Functional Analysis Of Fe-related Transcription Factor OsbHLH133&the Molecular Mechanism Of Ethvlene Svnthesis Induced By Fe Deficiency

Iron is an essential mineral element for plant growth. Although iron (Fe) is abundant in the earth’s crust, it is not available to plants because the low solubility of Fe in soil solution. On the other hand, excess accumulation of Fe can cause damage to plant cells. Studying on the molecular mechanism in response to Fe deficiency is very important for plants to adapting to the stress caused by Fe deficiency or Fe excess accumulation. It also plays a significant role in developing crop varieties with improved iron content.In this study, we characterized a novel basic helix-loop-helix (bHLH) transcription factor in rice, named OsbHLH133. OsbHLH133was targeted to the nucleus, and the transcript abundance of OsbHLH133was strongly up-regulated in roots under Fe-deficiency conditions. Using transgeneic rice expressing the GUS reporter gene driven by the OsbHLH133endogenous promoter, it was found that OsbHLH133is expressed throughout the whole roots, preferently in the stele of the roots and vascular budndles of the shoots in response to Fe deficiency. Phylogenetic analysis showed that OsbHLH133is not closely related to other known Fe-responsive bHLH transcription factors. Thus, OsbHLH133should be involved in an unknown mechanism related to Fe homeostasis.Study on OsbHLH133overexpression (OsbHLH133-OE) lines and T-DNA insertional mutant(bhlh133) plants showed that the leaf chlorophyll content (SPAD value) increased in bhlh133mutant while decreased in OsbHLH133-OE plants. Compared to wild type (WT), bhlh133showed growth retardation with enhanced Fe concentration seen in shoots and reduced Fe concentration in roots. OsbHLH133-OE had the opposite effect, which is resulted in an enhanced Fe concentration in roots and reduced Fe concentration in shoots. In addition, Fe concentration in xylem sap of OsbHLH133-OE also reduced. Therefore, alteration of the transcript abundance of OsbHLH133affected Fe distribution between roots and shoots.Microarray and quantitative RT-PCR analysis showed that the transcript abundances of some of the genes encoding Fe-related functions in OE, bhlh133and WT plants were related to the local Fe concentration. Expression of Fe deficiency response genes was repressed in the tissue with higher Fe concentration, and was Induced in the tissue with lower Fe concentration. Significant differential expression of a number of signalling pathways, including calcium signalling, was also seen in bhlh133plants compared to WT. It suggested a role for calcium signalling in Fe homeostasis of plants.The main purpose of this study is to improve the regulatory mechanism of Fe deficiency response in plants. In response to Fe-deficiency, ethylene production was induced. The preliminary study on the molecular mechanism of ethylene synthesis induced by Fe deficiency was conducted in this study. Analysis of the transcirpt abundances of the9authentic ACS genes in Arabidopsis showed that AtACS2/6/7/11was up-regulated by Fe deficiency in the leaves and roots. It suggested those four ACS genes might be involved in the pathway of ethylene synthesis which was induced by Fe deficiency. Additionally, the expression of AtMPK3and AtMPK6which can phosphorylate AtACS2/6was also induced by Fe deficiency.Time-course analysis of ethylene production under different period of Fe deficiency indicated that ethylene production was induced by Fe deficiency in Arabidopsis, and the induction level was up to1.7-2folds. Because of the function reductant of ACSs and less than2folds of ethylene production induced by Fe deficiency, it is difficult to identify the ACS which is involved in biosynthesis of ethylene that induced by Fe deficiency by measuring the ethylene production of ACS mutants. Analysis of the ethylene production of mpk3and mpk6mutant plants showed a significantly reduced ethylene production in mpk6and a slight reduced in mpk3compared to WT under Fe-deficiency condition. It suggested a role for AtMPK6in regulating ethylene production which is induced by Fe deficiency.Taken together, we characterized a novel bHLH transcription factor, named OsbHLH133in rice. This transcription factor is strongly up-regulated by Fe deficiency. Our results indicated that OsbHLH133acted as an important regulator of Fe distribution between shoots and roots and it might be involved in regulating the root-to-shoot Fe translocation. The ACS genes involved in the ethylene biosynthesis which is induced by Fe deficiency were identified in Arabidopsis and AtMPK6might be the regulator of this pathway.