Molecular Mechanism Of P And Fe Interaction And Development Of Transgenic Rice With Enhanced Iron Content And Bioavailibility

Fe is an essential micronutrient for plants.Although Fe is one of the most abundant elements in the Earth's crust,Fe deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability.On the other hand,because plant based food is the main diet for peoples from many developping areas,Fe deficiency is the most prevalent micronutrient deficiency.Therefore,understanding of the machanisms of the iron uptake,transport,metabolism,storage and their regulation is not only important for the improvement of crop production,but also for the development of crop varieties with densed Fe to alleviate the world-wide Fe deficiency problem.Interaction between Fe and P has been noted in the field of plant nutrition.To understand the physiology and molecular mechanisms of their interaction,we studied the growth performance,nutrient concentration,and gene expression profiles of root and shoot segments derived from 10 day old rice seedlings under four different nutrient conditions;ⅰ) full strength of Fe and P,(+Fe+P),ⅱ) full strength of P and no Fe(-Fe+P),ⅲ) full strength of Fe and no P(+Fe-P) andⅳ) without both Fe and P (-Fe-P).While removal of Fe in the growth medium resulted in very low shoot and root Fe concentrations,the chlorotic symptoms and retarded seedling growth were only observed on seedlings grown in the presence of P.Microarray data showed that in roots,7,628 transcripts were significantly changing in abundance in the absence of Fe alone.Interestingly,many of these were changes were reversed if P was also absent (-Fe-P),with only～15%overlapping with-Fe alone(-Fe+P).Analysis of the soluble Fe concentration in rice seedling shoots showed that P deficiency resulted in significantly increased Fe availability within the plants.The soluble Fe concentration under -Fe-P conditions was similar to that under +Fe+P conditions.These results provide evidence that the presence of P can affect Fe availability and in turn can influence the regulation of Fe responsive genes. The microarray analysis identified many Fe deficiency response genes,including many classical -Fe response genes,such as IRO2,OsIRT1,OsNAS1,OsYSL15,and many novel genes with unknown functions.These genes includes OPT transport family genes OsOPT1 and OsOPT2,bHLH transcription factors OsIRbHLH1 and OsIRbHLH2 and so on.Among these novel Fe regulated genes,the bHLH-domain contained putative transcripofion factor OsIRbHLH2 was studied.Nutrient deplete treatments show that the expression of OsIRbHLH2 was specifically induced by Fe deficiency conditions,but not by other nutrients(-N,-P,-K,-Zn,-Mn,-Cu). OsIRbHLH2 protein is localized into nucleus,supporting that OsIRbHLH2 is an putative transcription factor.The physiological and molecular responses of the OsIRbHLH2-overexpressing transgenic rice lines suggest that OsIRbHLH2 is a negative regulator in Fe deficiency responses in rice.Development of crop varieties with improved iron content and bioavailiablility is important approach for the alleviation of Fe deficiency problem world-wide.In this study,rice nicotianamine(NA) synthase gene(OsNAS1) was endosperm-specifically expressed in rice grain.The resulted transgenic rice accumulated a significant amount of NA in the grains.Bioavailability of iron from the high NA grain,as measured by ferritin synthesis in an in vitro Caco-2 cell model that simulates the human digestive system,was twice as much as that of the control line.When added at 1:1 molar ratio to ferrous iron in the in vitro system,NA produced two times more ferritin than that of ascorbic acid(AA),one of the most potent enhancers of iron bioavailability.These data demonstrated that NA is a novel and powerful promoter of iron utilization. Biofortifying polished rice with this compound has great potential in combating global human iron deficiency in people dependent on rice for their sustenance.