Functional Analyses Of Mir169o And Its Target Genes NF-YA In Populus Trichocarpa

Plants often suffer from a variety of abiotic stresses during their whole life such as drought,which seriously affect plant growth and development.Transcription factor,play an important role in the molecular mechanism of stresses by binding cis-elements on the promoter of target genes and then regulate the expression of them.NF-Y(Nuclear Factor Y)is composed of three subunits NF-YA,NF-YB,and NF-YC.NF-YA,targeted by the miR169,plays an important role in the stresses.Populus,acted as an extremely important economic tree species in China's artificial afforestation,plays an important role in the economic development and ecological environment construction.Therefore,screening and identifying the stress resistance genes of Poplar,and then to obtain the highly resistant varieties of Poplar by genetic engineering is of great significance.In our study,the functions of NF-YA transcription factor and miR169o were studied by transform them into Arabidopsis thaliana(Col-0)and 84K(P.alba × P.glandulosa).The main results are as follows:(1)Thirteen NF-YA transcription factor members were identified in the genome of P.trichocarpa.Among them,PtNF-YA12 and PiNF-YA13 are two newly discovered members.Three pairs of PtNF-YA1 and PtNF-YA3,PtNF-Y45 and PtNF-YA11,PtNF-YA10 and P1NF-YA13 were located in the genome replication regions,respectively.Combined with the cis-elements of promoter and RT-qPCR,most NF-YA family members were involved in stresses response,and their showed diversity expression patterns,which suggested that NF-YA family members may play different roles in the regulation of stress.In addition PtNF-YA1,2,3,5,6,7,8,10,and 11 have miR169 target site and their act as potential target genes of miR169.Therefore,this study showed that NF-YA participated in the regulation of plant stresses might mediated by miR169.(2)PtmiR169o,one of the miR169 large family members,which up-regulated under drought stress,was screened and cloned.The GUS transgenic Arabidopsis thaliana which driven by the promoter of PtmiR169o,deepen the GUS staining under drought,NaCl and ABA treatments.The seed germination,seedling growth of PtmiR169o transgenic Arabidopsis thaliana and plantlets growth of PtmiR169o transgenic Populus showed anti-mannitol osmotic stress.It also improved the net photosynthetic rate,reduced the water lose rate of leaves,which showed obvious drought resistance.In terms of growth,the overexpression of PtmiR169o Arabidopsis thaliana showed an increase in leaf diameter and leaf area,while the overexpression of PtmiR169o Poplar promoted the height of the plant and the length of the roots.Finally,PtNF-YA2 and PtNF-YA6,the target gene of PtmiR169o,were obtained by psRNATarget prediction,real-time PCR,tobacco co-injection and 5' RACE(3)The target gene of PtNF-YA6(Potri.006G145100)was cloned,which showed down-regulated pattern under drought stress,contrary to the expression pattern of PtmiR169o.PtNF-YA6 protein is located in the nucleus.Overexpression of PINF-YA6 transgenic Arabidopsis thaliana could slow down the growth of plants and reduce the area of leaves.Similarly,overexpression of PtNF-YA6 reduced the height and the photosynthetic efficiency of transgenic Poplar.The seed germination of PtNF-YA6 transgenic Arabidopsis thaliana and the plantlets growth of PtNF-YA6 overexpression Poplar showed resistance reduced under mannitol osmotic stress.Transgenic Arabidopsis thaliana seedling and transgenic Poplar plantlets increased in vitro water loss rate of leaves,thereby reducing the drought resistance of plants.Above all,the functions of PtNF-YA6 showed contrary to the functions of PtmiR169o,which is consistent with the functional characteristics of miRNA and its target gene.In conclusion,these results indicated that PtmiR169o acts on target gene PINF-YA6,thereby regulating plant growth and drought resistance(4)The biological functions of PtNF-YA9(Potri.011G101000),was first fully investigated PtNF-YA9 protein is located in the nucleus.The expression of PtNF-YA9 was reduced by mannitol.NaCl,and abscisic acid(ABA).The GUS staining of ProNF-YA9::GUS transgenic lines were also reduced by mannitol treatments.In the PtNF-YA9 overexpressed Arabidopsis(OxPtNA9)lines,OxPtNA9 lines exhibited sensitivity to simulated drought,ABA,and salinity stress during germination stage,and growth arrest emerged at post-germination stage.At vegetative stages,OxPtNA9 lines decreased in water loss via promoting stomatal closure and displayed high instantaneous water-use efficiency(WUE)of the leaf to exhibit enhanced drought tolerance.Furthermore,OxPtNA9 lines exhibited 1.5?1.73-fold length of primary roots in the half-strength Murashige-Skoog agar medium supplemented with NaCl and conferred strong tolerance in the soil under salt stress than WT.Additionally,PtNF-YA9 exhibited dwarf phenotype,short hypocotyl,small leaf area and biomass,and delayed flowering.Overexpressed PtNF-YA9 transgenic Poplar was further studied,there also showed drought resistance phenotype in both mannitol and drought treatment.In term of growth,over-expression PtNF-YA9 transgenic Poplar exhibited shorter height and smaller internode.Furthermore,under NAA treatment,NAA significantly promoted the growth of WT plants,but had no significant effect on PtNF-YA9 transgenic Poplar,indicating that PtNF-YA9 involved in the regulation of plant growth was mediated by NAA.Above all,different PtNF-YA members showed functional differentiation in the process of responding to abiotic stress.PtNF-YA9 was a positive regulator of stress resistance,while PtNF-YA6 is a negative regulator of stress resistance targeted by PtmiR169o.Our research proposes a model in which NF-YA or miR169 mediated NF-YA not only plays a key role in the regulation of plant growth but also play a primary role in the mechanism of an acclimatization strategy in response to adverse environmental conditions.Therefore,the functional study of miR169 and NF-YA transcription factors provides a new idea for the improvement of forest trees.