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Expression profiling of iron deficiency responsive microRNAs and gene targets in rice seedlings of Madhukar x Swarna recombinant inbred lines with contrasting levels of iron in seeds

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Abstract

Aims

To test if microRNAs are involved in iron (Fe) homeostasis in Oryza sativa.

Methods

Recombinant inbred lines (RILs) of rice with contrasting levels of iron in seeds (high iron line HL, low iron line LL) and parent Swarna were grown in Fe sufficient (+Fe) and deficient (−Fe) environment. miRNAs whose target genes underlie the QTLs mapped for iron concentration (mapped in our previous study) were identified using bioinformatics. The expression analysis of these miRNAs and their targets along with few other miRNAs involved in nutrient homeostasis was done in root and shoot tissue. Real time PCR was used to study the relative expression of miRNAs and their target genes.

Results

Out of nine miRNAs used in this study, 7 miRNAs-miR156, 168, 172, 162, 167, 171, and 398 showed down-regulation under Fe deficiency in root and shoot of high iron line when compared with Fe sufficient condition. Further, most of the miRNAs showed down-regulation while their target genes showed up-regulation under Fe deficiency in roots of all three genotypes (HL, LL and Swarna) suggesting roots are more responsive to Fe deficiency. Important role of miRNAs in iron homeostasis was analyzed by comparing the expression of these miRNAs in HL, LL and Swarna under + Fe and –Fe.

Conclusion

MicroRNAs showed differential expression in + Fe and –Fe environment. Further, their expression is more effectively regulated in root under Fe deficiency. This indicates that miRNAs might be playing regulatory roles in iron homeostasis in rice. This study suggests that Fe deficiency responsive miRNAs are involved in cross talk between other nutrients stress.

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Acknowledgments

The work was financially supported by the Indian Council of Agricultural Research, Govt. of India, Network project on transgenics and functional genomics of crops—project 3019 on rice micronutrients (NPTC/FG/05/2672/33).

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Authors and Affiliations

  1. Directorate of Rice Research, Hyderabad, India

    Surekha Agarwal, Satendra Kumar Mangrauthia & N. Sarla

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  1. Surekha Agarwal
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  2. Satendra Kumar Mangrauthia
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  3. N. Sarla
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Corresponding authors

Correspondence to Satendra Kumar Mangrauthia or N. Sarla.

Additional information

Responsible Editor: Jian Feng Ma.

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Supplementary Fig. 1

Real time expression of microRNAs in high iron line (HL) and low iron line (LL) rice seedlings under Fe sufficient and deficient condition compared to Swarna as control. Seven days old rice seedlings were transferred to plastic boxes containing nutrient solution for 10 days. For +Fe treatment, Fe-EDTA was present in the nutrient solution but for –Fe treatment, Fe-EDTA was not added. Seedlings were grown in hydroponics in a growth chamber at 30ºC/22ºC (day/night) temperatures with a 12-h-light/12-h-dark regime. Fold difference was calculated from 2−ΔΔCt method. Note: Y axis shows the fold expression and the scale is different in each graph. A- miR156; B- miR162; C- miR167; D- miR168; E- miR169; F- miR171; G- miR172; H- miR395; I- miR398. (JPEG 701 kb)

Supplementary Fig. 2

Real time expression of microRNAs in low iron (LL) rice seedlings under Fe sufficient (A) and deficient (B) condition compared to high iron (HL) as control. Seven days old rice seedlings were transferred to plastic boxes containing nutrient solution for 10 days. For +Fe treatment, Fe-EDTA was present in the nutrient solution but for –Fe treatment, Fe-EDTA was not added. Seedlings were grown in hydroponics in a growth chamber at 30ºC/22ºC (day/night) temperatures with a 12-h-light/12-h-dark regime. Fold difference was calculated from 2−ΔΔCt method. Y axis shows the fold expression and the scale is different in each graph. (JPEG 339 kb)

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Agarwal, S., Mangrauthia, S.K. & Sarla, N. Expression profiling of iron deficiency responsive microRNAs and gene targets in rice seedlings of Madhukar x Swarna recombinant inbred lines with contrasting levels of iron in seeds. Plant Soil 396, 137–150 (2015). https://doi.org/10.1007/s11104-015-2561-y

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