The Molecular Mechanism Of The Formation Of Allopolyploid Giant Cells In Populus Spp. (Section Tacamahaca)

Polyploid plants usually have giant cells in comparison with diploid plants.It is of great significance to study the molecular mechanism of gaint cells formation in Populus polyploids for promoting the basic theory and technical progress of tree polyploid breeding.In this paper,the F1 diploid and triploid obtained by the hybridization of(P.pseudo-simonii× P.nigra ‘Zheyin3#’(2n=2X=38))×(P.× beijingensis(2n=2X=38)),and the tetraploid obtained by doubling the F1 diploid somatic cells.And the leaves of three ploidy poplars at different developmental stages were used as experimental materials.Based on the measurement of leaf size,cell number,cell size and structure,and other physiological and biochemical indexes,transcriptome sequencing(RNA-seq)technology was used to analyze the differential expression of related genes and their regulatory networks during leaf growth and development,and to elucidate the molecular regulation mechanism of the formation of giant cells in polyploid Populus.The main results are as follows:(1)There were some differences in the number and size of leaf cells among different ploidy types of Populus,and the size of triploid and tetraploid leaf cells were significantly larger than diploid cells.The results showed that the content of auxin in polyploid leaves was significantly higher than that in diploid leaves,and it increased with the increase of leaf age in triploid,while decreased with the increase of leaf age in tetraploid.The content of zeatin in triploid leaves was higher than that in diploid leaves.The contents of endogenous hormones such as auxin and zeatin were positively correlated with the number and size of leaf cells.The number of cells in the leaves of triploid Populus was the largest and significantly more than that of diploid Populus.The number of cells in tetraploid leaves was slightly less than that in diploid leaves,but the difference was not significant.Both triploid and tetraploid leaf cells were significantly larger than diploid cells.Resulting in a significantly larger polyploid leaf area than diploid,and the net photosynthetic rate,chlorophyll content and carotenoid content of leaves aged 7 d were higher in polyploid leaves than those in diploid leaves.(2)Differential genes positively related to cell growth were significantly up-regulated in triploid leaves of Populus,while differential genes negatively related to cell growth were significantly down-regulated.Compared with diploid,there were 1783,1073 and 2935 differentially expressed genes in 1 d,7 d and 11 d leaves of Populus triploid,respectively.These genes are mainly concentrated in hormone synthesis and response,DNA replication,auxin response,cell wall modification,transmembrane transport,primary cell wall,plant hormone signal transduction,photosynthesis pathways.Among them,the auxin response factor genes ARF2,ARF5,ARF9,ARF10 and ARF11,which is positively related to auxin signal transduction,and the gene PIN1,which is related to auxin transport were significantly up-regulated.However,the expression of IAA4,IAA12,IAA19,SLR,IAA29,GH3.1,GH3.6and JAR1,which are negatively related to auxin transduction,were significantly downregulated.The expressions of growth regulating factors GRF1,GRF2,GRF5,GRF8,GRF9,which play an important role in cell growth and development,were significantly upregulated,thus the expressions of auxin response factors and cell wall loosening factors were significantly up-regulated.Meanwhile,genes related to brassinolide biosynthesis,such as CYP90D1,DWF1,BAS1,DWF6,BR signal kinase gene BSK3,and positive regulator of brassinosteroid signaling BZR1,were significantly up-regulated,while BRI1 kinase inhibitor BKI1 and brassinosteroid response gene BRH1,were significantly down-regulated.Therefore,brassinolide signaling pathway and genes related to cell wall loosening factors were significantly up-regulated.EXPA4,EXPA11,EXLB1,EXPA20,XTH5,XTH9,XTH16,XTH26,XTR4 and XTR6 were significantly up-regulated,which promoted cell wall loosening and cell expansion.