Functional Characterization Of A Rice Transcription Factor OsWRKY72 In Maintenance Of Phosphate Homeostasis And Plant Architecture

Rice is a staple food for nearly half of the world's population,with more than 10,000 rice varieties providing almost one-quarter of the global per capita dietary energy supply.Phosphorus is one of the major limiting factors for plant growth and development.The predominant form of P accessible to plant roots is inorganic orthophosphate(Pi;H2PO4-).However,Pi is readily fixed by certain cations depending on the soil condition,resulting in insufficient Pi availability.In order to cope with P-deficient environment,plants have evolved a suite of physiological,metabolic and morphological adaptations during which dozens of transcriptionally-regulated genes play important roles.Transcription factors(TF)are key regulators for plant growth and development as well as for plant responses to diverse biotic and abiotic stresses.They act as switches controlling the transcriptional regulation of the downstream genes by binding to the cis-regulatory elements present in their promoter regions.An increasing number of TFs have been reported to be responsive to Pi starvation stress and/or involved in maintenance of Pi homeostasis.These TFs could be grouped into at least nine families(AP2/EREBP,ARF,BES1,bHLH,bZIP,G2-like,MYB,WRKY,and Zinc Finger family)based on their DNA binding domain.The most extensively studied TF genes are those encoding PHR(Phosphate Starvation Response),which belongs to the MYB-CC subfamily.In rice,in addition to OsPHR2,OsPTFl,OsARF12/16 and OsMYB2P-1/4P,which belong to bHLH,ARF,and MYB family,respectively,have been reported to be involved in Pi starvation signaling and maintenance of Pi homeostasis.Among the P starvation-related TFs in rice plants,none of which is from WRKY family.In this study,a Pi starvation-responsive WRKY TF gene,OsWRKY72,was identified and functionally characterized.The main results acquired are listed as follows:1.OsWRKY72 localizes to chromosome 11,and encodes a C2H2-type WRKY TF.It consists of two exons and one intron.The open reading frame of OsWRKY72 is 729 bp in length,which is predicted to encod a protein of 242 amino acids.2.The subcellular localization and expression pattern of OsWRKY72 were analyzed by onion epidermal bombardment experiment and RT-qPCR(Reverse Transcriptase-quantitative Polymerase Chain Reaction),respectively.OsWRKY72 was localized to the nucleus,and expressed in all the organs tested.The expression level of OsWRKY72 is higher at seedling stage,and its expression was repressed exclusively in roots of wild-type(WT)rice plants challenged with Pi starvation stress,while remained stable in Pi-depleted shoots.Tissue localization analysis with transgenic rice plants harboring a GUS reporter gene driven by OsWRKY72 promoter showed that OsWRKY72 expression was restricted to the vascular bundles in leaves,while detectable in the meristematic zoon and the elongation zoon of root tip and also in the region neighbouring the lateral root emergence zoon toward root tip.3.Transgenic rice plants with overexpressed(OsWRKY72-Ox)or suppressed(OsWRKY72-Ri)expression of OsWRKY72 were generated by introducing the constructs into rice calli via Agrobacterium tumefaciens-mediated transformation.Overexpression of OsWRKY72 led to a phenotype with stunted growth and prostrate leaves.The fertility and seed-setting rate of the overexpression lines were both decreased as compared with that in the WT plants,which was not observed in OsWRKY72-Ri plants.In addition,heterologous constitutive expression of OsWRKY72 in Arabidopsis gave rise to a similar phenotype as observed in OsWRKY72-Ox rice plants,namely suppressed growth.4.In a hydroponic culture system,transgenic plants and WT plants were subjected to both high Pi(HP)and low Pi(LP)treatments.An impaired growth as reflected by shoot and root biomass was observed for both OsWRKY72-Ox and OsWRKY72-Ri plants as compared with WT plants irrespective of the Pi regime.The Pi concentration was increased in the leaf blades,but not in the roots of Pi-replete OsWRKY72-Ox plants.Such effect of elevated OsWRKY72 expression on Pi accumulation in leaves was not evident under LP condition,and no obvious variation of Pi accumulation was detected in OsWRKY72-Ri plants under both Pi regimes.On the other hand,OsWRKY72-Ox Arabidopsis plants showed impaired growth and thus decreased biomass as compared with WT plants,similar to that found in rice.In addition,overexpression of OsWRKY72 in Arabidopsis also gave rise to increased Pi concentration in shoots,but not in roots.5.Transcript abundance of PHT1(Phosphate Transporter 1)and PSI(Phosphate Starvation Induced)genes in OsWRKY72-Ox and OsWRKY72-Ri rice plants was examined by RT-qPCR.Expression of three most induced PHT1 genes by Pi starvation,OsPT3,OsPT6 and OsPT10,was suppressed in Pi-depleted OsWRKY72-Ri plants,and under Pi sufficient condition.Only one member of the PHT1 family,OsPT12,was dramatically increased in OsWRKY72-Ox plants as compared with that in WT plants.Several PSI genes,such as OsIPS1,OsSQD2,OsPAP10 and OsPHO2.2,were also down regulated in OsWRKY72-Ri plants under P-deficient condition.6.The root system architecture of the OsWRKY72-Ox lines and the WT plants grown in a hydroponic system under HP(200?M)and NP(no Pi)conditions was analyzed.The OsWRKY72-Ox plants showed a significant increase in primary root length under both Pi regimes.In contrast,the average length of lateral roots on the primary roots and adventitious root number of the OsWRKY72-Ox plants were decreased comparing with WT plants.These results indicate that the regulation of OsWRKY72 on these root traits is independent of P status.Moreover,overexpression of OsWRKY72 led to an increase in lateral root density only under NP condition,indicating that the regulation of OsWRKY72 on lateral root emergence might be Pi dependent.7.In an attempt to investigate the mechanism underlying the alteration of flag leaf inclination,a microarray analysis and RT-qPCR validation was conducted.Both results showed that OsIAA1 was up-regulate upon overexpression of OsWRKY72.Since it has been reported that OsIAA1 overexpressing plants resembles the phenotype of OsWRKY72-Ox plants,we presume that OsWRKY72 controls plant growth and flag leaf inclination at least partially,if not all,by up-regulating OsIAA1.