Elsevier

Phytochemistry

Volume 61, Issue 3, October 2002, Pages 221-294
Phytochemistry

Review
Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity

https://doi.org/10.1016/S0031-9422(02)00211-XGet rights and content

Abstract

A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl–syringyl (G–S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/“p-coumarate-3-hydroxylase” (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H activities. Such enzymes thus fulfill subsidiary processing roles, with all (except CCOMT) apparently being bifunctional for both H and G substrates. Their severe downregulation does, however, predictably result in impaired monolignol biosynthesis, reduced lignin deposition/vascular integrity, (upstream) metabolite build-up and/or shunt pathway metabolism. There was no evidence for an alternative acid/ester O-methyltransferase (AEOMT) being involved in lignin biosynthesis.

The G/S lignin pathway networks are operative in specific cell types in angiosperms and employ two additional biosynthetic steps to afford the corresponding S components, i.e. through introduction of an hydroxyl group at C-5 and its subsequent O-methylation. [These enzymes were originally classified as ferulate-5-hydroxylase (F5H) and caffeate O-methyltransferase (COMT), respectively.] As before, neither step has apparently any role in carbon allocation to the pathway; hence their individual downregulation/manipulation, respectively, gives either a G enriched lignin or formation of the well-known S-deficient bm3 “lignin” mutant, with cell walls of impaired vascular integrity. In the latter case, COMT downregulation/mutation apparently results in utilization of the isoelectronic 5-hydroxyconiferyl alcohol species albeit in an unsuccessful attempt to form G-S lignin proper. However, there is apparently no effect on overall G content, thereby indicating that deposition of both G and S moieties in the G/S lignin forming cells are kept spatially, and presumably temporally, fully separate. Downregulation/mutation of further downstream steps in the G/S network [i.e. utilizing 4CL, CCR and CAD isoforms] gives predictable effects in terms of their subsidiary processing roles: while severe downregulation of 4CL gave phenotypes with impaired vascular integrity due to reduced monolignol supply, there was no evidence in support of increased growth and/or enhanced cellulose biosynthesis. CCR and CAD downregulation/mutations also established that a depletion in monolignol supply reduced both lignin contents and vascular integrity, with a concomitant shift towards (upstream) metabolite build-up and/or shunting.

The extraordinary claims of involvement of surrogate monomers (2-methoxybenzaldehyde, feruloyl tyramine, vanillic acid, etc.) in lignification were fully disproven and put to rest, with the investigators themselves having largely retracted former claims. Furthermore analysis of the well-known bm1 mutation, a presumed CAD disrupted system, apparently revealed that both G and S lignin components were reduced. This seems to imply that there is no monolignol specific dehydrogenase, such as the recently described sinapyl alcohol dehydrogenase (SAD) for sinapyl alcohol formation. Nevertheless, different CAD isoforms of differing homology seem to be operative in different lignifying cell types, thereby giving the G-enriched and G/S-enriched lignin biopolymers, respectively. For the G-lignin forming network, however, the CAD isoform is apparently catalytically less efficient with all three monolignols than that additionally associated with the corresponding G/S lignin forming network(s), which can more efficiently use all three monolignols. However, since CAD does not determine either H, G, or S designation, it again serves in a subsidiary role—albeit using different isoforms for different cell wall developmental and cell wall type responses.

The results from this analysis contrasts further with speculations of some early investigators, who had viewed lignin assembly as resulting from non-specific oxidative coupling of monolignols and subsequent random polymerization. At that time, though, the study of the complex biological (biochemical) process of lignin assembly had begun without any of the (bio)chemical tools to either address or answer the questions posed as to how its formation might actually occur. Today, by contrast, there is growing recognition of both sophisticated and differential control of monolignol biosynthetic networks in different cell types, which serve to underscore the fact that complexity of assembly need not be confused any further with random formation. Moreover, this analysis revealed another factor which continues to cloud interpretations of lignin downregulation/mutational analyses, namely the serious technical problems associated with all aspects of lignin characterization, whether for lignin quantification, isolation of lignin-enriched preparations and/or in determining monomeric compositions. For example, in the latter analyses, some 50–90% of the lignin components still cannot be detected using current methodologies, e.g. by thioacidolysis cleavage and nitrobenzene oxidative cleavage. This deficiency in lignin characterization thus represents one of the major hurdles remaining in delineating how lignin assembly (in distinct cell types) and their configuration actually occurs.

The comprehensive analysis of the effects of genetic and mutational manipulations of monolignol pathway enzymes revealed fully predictable consequences on lignification and the vascular apparatus. Carbon allocation to the pathway is determined by Phe availability, and relative C4H and C3H activities. While the data obtained put to rest the claims of surrogate monomers being involved in lignin biosynthesis, new tools have been developed which now permit dissection of the lignin assembly process.