(3)The expression of positively related genes in tetraploid leaves of Populus was significantly up-regulated,while the expression of negatively related genes was significantly down-regulated.Compared with diploid,there were 1683,1034 and 1758 DEGs in tetraploid leaves at 1 d,7 d and 11 d age,respectively,which were mainly enriched in transcription factor activity,cell wall,plant hormone signal transduction,microtubules,cell wall modification,and cell wall and other processes.The auxin response factor genes ARF2,ARF5,ARF10,ARF11,which are positively related to auxin signal transduction,and PIN1 and LAX3,which are related to auxin transport,were significantly up-regulated in tetraploid leaves.However,the expressions of IAA4,IAA14,IAA19,IAA29,GH3.1 and GH3.6negatively correlated with auxin response were significantly down-regulated.The expressions of GRF1,GRF5 and GRF9,which play an important role in cell growth and development,were significantly up-regulated.And the expression of ARGOS,a gene related to cell size regulation,was significantly up-regulated in tetraploid.These DEGs together promoted the transduction of auxin signal to activate cell wall loosening factors.Meanwhile,genes CYP90D1,DWF1,BAS1,DWF4,BR6OX1 and DWF6 related to brassinolide biosynthesis,as well as BR signal kinase gene BSK3 and brassinosteroid signal positive regulator BZR1 were significantly up-regulated,while negatively related BR signal transduction genes BKI1 and BRH1 were significantly down-regulated,jointly promoting brassinosteroid signaling pathway and cell wall loosening factor genes up-regulating.EXPA4,EXPA11,XTH5,XTH9,XTH32,XTR4 and XTR6,which promoted cell wall loosening and cell expansion,were significantly up-regulated.(4)The key DEGs in the two pathways of Populus polyploid auxin signal response and brassinolide synthesis and signal transduction co-regulate the formation of giant cells.There were 35 co-DEGs related to cell growth and development in the immature leaves of triploid and tetraploid of Populus,all of which belong to the genes related to auxin and brassinolide biosynthesis and signal transduction,cell wall loosening factor genes and growth regulating factor genes.Among them,genes positively correlated with auxin and brassinolide hormone synthesis and signal transduction were significantly up-regulated in immature polyploid leaves,including auxin response factor genes ARF2,ARF5,ARF9,ARF10,ARF11,and brassinolide biosynthesis related genes DWF1,DWF6,BAS1 and CYP90D1,and positively regulating brassinolide signal transduction genes BZR1 and BSK3.However,genes negatively related to auxin and brassinolide synthesis and signal transduction were significantly down-regulated in immature polyploid leaves,including auxin and brassinolide signal transduction negatively related AUX/IAA protein genes IAA4 and SLR,and brassinolide signal transduction negatively regulating genes BKI1 and BRH1.The cell wall loosening factors including xyloglucan endotransglucosylase/hydrolase gene XTH9 and expasin genes EXPA4 and EXPA11 were significantly up-regulated in immature polyploid leaves,which promoted cell wall loosening and expansion.The genes GRF1,GRF5 and GRF9,which are related to cell growth and development,were significantly up-regulated in immature polyploid leaves.In summary,we proposed a molecular mechanism model for the formation of giant cells in Populus polyploid leaves.During the growth and development of polyploid leaves,the expression of auxin receptor protein gene TIR1 was significantly up-regulated,and the binding of TIR1 to Aux/IAA protein resulted in the decreased expression of Aux /IAA genes.However,several GRF genes,important transcription factors related to cell growth and development,were significantly up-regulated,which promoted the expression of the auxin response factor genes ARFs and auxin signal response.And it further promoted the expression of XTH9,EXPA4 and EXPA11,which are related to cell wall expansion,and significantly promoted cell expansion.At the same time,the DWFs that promotes brassinolide biosynthesis were significantly up-regulated,while the expression of BKI1 and BRH1,which inhibit brassinolide signal transduction,were down-regulated,which together promoted the up-regulation of the BR-signaling kinase gene BSK3 and brassinosteroid signaling positive regulator BZR1.Furthermore,the expressions of XTH9,EXPA4 and EXPA11 were significantly up-regulated,which can significantly regulate cell expansion and growth.These results indicated that the key DEGs in the two pathways of auxin response and brassinolide signal transduction in Populus regulate the formation of polyploid giant cells.