| Iron(Fe)is an essential micronutrient for plant growth and development.Despite the high content of Fe in the earth’s crust,it usually exists in the form of insoluble oxides.On calcareous soils with higher pH,Fe deficiency is a common environmental stress in crop production.In order to adapt to the Fe-deficient environment,plants have evolved two distinct strategies for Fe absorption.Non-grass plants use Strategy Ⅰ to absorb ferrous Fe while gramineous plants use Strategy Ⅱ to take up ferric Fe.Adapted to the flooded and upland growth condition,rice retain two types of iron absorption,and rice is the largest food crop in China.Therefore,it is of great theoretical and practical significance to study the physiological and molecular mechanisms of rice Fe uptake,transport and metabolism for enriching the physiological mechanism of rice iron stress,and effectively rice breeding towards to tolerance to Fe deficiency stress and Fe-rich in grains.A previous work in the laboratory identified a rice homologous gene of Arabidopsis FIT,OsbHLH156.The expression of this gene was significantly upregulated by Fe deficiency.It was known that OsbHLH156 can interact with IRO2 and help IRO2 to locate to the nucleus,and then activate the expression of the downstream genes in Strategy Ⅱ.However,it is unclear how OsbHLH156 mediates downstream genes after IRO2 enters the nucleus.In this paper,we mainly studied the expression patterns of OsbHLH156 and IRO2,constructed osbhlh156 and iro2 mutants and performed phenotypic and transcriptomic analysis of the mutants,and analyzed the interaction between OsbHLH156 and IRO2 proteins and the regulation of downstream genes.Through RT-qPCR analysis,we found that both OsbHLH156 and IRO2 genes were up-regulated after 1 day of Fe deficiency treatment,and the expression level was down-regulated after Fe re-supply,indicating that the two genes’ spatial and temporal expression patterns were basically synchronal.The osbhlh156 and iro2 gene editing mutants were obtained using the CRISPR/CAS9 system.The phenotypes of iro2 and osbhlh156 mutants were analyzed and compared under different Fe supplied conditions.Results showed that under normal Fe-supply conditions,the osbhlh156 and iro2 mutants had no obvious phenotypic changes from the wild type.However,under Fe deficient condition,both mutants showed significant chlorosis with reduced leaf SPAD values.Moreover,the plant growth was greatly inhibited.Transcriptome sequencing analysis found that the induction of Fe deficiency in the genes of Strategy Ⅱ was strongly suppressed in both osbhlh156 and iro2 mutants,while only IRT1 gene in Strategy Ⅰ pathway was affected in osbhlh156 and iro2 mutants.Bimolecular fluorescence complementation,immunoprecipitation and yeast twohybridization studies showed that OsbHLH156 and IRO2 can interact to form a complex.Transcription activation test in yeast showed that OsbHLH156 and IRO2 can activate the downstream genes,including OsIRT1,OsYSL15,OsYSL16,OsIDI2,OsNAS2,OsNAS3 and OsDMAS1.In summary,we found that OsbHLH156 and IRO2 have a consistent spatiotemporal expression pattern,and OsbHLH156 and IRO2 can form a protein complex and jointly activate the expression of downstream genes.The results of this study further improve the regulation network of rice Fe homeostasis.It will lay the foundation for rice breeding for tolerance to Fe-deficiency and enrichment of grain Fe in rice. |
