Laurence B. Davin, Ann M. Patten,

Michael Jourdes, and Norman G. Lewis

7.1 Lignin: molecular basis and role in plant adaptation to land

Life, as humanity understands it, has inextricably been tied since eons past to the successful evolutionary adaptation of aquatic flora to terrestrial environments. This conquest appar­ently first began with emergence of various “primitive” forms of, and/or forerunners to, tracheids (water-conducting elements) in the land-based plants during the late Ordovician to Silurian periods (>400 million of years) (1-3). Such land plant forms gradually became capable of efficient hydration and metabolism under “water-limited” conditions and hence attained an inherent ability to survive in a wide variety of habitats. Eventually, various other modified forms of plant cell walls also evolved, these containing — at least for the vascular apparatus — celluloses, hemicelluloses, lignins, and small amounts of proteins, as their main chemical/structural constituents. Ultimately, lignification provided the means by which large upright vascular plant forms could be produced, and which enabled some species to more successfully compete for photosynthetic energy. This, in turn, gave a molec­ular or structural basis for much of the plant biodiversity that humanity enjoys today in its many resplendent forms. Furthermore, in addition to competition for light, an upright growth habit (provided by a true vascular system), allowed for better spore/pollen dispersal, increasing the genetic variability and species range. Yet today, our knowledge of how plant cell wall assembly occurs is at the most rudimentary level.

This chapter focuses upon the lignins. Next to cellulose, they are Nature’s second most abundant organic substances, and are products of the phenylpropanoid pathway (Figure 7.1). Significantly, many of the monolignol/lignin-forming pathway steps apparently also evolved during transition of plants to the land habitat, providing broad adaptive advantage to some 350 000 or so distinct present-day vascular plant species (4). To put this pathway into the broader context of carbon allocation from photosynthesis, woody gymnosperm stems gen­erally have lignin contents ~28-30%, whereas those in woody angiosperms are of lower amount (~20-24%) (5). In both cases, this represents a significant commitment and des­ignation of the total carbon taken up during photosynthesis; indeed, the lignins are some of the most metabolically “expensive” of all plant products formed (6).

Studies of many different plant species (i. e., from gymnosperms to angiosperms) have established that their lignins proper, while often varying in monomeric compositions, are derived from the three monolignols 1, 3, and 5 (Figure 7.1); additionally, various grasses

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel © 2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6

СН2ОН	СН2ОН	СН2ОН	СН2ОН	СН2ОН
но у осн3 он 5-Hydroxyconiferyl alcohol (4)	н3со у осн3 он Sinapyl alcohol (5)
CAD	CAD
ОН 5-Hydroxyconiferyl aldehyde (22)	OH Sinapyl aldehyde (23)
CCR	CCR
OCH3
OH Sinapoyl CoA (18)
OH Sinapic acid (13)
Ferulicacid (11) 5-Hydroxyferulic acid (12)
Figure 7.1 Contemporary view of the phenylpropanoid pathway. pC3H, p-coumarate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; CAD, cinnamyl alcohol dehydrogenases; CCOMT, hydroxycinnamoyl CoA O-methyl — transferases; CCR, cinnamoyl-CoA oxidoreductases; 4CL, hydroxycinnamoyl CoA ligases; COMT, caffeic acid O-methyltransferases; F5H, ferulate 5-hydroxylase; HCT, hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase; HQT, hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase; PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase. Note that most of the current understanding of biosynthetic steps results from in vitro analyses; how the hydroxycinnamic acids 10-13 are formed via, for example, CoA esters or aldehydes needs to be fully established.

contain small amounts (~10% or so) of p-hydroxycinnamate esters 30-32 linked to the monolignols (Figure 7.2A). Lignins are also generally designated as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), based on their aromatic ring substitution pattern (Figure 7.2B). Regardless of monomeric compositions, lignins are considered to be complex, amor­phous, phenylpropanoid polymers within the lignocellulosic matrices of plant cell walls. This highly conserved monolignol deployment occurs irrespective of the remarkable dif­ferences encountered in both diverse body plans and architectures of the plant forms in existence today.

In spite of their overall abundance, the lignin biopolymers are quite challenging to work with. This is because of their aromatic and hence very hydrophobic character; their relative intractability due to difficulties to efficiently solubilize and characterize derivatives thereof and then only under quite drastic chemical degradation conditions (7); and their striking abilities to self-associate (8, 9) due to the strong electronic stabilization energies between the various subunits in the adjacent biopolymeric chains. In terms of their molecular ar­chitecture, the lignins are considered to be products of phenoxy-radical-derived coupling reactions as originally proposed by Erdtman in 1933 (10) in model studies using isoeugenol (33, Figure 7.2C); Figure 7.2D also illustrates several of the most abundant interunit linkage types known in lignins proper that can be at least partially quantified (discussed below).

It needs to be emphasized that the artistic depictions of lignin macromolecular config­uration have changed rather enormously over a time span of nearly five decades, i. e., from originally being envisaged as a complex three-dimensional (Bakelite-like) phenolic poly­mer (11, 12) (Figure 7.3A), to that amenable for a computer-programmable simulation (Figure 7.3B) (13,14), to that now more recently of essentially more linear macromolecular entities (e. g., Figure 7.3C) (15). All of these models though represent simply artistic and consequently quite artificial depictions of native lignin structure(s). Indeed, several quite abundant substructures (e. g., 8-1′ or their forerunners, dibenzodioxocin, etc.) are even absent in Figures 7.3A and 7.3B; moreover, the very changing nature of such depictions un­derscores the fact that little truly systematic research has yet been carried out to investigate (and develop methodologies for) the study of lignin primary structure(s). Furthermore, there maybe no other biopolymeric entities whose structure(s) has (have) been depicted in such quite arbitrary ways, particularly since experiments had neither been conducted nor devised to investigate both the actual biochemical mode of assembly and the structure(s) so obtained. Yet this has been the situation for lignins for almost a century now.

Interestingly, in the 1970s and 1980s, there was also much enthusiasm in identification of lignin-degrading enzymes, with various laccases and peroxidases (specifically manganese and lignin peroxidases) reported as the main degradative enzymes involved (16-19). Today, it is doubtful that either class has such a function, and neither has fulfilled the optimistic promise for industrial application once envisaged over two decades ago (20). Moreover, our understanding today of how lignin deconstruction (so-called biodegradation) occurs in vivo is still at a most rudimentary level. For this reason, research activities are now being directed and/or initiated toward identifying the nature of true lignin degrading enzymes, e. g., putative lignin depolymerases acting on specific linkage types (21).

Nevertheless, the fantastic diversity of the extant vascular plant species — in terms of not onlytheir remarkable differences in size, shape, and growth rates, but also as to whether they have woody or non-woody character, etc. — has all depended upon the successful formation of the lignified vascular apparatus.