Differential Expression of Iron Deficiency Responsive Rice Genes under Low Phosphorus and Iron Toxicity Conditions and Association of OsIRO3 with Yield in Acidic Soils

doi:10.1016/j.rsci.2022.07.009

Abstract

Abstract:

With the hypothesis that iron (Fe) deficiency responsive genes may play a role in Fe toxicity conditions, we studied five such genes OsNAS1, OsNAS3, OsIRO2, OsIRO3 and OsYSL16 across six contrasting rice genotypes for expression under high Fe and low phosphorus (P) conditions, and sequence polymorphism. Genotypes Sahbhagi Dhan, Chakhao Poirieton and Shasharang were high yielders with no bronzing symptom visible under Fe toxic field conditions, and BAM350 and BAM811 were low yielders but did not show bronzing symptoms. Hydroponic screening revealed that the number of crown roots and root length can be traits for consideration for identifying Fe toxicity tolerance in rice genotypes. Fe contents in rice roots and shoots of a high-yielding genotype KMR3 showing leaf bronzing were significantly high. In response to 24 h high Fe stress, the expression levels of OsNAS3 were up-regulated in all genotypes except KMR3. In response to 48 h high Fe stress, the expression levels of OsNAS1 were 3-fold higher in tolerant Shasharang, whereas in KMR3, it was significantly down-regulated. Even in response to 7 d excess Fe stress, the transcript abundances of OsIRO2 and OsNAS3 were contrasting in genotypes Shasharang and KMR3. This suggested that the reported Fe deficiency genes had a role in Fe toxicity and that in genotype KMR3 under excess Fe stress, there was disruption of metal homeostasis. Under the 48 h low P conditions, OsIRO2 and OsYSL16 were significantly up-regulated in Fe tolerant genotype Shasharang and in low P tolerant genotype Chakhao Poirieton, respectively. In silico sequence analysis across 3 024 rice genotypes revealed polymorphism for 4 genes. Sequencing across OsIRO3 and OsNAS3 revealed nucleotide polymorphism between tolerant and susceptible genotypes for Fe toxicity. Non-synonymous single nucleotide polymorphisms and insertion/deletions (InDels) differing in tolerant and susceptible genotypes were identified. A marker targeting 25-bp InDel in OsIRO3, when run on a diverse panel of 43 rice genotypes and a biparental population, was associated with superior performance for yield under acidic lowland field conditions. This study highlights the potential of one of the vital genes involved in Fe homeostasis as a genic target for improving rice yield in acidic soils.

Key words: acidic soil, low phosphorus, iron toxicity, OsNAS1, OsIRO3, rice, yield

Ernieca Lyngdoh Nongbri, Sudip Das, Karma Landup Bhutia, Aleimo G. Momin, Mayank Rai, Wricha Tyagi. Differential Expression of Iron Deficiency Responsive Rice Genes under Low Phosphorus and Iron Toxicity Conditions and Association of OsIRO3 with Yield in Acidic Soils[J]. Rice Science, 2023, 30(1): 58-69.

Figures/Tables 5

Fig. 1. Screening under acidic lowland soils and hydroponic conditions. A, Leaf bronzing scores (0?5, from left to right) given to rice genotypes grown in the lowland fields depending on the intensity of leaf bronzing symptoms on foliage. B, Fe plaque accumulation in an experimental field. C, Fe content evaluated on selected representative genotypes harvested from lowland fields. Data are Mean ± SD (n = 20). Statistically significant differences between the root and shoot were determined by the Student’s t-test (*, P < 0.05). D?F, Root length (D), shoot length (E) and root fresh weight (F) under hydroponic conditions. The histograms represent average phenotypic values under control (0.0284 mmol/L Fe2+ and 0.3500 mmol/L Pi) and treatment (3.6000 mmol/L Fe2+ and 0.3500 mmol/L Pi) conditions. Data are Mean ± SD (n = 10). Statistically significant differences between the control and treatment were determined by the Student’s t-test (*, P < 0.05). G, Cross-section of roots after staining with Perl’s blue stain viewed under a Leica DM750 microscope. Scale bars, 1 mm. SH, Shasharang; LR15, Priya; LR18, Paijong; CP, Chakhao Poirieton; Kas, Kasalath.

