Lignin pathway evolution, deposition, and function in vascular anatomical development

7.2.1 Vascular plant diversification and iignification

The highly diversified terrestrial (plant) environment found today originated with the shift of photosynthetic organisms from water to land some 475 million years ago (1-3). After small flora with prevascular water-conducting cells had been established in coastal wetlands, plants next developed tracheids (xylem) (22, 23), and the ability to fortify these cell walls with lignin. This evolutionary step thus gave plants a profoundly different and more efficient way to control water uptake, use and storage, thereby allowing them to become indepen­dent of wetlands and diversify onto most landscapes on earth. Further evolution of the phenylpropanoid pathway also led to the development of different types of both lignins and lignified cells, which in turn allowed plants to evolve arborescent growth in order to compete for light and space. By developing diverse height and water usage strategies, vascular plants thus created a myriad of environments that other organisms were then able to inhabit and co-evolve within. While this diversity in cell wall structure is still not fully understood, it reflects a rich source of genetic information that is central to finding methods to alter plant structure for human use without adversely affecting the subject plants (24).

The evolutionary appearance of plant vascular anatomy is quite well represented in both the fossil record and in living plants. General trends in tracheid (i. e., water-conducting cells) secondary cell wall thickenings related to the development of lignification can be seen throughout the vascular plant lineages including the extant lineages shown in Figure 7.4 (in the order their ancestors appeared in the fossil record) (2, 25-27): 1) Lycopodiophyta, represented by Lycopodium tristachyum, 2) Equisetophyta and Psilotophyta, with Psilotum nudum as an example, 3) Filicophyta, with Pteridium aquilinum representing higher ferns, 4) gymnosperms, e. g., loblolly pine (Pinus taeda), and 5) angiosperms, as represented by alfalfa (Medicago sativa). Tracheids with simple (limited) secondary thickenings, such as the annular and helical formations of proto — and meta-xylem are found in all these plant groups, with the reticulate thickening form found more often in the older lineages, such as the Filicophyta (arrowheads in corresponding images, right-hand column, Figure 7.4). Secondary cell wall thickenings with scalariform pitting also appeared throughout vascular plants, but with greater prevalence within the earlier lineages (through the Filicophyta); scalariform pitting has a ladder-like appearance due to a broad surface area of secondary thickening with elongated pits (arrowhead, Figure 7.4). This form is intermediate to the an — nular/helical forms described above and the more continuous secondary cell wall thickenings with simple (to more elaborate; not shown) circular pitting of tracheids in gymnosperms (e. g., Pinus taeda) and vessels of (secondary growth) angiosperms (28), such as in alfalfa (see corresponding simple pitting figure). Additionally, non-water conducting cell types with variable amounts of lignification/secondary cell walls include sclerenchyma and other structural fibers, these bearing an important mechanical function in stem and branch sup­port. Thick-walled sclerenchyma are also found in some species of the Lycopodiophyta (e. g., the sclerified cortex of L. tristachyum), Psilotophyta (e. g., the sclerified outer cortex of P. nudum), Filicophyta (e. g., the sclerified hypodermis of P. aquilinum), gymnosperms, and angiosperms. Fibers with true lignin are found only in the gymnosperms and angiosperms (e. g., sclerified fibers of M. sativa, Figure 7.4).

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Figure 7.4 Lignified vascular and sclerenchyma anatomical structures in extant plant lineages. Proto — and metaxylem of the major plant lineages: Lycopodiophyta (e. g., Lycopodium tristachyum), Psilophyta (e. g., Psilotum nudum), Filicophyta (e. g., Pteridium aquilinum), gymnosperms (e. g., Pinus taeda), and angiosperms (e. g., Medicago sativa) have very similar annular and helical secondary cell wall thickenings. Some members of each group, but especially the Filicophyta, have reticulate tracheid cell wall thickenings, a more complex form than the helical form. Secondary cell wall thickenings with scalariform pitting may be found in all plant lineages and represent an intermediate form to the simple pitting found in higher plants (i. e., gymnosperms and angiosperms). Brightfield microscopy images of L. tristachyum, P. nudum, and P. aquilinum were taken of hand-cut fresh sections stained with phloroglucinol-HCL to reveal patterns of phenolic deposition (red) in the xylem and sclerified fiber cells. Epifluorescent confocal images of P taeda and M. sativa were made using cryosections of fresh tissue stained with a combination of acridine orange and ethidium bromide to reveal patterns of lignification. Examples of secondary cell wall thickenings were recorded using brightfield microscopy and hand-cut longitudinal sections of fresh unstained xylem from Equisetum telmateia (a member of Equisetophyta for annular and helical examples), Ophioglossum reticulatum (a member of the Filicopsida, for the reticulate example), P. aquilinum (another member of the Filicopsida, for the scalariform-pitted example), and M. sativa (an angiosperm, for the example of vessel secondary cell wall thickenings with simple pitting). (Reproduced in color as Plate 16.)

