Variation Characteristics And Regulation Mechanism Of Ginkgo Biloba L.cylindrical Leaf Shape

Ginkgo biloba L.,one of the relic tree species,has survived in China after the Quaternary glaciation.It has a long evolutionary history since the late Paleozoic era and is known as a "living fossil" in the plant kingdom.As a monotypic plant in the Ginkgoaceae family,G.biloba typically has bilobed fan-shaped leaves.In this study,we focused on the unique "tubular" leaf morphology of G.biloba and compared it with the normal fan-shaped leaves.Through phenotypic,anatomical,transcriptomic,and metabolomic analyses,we investigated the leaf morphological variation and regulatory mechanisms in tubular-leaved G.biloba,providing new insights into the mechanisms underlying leaf variation in G.biloba.The successful progress of this study is of great significance for the development of G.biloba ’s ornamental value and the promotion of breeding programs for ornamental G.biloba varieties.It also complements the understanding of molecular mechanisms underlying plant morphological variations.The main results of this study are as follows :（1）Differences analysis of phenotypes between tubular and normal G.biloba leavesThe overall size of tubular G.biloba leaves is smaller than that of normal fanshaped leaves.The cross-section of the leaf base in tubular leaves forms a circular ring shape,while in normal fan-shaped leaves,it appears as a strip shape.The individual cell volume of the epidermis on the ventral side of tubular leaves is larger than that of fan-shaped leaves,while the individual cell volume of the epidermis on the dorsal side is smaller than that of fan-shaped leaves.（2）Differences in physiological and biochemical indicators between tubular and normal G.biloba leavesThe chlorophyll content and net photosynthetic rate of tubular G.biloba leaves are lower than those of fan-shaped leaves.However,the activities of SOD（superoxide dismutase）and POD（peroxidase）in tubular leaves are higher than in normal leaves.The content of three jasmonic acids（OPC-4,OPDA,JA）,two cytokinins（m T,Me Sc Z）,one strigolactone（Strigol）,and one auxin（3-Indole acetamide）are all higher in tubular leaves compared to normal fan-shaped leaves.This indicates that the aforementioned plant hormones are related to the formation of leaf shape,and the accumulation of hormone content within the leaves can lead to changes in the structure and organization of G.biloba leaf tissues and cells,thereby affecting intercellular forces and resulting in leaf curling（3）Differences in transcriptome and metabolome profiles between tubular and normal G.biloba leavesA total of 589 differentially expressed genes were identified through transcriptomic sequencing.KEGG analysis annotated the differentially expressed genes to secondary metabolite biosynthesis,phenylpropanoid biosynthesis,and the plant MAPK signaling pathway.Additionally,WGCNA analysis identified one module,called "blue," which contains known key transcription factors involved in jasmonic acid synthesis pathway,providing further insights into the mechanisms underlying G.biloba leaf formation.Metabolomic analysis of tubular and normal G.biloba leaves detected a total of 1302 metabolites,and after screening,40 differentially abundant metabolites were identified,mainly belonging to flavonoids and phenolic acids.Except for one lignin compound that exhibited higher abundance in normal leaves,the remaining differentially abundant metabolites were found to be higher in tubular leaves compared to normal leaves.The changes in the levels of flavonoids and phenolic acids reflect the adaptations made by tubular leaves to their environment.（4）Mechanism of tubular leaf formation in G.bilobaBy integrating the phenotypic,anatomical,transcriptomic sequencing,and metabolomic analysis results,the mechanism underlying tubular leaf formation in G.biloba has been elucidated.Among them,the transcription factor MYC2（Gb₀4842）involved in plant hormone signal transduction and the MAPK pathway was found to be highly expressed in tubular G.biloba leaves,positively regulating the synthesis of jasmonic acid（JA）and leading to an increase in JA content.In the tryptophan metabolism pathway,a negative feed back from TAA1（Gb₀8392）on indole-3-acetamide,a type of auxin,resulted in increased levels of auxin in tubular leaves.Additionally,the high expression of the GA receptor GID1 in the plant hormone signal transduction pathway inhibited the synthesis of GA in tubular leaves.The synergy between JA and auxin in tubular leaves inhibited cell division,extended the cell cycle,and consequently resulted in fewer cells in tubular leaves.Conversely,the low concentration of GA in tubular leaves promoted cell elongation and increased cell volume.The larger cell volume in the ventral side of tubular leaves increased intercellular mechanical forces,causing cell compression inward,while the looser cell arrangement in the dorsal side reduced intercellular interactions.Ultimately,these factors led to the inward curling and formation of tubular leaves.