Study On Function And Regulation Of Key Genes HA6 And IRT1 For Iron Absorption In Citrus Rootstock

Citrus producing areas in western China often cause iron deficiency and yellowing of leaves due to alkaline soil,which seriously affects the yield and quality of fruits.Rootstocks are very important for the growth of citrus fruit trees,and the use of irontolerant rootstocks can effectively correct iron deficiency yellowing of plants.In this study,we compared the process of iron absorption in roots of iron intolerant stock TO(Poncirus trifoliata(L.)Raf,TO)and iron tolerant stock ZQ(Citrus Wilsonii Tanaka,ZQ),explored the key genes of iron nutrition and clarified their molecular regulation mechanism.The main findings are as follows:1.Analysis of the transcriptional mechanism of citrus HA6 gene regulating root iron activation.In alkaline soils,citrus roots activate soil insoluble iron by secreting H+ and promote iron absorption by plants.Therefore,the differences of iron activation in ZQ and TO were first compared.The research showed that the H+ secretion capacity of roots of ZQ was significantly higher than that of TO under +Fe or-Fe medium.It shows that ZQ has a strong iron activation ability.The q RT-PCR technique was used to analyze 10 H+-ATPase family genes responsible for H+ secretion in ZQ and TO.It was found that ZQHA6 of was highly expressed in-Fe conditions,while the upregulated expression amplitude and transcript accumulation of the TOHA6 were were smaller than those of ZQHA6.As for other HA genes,they are not induced by-Fe or it shows a low degree of induction.The results of subcellular localization showed that HA6 was mainly localized to the plasma membrane.Phylogenetic analysis showed that HA6 of citrus was homologous to AHA2 gene in Arabidopsis thaliana,and found that ZQHA6 could save the yellowing symptoms in leaves of aha2 mutant and increase the iron content in leaves under-Fe.Overexpression of ZQHA6 also can significantly increase the iron content in TO leaves.A yeast one-hybrid showed a transcription factor MYB308 can bind and activate ZQHA6 was screened,but MYB308 did not bind and regulate TOHA6.The analysis showed that the binding site of MYB308 had a two-base difference on the ZQHA6 and TOHA6 promoters,and this variation may be related to the adaptation of the ZQ to alkaline iron deficiency stress.Furthermore,The ZQHA6 gene activated the mass expression of MIOX4 gene related to cell wall pectin synthesis.Furthermore,MIOX4 was overexpressed in TO roots,and it was found that the transgenic plants significantly increased the iron content of leaves under iron deficiency conditions,but the iron content of roots was no different from that of wild-type controls,indicating that HA6 may be involved in the transport of iron from roots to leaves by activating MIOX4.2.Analysis of the transcriptional mechanism of citrus IRT1 gene regulating root iron transport.After the iron of plant roots is activated and reduced to Fe2+,it enters the root cells under the action of IRT1 transporter,so this study also compared the expression differences of IRT1 gene between ZQ and TO.Subcellular localization showed that IRT1 of citrus was mainly localized in the cytoplasm and plasma membrane.Overexpression of IRT1 gene in roots of TO can significantly increase the iron content in leaves.It also increases the iron content of the root under-Fe conditions,although the iron content is reduced during the iron addition treatment and may be antagonized by other metals.Since the expression of the IRT1 gene in ZQ is significantly lower than in TO,its upstream regulatory sequence is cloned.It was found that the promoters of ZQIRT1 were in a heterozygous state with lengths of 1383 bp and 1779 bp,respectively,while the promoter of TOIRT1 was 1018 bp in length.Compared with TO,ZQ has a385 bp MITE1 and a 778 bp MITE2 respectively,and the Promoter::GUS experiments have shown that MITE can inhibit the activity of promoters.A yeast one-hybrid revealed that an ethylene-responsive transcription factor ERF12 binds and activates the MITE1 element,which may play the role of transcriptional activation of IRT1 under-Fe medium.PCR amplification of IRT1 promoters of different citrus species showed that "orange" contained MITE1,"pomelo" contained MITE2.The iron tolerance of orange and pomelo is stronger than that of TO,and iron tolerant stock ZQ and pomelo.both contain MITE,so the existence of MITE may be closely related to the iron tolerance.Therefore,we identified germplasm resources of TO in China,and found that TO No.1,No.58 and No.130 in southern Shaanxi contained MITE1,the next step is to evaluate its iron deficiency resistance.In conclusion,this study identified the key genes of ZQ and TO in the process of iron activation and iron transport,and analyzed the regulatory mechanism of ZQ tolerance to iron deficiency.The precise regulation of root iron absorption in the iron environment provides an important theoretical basis.