Abstract
Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.
Highlights
Abbreviations 4-coumarate-CoA ligase (4CL) 4-Coumarate-CoA ligase C3′H p-Coumarate 3-hydroxylase C4H Cinnamate 4-hydroxylase CAD Cinnamyl alcohol dehydrogenase CaMV35S Cauliflower Mosaic Virus 35S CCoAOMT Caffeoyl-CoA O-methyltransferase CCR Cinnamoyl-CoA reductase CESA Cellulose synthase COMT Caffeic acid O-methyltransferases cafferoyl shikimate esterase (CSE) Cafferoyl shikimate esterase CSL Cellulose synthase-like
Homologous genes of Arabidopsis thaliana VASCULAR-RELATED NAC DOMAIN (VND)[9], NAC SECONDARY WALL THICKENING PROMOTING FACTOR/SECONDARY WALL-ASSOCIATED NAC DOMAIN (NST/SND)[10,11] and SOMBRERO (SMB) proteins designated as VNS (VND, NST/SND,and SMB RELATED) p roteins[12] are key regulators of secondary cell wall formation in xylem fibers, phloem fibers, and xylem ray parenchyma cells in Populus[12,13,14,15,16]
Toward reducing wood recalcitrance and improving its enzymatic degradability, we focused on transcriptional factors (TFs) genes associated with wood formation as xylem cell wall modification targets, since most previous studies have focused on genes involved in cell wall biosynthesis or cell wall-degrading/-modifying enzymes in Populus[26,27]
Summary
Abbreviations 4CL 4-Coumarate-CoA ligase C3′H p-Coumarate 3-hydroxylase C4H Cinnamate 4-hydroxylase CAD Cinnamyl alcohol dehydrogenase CaMV35S Cauliflower Mosaic Virus 35S CCoAOMT Caffeoyl-CoA O-methyltransferase CCR Cinnamoyl-CoA reductase CESA Cellulose synthase COMT Caffeic acid O-methyltransferases CSE Cafferoyl shikimate esterase CSL Cellulose synthase-like. VNS09, VNS10, VNS11 and VNS12 corporately play important roles as a master switch regulators of cell wall formation in Populus[17,18], but their downstream genes including several TFs are functionally yet uncharacterized. In addition to these studies of A. thaliana TF homologs in Populus species, more comprehensive analyses have focused on TFs expressed during the wood formation processes including secondary xylem differentiation, cell expansion along with PCW deposition, SCW deposition, and programmed cell death along with further lignification[19,20,21]. Our understanding of the TFs involved in the regulatory network system of Populus xylem cell wall formation and the potential for application in a bioengineering context is still limited
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