Molecular Mechanism Of Vegetative Growth Advantage In Allotriploid Populus Spp.(section Tacamahaca)

Allotriploid Populus has prominent advantage of vegetative growth.This study used leaves of various leaf positions in three triploid Populus populations with three different kinds of 2n gamete source and one diploid population as experimental materials.These four populations were generated by the same female(P.pseudo-simonii x P.nigra var.italica)and male(P.×beijingensis)parent.The characteristic of gene expression changes and regulation of gene expression by microRNA were analyzed on the different leaf positions through the measuring of leaf photosynthesis,cell ultrastructure,physiological and biochemical characteristic,gene expression and the miRNA expression using the transcriptome sequencing(RNA-seq)and microRNAs high-throughput sequencing technology.The study revealed the molecular mechanism of vegetative growth advantage in allotriploid Populus.The main results were as follows:(1)The plant height,diameter at the base,photosynthesis,chlorophyll content,leaf area and photosynthetic efficiency of the whole leaf in allotriploid population were significantly higher than those in diploid population in Populus.The majority of genotypes with mass growth had higher photosynthesis,larger leaf area and more chlorophyll.These were considered to be higher photosynthesis and fast-growing plants type.While a little genotype which had mass growth with lower photosynthesis,smaller leaf area and more leaf(lower photosynthesis and fast-growing plants type)were discovered.Growth parameters showed significantly positive correlation with photosynthetic parameters,chlorophyll content and leaf area(p<0.01).The photosynthetic rate was higher in triploid than diploid on the different leaf positions,even on the lower of plants,indicating that the leaf senescence was slower in triploid than in diploid.(2)The characteristic of gene expression in allotriploid was temporal difference.In the metabolic pathways related to the vegetative growth advantages,only part of genes showed the gene dosage effect.While there were various genes appeared in the metabolic pathways in different triploid types and the leaf positions,without obvious regularity,indicating the gene transcription and expression in the triploid cell showed temporal difference.The dosage effect genes which generated the enzyme protein in metabolism pathways depended on the product formed in the previous period.And the gene product was the substrate of downstream gene in the next metabolic pathways.(3)The differently expressed genes of leaves positions with various chromosome doubling sources and photosynthesis types showed differences.10275 genes were differently expressed between triploid and diploid on the higher photosynthesis and fast-growing plants type.While there were 16394 differently expressed genes between the triploid and diploid in the lower photosynthetic and fast-growing plant type.There were almost the same genes differently expressed between triploid and diploid in lower compared to higher photosynthetic and fast-growing type plant,but there are differences in number.The fast growth of low photosynthetic plant might be due to the synthesis and signal transduction of cytokinins,zeatin and gibberellin were stronger,leading to more leaves.(4)The chlorophyll synthesis ability was stronger and the chloroplast aging rate was slower in allotriploid compared to diploid Populus.From the perspective of related gene expression of the synthesis and degradation of chlorophyll,the PORA related to chlorophyll synthesis was up-regulated in triploid than diploid.At the same time,the number of chloroplast and chlorophyll content in triploid was significantly higher,and the chlorophyll enzyme activity was significantly lower than those in diploid through the physiology measuring.Triploid were 1.3 and 1.2 times higher than diploid,respectively.The chloroplast ultrastructure showed almost no difference between triploid and diploid on the fifth and tenth leaves.While on the twenty-fifth leave of diploid,the osmiophilic granules were more and stroma lamella was scattered than that in triploid by electron microscopy observation,indicating that the disintegration of chloroplast was much more serious in diploid compared in triploid.(5)The light capture,photosynthesis,carbon fixation efficiency sugar and starch synthesis and decomposition were higher in triploid than that in diploid in allotriploid Populus.Compared with diploid,the LHC family genes involved in light capture,most of the members of two subunits protein complexes Psa and Psb gene family in thylakoid membrane and the key genes involved in the Calvin cycle process,such as GAPDH,FBP,PFK were up-regulated in triploid.The photosynthetic verification showed that the photosynthetic rate at different leaf canopy positions in triploid was significantly higher than that of diploid.This was almost consistent with the results of gene expression.Compared to diploid,the key genes of SUS,SPS involved in the formation and decomposition of sucrose,key genes of SS,ISA,AMS,DBE,BAM,AMY,EMB involved in starch synthesis and decomposition were up-regulated expression in triploid.Important products of carbon assimilation of sugar,starch content and related synthesis and decomposition enzyme(SUS,SPS,AMS)activity were determined,and the results showed the content of starch,sucrose,glucose and fructose,and related enzyme activity were significantly higher in triploid than those in diploid.(6)The endogenous hormone synthesis and signal transduction process were more active in allotriploid Populus than diploid.Some related genes,containing the auxin response regulon ARR,main transcription factors in the process of BR signal BZR1,cytokinin oxidase gene CKX participatiing in cytokinin synthesis,gibberellic acid oxidase gene GA20ox,GA3ox involving in GA3 synthesis,genes involved in cell cycle regulation of CYCD3,ABA receptor proteins PYL,response element ABF3 genes and so on.In the meanwhile,the contents of IAA,GA3,ZT were higher,while the ABA content was lower compared triploid to diploid,which was consistent with the results of gene expression.(7)Analysis of miRNA expression between triploid and diploid Populus showed 59 differently expressed miRNAs were detected.The differently expressed miRNAs were targeted to 22 differentially expressed genes combining with the transcriptome data in the same period.Of these,there were 19 negative control of miRNA-mRNA.Differentially expressed target genes mainly involves in related biological process of salt stress,sulfur stress,lignin synthesis.And no differentially expressed target genes directly involved in vegetative growth traits were detected,which indicating the miRNAs expression that related to vegetative growth traits had no dosage effect,making the target genes has relatively less negative control.This was supposed to be one of important epigenetic mechanisms that the allotriploid Populus had vegetative growth advantage than diploid.Compared to diploid,expression of circadian clock repressor LHY,regulon TOC1 and GI showed differences between higher and lower photosynthesis plant types,suggesting that the Populus allotriploid might not follow the circadian clock pattern similar to Arabidopsis tetraploid.In conclusion,the miRNAs directly related to vegetative growth traits in triploid Populus showed no dosage effect,causing a relatively weaker negative regulation.At the same time,the gene expression show dosage effect,resulting in the photosynthesis,starch and sucrose synthesis and metabolism,and the endogenous hormone content were significantly higher in triploid than diploid.In addition,the triploid plant had a larger photosynthetic area and a relatively slower chloroplast degradation rate,resulting vegetative growth advantages in allotriploid than the diploid in Populus.