two genes in Dongdao-4 plants (Fig. 6B–D).

Effect of saline-alkaline stress on secretion of phytosiderophores

To test whether the two genotypes differ in their capacity to exude phytosiderophores under Fe-deficient conditions, we compared the effects of saline-alkaline stress on their phytosiderophore release rates. The two genotypes had comparable low levels of phytosiderophore release rates in control medium. Exposure to the saline-alkaline medium led to significant increases in phytosiderophore release in both genotypes; the increase in Dongdao-4 was greater than in Jigeng-88 (Fig. 7).

Effects of saline-alkaline stress on expression of Fe deficiency-related genes

The greater capacity of the Dongdao-4 genotype to acquire Fe under saline-alkaline conditions suggests that it is more tolerant to Fe deficiency than Jigeng-88. To further explore the physiological mechanisms underlying the greater accumulation of Fe in Dongdao-4 plants, we monitored changes in the expression patterns of genes involved in Fe acquisition, includingOsIRO2,OsIRT1,OsNAS1,OsNAS2,OsYSL15, andOsYSL2. Among these genes, the expression levels in Dongdao-4 were greater than in Jigeng-88 under both control and saline-alkaline conditions, except forOsIRT1, which exhibited a comparable expression level in the two genotypes in control medium, and a higher expression level in Dongdao-4 than in Jigeng-88 in saline-alkaline medium (Fig. 8). Expression levels of the six genes were markedly up-regulated by saline-alkaline treatment; the treatment-induced up-regulation was greater in Dongdao-4 than in Jigeng-88, such that a significantly higher expression level in Dongdao-4 plants was observed under saline-alkaline conditions (Fig. 8).

Effect of Fe deficiency on Dongdao-4 and Jigeng-88

The observation that Dongdao-4 plants can maintain Fe homeostasis under saline-alkaline conditions suggests that the Dongdao-4 genotype is highly efficient at Fe acquisition. To test this hypothesis, the effects of Fe deficiency on Dongdao-4 and Jigeng-88 plants were evaluated and compared. No differences in phenotype were observed when the two genotypes were grown in Fe-sufficient medium. Growth of Jigeng-88 was significantly suppressed upon transfer into Fe-deficient medium; in contrast, the growth of Dongdao-4 was unchanged by exposure to Fe-deficient medium (Fig. 9A). Jigeng-88, but not Dongdao-4, exhibited foliar chlorosis when exposed to Fe deficiency (Fig. 9B). In addition, there were no differences in the height, shoot biomass, root biomass, or SPAD value of Dongdao-4 plants grown in Fe-sufficientversus Fe-deficient solution. In contrast, Jigeng-88 plants exhibited significant reductions in height, shoot biomass, root biomass, and SPAD value when exposed to Fe-deficient medium (Fig. 9C–F). These results confirm that Dongdao-4 plants are more tolerant to Fe deficiency than Jigeng-88 plants.

Effects of Fe deficiency on Fe accumulation

We further compared Fe concentrations in shoots and roots of the two genotypes under Fe-sufficient and Fe-deficient conditions. Under Fe-sufficient conditions, there was no significant difference between the two genotypes in shoot Fe concentration (Fig. 10A). After exposure to Fe-deficient medium there were significant reductions in shoot Fe concentrations in both genotypes; the reduction in shoot Fe concentration in Jigeng-88 was greater than that in Dongdao-4, leading to a significantly higher shoot Fe concentration in Dongdao-4versus Jigeng-88 plants (Fig. 10A). Root Fe concentrations in the two genotypes were comparable under Fe-sufficient conditions; exposure to Fe-deficient medium led to similar reductions in root Fe concentration in both genotypes (Fig. 10B).

Effect of Fe deficiency on rhizosphere acidification

Upon exposure to Fe-deficient medium, there were significant reductions in medium pH for both rice genotypes (Fig. 11). The Fe-deficiency-induced reduction in medium pH of Dongdao-4 was greater than that of Jigeng-88, resulting in a significantly higher medium pH for Jigeng-88 (Fig. 11A). These results indicate that Fe-deficiency-induced acidification is greater in Dongdao-4 than in Jigeng-88 plants (Fig. 11A).

We further monitored changes in the expression of the H⁺-ATPase-encoding genesOs12g0638700,Os03g0100800, andOs03g0689300 in the two genotypes. Levels of expression ofOs12g0638700 were comparable in the two genotypes under both Fe-sufficient and Fe-deficient conditions (Fig. 11B). Under Fe-sufficient conditions, expression levels ofOs03g0100800 andOs03g0689300 were comparable in Dongdao-4 and Jigeng-88 plants (Fig. 11C,D). The expression ofOs03g0100800 was markedly up-regulated by exposure to Fe deficiency in Dongdao-4 plants, whereas the expression level in Jigeng-88 plants exposed to Fe-deficient medium remained relatively unchanged, leading to significantly higher expression levels in Dongdao-4 plants (Fig. 11C). Expression ofOs03g0689300 was markedly up-regulated by Fe deficiency in both genotypes; the up-regulation was more pronounced in Jigeng-88 plants, such that significantly higher expression in Jigeng-88versus Dongdao-4 plants was observed in Fe-deficient conditions (Fig. 11D).

Effect of Fe deficiency on secretion of phytosiderophores

As shown inFig. 12, no difference between the two genotypes in the rate of phytosiderophore secretion from the roots was detected under Fe-sufficient conditions. Exposure to Fe-deficient medium led to a significantly greater increase in phytosiderophore release from Dongdao-4 than from Jigeng-88 roots (Fig. 12).

Effects of Fe deficiency on expression levels of Fe deficiency-responsive genes

To investigate the mechanisms underlying the different tolerance of the two genotypes to Fe deficiency, we monitored changes in the expression ofOsIRO2, OsIRT1, OsNAS1, OsNAS2, OsYSL15, andOsYSL2 in roots. Under Fe-sufficient conditions, the levels of expression ofOsIRT1 in roots of Dongdao-4 and Jigeng-88 plants were comparable (Fig. 13A). When treated with Fe-deficient medium, the expression level ofOsIRT1 was up-regulated in both genotypes; the increase was greater in Dongdao-4 than in Jigeng-88 (Fig. 13B). The levels of expression ofOsIRO2, OsNAS1, OsNAS2, OsYSL15, andOsYSL2 in both genotypes were also up-regulated by exposure to Fe-deficient medium (Fig. 13A,C–F). Similar toOsIRT1, the magnitude of Fe deficiency-induced increases in expression ofOsIRO2, OsNAS1, OsNAS2, OsYSL15 andOsYSL2 in Dongdao-4 plants were higher than those in Jigeng-88 plants (Fig. 13A,C–F).

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