Multiscale 3D Reconstruction,Metabolism And Transcriptome Analysis Of Ulmus Pumila Seeds During Late-maturation Stage

Seeds are the reproductive organs of plants and play an important role in the continuation of species and the reproduction of offspring.At present,the research on Ulmus pumila seeds mainly focuses on the germination,aging and programmed cell death(PCD)of mature seeds after they were harvested.However,there are few reports on the cytological structure of seeds,the expression differences of transcripts,and the distribution of lipid metabolites before harvesting,particularly at the late-maturation stage of seed development.As the reconstruction technology can be used to analyze the internal microstructure of the tissues three-dimensionally,RNA sequencing(RNA-seq)can accurately characterize the type and abundance of gene expression in tissues at different phases,and chemical constituents of the tissues can be detected in situ by using the mass spectrometry imaging technology,these tecchniques can be combined together to analyze the cytological structure and chemical composition distribution of the tissue,which is valuable for the study on structure-function relationship of Ulmus pumila seeds.In this study,X-ray microscopy imaging and LSFM(light sheet fluorescence microscopy)were used to determine the 3D morphological and cytological variations of Ulmus pumila seeds.Moreover,in situ chemical constituents were analyzed by means of matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry(MALDI-TOF/TOF MS)and ultra-performance liquid chromatographyquadrupole time-of-flight mass spectrometry(UPLC-QTOF MS).Finally,RNA-seq analysis was conducted to reveal pathways involved in seed development.The main results are as follows:(1)X-ray microscopy imaging showed that the seeds contained two hypertrophic cotyledons,hypocotyl,radicle,shoot apical meristem and no endosperm.With the development of Ulmus pumila seeds,the epidermal cells were much denser,the parenchyma cells were loosely arranged,the intercellular spaces were clearly visible,and the original vascular tissue can be clearly distinguished by Imaris.(2)The LSFM imaging of Ulmus pumila seeds revealed that the epidermal cells were rectangular in shape and arranged neatly and densely,the parenchyma cells were polygonal,arranged closely but disorderly,and the provascular cells were connected to each other to form a network structure.(3)The observation of tissue semi-thin sectionings showed that the anatomical structures of different tissues had different structural characteristics,indicating that the internal structures of various cells changed significantly during development.Electron microscopy revealed that during phase I and phase II,the membrane structure of the cells was distinct,while the cytoplasm was relatively dense.In addition,the intact nuclear membrane was clearly visible and nucleoli can be also observed,during which phases the liposomes and protein bodies were continuously accumulated.In phase III,the protein bodies occasionally become irregular or even partially disintegrated.(4)SR-Tesseler morphological analysis of cotyledon cells showed that the distance and area between provascular cells were significantly smaller than those of surrounding cells,while the local density was higher.With the development of cotyledons,the local density became decreased.The density between cells in phase III was significantly lower than that in phase I,and the average distance and area between cells changed significantly.(5)Detection and imaging of endogenous lipid metabolites in Ulmus pumila seeds showed that phospholipids,glycerides and sterols were concentrated in the hypocotyls of seeds;fatty acids were concentrated in the cotyledons of seeds;waxes were distributed in the cotyledons of peripheral edge.(6)RNA-seqencing analysis showed that differentially expressed genes in the three developmental phases of the late-maturation stage were mainly enriched in biological processes such as cell division,cell wall biosynthesis,carbohydrate and starch metabolism,and plant hormone signal transduction.In summary,this study demonstrated the dynamic characteristics of Ulmus pumila seed development through the cytological observation and three-dimensional reconstruction technology,characterized the distribution of endogenous lipid metabolites in Ulmus pumila seeds by mass spectrometry imaging in situ,and revealed the cell physiological mechanism during development by means of transcriptome technology.These results provide a theoretical basis for exploring the mechanism of plant seed development and seed harvesting.