CAD1 and CCR2 protein complex formation in monolignol biosynthesis in Populus trichocarpa.

Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis in planta.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

Wood cellulose microfibrils have a 24-chain core-shell nanostructure in seed plants.

Wood cellulose microfibril (CMF) is the most abundant organic substance on Earth but its nanostructure remains poorly understood. There are controversies regarding the glucan chain number (N) of CMFs during initial synthesis and whether they become fused afterward. Here, we combined small-angle X-ray scattering, solid-state nuclear magnetic resonance and X-ray diffraction analyses to resolve CMF nanostructures in native wood. We developed small-angle X-ray scattering measurement methods for the cross-section aspect ratio and area of the crystalline-ordered CMF core, which has a higher scattering length density than the semidisordered shell zone. The 1:1 aspect ratio suggested that CMFs remain mostly segregated, not fused. The area measurement reflected the chain number in the core zone (Ncore). To measure the ratio of ordered cellulose over total cellulose (Roc) by solid-state nuclear magnetic resonance, we developed a method termed global iterative fitting of T1ρ-edited decay (GIFTED), in addition to the conventional proton spin relaxation editing method. Using the formula N = Ncore/Roc, most wood CMFs were found to contain 24 glucan chains, conserved between gymnosperm and angiosperm trees. The average CMF has a crystalline-ordered core of ~2.2 nm diameter and a semidisordered shell of ~0.5 nm thickness. In naturally and artificially aged wood, we observed only CMF aggregation (contact without crystalline continuity) but not fusion (forming a conjoined crystalline unit). This further argued against the existence of partially fused CMFs in new wood, overturning the recently proposed 18-chain fusion hypothesis. Our findings are important for advancing wood structural knowledge and more efficient use of wood resources in sustainable bio-economies.

Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis.

A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.