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Section snippets

Introduction: overall scope of the analysis

In just over a decade, a number of studies have been conducted to manipulate gene expression in the monolignol (13) pathway, with important ramifications for control of subsequent lignin assembly(ies) (see Fig. 1). These studies have usually been done with the overall aim of modifying lignin contents and/or their compositions in plants. The anticipated benefits to humanity are potentially economic and environmental, in terms of cheaper and more process-amenable trees for pulp and paper

The functional basis for organized heterogeneous lignin assembly

From an evolutionary perspective, the monolignol-derived lignins are found in the pteridophytes (ferns), gymnosperms and angiosperms. [Fig. 1 summarizes our current understanding of the biosynthetic pathway to the monolignols (13) from Phe (4) and/or Tyr (5).] With few exceptions, it has long been known (Lewis and Yamamoto, 1990) that the monolignol pathway affording the corresponding lignins in pteridophytes and gymnosperms only utilizes p-coumaryl (1) and coniferyl (2) alcohols, whereas in

The control of monolignol ratio and lignin composition: metabolic and transcriptional profiling

Why do vascular plants consistently produce various types of cell walls with lignins from either two or three monolignols, and how does this occur in vivo? Furthermore, why are these moieties also differentially deposited into specific regions of developing cell wall types? To begin to answer such questions, two approaches have been taken: one is via metabolic flux and transcriptional profiling studies, and the second involves analysis of transgenic plants and/or mutants, where levels of

Technical limitations in current lignin analysis

Given that lignin deposition and composition can vary with cell wall layer, cell wall type and species, it is necessary to consider the efficacy and limitations of the lignin analytical procedures currently employed. Typically, these include methods of lignin quantification, estimates of lignin monomeric compositions, the isolation of lignin-derived preparations, and their spectroscopic analyses. None of these methods, however, are cell specific. Furthermore, each has serious limitations, most

Does perturbing lignin assembly always occur at the expense of vascular integrity?

To date, many transgenic plants and mutants have only been very preliminarily characterized, and thus the overall effects of genetic alteration are not well understood. This is especially true in terms of determining the effects on lignin assembly proper and the differential regulation of compartmentalized metabolic segments in the phenylpropanoid network. Nevertheless, it is instructive to consider what has been obtained thus far from studies directed towards lignin modification, and to

Concluding remarks

This comprehensive analysis summarizes what is known thus far as regards the preliminary characterization of plant tissues, following either up- or downregulation and/or mutation of various steps in the monolignol-forming pathway. The data obtained reveal a much more complex pattern of lignin assembly in different cell types and cell wall layers than hitherto recognized. No evidence, at any level of inquiry, indicated that the process of lignification, including biopolymer assembly, involved

Acknowledgements

The authors wish to thank the Department of Energy (DE-FG03-97ER20259), the National Aeronatutics and Space Administration (NAG2-1198 and NAG-1513), the US Department of Agriculture (99-35103-8037), McIntire-Stennis and the G. Thomas Hargrove and Lewis and Dorothy B. Cullman for generous support of the studies related to this review. The authors are also indebted to Drs. Richard A. Dixon and Jacquline Grima-Pettenati for PAL and CCR figures, respectively, and to Dr. Clint Chapple for provision

Norman G. Lewis is the Director of the Institute of Biological Chemistry and the Arthur M. and Katie Eisig-Tode Distinguished Professor at Washington State University. He was initially trained in Chemistry (BSc Honors, 1973, University of Strathclyde) before completing a PhD (1977) in organic chemistry (alkaloid biosynthesis) at the University of British Columbia (Vancouver, BC). Postdoctoral training was in alkaloid/Vitamin B12 biosynthesis with Sir Alan R. Battersby. Professor Lewis' current

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    Norman G. Lewis is the Director of the Institute of Biological Chemistry and the Arthur M. and Katie Eisig-Tode Distinguished Professor at Washington State University. He was initially trained in Chemistry (BSc Honors, 1973, University of Strathclyde) before completing a PhD (1977) in organic chemistry (alkaloid biosynthesis) at the University of British Columbia (Vancouver, BC). Postdoctoral training was in alkaloid/Vitamin B12 biosynthesis with Sir Alan R. Battersby. Professor Lewis' current research interests are mainly associated with biosynthesis of plant phenolics, including how their ordered deposition into various cell wall types is achieved. He is a Regional Editor of Phytochemistry, as well as serving on other editorial boards.

    Aldwin M. Anterola holds a PhD (2001) degree in Plant Physiology from Washington State University. He graduated cum laude from the University of the Philippines, with a BS Degree in Agricultural Chemistry (1994). He worked for a year in the same University, as a junior faculty teaching laboratory classes in general chemistry, organic chemistry and biochemistry. As a graduate student, he received the Helen and Loyal H. Davis Fellowship, a Student Award for Best Paper from the Phytochemical Society of North America, and an American Chemical Society (Cellulose and Paper Division) Graduate Student Award for his research. He currently works as a Scientific Editor for Phytochemistry, and as a part-time Postdoctoral Research Associate in the co-author's laboratory.

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