| Cellulose is the most abundant biomass resource on the earth and wood is one of the most abundant cellulose raw materials in nature.Because of lacking gene knockout mutants in trees,an overall identification and functional analysis of wood secondary cell wall cellulose synthases(SCW CesAs)are limited.In Populus,cellulose was assumed to be synthesized by SCW PtrCesAs(PtrCesA4,7A,7B,8A,and 8B).Herein,we established a Cas9/gRNA-edited mutagenesis system in Populus trichocarpa,the first sequenced tree and produced the Cas9/gRNA-edited knockout mutants of 5 SCW PtrCesA genes.Based on anatomic,immunohistochemical and wood composition evidence,we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level.The main results are listed as following:Agrobacteria-mediated the hygromycin selection method delivered Cas9/gRNA expression cassettes into the Nisqually-1 genome.The Cas9/gRNA-edited mutations were characterized in single PtrCHLI and two PtrCHLI1/2 genes.Of 18 PtrCHLI1-edited plants,11 lines were biallelic/homozygous(61.1%)mutations with albino phenotypes.20 PtrCHLI1/2-edited plants also generated the majority of homozygous/biallelic mutations(50-91.7%).These data indicate that the knockout of one or two genes mediated by Cas9/gRNA-edited is high-efficiency in Populus.The Cas9/gRNA-edited knockouts of PtrCesA4,7A,7B,8A and 8B genes were produced in Populus trichocarpa using this Cas9/gRNA system.We obtained 54 lines for the PtrCesA4,7A,7B,8A,8B,7A/B,and 8A/B single or two genes.In most cases,Cas9/gRNA-editing at targetsites causes frameshift mutations in protein-coding sequences,and no mutation was detected in potential off-target sites among the tested samples.Furthermore,we assessed the edited sites in progenies of ptrcesa4,7ab and 8ab mutants,showing the Cas9/gRNA-edited mutations were stably heritable in progenies of apical and axillary bud.In the phenotypes,ptrcesa4,7ab and 8ab mutants were completely prostrate and lost apical dominance,thus resulting in weeping bonsai trees,as well as decreased the sizes of xylar vessel,xylar fiber,the pavement,guard and pith cell,and ptrcesa7a,7b,8a and8b mutants showed slight growth defects,indicating redundant roles for PtrCesA7A and 7B,PtrCesA8A and 8B,and the identical and nonredundant roles of PtrCesA4,7A/B and 8A/B in Populus.PtrCesA4,7A/B or 8A/B protein levels were not examined in the ptrcesa mutants,suggesting that they are null mutants.Deletion of one class of the SCW PtrCesAs(PtrCesA4,7A/B,or 8A/B)diminished the protein levels of the other two classes of the SCW PtrCesAs,suggesting that three classes of SCW PtrCesAs(PtrCesA4,7A/B and 8A/B)constitute the core components of xylem SCW cellulose synthase complex.Scanning and transmission electron microscope analysis showed serious defects of ptrcesa4,7ab,and 8ab mutants in wood wall structures and one-layer thinner fiber wall.Cellulose contents in the mutant woods were approximately 4%compared to normal wood 41%,and conversely,lignin contents and hemicellulose polysaccharides were increased by two fold.The results showed the crucial roles of PtrCesA4,7A/B,or 8A/B in wood cell wall structure,and PtrCesA4,7A/B,and 8A/B are completely responsible for wood cellulose synthesis.In the leaning stems of ptrcesa4,7ab,8a,8b,and 8ab mutants,ptrcesa4,7ab and 8ab showed eccentric growth,a reduction in the number of vessels and thinner fiber S-layers,but no G-layers in fiber,and the ability of G-layer formation was slightly impaired in ptrcesa8a and 8b mutants.The results showed that CSC for G-layer cellulose biosynthesis in also recruited three chasses of SCW PtrCesAs(PtrCesA4,7A/B and 8A/B).In addition,the primary and secondary phloem fibres of ptrcesa4,7ab,and 8ab mutants lost the n(G+L)-and G-layers and retained one-thicker S-layers.Together with polysaccharide immunolocalization data,these findings suggest PtrCesA4,7A/B and 8A/B are indispensable for the G-layer formation of TW fibres and unique wall structure of phloem fibres. |
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