Functional Analysis Of PdbTCP9 In Drought Stress Responses And Secondary Wall Biosynthesis In Populus Davidiana×Populus Bolleana

Plant-specific TCP transcription factors play important roles in plant biological processes and stress responses.Here,based on transcriptome,weighted gene co-expression network analysis(WGCNA)and gene family analysis,PdbTCP9 gene was cloned from "Shanxinyang"(Populus Davidiana×Populus Bolleana),which belongs to TCP transcription factors family and may be associated with plant drought responses and secondary wall biosynthesis.In this study,functional roles of PdbTCP9 as a transcriptional regulator of both drought stress responses and secondary wall biosynthesis were mainly characterized.This research provides foundational knowledge for unraveling the transcriptional regulation underlying drought responses and wood formation in "Shanxinyang".The main results are listed as below:(1)Through transcriptome and WGCNA analysis,we found some TCP transcription factors were in response to drought stress,and through gene family analysis,we found that the candidate gene PdbTCP9 may be related to the drought response of "Shanxinyang".(2)The expression of PdbTCP9 was induced by drought stresses,suggesting an involvement in drought stress responses.PdbTCP9 is expressed in roots,stems and leaves,and has the higher expression in stems.Yeast transformation experiments revealed that PdbTCP9 serves not as a self transcriptional activator.The PdbTCP9 gene was fused with GFP driven by the CaMV 35S promoter,and introduced into onion epidermal cells by particle bombardment.Subcellular localization analysis indicated that PdbTCP9 is a nuclear protein.(3)The transgenic "Shanxinyang" plants that overexpressed PdbTCP9 or had PdbTCP9 suppressed by RNA interference(RNAi)were generated using Agrobacterium-mediated transformation.Under osmotic stress treatments,PdbTCP9 suppression transgenic"Shanxinyang" lines had higher fresh weight and root length,lower cell membrane damage than those of the Vector control lines.Silencing PdbTCP9 is able to enhanced SOD and POD activities and increased proline content,thus to improve ROS scavenging capability,resulting in enhanced osmotic stress tolerance in PdbTCP9 suppression lines.Compared with Vector control plants,PdbTCP9 suppression lines displayed significantly reduced stomatal apertures and water loss rates,showing enhanced drought resistance,the further sap flow experiment showed that the difference of the stem cell secondary wall may lead the enhanced drought defence ability.(4)The basal parts of stems from PdbTCP9 overexpression,RNAi-suppression,and vector control plants were cross-sectioned to examine the secondary wall deposition.The results showed that pith and xylem were larger in PdbTCP9 suppression lines and the presence of the three major secondary wall components,lignin,cellulose,and xylan,were examined in the cross-sections of stems.Meanwhile,suppression of PdbTCP9 resulted in ectopic secondary wall deposition of lignin and cellulose in the stem epidermis.(5)A scanning electron microscope(SEM)was used to detect the secondary wall thickness,and the results showed that suppression of PdbTCP9 caused a drastic increasing in the secondary wall thickening of stem fibers;while,overexpression of PdbTCP9 caused a reduction in the secondary wall thickening of stem fibers.Which indicating that the PdbTCP9 regulates the secondary wall thickening of stem fibers.(6)Under normal and osmotic stresses conditions,elicited lower expression levels of PdbTCP9 leading to elevating lignin accumulation,indicating that PdbTCP9 negatively regulates drought stress responses in part by enhanced lignification.