Functional Analyses Of Poplar Secreted Peptides PdEPFL6 And PdEPFL9 In Regulating Stomatal Development And Drought Tolerance

Populus has been listed as one of the raw materials for the bio-energy industry,which has important ecological functions and economic value.Drought is the main problem faced by agricultural production in China,which seriously affects plant growth and crop production.Therefore,the cultivation of new drought-resistant poplar varieties has important application prospects.An effective strategy to cope with drought stress is to reduce transpirational water loss by regulating stomatal opening and closing and stomatal density/size,thereby improving plant water use efficiency(WUE)and drought tolerance.At present,the relationship between stomatal opening and plant drought resistance has been deeply studied,while the mechanism of stomatal development mediating drought resistance of poplar remains to be explored.Therefore,the physiological and molecular mechanisms of drought response regulated by stomatal development in poplar are investigated.It is of great significance for the directional improvement of poplar resistance to drought by genetic engineering.EPF(EPIDERMAL PATTERNING FACTOR)members play an important role in plant stomatal development and participate in plant growth and stress response.Combined with previous laboratory studies,we cloned two genes,Pd EPFL6 and Pd EPFL9,from Populus NE-19 [Populus nigra ×(P.deltoides × P.nigra)].Using experimental techniques such as morphology,physiology and molecular biology,explored its function in drought stress in Arabidopsis thaliana and 84 K poplar(P.alba × P.glandulosa).The main research results are as follows:(1)Using EPF family member sequences and protein conserved domains as informative probes,15 EPF family members were identified in the poplar genome through database search and manual screening.A phylogenetic tree was constructed using the neighbor-joining method and divided into 4subclades(EPF1-EPF2-EPFL7,EPFL1-EPFL3-EPFL8,EPFL4-EPFL6 and EPFL9).The chromosomal location,gene structure,conserved domain and evolutionary relationship of homologous genes of EPF family members were further analyzed.Through the prediction analysis of family gene promoter elements,EPF family members contain a variety of growth and development,hormone response and stress response elements,it is speculated that they have important functions in plant growth and development and stress.(2)Combined real-time fluorescence quantification(RT-q PCR)and Pd EPFL6Pro:GUS transgenic Arabidopsis seedling histochemical staining(GUS)results showed that Pd EPFL6 could be significantly induced under dehydration stress,and the subcellular localization results showed that Pd EPFL6 was located in the cell membrane.To investigate the potential biological functions of Pd EPFL6,we obtained Pd EPFL6-overexpressing Arabidopsis and poplar lines by genetic transformation.Under normal conditions,the transgenic lines had similar net photosynthetic rates and lower transpiration rates,and thus higher WUE than that of wild type(WT);Under osmotic stress simulated by mannitol,the growth of Ox Pd EPFL6 poplar was less inhibited;Under short-term drought treatments,Ox Pd EPFL6 poplar maintained a high photosynthetic rate and reduced leaf water loss,thereby improving drought resistance.Exploring the physiological mechanism of its enhanced drought tolerance found that the stomatal density of the transgenic plants was significantly reduced,which led to a decrease in the transpiration rate of the transgenic lines.Further,through yeast two-hybrid(Y2H),bimolecular fluorescence complementation(Bi FC)and real-time fluorescence quantitative(RT-q PCR)experimental analysis,it was found that Pd EPFL6 ligand peptide can interact with the downstream receptor-like protein kinase Pd ERECTA and receptor-like protein Pd TMM.Overexpression of Pd EPFL6 regulated the expression levels of MAPK-associated genes and significantly decreased the expressions of stomatal development-related transcription factors(Pd SPCH and Pd MUTE)under normal and drought conditions,leading to a decrease in stomatal density.Taken together,these results suggest that the Pd EPFL6-Pd ERECTA/Pd TMM module signaling plays a crucial role in poplar stomatal development and that the ligand-receptor pair plays an active role in regulating poplar drought resistance by reducing stomatal density.(3)RT-q PCR results showed that the Pd EPFL9 gene was highly expressed in poplar young leaves,and its transcriptional abundance was inhibited by drought stress and exogenous application of brassinolide(BR).Compared with WT,the stomatal density of Pd EPFL9 transgenic Arabidopsis and transgenic poplars leaves increaed,indicating that Pd EPFL9 can promote stomatal production and is a positive regulator of stomatal development.The GUS staining of Pd EPFL9Pro:GUS transgenic Arabidopsis became lighter under BR treatment.Furthermore,by overexpressing Pd EPFL9 in poplar,it was found that the tolerance of transgenic lines to water loss was weakened,while the CRISPR/Cas9-mediated poplar mutant CRISPR-EPFL9 had enhanced tolerance under osmotic stress.Mannitol was superimposed on BR treatment,and it was found that BR could alleviate the reduction of drought resistance caused by Pd EPFL9.Taken together,Pd EPFL9 can promote stomatal formation and reduce plant drought tolerance.Pd EPFL9 is negatively regulated by BR.These results provide new information for revealing the interaction of BR and peptide hormone signaling,and provide theoretical basis and candidate genes for improving crop drought resistance through gene editing.In conclusion,studies on the functions of EPF family members Pd EPFL6 and Pd EPFL9 genes in Arabidopsis thaliana and poplar indicate that these two genes have functional differentiation in stomatal development and response to drought stress.Overexpression of Pd EPFL6 can reduce stomatal density and enhance plant drought tolerance,and we revealed the molecular mechanism of Pd EPFL6 gene improving poplar drought tolerance;overexpression of Pd EPFL9 promotes plant stomatal production and negatively regulates plant drought resistance.In this study,we investigated the physiological and molecular mechanisms of two members of EPF family response to drought,providing good candidate genes for molecular genetic improvement of plant water use efficiency and drought tolerance.