PagUNE12 Is Involved In The Regulation Of Vascular Tissues Development In Poplar

Secondary growth is an important biological process occured after primary growth.The secondary growth generated new cells and tissues by the activities of vascular cambium,a major of secondary meristem,and drove the radial expansion of stems and roots.Increasing evidence indicated the secondary growth are regulated by various of endogenous factors,including transcription factors.bHLH transcription factors are a large family of transcription factors,which are necessary for plant normal growth and development.Due to the large quantities and complexity,the functions of plant bHLH family remained to be further determined.This study used the model woody plant—poplar as material to investigate the biological functions of PagUNE12 in poplar secondary growth.From hybrid poplar P.albaxP.glandulosa Uyeki(84K),an bHLH transcription factor PagUNE12 was cloned.An efficient poplar transformation system for 84K was established,and vairous of transgenic poplars with high expression levels of PagUNE12 were obtained through this transformation system.The biological functions of PagUNE12 in poplar secondary growth were analyzed by using genetic,bioinfonnatics and cytological analysis together with muti-omics studies.The main results are as follows:After optimizing seveal independent factors of the poplar transformation system,the transformation efficiency was improved significantly by using juvenile leaf explants,an Agrobacterium suspension with the OD600=0.6 for 15 min,and co-cultured in dark conditions for 3 days.Using the improved poplar transformation system,twelve transgenic poplar lines were obtained.Quantitative real time PCR and Western bloting assay confirmed that the transgene can be translated into proteins steadily.Tissue-specific expression profile showed that PagUNE12 was widely expressed in all of the vascular tissues,including primary phloem,primary xylem,secondary phloem and secondary xylem.Subcellular localization analysis showed that PagUNE12 was localized in cell nucleus.Yeast hybrid assay showed that PagUNE12 had autoactivation.Phenotypic analysis revealed that the plant height of overexpressed PagUNEl2 poplar plants were significantly lower than wild type plants,with more internode numbers,shorter internodes and obvious leaf shrunk.The transgenic plants displayed higher photosynthetic parameters than wild type plants with more net photosynthetic rate,stomatal conductance,water-use efficiency and transpiration rate.Anatomical analysis of cross sections showed that overexpression of Pag UNE12 can promote the development of secondary xylem.Furthermore,transmission electron microscopy(TEM)revealed that the thicker secondary cell walls occurred in the stem of overexpressed PagUNE12 plants.These results suggested that PagUNE12 is involved in the regulation of morphogenesis of the secondary xylem in poplar.FTIR(fourier transform infrared spectroscopy)analysis showed that the lignin content of secondary xylem cells in overexpressed PagUNE12 plants was higher than that in wild type plants.From the lignin distribution of secondary xylem cell walls as revealed by confocol Raman microspectroscopy,a label free and in situ microscopic imaging technology,it is clear that the fluorescence intensity of the secondary cell walls in overexpressed PagUNE12 plants was higher than that in the wild type plants.Using 2D NMR spectroscopy to analyz the lignin composition of secondary cell walls,it was found that the lignin in overexpressed PagUNE12 plants had a lower relative abundance of S lignin units and a higher relative abundance of G lignin units.Transcriptome sequencing data showed that the expression level of genes encoding for phosphoenolpyruvate carboxykinase(CCR)and phosphoenolpyruvate carboxykinase(PEPCK)were significantly increased in overexpressed PagUNE12 plants.Actually,CCR is a key enzyme in lignin biosynthesis,and PEPCK is an enzyme involved in the synthesis of pyruvic acid,which is an intermediate in carbohydrate metabolism in plants.Combinative analyses of transcriptome sequencing and miRNA sequencing data revealed that the plants displayed higher expression level of genes encoding for starch synthase 1(SS1),an enzyme involved in the biosynthesis of starch,by reducing the expression level of miR6462c,suggesting that PagUNE12 can promote the matebolism of photosynthetic products and lignin biosynthesis.In conclusion,by using 84K hybrid popalr as material,the current study demonstrated that the overexpression of PagUNE12 promoted the development of secondary xylem and increased the lignin concent of secondary xylem in 84K,suggesting that PagUNE12 was involved in regulating the vascular morphogenesis and lignin polymerization in 84K poplar.These results lay the foundation for the further investigation on the mediation mechanism of the PagUNE12 involved in secondary growth in plants.