Mechanism Of Drought Response Regulated By A Long Intergenic Noncoding RNA 13853 And RBP314 In Arabidopsis Thaliana

Plants have different sensitivities to water demand and drought stress at different stages of growth and development.Drought seriously reduces crop yields through negatively affecting plant growth,physiology and reproduction.In order to cope with the severe impact of drought,plants usually change their growth patterns and physiological processes.Drought can change water content,hormone content and gene expression level in plants.Studies regarding the physiological and biochemical mechanisms of plant responses to drought stress in Arabidopsis thaliana are great important for improving plant drought tolerance through the combination of genetic engineering and traditional breeding methods.Numerous studies have shown that lnc RNAs can regulate plant adaptation for drought stress.Long noncoding RNAs(lnc RNAs)are involved in plant responses to drought through complex cellular pathways,such as transcriptional and chromatin regulation,hormone signaling and gene regulation.However,their regulatory roles and molecular mechanisms are still unclear.Our group had applied yeast three-hybrid screening to obtain a member of ribonuclease H(RNase H)family,RBP314,which interacts directly with linc RNA13853.The aim of this research is to study both function and mechanism of RBP314 together with linc RNA13853 in plant drought stress response.The main results are as follows:1.The interaction of linc RNA13853 and RBP314.The interaction between RBP314 and linc RNA13853 was verified by both yeast three-hybrid and t RSA RNA pull-down in vitro.In addition,RBP314 was found to locate in both nucleus and cytoplasm by means of subcellular localization experiment in Arabidopsis protoplast.Further applying trimolecular fluorescence complementation(Tri FC)and RNA immunoprecipitation(RIP)assays,we showed that RBP314 can interact with linc RNA13853 in the plant nucleus.2.RBP314 positively regulates drought response in plant.We have successfully obtained two independent rbp314 loss-of-function homozygous mutants by CRISPR/Cas9.Comparing with Col-0,rbp314 mutant plants exhibited increased sensitivity to PEG treatment,decreased drought tolerance,increased water loss and enlarged stomatal aperture index,which is consistent with phenotypes presented by rbp314 mutants.Therefore,RBP314 can positively regulate plant responses to drought stress.3.RBP314 joints with linc RNA13853 to regulate the target gene ABA2.Under drought stress,plants accumulate ABA content by increasing ABA biosynthesis and decreasing ABA catabolism.The q RT-PCR assay shown that ABA2,the key gene of ABA biosynthesis,was significantly down-regulated in both rbp314 and linc RNA13853 mutants.Both RBP314 and linc RNA13853 can directly bind to ABA2 chromatin region through chromatin immunoprecipitation(Ch IP)and chromatin isolation by RNA purification(Ch IRP)experiments.Moreover,mutated RBP314 could not impair the linc RNA13853 recognition of ABA2.In summary,linc RNA13853 interacts with RBP314 in plant nucleus.They form a RNA-protein complex,and then directly bind to ABA2 locus,which positively regulate the transcriptional levels of ABA2.RBP314 belongs to the RNase H family,which can specifically degrade RNA in RNA/DNA hybrid chains,and its activity affects the level of R-loop.R-loop plays an important role in gene transcription,DNA replication,epigenetic modification,and DNA damage repair.Therefore,we speculate that RBP314 together with linc RNA13853 may regulate ABA2 expression by influencing the R-loop of the ABA2 locus,thereby participating in plant drought stress response.Our study not only enriches the function and mechanism of lnc RNA action,but also provides theoretical basis and support for improvement of drought-tolerant crops by using genetic engineering method.