Ann M. Patten

Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.

Arogenate Dehydratase Isoenzymes Profoundly and Differentially Modulate Carbon Flux into Lignins

Background: The plastid-localized arogenate dehydratase (ADT) gene family is hypothesized to differentially control carbon flux for lignin deposition, with lignin being the main contributor to lignocellulosic recalcitrance. Results: Single and multiple ADT knock-outs resulted in differential control over lignin content/composition. Conclusion: The first evidence for Phe upstream metabolism differentially controlling carbon flux into distinct secondary cell wall types was discovered. Significance: Upstream metabolic networks regulate secondary cell wall formation. How carbon flux differentially occurs in vascular plants following photosynthesis for protein formation, phenylpropanoid metabolism (i.e. lignins), and other metabolic processes is not well understood. Our previous discovery/deduction that a six-membered arogenate dehydratase (ADT1–6) gene family encodes the final step in Phe biosynthesis in Arabidopsis thaliana raised the fascinating question whether individual ADT isoenzymes (or combinations thereof) differentially modulated carbon flux to lignins, proteins, etc. If so, unlike all other lignin pathway manipulations that target cell wall/cytosolic processes, this would be the first example of a plastid (chloroplast)-associated metabolic process influencing cell wall formation. Homozygous T-DNA insertion lines were thus obtained for five of the six ADTs and used to generate double, triple, and quadruple knockouts (KOs) in different combinations. The various mutants so obtained gave phenotypes with profound but distinct reductions in lignin amounts, encompassing a range spanning from near wild type levels to reductions of up to ∼68%. In the various KOs, there were also marked changes in guaiacyl:syringyl ratios ranging from ∼3:1 to 1:1, respectively; these changes were attributed to differential carbon flux into vascular bundles versus that into fiber cells. Laser microscope dissection/pyrolysis GC/MS, histochemical staining/lignin analyses, and pADT::GUS localization indicated that ADT5 preferentially affects carbon flux into the vascular bundles, whereas the adt3456 knock-out additionally greatly reduced carbon flux into fiber cells. This plastid-localized metabolic step can thus profoundly differentially affect carbon flux into lignins in distinct anatomical regions and provides incisive new insight into different factors affecting guaiacyl:syringyl ratios and lignin primary structure.

Trees: A Remarkable Biochemical Bounty

Our enormous dependence on the biochemical bounty from trees is seldom fully appreciated. Yet, the evolution of the woody plant cell wall eventually provided humanity with the means for pulp/paper, wood and lumber, musical instruments, nanocomposites, structural dwellings, sailing vessels, and so forth that we often take for granted. However, in addition, woody plants provide us with an enormous array of medicinals, commodity chemicals (gums, rubbers, resins, essential oils), spices, specialty chemicals, fruits, nuts, cork and bark products, etc. through their formation and deposition in highly compartmentalized phytochemical factories within various anatomical structures. This chapter addresses the importance of the woody plant habitat to our lives, with an initial emphasis on how the woody plant forms in trees evolved. A major emphasis is then placed upon the types of anatomical structures that store the different plant phytochemicals, and the paucity of knowledge that we have about how the latter are produced. The various types of phytochemical classes, and our current knowledge of their biochemical pathways, are discussed. It should be emphasized though that humanity’s dependence on these life forms must be tempered by the unsatisfactory paucity of knowledge as to how both these substances, and the anatomical structures that contain them, are generated.

Relationship of dirigent protein and 18s RNA transcript localization to heartwood formation in western red cedar.

Western red cedar (Thuja plicata) heartwood contains abundant amounts of structurally complex plicatic acid-derived lignans that help confer protective properties and longevity to this tissue type. Although the lignan biochemical entry point is dirigent protein-mediated, the formation of heartwood and its associated lignans in some species remains poorly understood due to technical difficulties of working with the former. To begin to address such questions, this study therefore focused on the anatomical localization of dirigent protein and 18s rRNA (control) gene transcripts within recalcitrant woody tissues, including heartwood. This in situ mRNA hybridization approach enabled detection of dirigent protein transcripts in cork cambia, vascular cambia and ray parenchyma cells of the sapwood, but not the heartwood under the conditions employed. By contrast, the hybridization of the 18s rRNA (control) transcript resulted in its detection in all tissue types, including radial parenchyma cells of apparently preformed heartwood. Application of in situ hybridization to such recalcitrant tissues thus demonstrates the utility of this technique in identifying specific cell types involved in heartwood formation, as well as the relationship of dirigent protein localization to that of heartwood metabolite generation.

Vascular Plant Lignification: Biochemical/Structural Biology Considerations of Upstream Aromatic Amino Acid and Monolignol Pathways

This chapter provides both a timely comprehensive and critical analysis of how aromatic amino acid metabolism and monolignol biosynthesis occur in vascular plants, with a particular emphasis upon the biochemistry and structural biology of the pathway steps. Ultimately, these lead to cell wall polymeric lignins, lignans, and allyl/propenyl phenols. In particular, the contribution compares and contrasts distinctions between how plants and bacteria differentially produce the essential amino acids phenylalanine and tyrosine, and what is currently known about their most interesting biochemical mechanisms. The progress toward understanding each of the biochemical steps involved in monolignol biosynthesis is considered comprehensively in an analogous manner, in terms of the enzyme kinetics and proposed mechanisms, the structural biology involved, and the limited substrate degeneracy of the enzymes.