Study On The Mechanism Of Iron-deficient Response In Blueberry

Under the condition where other plants can grow normally,blueberry(Vaccinium spp.)is prone to be chlorosis induced by Fe deficiency,which named Fe inefficiency,however,the related mechanism is still not clear.At present,the limited literatures showed that the Fe absorption in blueberry includes the activation,reduction and absorption of Fe,the same three steps found in Arabidopsis thaliana.But which steps resulted in the Fe-inefficiency in blueberry remains unclear.In addition,the pivotal regulation transcription factors involved in response to Fe deficiency of blueberry,as well as the their target genes have not been identified.In the present study,through hydroponic culture experiments,the biological responses as well as the transcriptional levels of iron metabolism related genes were assayed in blueberry seedlings after being subjected to Fe-deficit and non-acid rhizospheric treatments.The aim was to elucidate the influence of iron deficiency and non-acid rhizosphere to the iron nutrition metabolism of blueberry.Furthermore,based on the isolations of full-length cDNA of FER(Iron deficiency-induced transcription factor,FER),FR02(Ferric reduction oxidase 2,FR02)and IRT1(Iron-regulated transporter 1,IRT1),the expression vector of these three genes were constructed and transformed into the homologous gene mutant of Arabidopsis thaliana.Then,the physiological response relative to Fe metabolism in transgenic lines were assayed to uncover the mechanism involved in Fe inefficiency of blueberry.The study will facilitate the solving of Fe deficiency widely existing in blueberry cultivation and plays an active role in nutritional management and biotechnological breeding of blueberry.The specific research results are as follows:Under the condition of Fe deficiency,which firstly negatively affected the Fe nutrition supply in the new leaves and led to blocked of blueberry chlorophyll synthesis indicated by the severe chlorosis in leaves,The chlorophyll content decreased to about 55%of the control group.the root activity of blueberry dramatically decreased to 66.7%of the control.The Fe contents in roots decreased to 33.95%of that grown in the normal Fe condition.The FRO activity in roots and leaves were 1.26 and 1.38 times of the control group,which indicated that the expression and protein activity of VcFRO2 still increased in blueberry.In non-acid rhizospheric condition,the browning was observed in blueberry roots which paralleled by the significantly decreased root vigor to 1/3 of the control group,meanwhile,the chlorosis appeared in the newly sprout leaves.The results of qRT-PCR showed that,the expression levels of VcFER were significantly after being exposed to Fe-deficiency,and kept in fairly high level.But when being exposed to Fe-deficiency 10 d,the expression quantity of VcFRO2 and VcIRT1 were not significantly upregulation,which indicated that the upregulation of the downstream genes of VcFRO2 and VcIRTl obviously lagged.Meanwhile,the transcription of fer2(Ferritin2,Fer2)was significantly declined,implying the reduced cellular Fe storage.In contrast,the relative expression of intracellular Fe transport-related gene Nramp3(Natural Resistance-Associated Macrophage Protein 3,Nramp3)was significantly up-regulated to more than 4 times by the Fe-deficit treatment for 20 d,which facilitate the transport of iron from the vacuoles into the cytoplasm.No difference in VcHA(H+-ATPase,HA)expression was induced by the Fe-deficit treatment,indicating an obstacle existed in the rhizospheric acidification through H+ secreting involved in Fe-deficiency mechanism in blueberry.The cDNA of VcFER,VcIRT1 and VcFRO2,with the length as 939,1071 and 2106 bp,respectively,were amplified and cloned for the genetic transformation of Arabidopsis homologous mutants.The bioinformatics analysis showed that VcFER belongs to the HLH transcription factor superfamily.It is predicted that VcFRO2 belongs to the iron chain protein reductase superfamily.Similarly,IRT1 belongs to the ZIP superfamily,which is a conservative metal transporter.The low similarity existed in VcFER and VcIRTl with their homologies of other species may be an important reason account for its inefficient iron nutrition.The genetic transformation experiment showed,compared with Atfit Arabidopsis mutant,the chlorosis was greatly alleviated despite its biomass growth was still lower than WT.The alleviation in chlorosis also found in Atirt/VcIRT1 transgenic plants,with a relatively robust growth.However,when compared with WT,the Fe-deficiency induced chlorosis also occurred at the flowering stage.Sever chlorosis was also observed in Atfro/VcFR02 transgenic lines,which finally died after the leaves turned into white.It my be deduced that VcFR02 had a fairly low reducing power on ferric ions.In summary,the lack of mechanism of rhizosphere acidification,the low efficiency of transcription factor VcFER,as well as the low reducing power of VcFR02,may be the key reasons account for the Fe inefficiency in blueberry.