The physiological functions of the lignins are thus quite distinct from those of either the cell wall celluloses or hemicelluloses. They are formed as biopolymeric entities within the cell wall, and help to reinforce the plant walls of the vasculature. In this way, they enable vascular plants to both form their water/nutrient conducting cells and also to provide a means of withstanding compressive forces acting on the overall plant body. Additionally, generation of the lignified plant cell wall matrices provides a relatively formidable physical barrier to opportunistic pathogens and to other encroaching organisms.

While the “lignin” chemistry of “primitive” plants is still yet very poorly understood, it is well established that lignin composition in higher plants is essentially only derived from the three monolignols 1, 3, and 5 (Figure 7.1) as indicated earlier (5, 29-31). To a much lesser extent, lignification can also involve some limited participation of related p-hydroxycinnamyl alcohol-monolignol esters 30-32 (Figure 7.2A), such as in grasses (5, 30, 31). This conclusion follows many detailed and exhaustive analyses of a large number of different plant species over a period encompassing 5-10 decades (32, and references therein); this established that a very strong evolutionary pressure had emerged to form lignins from these moieties and not from other non-monolignol phenolics (33). Interestingly, two of the monolignols, p-coumaryl (1) and coniferyl (3) alcohols, which differ only in methoxylation substitution pattern at carbon-3, are the precursors of lignins in some of the primitive (extant) plants, e. g., Psilotum nudum, as well as gymnosperms, e. g., loblolly pine (Pinus taeda) (34, 35). These moieties thus became the p-hydroxyphenyl (H) and guaiacyl (G) aromatic ring components of their lignins (Figure 7.2B). Various gymnosperms are, of course, widely employed as commercial sources of wood for lumber and pulp/paper production, respectively, e. g., loblolly pine, black spruce, etc.

The subsequent evolution of the angiosperms (flowering plants), by contrast, resulted in additional forms of lignified cell-wall architecture (e. g., true vessels and various fiber types), as well as the elaboration of the monolignol-forming pathway to afford the dimethoxylated monolignol, E-sinapyl alcohol (5), and ultimately the syringyl(S) aromatic ring component of the lignin biopolymers (36) (Figure 7.2B). Interestingly, S-moieties have also been re­ported to occur in some extant primitive Selaginella plant species (37-39), as well as in other isolated non-angiosperm families including the Dennstaedtiaceae (40) and Podocarpaceae [reviewed by Gibbs (41)]. While the evolutionary significance of such observations is not yet well understood, they may represent an example of convergent evolution.

The angiosperms are also widely used by humanity; for example, many hardwoods are utilized for lumber/pulp and paper production, whereas others (e. g., rice, corn, soy­bean, etc.) are food crops. In addition, three other angiosperms, thale cress (Arabidopsis thaliana), hybrid poplar (Populus sp.), and alfalfa (Medicago sativa) are currently of con­siderable scientific interest: the first as a “model” plant species, the second as a possible vehicle for generating biotechnologically modified fast-growing woody crops for fiber, bio­fuel applications, cellulose to ethanol, etc., and the third as a source of animal nutrition/ feedstock.

Localization of lignin in xylem and fiber cells has also previously been studied in a limited number of gymnosperm and angiosperm species (36,42-49). The earlier work indicated that lignification in gymnosperms begins in the cell wall corners and then proceeds throughout the cell wall, where p-coumaryl alcohol (1, H-unit) is differentially laid down in the cell corners/middle lamella and the coniferyl alcohol (3, G) residues are mainly in the secondary wall layers (42, 44, 46, 47). Syringyl moieties, by comparison, in angiosperms are deposited

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Figure 7.5 Lignification and cell wall development in loblolly pine cambial tissue (A and B) show de­position of lignin in both transverse and longitudinal sections (Y. Nakazawa, unpublished). In Fig. 5A, lignin is differentially initiated at sites a, a’, b, b’ and c, c’. In Fig. 5B, the particular cell has initiated lig­nification at cell corners c, c’, but not yet at a, a’. Lignification is initiated at 3 points, c, c’, b, b’ then a, a’ in a symmetrical but differential manner. [Cellulose/hemicellulose regions are visualized in green, and lignin is orange/yellow.] (C) Idealized depiction of differential lignin deposition in one tracheary cell type. ^ in A and • in C = lignin initiation sites. A dual-stain method using acridine orange (AO) and ethidium bromide (EB) was used to visualize lignin as a red-orange epifluorescence. The merged confocal images were individually recorded using detection filters specific to 488 nm (Emission) and 522 nm (Excitation) for AO and 568 nm (Ex.) and 598 nm (Em.) for EB. (Reproduced in color as Plate 17.)

mainly in fiber cell walls with mixed guaiacyl-syringyl lignins found in vessel cell walls (36, 45).

