The Function Of FmTCP2 And FmTCP15 In Leaf Development And Drought Tolerance In Fraxinus Mandshurica

TCP transcription factor family is a novel plant-specific TF family and plays an important role in plant morphogenesis and environmental adaptation.However,most of the previous studies often focused on its role in regulating plant development but ignored the characteristics of which were participating in the stress response.Recent studies have found that TCP transcription factors could regulate the balance between growth,development,and stress throughout GA signaling pathway from multiple perspectives such as the GA synthesis and degradation.So far,the studies on TCP transcription factors are mainly focused on Arabidopsis,tobacco,cotton,and other herbaceous plants.However,herbs and trees have undergone different evolutionary processes,and their gene functions will also change greatly accordingly.Fraxinus mandshurica is an important economic and ecological tree species in China,and with strong stress tolerance.To reveal the role of the TCP family in development and adaptation to stress tolerance in F.mandshurica,will provide more significant gene resources for forest molecular breeding,and enrich the theory of stress molecular biology,and guide the breeding progress of F.mandshurica.In this research,the TCP transcription factor family of F.mandshurica was identified and screened.And FmTCP2 and FmTCP15 were set as representatives of TCP family Class Ⅱ and Class Ⅰ,respectively.The gene function of FmTCP2 and FmTCP15 were fully revealed.We illustrated that the FmTCP2 and FmTCP15 could interact with DELLA and regulate plant growth,development,and drought stress throughout the gibberellin signal pathway.The main results are as follows:(1)Through the conjoint analysis of the F.mandshurica genome and transcriptome database,all members of the TCP transcription factor family were screened,identified,and cloned.The TCP family of F.mandshurica was consisted of 27 members and divided into Class Ⅰ and Class Ⅱ according to the conservative structural analysis.Class I,also known as PCF,was composed of 16 members.Class II was further divided into CIN and CYC/TB1.The TCP family members all contained a TCP domain composed of 57 amino acid residues.Combined with bioinformatics prediction,gene expression analysis,and promoter cis-elements analysis,we found the TCP members of F.mandshurica could respond to the regulation of growth and development,hormone response,and stress response.The TCP members could respond to plant hormones such as ABA,GA3,and SA,and were involved in the regulation of stress tolerance including low temperature,salt,drought,alkali,and heavy metals.And Fm TCP2 and FmTCP15 were selected as the key genes,which represented Class II and Class I for further study.(2)The expression patterns of FmTCP2 and FmTCP15 showed that both FmTCP2 and FmTCP15 could respond to the gibberellin signal and participate in the regulation of drought and high salinity,but FmTCP2 was mainly expressed in leaves,and FmTCP15 was mainly expressed in stems.(3)The technology of the isolation and transform of tender branch cortex protoplasts of F.mandshurica were established,which could effectively carry out subcellular localization of FmTCP2 and FmTCP15.And this technique will enrich the molecular biology research tools of F.mandshurica.(4)The overexpression of FmTCP2 and FmTCP15 in tobacco and F.mandshurica cause the morphological changes of transgenic plants.Similarities:Overexpression of FmTCP2 and FmTCP15 resulted in decreased epidermal cell size and increased mesophyll cell density in transgenic tobacco leaves,resulting in narrower leaf shape,lower leaf surface area,and shorter plant height.Difference:Overexpression of FmTCP2 resulted in the thicker and narrower size of leaves,and partial leaf symmetry destruction,increased epidermal hairs,and thicker veins,but with stomatal index not changed.However,overexpression of FmTCP15 increased stomatal index but did no effect on leaf thickness,symmetry,epidermal hairs,or veins.(5)The overexpression of FmTCP2 and FmTCP15 had an important relationship with the regulation of the gibberellin pathway in tobacco phenotype.Firstly,the endogenous gibberellin content in transgenic plants was significantly reduced.Secondly,exogenous gibberellin could also recover the phenotype changes caused by the overexpression of FmTCP2 and FmTCP15 in tobacco.(6)Overexpression of FmTCP2 enhanced abiotic stress tolerance of transgenic tobacco,such as drought tolerance,while overexpression of FmTCP15 decreased stress tolerance of transgenic tobacco.The stress experiments with leaves and seedlings showed that,under drought stress,FmTCP2 overexpression tobacco lines could maintain high chlorophyll content,low ROS content,electrolyte permeability,and MDA content,while the proline content was maintained high,which showing a strong stress tolerance.Correspondingly,the leaves of FmTCP15 overexpression tobacco lines rapidly faded white,even died of water loss,and the endogenous defense response signal also confirm its weak stress tolerance characteristics.(7)FmTCP2 and FmTCP15 could interact and regulate the expression of DELLA family members,indicated that FmTCP2 and FmTCP15 could participate in the gibberellin pathway through protein-protein interaction and regulate the balance of plant growth,development,and abiotic stress tolerance.In summary,the TCP family of F.mandshurica was screened and identified,and the function of Fm TCP2 and FmTCP15 was fully recovered,which belonged to Class Ⅱ and ClassⅠ,respectively.FmTCP2 and FmTCP15 could interact with DELLA family members and participate in the regulation of leaf development throughout the gibberellin signaling pathway.FmTCP2 and FmTCP15 could regulate the homeostasis of endogenous gibberellin,changing the development of leaf cells(including leaf epidermal cells,epidermal hairs index,etc.),and further changing leaf morphological characteristics(including leaf size,thickness,symmetry,etc.).The FmTCP2 and FmTCP15 also differently regulated abiotic stress tolerance.Class ⅡFmTCP2 enhanced plant tolerance to drought stress,while Class I FmTCP15 showed the opposite result.This research revealed the biological functions of FmTCP2 and FmTCP15 in regulating leaf development and drought tolerance,providing significant gene resources for tree molecular breeding,enriching the theory of stress molecular biology,and guiding the breeding and breeding of F.mandshurica.