Functional Analyses Of Poplar PdGATA19 And PdGATA13 In Response To Low-nitrogen And Drought Stress

Populus,acted as an extremely important economic tree species in China's artificial afforestation,which has great social and ecological benefits.Water and nitrogen are two important factors limiting the growth of poplar Exploring the molecular response mechanisms of poplar to drought and low nitrogen stress have important theoretical significance for breeding of new poplar varieties by genetic engineering technology.In this study,PdGATA19 and PdGATA13 were up-regulated in response to drought stress and nitrogen deficiency,respectively.The molecular mechanism of B-GATA transcription factors PdGATA19 and PdGATA13 responding to abiotic stress in poplar and Arabidopsis thaliana was analyzed by morphological,physiological,molecular biology and so on.The result were showed as follows:(1)Yeast one-hybrid assay(Y1H),electrophoretic mobility shift assays(EMSA)and dualluciferase reporter assay(DLR)showed that the PdGATA19 directly activated the promoter of Pd HXK1 by binding the GATC element,a hexokinase(HXK)synthesis key gene.Here,PdGATA19,a member of the GATA transcription factor family,was greatly induced by ABA and dehydration.Overexpressing PdGATA19 in poplar(Populus clone 717,Populus tremula×Populus alba clone 717-1B4)resulted in reduced stomatal aperture with greater water use efficiency(WUE)and increased water deficit tolerance.By contrast,CRISPR/Cas9-mediated poplar mutant gata19 exhibited increased stomatal aperture and water loss with reducing drought resistance.PdGATA19 activates Pd HXK1(a hexokinase synthesis key gene),resulting in a remarkable increase in hexokinase activity in poplars subjected to water deficit.Furthermore,hexokinase promoted nitric oxide(NO)and hydrogen peroxide(H₂O₂)production in guard cells,which ultimately reduced stomatal aperture and increased drought resistance.Together,PdGATA19 confers drought stress tolerance by reducing stomatal aperture caused by NO and H₂O₂ production via the direct regulation of Pd HXK1 expression in poplars.(2)Plants have evolved complex mechanisms to cope with fluctuating environmental nitrogen availability.However,more potential genes modulating the response to nitrate have yet to be characterized.Here,a poplar GATA transcription factor gene PdGATA19 was greatly induced by low nitrate.Overexpressing PdGATA19 in poplar(Populus clone 717)significantly improved nitrate uptake and remobilization with higher nitrogen-use efficiency(NUE)and faster growth,particularly in low nitrate condition.Conversely,CRISPR/Cas9-mediated poplar mutant gata19 exhibited decreased nitrate uptake and relocation with lower NUE and slower growth.Yeast one-hybrid assay,electrophoretic mobility shift assays and dualluciferase reporter assay showed that PdGATA19 directly activated the promoter of the gene Pd NRT2.4b(a high-affinity nitrate transporter)resulting in a significant increase in nitrate uptake and remobilization in poplars at low nitrate supply level.Expectedly,the enhanced NUE promoted the growth under low nitrate availability.Taken together,PdGATA19-NRT2.4b module plays an important role in regulation of NUE and growth by improving nitrate acquisition and remobilization in poplars.(3)In the present study,PdGATA13,B-GATA transcription factor,was isolated from the NE19[P.nigra×(P.deltoides×P.nigra)].In this paper,the promoter sequence of PdGATA13 was analyzed,and it was found that there were stress-related elements and cis-acting elements related to ABA signals such as ABRE,CAAT and TATA-motif in its promoter region.PdGATA13 was preferentially expressed in the adult leaves and was significantly upregulated during dehydration and ABA treatments.PdGATA13 was localized to the nucleus.In the model plant Arabidopsis thaliana,ox PdGATA13 plants and complementary line gata16/ox PdGATA13 were obtained by the method of inflorescence impregnation.PdGATA13 overexpression significantly reduced stomatal aperture,improved water use efficiency(WUE)and enhanced drought tolerance.