Functional Studies On Populus Euphratica BZIP Transcription Factor PeABF3 And PeTGA1 In Response To Drought Stress And Pathogen Infection

Populus is an important industrial tree species in the world as well as the important afforestation tree species in north China.They not only produce economic benefits,but also provide ecological benefits.However,drought stress and various pathogens seriously influence the survival and growth of poplars,causing considerable economic losses.P.euphratica is well adapted to extreme desert environments,including abiotic and biotic stresses,and is the only tree species that can grow in desert.The basic leucine zipper(b ZIP)protein family is an important family in plants.Studies on the functional and molecular mechanism of P.euphratica b ZIP transcription factor family members in regulating drought stress and pathogen infection provide a theoretical basis for improving the stress resistance of poplar,and have important theoretical significance in breeding excellent stress-resistant poplar varieties.In this study,based on the previous transcriptome and RT-q PCR data from the laboratory,P.euphratica ABA-responsive element binding factor(Pe ABF3)and TGACG-binding(TGA)transcription factor(Pe TGA1),which were up-regulated in response to drought stress and pathogen infection,respectively,were selected and then study their function and molecular mechanisms in response to drought stress and biotic stress in poplars.The main research results are as follows:(1)Water availability is an important factor for affecting plant growth,development,and distribution.Stomatal movement mediated by abscisic acid(ABA)is particularly important for drought adaptation,but the molecular mechanisms in trees are largely unclear.In this study,we isolated Pe ABF3 in P.euphratica.Pe ABF3 was preferentially expressed in xylem and young leaves,and can be induced by dehydration and ABA treatments,and down-regulated by waterlogging stress.Pe ABF3 showed transactivation activity in yeast cells and was specifically localized to the nucleus.Transgenic triploid white poplars(Populus tomentosa‘Yi Xian Ci Zhu B385’)overexpressing Pe ABF3(Ox Pe ABF3)enhanced significantly sensitivity to exogenous ABA,including inhibiting the growth of poplar lateral shoots in stem segment and promoting ABA-induced stomatal closure.Ox Pe ABF3 improved instantaneous water use efficiency.When subjected to drought stress,Ox Pe ABF3 plants had higher leaf water content,higher net photosynthetic rate and PSII maximum quantum yield(Fv/Fm)to maintain higher photosynthetic activity.Ox Pe ABF3 plants had lower malondialdehyde content and relative electrical conductance,which promoted the integrity of cell membrane.In addition,Ox Pe ABF3 plants accumulated more proline and maintained higher activity of superoxide dismutase(SOD),resulting in increased drought tolerance in poplars.(2)Furthermore,the molecular mechanism of Pe ABF3 in response to drought stress was analyzed.Yeast two-hybrid and bimolecular fluorescence complementation experiments revealed that Pe ABF3 could interact with Pe OST1,which is the core component of ABA signaling pathway.RT-q PCR and cis-element analysis of promoter sequence indicated that P.euphratica Actin-Depolymerizing Factor 5(Pe ADF5)might be a potential target gene of Pe ABF3.Yeast one-hybrid(Y1H)and electrophoretic mobility shift assay(EMSA)demonstrated that Pe ABF3 could target the promoter of Pe ADF5,and Pe ABF3 could activate the expression of Pe ADF5.GUS staining deepen in stomata and roots of GUS transgenic Arabidopsis which driven by Pe ADF5 promoter under D-Mannitol and ABA treatment.Pe ADF5 can be induced by drought and dehydration stress.Pe ADF5-m Cherry and microfilament marker f ABD2-GFP had co-localization signals in stomata.These results suggest that Pe ADF5 may promote stomatal closure by regulating actin cytoskeleton remodeling.In summary,Pe ABF3 can interact with Pe OST1 to participate in ABA signaling pathway,and promote ABA-induced stomatal closure by directly regulating downstream Pe ADF5 expression,resulting in enhancing drought tolerance in poplar.(3)Colletotrichum gloeosporioides seriously affects fruit quality and healthy growth of plants.However,the molecular mechanism of b ZIP transcription factor in regulating disease resistance in poplar is rarely studied.The present study isolated Pe TGA1 in P.euphratica.Pe TGA1 belongs to subgroup D of the b ZIP family and was located to the nucleus.Pe TGA1 was homologous to Ptr TGA1 and At TGA1.Pe TGA1 overexpressing poplar lines showed the higher effective defense response to C.gloeosporioides than wild-type poplars.Y1 H and EMSA revealed that Pe TGA1 could directly bind the Pe SARD1(P.euphratica SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1)promoter,an important regulator for salicylic acid synthesis.Luciferase and RUBY analysis indicated that Pe TGA1 activated the expression of Pe SARD1,and PR1(PATHOGENESIS-RELATED 1),a SA marker gene involved in SA signaling.Moreover,we observed that the Pe TGA1 overexpression lines showed elevated SA levels,thereby resulting in the increased resistance to C.gloeosporioides.Our results indicated that Pe TGA1 may exert a key role in plant immunity by targeting Pe SARD1 to participate in the SA biosynthesis pathway and involve in SA signal pathway via activating PR1 expression.In conclusion,P.euphratica transcription factor Pe ABF3 can directly regulate Pe ADF5 gene,participate in ABA signaling pathway,and promote ABA-induced stomatal closure to enhance drought tolerance in poplar.Pe TGA1 responds to SA signals by targeting Pe SARD1 and regulating the expression of Pe PR1 and Pe SARD1,thus positively regulating the defense response of poplar to pathogens.This study provides a new theoretical basis and idea for poplar b ZIP family members in tree variety improvement.