Study On The Regulation Mechanism Of Iron Accumulation In Grains Of "Luotian Red Rice"

Iron（Fe）is one of the important nutrients required by living organisms.As a major food crop,the iron nutrition level of rice is closely related to human health.The group has developed an iron-rich line“Luotian red rice”and used foliar spraying of FeSO₄ to increase the brown rice iron content of this red rice and common rice 9311 by 150%,but the mechanism involved in the regulation of seed iron enrichment is not clear.In this study,we continued to use"Luotian red rice"and 9311 varieties as materials,planted in pots and treated with 0.1%FeSO₄ foliar spray at the gestation and tasseling stages to study the changes of iron storage location,accumulation pattern and other trace elements of zinc（Zn）,copper（Cu）,manganese（Mn）and cadmium（Cd）content in the grain.In addition,the expression and regulation of Fe（II）-NA and Fe（III）-DMA,the major chelated Feforms and their transport molecules YSL in rice grain Fetransport,were investigated together with transcriptome analysis to clarify the regulatory effect of foliar FeSO₄ application on Feaccumulation in rice grain and its relationship with Zn,Cu,Mn and Cd accumulation.This study provides a basis for the analysis of the molecular basis of Fetransport and regulation in rice seeds and the development of rice variety improvement and high-quality cultivation techniques.The main results were obtained:1.Analysis of FePerls Blue staining of seeds at different developmental stages from15 to 35 d after flowering showed that 0.1%FeSO₄ spraying treatment of red rice and 9311grain-filled seeds and mature brown rice had deeper tissue Festaining,especially epidermal and endosperm tissue staining was significantly deeper,which clearly showed that both FeSO₄ spraying treatments promoted Feaccumulation in red rice and white rice seeds and effectively improved The level of Feaccumulation in epidermis and endosperm was effectively increased.2.The analysis of elemental content determination by ICP-AES/MS showed that the Fecontent in all parts of mature red rice seeds,including brown rice,polished rice and pericarp（including embryo）,increased significantly after 0.1%FeSO₄ spray treatment,and the Fecontent in brown rice and semolina increased more than 1.6 times,which was consistent with the staining results.Meanwhile,Zn,Cu and Mn contents in mature brown rice of red rice after iron spraying treatment were significantly increased by 13.1%,7.9%and 8.8%.However,the Cd content of fine rice was significantly reduced by 30%,and the Cd content in all tissues of the seeds was much lower than the national safety standard(0.2mg·kg^-1).The present results further clarify that a foliar spraying of 0.1%FeSO₄ at each of the gestation and tasseling stages of Luotian red rice growth increased the accumulation of Fein mature rice,especially in fine rice,and simultaneously increased the contents of Zn,Cu and Mn in brown rice and significantly reduced the Cd content in fine rice.The levels of the above metals in all tissues were also lower than the maximum limits of metal elements in foods set by international agencies.3.Analysis of seed transcriptome data from 5 to 15 d after flowering revealed significant differential expression of genes GO enriched in the transmembrane transport process of iron and the nicotinic acid（NA）synthesis pathway that assists in metal element transport,including the iron transport-related genes OsYSL12,OsYSL14,OsYSL2,OsFRDL1,and OsNAMP5,as well as the iron chelator NA and 2’deoxymaltic acid（DMA）synthesis-related genes OsNAS1,OsNAS2,OsNAS3,OsNAAT1 and OsNAAT2.The results of qPCR validation showed that metal transport-related genes OsYSL12,OsYSL14 and OsFRDL1 were significantly up-regulated in DAF5-15 seeds of Luotian red rice after 0.1%FeSO₄spraying,while OsYSL2 and OsNRAMP5 were significantly increased at DAF15.The expression of the chelate NA and DMA synthesis genes OsNAS1,OsNAS2,OsNAAT1and OsDMAS1,which assist in metal transport,was also significantly increased from DAF5to 15.It indicates that foliar spraying of FeSO₄ plays a certain role in promoting the expression of genes related to Fetransport and chelate synthesis in red rice,which is beneficial to the accumulation of Fein Luotian red rice seeds.4.The expression of related genes in 9311 was measured,and the results showed that only OsYSL2 and OsFRDL1 were more strongly up-regulated in response to iron spraying after 0.1%FeSO₄ treatment in 9311 compared with red rice,while two uncharacterized functional genes,OsYSL12 and OsYSL14,were both strongly up-regulated in response to NA and DMA synthesis-related genes in red rice and DMA synthesis-related genes.Meanwhile,combined with the comprehensive bioinformatics analysis of the members of OsYSLs,it was found that both OsYSL12 and OsYSL14 were located in the same subfamily as OsYSL13,AtYSL5 and AtYSL7,which are related to the balance of iron distribution in seeds,in the phylogeny.Analysis of promoter cis-acting elements revealed that both OsYSL12 and OsYSL14 have seed-specific regulatory elements.Taken together,it is hypothesized that OsYSL12 and OsYSL14 have important roles in seed iron transport and regulation,and are potential characterizing molecules for iron enrichment and regulation in the seeds of Luotian red rice.In conclusion,foliar spraying of FeSO₄ can effectively increase the Fecontent in red rice,which is an economic and effective cultivation measure for iron enrichment in rice.Atthe same time,the molecular basis of iron accumulation in red rice seeds was understood,which provided some theoretical basis for the subsequent study of iron enrichment mechanism in red rice.