Fig. 2. Transcript levels of five rice genes involved in Fe deficiency responses in shoots of six rice genotypes after treatments of low P and high Fe for 24 h. A?E, Expression levels of OsIRO2 (A), OsIRO3 (B), OsNAS1 (C), OsNAS3 (D) and OsYSL16 (E) under low P and high Fe conditions for 24 h. Plants were grown using sand culture supplemented with Yoshida solution in control, low P and excess Fe treatments at pH 5.4. Shoots were harvested and used for qRT-PCR. Rice β-tubulin transcript level was used for normalization and transcript abundance was expressed as a ratio relative to the levels in control (Mean ± SD, n = 4). Y axes on the left and right represent normalized relative expression in the P and Fe experiments, respectively. CK, Control (0.3500 mmol/L Pi and 0.0284 mmol/L Fe2+); T, Treatment (low Pi: 0.0150 mmol/L Pi and 0.0284 mmol/L Fe2+; high Fe2+: 0.3500 mmol/L Pi and 3.6000 mmol/L Fe2+). SD, Sahbhagi Dhan; CP, Chakhao Poirieton; B350, BAM350; B811, BAM811; SH, Shasharang.

Fig. 3. Transcript levels of five rice genes involved in Fe deficiency responses in shoots of six rice genotypes after treatments of low P and high Fe for 48 h. A?E, Expression levels of OsIRO2 (A), OsIRO3 (B), OsNAS1 (C), OsNAS3 (D) and OsYSL16 (E) under low P and high Fe conditions for 48 h. Plants were grown using sand culture supplemented with Yoshida solution in control, low P and excess Fe treatments at pH 5.4. Shoots were harvested and used for qRT-PCR. Rice β-tubulin transcript level was used for normalization and transcript abundance was expressed as a ratio relative to the levels in control (Mean ± SD, n = 4). Y axes on the left and right represent normalized relative expression in the P and Fe experiments, respectively. CK, Control (0.3500 mmol/L Pi and 0.0284 mmol/L Fe2+); T, Treatment (low Pi: 0.0150 mmol/L Pi and 0.0284 mmol/L Fe2+; high Fe2+: 0.3500 mmol/L Pi and 3.6000 mmol/L Fe2+). SD, Sahbhagi Dhan; CP, Chakhao Poirieton; B350, BAM350; B811, BAM811; SH, Shasharang.

Fig. 4. Transcript levels of five rice genes involved in Fe deficiency responses in shoots of two genotypes after exposure to 7 d of high Fe treatment. A?E, Expression levels of OsIRO2 (A), OsIRO3 (B), OsNAS1 (C), OsNAS3 (D) and OsYSL16 (E) under high Fe conditions for 7 d. Plants were grown using sand culture supplemented with Yoshida solution in control (CK, 0.3500 mmol/L Pi and 0.0284 mmol/L Fe2+) and excess Fe (T, 0.3500 mmol/L Pi and 3.6000 mmol/L Fe2+) treatments at pH 5.4. Shoots were harvested and used for qRT-PCR. Rice β-tubulin transcript level was used for normalization and transcript abundance was expressed as a ratio relative to the levels in control (Mean ± SD, n = 4). Y axis on the left represents normalized relative expression under Fe experiment. SH, Shasharang.

Fig. 5. Marker development and validation of OsIRO3 insertion/deletion (InDel) marker. A, A 25-bp InDel in 3°-UTR (untranslated region) of OsIRO3 was targeted for marker development. Schematic diagram of OsIRO3 gene along with primers designed to amplify the 3°-UTR region is shown. Dark grey triangles and lines indicate introns and exons, respectively. The arrow and star in the top indicate transcription start and stop positions, respectively. The positions of forward and reverse primers (33-3F/R) are given below the gene diagram. Allelic polymorphisms across OsIRO3 gene for five rice genotypes are given in the bottom. Dark grey and white colours indicate reference and novel alleles, respectively. B, Representative polymorphisms observed in a panel of 43 diverse rice genotypes and ULRC34 recombinant inbred lines. C, Associations of OsIRO3 InDel in two populations with major traits of interest in Fe toxic acidic lowland field. The difference in the phenotypic means of the two allelic classes was tested using the t-test. Bars in the first two graphs represent significance of differences. Single and double asterisks on the top of bars indicate significance of phenotypic difference between the two allelic classes at 0.1 and 0.01 levels of significance. Bars in the third graph indicate phenotypic means of genotypes carrying SD and CP type alleles in the panel of 43 diverse rice genotypes. Error bars indicate confidence interval. TN30, Tiller number at 30 d after transplanting; TN60, Tiller number at 60 d after transplanting; PL, Panicle length (cm); LA, Leaf area (cm2); HGW, 100-grain weight (g); GYPP, Grain yield per panicle (g); SF, Spikelet fertility rate (%); SPP, Spikelet number per panicle; PUE, Phosphorus use efficiency at harvest (%); BY, Biological yield (g); FG, Filled grain number per plant. M, Marker; SH, Shasharang; SD, Sahbhagi Dhan; CP, Chakhao Poirieton.

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