Study On OsZIP4 Is Responsible For Distribution Of Zn To Meristems In Rice

Zinc(Zn)is an essential micronutrient for plant growth and development.It acts as a cofactor for a wide range of proteins involved in many important biochemical pathways including protein,carbohydrate,nucleic acid and lipid metabolism,auxin biosynthesis,cell division,protein synthesis,gene expression regulation,and so on,therefore,Zn deficiency results in stunted growth and ’little leaf’.Nearly one-third of the world’s cereal-growing area is affected by soil Zn deficiency,the rice,one of the world’s main staple foods,is more sensitive to zinc deficiency.Zinc deficiency will seriously affect favorable yield and grain Zn quality of rice.Zn deficiency will seriously affect the yield and quality of rice.The rice meristem needs to be provided more Zn to maintain cell division and growth during the tillering stage.Therefore,a tightly regulated system is required for maintaining Zn homeostasis,which is achieved by regulating different genes involved in the uptake,sequestration,translocation between different organs,especially the regulation of the efficient transport and distribution of Zn in the rapid growth period such as the tillering stage is particularly important.However,the molecular mechanisms for these Zn transport systems and their regulation are still poorly understood.In order to find genes involved in Zn transport systems and their regulation in the tillering stage of rice,we performed the following experiments:(1)Through Zn deficiency experiments,we confirmed the effect of Zn on TB elongation.To better understand the complex mechanisms and find transport regulating Zn homeostasis during early stage of tiller bud development,we performed a transcriptomic profile of the tiller bud samples in control and-Zn treatment.(2)The expression patern and tissue localization of OsZIP4 were analyzed.The phenotype analysis of mutants to clarified the function of OsZIP4 to transport Zn to AM and other tissues or other developmental stages.(3)Using LA-ICP-MS and μ-XRF to image the Zn distribution at a cellular level in rice nodes,SAM,AM and young leaf to explore the regulation mechanism of OsZIP4.The main results are summarized as follows.(1)We performed a mineral nutrients dependent tiller growth assay to examine the effect of each element on rice TB elongation.Nitrogen deficiency showed a significantly effect on the TB elongation that almost stopped the TB elongation,Zn deficiency significantly hampered TB elongation,showed the most similar phenotype to-N.We performed a RNA-seq of the TB samples to find transport regulating Zn homeostasis during early stage of tiller bud development,OsZIP4,a member of the ZIP gene family,showed the highest expression level among all ZIP genes.(2)We further performed a quantitative real-time-PCR to examine the tissuesspecific expression of OsZIP4.The expression of OsZIP4 is also highly induced by Zn deficiency in TB.OsZIP4 was expressed at a higher level in the nodes.Immunostaining with an antibody against GFP showed that OsZIP4 was mainly expressed at the AM and SAM at their early stage development in the basal stem.OsZIP4 was also mainly expressed at the phloem region of DVB in the basal node,DVB and(TVB)of node I.To investigate the role of OsZIP4 in axillary bud development,two knockout mutants of OsZIP4(oszip4-1,oszip4-2)were obtained from rice mutant databases We also generated two independent complementation lines of OsZIP4 in oszip4-1 background for further confirmation of the functional role of OsZIP4.During early TB elongation,mutants showed significantly shorter length in TBs,as compared to wild type control hydroponically cultivated in a normal nutrient solution.Both the mutants contained less Zn in their AM and SAM compare with their corresponding wild type and complementation lines.This is consistent with our Zn concentration and distribution results that mutation of OsZIP4 blocked the transport of Zn to the axillary bud cells that lead to Zn deficient phenotype and transcriptome.These gene expression pattern further indicated that under control condition of the oszip4-1 mutant background,already showed very similar Zn deficiency transcriptome of wild type.This is consistent with our Zn concentration and distribution results that mutation of OsZIP4 blocked the transport of Zn to the TB that lead to Zn deficient phenotype and transcriptome.(3)Both mutants accumulate significantly higher concentration of Zn compared with wild type in the basal stem.We used LA-ICP-MS to image the Zn distribution at a cellular level in rice basal stem and SAM.Zn was less in the SAM but highly accumulated Zn was observed at the connection point between the basal stem and the AM of the oszip4-1 mutant.We also used μ-XRF scanning method to determine the Zn distribution in nodes and AM of the oszip4-1 mutant and wild type at higher resolution.In a cross section of the middle part of the basal node,in different to the evenly distribution of Zn in wild-type rice,Zn was highly accumulated and restricted in theTVB and some EVB in oszip4-1 mutant.When we looked at the cross section of the upper part of the basal node,Zn was preferentially distributed to the young leaf,however,this pattern was abolished in the oszip4-1 mutant.The Zn concentration of different tissues in wild type and the oszip4 mutants were analyzed,the two nodes(Ⅰand Ⅱ)of mutants accumulate significantly high levels of Zn than wild type at the reproductive stages.In conclusion,OsZIP4 is highly expressed and induced by Zn deficiency in the TB during early development stage.Its major role is distribution of Zn from basal node to TB through intercellular transport pathway,which is required for TB elongation.In addition,we also provide evidence that OsZIP4 is involved in the DVB and TVB mediated preferential distribution of Zn from lower node to upper node which is essential for delivering Zn to SAM and young leaf that is essential for flower and panicle development at reproductive stage in rice.