Functional Characterization Of OsSKIPa-interacting Proteins OsTMF And OsARID3 In Stress Resistance And Development In Rice

Abiotic stresses significantly inhibit plant growth and crop productivity.Rice(Oryza sativa L.)is one of the most important food crops in the world.In order to improve the tolerance of rice to various abiotic stresses,thus to expand the planting area and to increase the crop yield,it is important to study the mechanisms of rice in the response to abiotic stresses.In our previous research,we identified OsSKIPa as a key regulator of plant growth and abiotic response in rice.Suppression of OsSKIPa(OsSKIPa-RNAi)resulted in reduced cell viability and growth arrest,while over-express ion of OsSKIPa(OsSKIPa-OE)significantly improved drought tolerance through enhancing the cell viability and influencing the expression of many important stress-related genes.Moreover,OsSKIPa-OE plants showed increased sensitivity to cold stress.In order to elucidate the molecular basis of OsSKIPa regulating plant growth and abiotic stress response,we analyzed the functions of two OsSKIPa-interacting transcription factors,OsTMF and OsARID3,in abiotic stress response and plant growth.OsTMF is the single homo log of human TATA element modulatory factor(TMF)in rice.It interacts with OsSKIPa through the TMF_TATA_bd domain in the C-terminus.Transgenic rice over-exqpressing OsTMF(OsTMF-OE)showed no obvious difference to wild type rice Zhonghua 11 under normal conditions and after drought,salt and heat stress treatments.Under cold stress,however,OsTMF-OE plants showed significantly increased sensitivity,while the proline content was also increased.Through gene chip analysis,we found that,under the cold stress,expression of many genes involved in cold stress response(eg.OsDREB1s)were significantly up-regulated in OsTMF-OE plants,while the expression of several peroxidase(POX)genes which encode the reactive oxygen species(ROS)-scavenging enzymes were down-regulated.Correspondingly,the ostmf mutant showed enhanced cold tolerance compared to the wild type,while the pro line content was decreased;and the expression of OsDREB1s and POX genes showed an opposite pattern to that in the OsTMF-OE plants.Moreover,OsTMF-OE plants also showed increased sensitivity to oxidative stress.These results suggest that OsTMF is a positive regulator of cold stress signaling and response.It promotes the cold-induction of the expression of OsDREB1s genes and of pro line production,and also modulates ROS level through influencing the expression of POX genes,thus to promote the ROS-dependent signal transduction of cold stress.The increased cold-sensitivity of OsTMF-OE plants might be mainly resulted from oxidative stress that caused by the over-accumulation of ROS.Furthermore,we analyzed the pro line content and gene expression pattern in(OsSK1Pa-OE plants during cold stress,and it showed similar results to that of OsTMF-OE plants.This suggests that the molecular mechanism of the increased cold-sensitivity in OsSKIPa-OE plants might be similar or related to that in the OsTMF-OE plants.In addition,sub-cellular localization analyses showed that OsTMF is localized in both Golgi apparatus and nucleus.OsTMF also interacts with the Rab GTPase through its C-terminus.The Golgi-localization and the interaction with Rab GTPase of TMF homologs are highly conserved in human,yeast and Arabidopsis.Through analyzing the function of OsTMF in cold stress response,we not only provided insights into the functions of TMF homologs in plants for the first time,but also raised a new relationship between proline or DREBls genes and the cold stress phenotype,and provided molecular basis for clarifying the mechanism of the increased cold-sensitivity of OsSKIPa-OE plants.OsARID3 is an ARID(AT-rich Interaction Domain)-containing DNA-binding protein.It interacts with OsSKIPa through the HSP20 domain in the C-terminus and localizes in nucleus.Through Chromatin I mmunoprec ip itat io n analyses and Electrophoretic Mobility Shift Assays,we demonstrated that OsARID3 can specifically binds to the AT-rich DNA sequences in the target genes,including one of the KNOXI(Class I KNOTTED1-LIKE HOMEOBOX)family genes,whichplay indispensible roles in shoot apical meristem(SAM)development,some auxin synthetic YUCCA(YUC)genes,and some cytokinin(CK)synthetic IPT genes.The osarid3 mutant exhibited severe developmental defects,including abnormal development of embryo and SAM,early seedling lethality,reduced cell viability,and failure of callus to regenerate shoot.The osarid3 mutant calli possessed significantly reduced expression of several KNOXI genes and had increased auxin but decreased CK levels,which are resulted from the misexpression of several YUC and IPT genes.Moreover,gene expression analyses showed that disruption of OsARID3 also influences the expression of many genes that implicated in phytohormone synthesis,metabolism,signal transduction and transport,as well as many transcription factor genes.These results suggest that OsARID3 positively regulates SAM development and cell viability,through regulating the expression of many hormone-related genes and some key transcription factor genes such as KNOXI.OsARID3 is also required for modulating the auxin and CK levels,which are very important for both embryo and SAM development,through directly regulating the expression of genes for auxin and CK biosynthesis.Since the OsSKIPa-RNAi plants also showed severe developmental defects such as growth arrest and reduced cell viability,which are very similar to the phenotypes of the osarid3 mutant,we proposed that the function of OsSKIPa in regulating plant development and cell viability may be related to OsARID3,through the protein interaction.Furthermore,transgenic rice over-expressing OsARID3 exhibited increased sensitivity to cold stress,which is consistent to the phenotypes of OsSKIPa-OE and OsTMF-OE plants.These results imply that,in the cold stress response,OsSKIPa,OsTMF and OsARID3 may function together and they may be involved in the same transcriptional regulation complex.Through the functional analyses of OsARID3,our findings not only shed new light on the molecular mechanism of plant SAM development and elucidate the molecular basis of OsSKIPa regulating plant growth and cell viability,but also further demonstrate the developmental significance of plant ARID proteins.