Identification,function And Regulation Of TaPHT1;9-4B Transporter And Development Of Its Functional Marker

Phosphorus is one of the most indispensible macronutrients for plant growth and development,because it is a major component of phospholipids and nucleic acids.Generally,Pi concentration in soil solution is frequently below the critical level needed by plants and Pi deficiency severely limits crop growth and yield.To improve crop yields,millions of tons of Pi fertilizers are applied annually to agricultural fields worldwide.However,only 10~20% of the applied Pi is effectively absorbed by plants due to the low utilization efficiency of Pi fertilizers in crops,while the remaining is lost,which not only increases agricultural costs but also causes environmental pollution.Therefore,improving the phosphate utilization efficiency of crops is greatly important to ensure the sustainable development of agriculture and environmental protection.Although bread wheat is the most widely cultivated and important staple food crop in the world,it is a complex polyploid model plant,and underlying molecular mechanism respone to Pi deficiency is still poorly understood.In this study,we applied proteomic approach to identify Pi deficiency-responsive protein species(PDPSs).Next,we identified and functionally characterized the Ta PHT1;9-4B transporter,and isolated its upstream transcription factors by using a series of molecular biological methods.Finally,we further identified the favored haplotype of Ta PHT1;9-4B in modern bread wheat cultivars and developed its functional molecular marker.The major results were summarized as follows:1.Identification of Ta PHT1;9 transporter.The wheat seedlings cultured in Pi-deficient conditions showed inhibited leaf growth but enhanced root length compared with the Pi-sufficient plants.Quantitative analysis of growth parameters(plant height,root length,shoot and leaf dry weight)and P concentrations further confirmed these changes.We applied the high throughput i TRAQ-based proteomic approach to survey the protein expression patterns of roots and leaf in the Pi-deficient and-sufficient wheatseedlings.A total of763 protein species(363 in root,400 in leaf)with significantly altered abundance(≥1.2-fold or ≤ 0.83-fold change)were identified and they were functionally involved in many biological processes(e.g.,transportation,metabolism,signal transduction,stress and defense,and function unknown).Moreover,there were significant differences between the PDPSs in roots and leaves,which indicated that the response mechanisms of Pi deficiency between the root and leaf organs were different to some extent.Genomic alignment of the identified protein species was studied using the genome assembly of Chinese Spring(CS).Among the4,013 identified protein species,1015 were assigned specifically to the unique homoeologs.A large proportion(78.5%,797/1015)of these protein species showed asymmetric expression pattern under Pi deficiency stress,including single homoeolog specific expression and multi homoeolog differential expression.Among the identified PDRPs,the abundance of four high affinity PHT1 proteins(Ta PHT1;3-5B,Ta PHT1;6-5B,Ta PHT1;9-4B,and Ta PT2)was significantly increasedin the roots of Pi-deficient wheat seedlings,with the average fold of induction being highest for Ta PHT1;9-4B,These results indicated that Ta PHT1;9-4B might play key role in response to Pi deficiency in wheat.2.Function of Ta PHT1;9 transporter.Ta PHT1;9-4B was a high-affinity Pi transporter characterized with 12 putative transmembrane(TM)domains and it localized at the plasma membrane.Three Ta PHT1;9 homoeologs were present on the 4A,4B,and 4D chromosomes of CS,respectively.In the MB192 yeast mutant strain defective in Pi absorption,the expression of Ta PHT1;9-4B restored its growth under low Pi conditions with the highest degree of complementation observed at p H 6.0,indicated that Ta PHT1;9-4B protein was characterized with Pi transport activity.The ectopic expression of Ta PHT1;9-4B in rice improved Pi uptake and transgenic plants growth under both Pi replete and deprived conditions,whereas the transient silencing of the Ta PHT1;9 gene in the wheat seedling by using barley stripe mosaic virus mediated gene silencing(BSMV-VIGS)decreased the Pi absorption and impaired wheat growth.In the pot experiment with four levels of P fertilizer,the grain yields and biomass of Ta PHT1;9-4B ectopic transgenic rice plants were also significantly higher than those of WT plants under P fertilizer-insufficient suppliment,and the P concentration in the root and straw of transgenic plants were also significantly higher than WT.These suggested that the Ta PHT1;9 was a candidate gene for the cultivation of phosphate use efficiency crops.3.Isolation of the upstream transcription factor for Ta PHT1;9 transporter.The promoter fragment of Ta PHT1;9-4B was used as bait to screen the c DNA library derived from Pi-deficient wheat roots by using yeast one hybrid(Y1H),and a R2R3-type MYB transcription factor Ta MYB4-7D was obtained.Its coding gene was located on 7D chromosome and the peotein phylogenetically related to At MYB4 transcription factor.Ta MYB4-7D transcription factor localized in the nucleus and possessed transcriptional activation activity.The Y1 H and dual-luciferase assay experiment furtherconfirmed that Ta MYB4-7D activated Ta PHT1;3-5B,Ta PHT1;6-5B,Ta PHT1;9-4B and Ta PT2 by directly binding to MBS elements in their promoter.Silencing Ta MYB4 by BSMV-VIGS conferred more sensitivity to low Pi or Pi deficiency and downregulated the transcript levels of the Ta PHT1;3,Ta PHT1;6,Ta PHT1;9 and Ta PT2 genes.4.Development of the Ta PHT1;9 molecular marker.The promoter and coding region of Ta PHT1;9-4B,Ta MYB4-7D and their orthologs were resequence in the 62 common wheat cultivars,11 tetraploid,and 28 diploid wheat ancestral relatives.Sequence analysis showed that there were only several single nucleotide polymorphisms(SNPs)in the promoterand coding region of MYB4-7D in diploid species,and no polymorphism was found in hexaploid wheat,implying highly conservation of the MYB4-7D in the evolutionary process.However,a large number of polymorphic sites(SNP and indel)were detected in the promoter and coding region of Ta PHT1;9-4B and its ortholog genes.Systematic evolutionary analysis suggested that genetic diversity of PHT1;9-4B decreased significantly during the evolution from diploid to polyploid species,and genetic diversity of promoter region was significantly higher than coding region.Four promoter haplotypes were identified for Ta PHT1;9-4B in modern wheat cultivars.Hap3,the favored haplotype,showed significant positive associations with Ta PHT1;9-4B transcript level and phosphorus content in wheat plants,and Hap3 promoter had stronger activity.A functional marker CAPS-799 was developed to discriminate Hap3.In summary,this study uncovered a molecular module composed of Ta MYB4-7D transcription factor and four PHT1,which required for maintaining efficient Pi acquisition and plant growth under Pi limiting conditions.Our work sheds new light on the molecular mechanism controlling Pi acquisition and utilization in bread wheat.In addition,the favored haplotype of Ta PHT1;9-4B promoter and the developed CAPS-785 markermay facilitate the development of P efficient wheat cultivars in the future.