| Populus Aigeiros is one of important and economic tree species because of its characteristic of rapid growth.Especially Populus euramerica hybrid clones,which have been introduced from other countries or cultivated in China,are the primary tree species in northern China.The wood formation results from tree stem radial growth,which is caused by cell division and differentiation of plant vascular cambium.Stem radial growth of trees represents a complex yet fascinating physiological process,the detailed understanding of which can optimize strategies for understanding of tree radial growth and the full exploitation of woody biomass.To elucidate the molecular basis of tree stem radial growth,two Populus genotypes Neva(Populus x euramericana cv.’Neva’)and 1-214(Populus x euramericana cv.’1-214’)were used for physiological comparison of growth difference.RNA-Seq was employed for the transcriptional network model and comparative analysis for two physiologically different Populus genotypes.We also constructed the gene coexpression network,and discovered and analyzed the modules which had the high correlation with traits of radial growth.A novel gene about genotype trait was cloned and characterized by overexpression and mutation induced by CRISPR/Cas9 system.The results were shown as follows:(1)We used the dendrometer to real-time monitor the stem radial growth of two genotypes during growth time(June to September).The results showed that two genotypes displayed the same growth trend of S style and Neva grew faster than 1-214.Additionally,Neva had higher photosynthetic rate than 1-214 and accumulated more organics utilized for plant growth.At the pre-growth stage(the beginning of June),Neva showed higher transpiration rate and more maximum daily shrinkage(MDS)than 1-214.At the both fast-growth(the beginning of August)and post-growth stages(the end of September),conversely,I-214 had higher transpiration rate and more MDS.During the entire growth season,1-214 had higher stem relative water content than Neva,suggesting that Neva had more dry substance accumulation.(2)We analyzed the cambial transcriptomes of genotypes Neva and I-214 by Illumina sequencing platform,and harvested 286,612,666 and 297,145,856 clean reads with length of 100 bp.The gene functional annotation after reads assembly showed the expressed genes were mostly clustered in biological processes about transcription,posttranslational modification,signal transduction,carbohydrate transport and metabolism,and intracellular trafficking,secretion and vesicular transport.These processes were closely relative to programs about cell cycle,growth,and cell wall development.The analysis of differential expressed genes showed genes were mainly clustered in inositol biosynthesis,defense response,and carbohydrates metabolism like alpha-amylase activity,cellular glucan metabolism,and xyloglucan-xyloglucosyl transferase activity.These results indicated that the growth differences between two genotypes were carbohydrate metabolism processes.(3)Populus cambial gene expression profiles were analyzed at three distinct growth stages.Total 34937 expressed genes were identified by mapping clean reads to referenced Populus genome.By analyzing differentially expressed genes between diffeerent stages,we found that two genotypes displayed the analogous gene functions and biological processes.At the transition from the pre-growth to fast-growth stage,abundant genes were up-regulated in cell cycle-related processes.At the transition from fast-growth to post-growth stage,genes were up-regulated in metabolism regulation and stimulus response,and down-regulated in biosynthesis of cellular components.Moreover,1542,2295,and 21 10 genes were differentially expressed between two genotypes at the pre-growth,fast-growth,and post-growth stage,respectively.At the pre-growth stage,faster-growing genotype Neva had up-regulated genes in cell wall biosynthesis,and 1-214 had genes in stress and defense response.When getting into the fast-growth stage,Neva clustered up-regulated genes in cell communication and signal transduction,while for 1-214,several cell wall biogenesis processes emerged and stimulus and defense response remained as before.Until the post-growth stage,Neva enriched more genes in various regulatory processes of circadian rhythm,while some functions involved light response had more genes in 1-214.Moreover,Weighted Gene Coexpression Network Analysis represented a global view of transcriptional patterns of two genotypes across the growth period.Total 18 modules were identified.Based on gene expression patterns of two genotypes and GO enrichment annotation,we found that numerous genes of interest clustered in module blue were determined to participate in cell cycle and division,and displayed the expression abundance profiles which was consistent with the variation of radial growth rates.This indicated these genes were related to growth rate variation,and conserved in both two genotypes.The analysis of cell wall-related module tan showed that module genes were differentially expressed at the pre-growth stage,and participated in processes of cell wall development,and biosynthesis of lignin and polysaccharides.The genotype-specific module red revealed several important genes with significantly differential expression between two genotypes.(4)We firstly identified 35 TALE transcription factor genes from Populus genome,including 20 BELL subfamily genes and 15 KNOX subfamily genes.Analysis of chromosome location and gene duplication showed whole-genome segmental duplication resulted in extension of Populus TALE family members.The phylogenetic relationship indicated that each was further divided into two clades after separation of BELL and KNOX subfamilies.Combining with data of Populus gene expression,we inferred that different clades had various roles in development and growth of Populus vascular tissues.(5)We characterized a novel BELL transcription factor PdnBELL15 from fast-growing Populus genotype Neva.This PdnBELL15 gene was up-regulated in the faster-growing Neva during the growth season,and tissue-specific expressed in stems.The cross-section analysis of transgenic Populus plants expressing the PdnBELL15 driven by a 35S promoter indicated that PdnBELL15 overexpression could improve lignification of phloem fibers and xylem cell,and expedite the establishment of secondary vascular tissues.Moreover,the CRISPR/Cas9 system successfully edited two target sites in Populus BELL15 to knock out the gene.However,the knock-out Populus plants displayed no significant difference with wild type in morphology,suggesting that Populus BELL15 had redundant functions with other genes.In this study,we compared two Populus genotypes in physiological traits,analyzed gene expression profiles of two genotypes,constructed the gene coexpression network,and evaluated the correlation of gene expression patterns with physiological traits.We found that the more stem radial increment is mainly attributed to more vigorous vascular cambium cell division,expansion,and earlier establishment of secondary vascular tissues.We also identified and characterized a novel Populus BELL transcription factor gene PdnBELL15,and revealed its potential roles in development and growth of secondary vascular tissues.Altogether,these works provided the practice experience and settled the foundation for understanding of the relationship between tree growth profiles and gene expression patterns and molecular mechanisms of tree growth and development. |
PDF Full Text Request