A Mechanistic Study Of Rice Inositol Polyphosphate Kinase Gene(OsIPK2) In Auxin-regulated Growth And Development

Inositol polyphosphate kinase has broad-specificities and it converts inositol 1,4,5-trisphosphate(IP3)into inositol 1,4,5,6-tetrakisphosphate(IP4)and subsequently into inositol 1,3,4,5,6-pentakisphosphate(IP5).Mammalian inositol polyphosphate multi-kinase has diverse physiologic activities that are independent of its catalytic activities,such as binding to mTOR and stabilizing it,strengthening p53-mediated transcription and cell death,binding to and regulating the recruitment of the histone acetyltransferase CBP to the c-fos promoter.In yeast,as an indispensable component of the ArgR-Mcml transcriptional complex,IPMK/IPK2 activates genes responsive to arginine and phosphate disposition by the kinase-independent stabilization determinant for Mcml and/or ArgR-Mcml complexes.There are two IPMK in Arabidopsis,AtIPK2a and AtIPK2?.Our previous studies showed that AtIPK2? is an auxin-responsive gene and is involved in axillary shoot branching through auxin signaling pathway.AtIPK2a and AtIPK2? also play a redundant and essential role in pollen development,pollen tube guidance and embryogenesis in a kinase activity-dependent manner.Auxin is one kind of plant hormone that plays an important role in the process of cell division,enlongation and apoptosis,tissue initiation,organ morphogenesis,response to environmental stimulation.Components and molecular interactions in auxin signaling pathway have been revealed in dicot plant Arabidopsis.In recent years,many auxin signal componets also have been characterized and functionally analyzed in monocot plant rice and corn.Studies in plant inositol polyphosphate multi-kinase and its relevant metabolites in this decade indicate that inositol phosphate signal is related to auxin signaling pathway.The molecular cloning of a rice inositol polyphosphate kinase(OsIPK2)gene have been reported in 2007,however,molecular or physiological functions of OsIPK2 have not been revealed.In this study,we used the model plant rice to clarify the physiological and biochemical roles of the inositol polyphosphate multi-kinase and possible regulatory mechanism in auxin signal transduction.The main results are the following:1.OsIPK2 is ubiquitously expressed in rice:The GUS or the GFP signal of OsIPK2-GUS or OsIPK2-GFP transgenic rice was clearly detected in the vascular tissues of roots,leaves,shoot base,sheath,nodes,panicle,and pollen.2.OsIPK2 is involved in auxin-regulated plant development:Several hromone responsive elements are found in the promoter of OsIPK2.OsIPK2 was down-regulated by auxin stimuli.OsIPK2 overexpression transgenic rice or Arabidopsis exhibited a growth retardation phenotypes,including fewer lateral roots,shorter plant height,delayed heading or bolting stage.OsIPK2 knockdown plants by RNA interpherence showed longer primary roots and shoot.3.OsIPK2 loss-of function plants are more sensitive to auxin stimuli:qRT-PCR showed that the auxin responsive level of early auxin-responsive genes are higher in RNAi plants.Also,the auxin sensitivity of OsIPK2 loss-of-function plants were also altered.A phenotype of higher sensitivity to auxin-inhibited primary root enlongation was observed in Osipk2 mutant rice,of which a T-DNA was inserted into the-471 bp sites of OsIPK2 promoter.4.OsIPK2 can directly bind to Aux/IAA:Bimolecular fluorescence complementation(BiFC)and yeast two-hybrid assays showed that multiple Aux/IAA proteins interact with OsIPK2.Subsequent studies were focused on OsIAA11,a member of Aux/IAA in rice.Pull-down and Co-IP assays verified the direct interaction between OsIPK2 and OsIAA11.5.OsIPK2 interacts with OsIAA11 through the degron region:The mutations of the key residues in the domain II weakened the interaction between OsIPK2 and OsIAA11 in Y2H and the Pull-down assays.The Y2H assays of truncated OsIPK2 or OsIAA11 showed that the N-terminal domain of OsIPK2 can interact with the DII-DIII of OsIAAll.OsIPK2 can also interact with AtIAA16,while AtIPK2?neither can interact with OsIAA11 nor AtIAA16.6.OsIPK2 protects OsIAAll from degradation:The 26S proteosome-mediated degradation of OsIAA11 is regulated by TIR1 and auxin.By transiently expressing Myc-OsIAA11 and GFP-OsIPK2 fusions in rice protoplasts,we found that OsIPK2 inhibited the auxin-induced degradation of OsIAA11.OsIAA11 was much more unstable in OsIPK2-RNAi or Osipk2 mutant plants.7.OsIPK2 competes with OsTIR1 to bind OsIAA11:With the incubation of wild-type or Osipk2 plants extracts,pull-down assays indicated that OsIPK2 proteins inhibits the interaction between OsTIR1 and OsIAA11.Quatification of the free auxin levels in OsIPK2-RNAi transgenic rice by HPLC-MS/MS showed that auxin level is lower than wild-type.8.The RNA interference of OsIPK2 partially resuces the developmental defect of Osiaall mutant:Osiaa11 mutant rice showed a serials of severe growth defects,including blocked lateral roots initiation.Kockdown the transcript of OsIPK2 in Osiaall mutant partially recovered the lateral roots formation.The expression level of auxin-responsive OsPIN1b,OsPIN10a genes were also partially recovered in OsIPK2-RNAi/Osiaall plants.OsIPK2 influenced the expression level of OsIAA11 in transgenic plants.OsIPK2 is also involved in the complex transcriptional regulation among Aux/IAA family genes.9.OsIPK2 is involved in the H3K27me3 modification on the loci of auxin-responsive genes:ChIP assays showed that the H3K27me3 level on the locus of OsIAA9,OsIAA20 and OsGH3-2 genes are down-regulated in OsIPK2-RNAi transgenic plants.10.IP6 can affect auxin response in rice:The direct application of IP6 can supress the DR5-GUS signal and the transcription of early auxin responsive genes in rice.The results above suggested that OsIPK2 plays an important role in auxin signal transduction and in the auxin-mediated degradation of Aux/IAA.This work provides a direct clue for the cross-talk between inositol phosphate pathway and auxin signaling pathway.