Functional Characterization Of Cellulose Synthesis Genes In Secondary Wall Thickening Of Xylem Cells During Wood Formation In Populus Trichocarpa

Cellulose is an abundantly produced biopolymer in secondary cell walls in tree species.It is being employed in cosmetics,pharmaceutical,paper,pulp and potentially biofuel industry.Due to fastest growing nature,poplar is best source of cellulose production.Study of the mechanism of secondary cell wall formation during wood formation would promote wood utilization.Cellulose synthases(CesAs)are the key enzymes catalysing cellulose biosynthesis.Studies in Arabidopsis show that two groups of CesAs are involved in the cellulose synthesis in primary and secondary cell walls,respectively.CesA4,CesA7 and CesA8 have been characterized as the enzymes involved in cellulose synthesis in secondary cell walls.Study of the CesA functions in the secondary cell wall formation during wood formation would promote wood utilization.In this study,the functions of cellulose synthase genes Ptr CesA4,Ptr CesA7-A/B and Ptr CesA8-A/B were characterized in Populus trichocarpa.Their expression was perturbed by RNAi strategy.RNAi transgenic plants exhibited stunted growth,narrow leaves,early necrosis,dismantled stature,collapsed vessels,thinner cell walls in fiber cells,and prolonged fiber lumen diameter.Stems of transgenic plantsexhibited reduced mechanical strength,with reduced modulus of rupture(MOR)and modulus of elasticity(MOE).A dramatic decrease in cellulose contents,such as 14.9% in Ptr CesA4,6.8% in Ptr CesA7 and 3.1% in Ptr CesA8 was observed in transgenics.On the other hand,lignin and xylan contents were increased.The wood composition changes were confirmed by solid-state NMR,two-dimensional(2D)solution-state NMR,X-ray diffraction(XRD),and sum-frequency-generation vibrational(SFG)analysis.Although XRD showed a decreased crystallinity of cellulose in transgenic plants,both solidstate NMR and SFG analyses demonstrated that crystallinity index of cellulose was not affected.SFG analysis revealed a good correlation between CH/CH2 and cellulose content.Un-uniaxial alignment of cellulose microfibrils,and meso-scale variation in packing of crystalline cellulose domains.The results showed the thinner cell walls in transgenic fiber cell walls,which may be the reason for the reduced stem mechanical strength.The collapsed vessels in xylem of transgenics may lead to a severe halt in water and mineral transport in xylem towards aerial parts and thus causing early necrosis.Our results provide the evidence for the involvement of Ptr CesA4,Ptr CesA7-A/B and Ptr CesA8-A/B in secondary cell wall formation in wood and demonstrate the pleiotropic effects of their perturbations on wood formation.This study provides important basis for further studying transcriptional control of CesA regulation.