More specifically, the process of lignin deposition occurs in a highly organized and con­trolled manner, whereby the cells undergo a polarized deposition of the lignin monomers at different rates in different cell corners (as in loblolly pine developing stems, Figure 7.5A, Y. Nakazawa etal., unpublished, this laboratory). Thus, as incipient xylem and sclerenchyma differentiate, lignin initiation sites at the cell corners/S1 sublayers of the lignifying matrix develop during maturation (see Figure 7.5A). Lignification is extended uniformly, in a con­tinuous thread-like pattern, down the entire length of the developing tracheid toward the cambium (Figure 7.5B). This, in turn, has important biological ramifications, in terms of both monolignol transport and monolignol (radical) alignment, and is apparently consis­tent with the proposed template polymerization process (discussed below) on preformed primary lignin chains (31, 50-52). Additionally, cell corners furthest from the cambial zone undergo both lignin initiation and lignification prior to those adjacent to the cambium, with the enlarging lignified domains in the former again being uniformly evident down the length of the tracheary element. The exact biochemical processes occurring at the lignin initiation sites is currently a subject of considerable scientific interest, as regards overall control of lignin macromolecular configuration (29).

Figure 7.5C thus shows an idealized lignification model in one cell adjacent to the cam­bium, whose cell corners are specified as a, a7, b, b7, and c, c7, respectively. In this diagram, only cell corners (c, c7) closest to the xylem have begun to lignify. Lignin deposition at these furthermost cell corners then extends symmetrically along the S1 sublayer from points c and c! until the two developing zones coalesce, as well as concomitantly extending upwards to the next two adjacent cell corners (i. e., b, b7 in this case). Lignin deposition is subsequently initiated at points b, b7 and symmetrical deposition occurs again in a likewise manner. Finally, lignin deposition in the remaining two corners (a, a7) closest to the cambium is initiated at the last phase, and these zones also begin to expand uniformly — albeit in a delayed man­ner — until eventually coalescence of lignin within the entire wall is achieved. [Figures 7.5A and 7.5B depict the asymmetry in cell wall thickness/lignin deposition as cell wall develop­ment continues, i. e., wall c, c7 is thicker and more heavily lignified than a, a7 at this stage.] Additionally, when cell wall development has occurred, the adjacent unlignified cell (closest to the cambium) is next “conscripted” to undergo lignin assembly/cell wall thickening as before. Such observations thus do not appear to be in agreement with Freudenberg’s original hypothesis of (random) diffusion of monolignol (glucoside) precursors into the cell walls undergoing lignification (53, 54).

In addition to their structural roles in plant stems, lignins provide a physical barrier to opportunistic pathogens (31), and various specialized structures throughout the plant body (e. g., trichomes, harboring an arsenal of plant defense compounds) apparently contain a lignified base. To put this into a more holistic perspective, Figure 7.6 illustrates some of the various tissues and cell wall types that are considered to contain lignified elements in Arabidopsis; these can be readily visualized through expression of the GUS-cinnamyl alcohol dehydrogenase (AtCAD4 and 5) promoter fusion product in the vascular apparatus (55), with CAD encoding the final step in monolignol biosynthesis (31). In this regard, it should also be noted that in this species these two CAD genes (AtCAD4 and 5) are considered to be largely responsible for the penultimate step(s) leading to lignification (discussed later) (56, 57). The main point is that any adverse effect on stem lignification could thus also potentially disrupt other physiological processes in these different tissues and organisms, i. e., whether in terms of structural support and/or in defense system impairment.

Interestingly, for almost three quarters of a century, various lignin mutants (beginning with the brown-midrib mutants in maize) have been described (58-67). Essentially, none currently find application as commercial cultivars, because of the deleterious effects, for example, on the overall plant vasculature, the reproductive system, and so forth. However, recent studies have begun to shed important light on the various genes and enzymes involved in each mutation, and how they affect lignin deposition processes proper. With the recent advances in lignin pathway modulation, biomechanics approaches are also beginning to be increasingly applied in order to begin to correlate such modulations with that of alterations

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Figure 7.6 Cinnamyl alcohol dehydrogenase gene expression in the vascular apparatus of Arabidopsis thaliana (55). Selected examples of GUS-visualized expression of AtCAD5 (A, C-F) and AtCAD4 (B). (A) In vascular apparatus, including hydathodes of 2-week-old leaf tissue;(B) at the base of trichomes;(C) in primary and secondary roots;(D) in sepal/petal veins, style, anthers, and stamen filaments of the flower; (E) in the abscission and style regions of the silique;and (F) in vascular cambium, interfascicular cambium, and the developing xylem of the inflorescence stem. (Reprinted from Phytochemistry, vol. 68, Kim, S.-J., Kim, K.-W., Cho, M.-H., Franceschi, V. R., Davin, L. B. & Lewis, N. G., Expression of cinnamyl alcohol dehy­drogenases and their putative homologues during Arabidopsis thaliana growth and development: Lessons for database annotations? pp. 1957-1974, Copyright 2007, with permission from Elsevier.) (Reproduced in color as Plate 18.)

of plant structural integrity (68-72). As more comprehensively discussed below, this is an essential first step toward understanding to what extent lignin composition and content can be manipulated.