Abnormal Lignins - American Chemical Society

Chapter 25

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The 'Abnormal Lignins': Mapping Heartwood Formation Through the Lignan Biosynthetic Pathway David R. Gang, Masayuki Fujita, Laurence B. Davin, and Norman G. Lewis Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340

A significant portion of 'non-lignin' phenolic extractives in heartwood tissues requires solubilization conditions normally used for lignin dissolution. Although they have incorrectly been characterized as 'abnormal' or 'secondary' lignins, their formation and accumulation differs profoundly from that leading to the lignins in three primary ways: first, they are transported through specialized cells (such as ray parenchyma) and are infused into surrounding pre-lignified cells; second, they are formed via distinct biochemical pathways, affording lignan or (iso)flavonoid-derived substances of various molecular sizes; third, their accumulation begins in the pith and over time extends out centrifugally towards the cambial regions, unless formed locally elsewhere as an inducible response. By contrast, lignification results via direct monomer transport from the cytoplasm of a lignifying cell into its polysaccharide-rich cell wall with subsequent polymerization; this process represents the first and final committed step to lignification, being primarily initiated and completed in maturing cell walls not far from the cambial zone. In this study, western red cedar, western hemlock and loblolly pine were examined to establish how their species -specific,non-lignin heartwood substances are biosynthesized. Specific enzymes and genes were obtained for pivotal steps leading to plicatic acid (western red cedar), α-conidendrin (western hemlock) and dihydrodehydrodiconiferyl alcohol and related structures (loblolly pine). In each case, coniferyl alcohol served as the initial precursor, being subsequently metabolized with precise regio- and stereochemical control to first give species-specific lignans from which the 'secondary', or 'abnormal lignins' derive. The results again underscore the need to systematically determine the precise temporal and spatial biochemical processes involved in phenolic coupling, as well as any subsequent metabolic events in planta. These findings pave the way to a full delineation of the temporal, spatial, tissue- and cell-specific events involved in heartwood formation. Ultimately this will offer new strategies for biotechnological manipulation. It will also lead to the adoption of a coherent terminology to define these non-lignin phenolics.

©1998 American Chemical Society

In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

389


390 The durability, longevity and resistance to rot of different tree species are, in large part, dictated by their particular heartwood properties (7). It is also this portion of trees that either ends up as lumber, solid wood products, building materials and fine furniture or becomes used for pulp and paper production. Heartwood, which can account for more than 95% of the merchantable bole of mature wood, is often distinguished from neighboring sapwood by the amounts of so-called 'extractive' components. Depending on the species, these primarily consist of significant levels of colored phenolic compounds derived from lignans, tannins and (iso)flavonoids, although certain species have additional substances such as isoprenoids and alkaloids. The 'extractives' can often make up a considerable proportion of heartwood (e.g. the lignans in western red cedar [Thuja plicata]) (2-10). They also significantly help define the distinguishing features of particular woods, such as durability, color, quality, odor and texture. On the other hand, the presence of heartwood compounds can often adversely affect wood-pulping processes, leading to increased production costs associated with their removal. The term 'extractive' is a misnomer, however, since many heartwoods contain colored phenolic substances which cannot be fully removed by simple aqueous/solvent extraction; rather, they require more rigorous procedures normally used for lignin removal. Thus, many of these lignan or (iso)flavonoid-derived phenolics have been erroneously described as 'abnormal lignins' or 'secondary lignins', even though they are produced via distinct biochemical pathways. Determining how heartwood formation occurs represents an important biotechnological goal, given that the specific characteristics of many woods (and hence biodiversity itself) are defined in large part by the nature of their particular 'extractive' constituents. Put in another way, any biotechnological endeavor to modify wood quality must also target heartwood, whose formation represents one of the largest unsolved questions in plant science. Heartwood's significance becomes even more apparent when its commercial value for all purposes, prior to mill processing, is taken into account: this value exceeds $135 billion annually in the U.S. alone (1990 figures). That is, heartwood is the largest contributor to all plant (agricultural and forestry) derived income. The purpose of this chapter is to summarize our recent advances in the definition of lignan biosynthetic pathways, and how this is shedding light on the general processes involved in heartwood formation. Selected woody plants, i.e. western red cedar (Thuja plicata), western hemlock (Tsuga heterophylla) and loblolly pine (Pinus taeda), were chosen for investigation, each generating different lignan-derived heartwood metabolic products. Theories of Heartwood Formation Within living, growing trees exists the remnant of years, and in some cases, centuries past: the heartwood. An excellent and striking example of this can be seen from the cross-section of a mature tamarack larch (Larix lancina) stem that primarily contains darker heartwood (Figure 1). This contrasts markedly to the pale outer few centimeters of sapwood and the even paler thin ring (the so-called 'transition' zone) between both tissue types. Not all woody species, however, biosynthesize heartwood components to the same extent, or even use the same range of metabolic products. Consequently, woody plants can often visually differ dramatically depending on their particular heartwood constituents, such as western red cedar (red-brown), ebony (black) and southern pines (yellow-orange). Regardless of the amount of metabolites actively deposited in heartwood, this tissue no longer participates in any of the growth processes associated with the living part of the tree. That is, it does not conduct photosynthate to the roots, which is transported in the phloem of inner bark. Nor does it conduct minerals and water upward to the leaves, which is the main function of sapwood, lying just inside the growing, cambial region. Nor does it directly inhibit growth of disease-causing organisms which primarily attack living, nutrient-rich, sapwood, since the latter In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


391

Figure 1. Cross-section of a tamarack larch (Larix lancina) log revealing both (dark, inner) heartwood and the (lighter, outer) sapwood zones.



392 effects are minimized by the compartmentalizing ability of trees, which wall off attacking organisms from living sapwood, cambium and phloem (11-15). Heartwood does, however, play a significant role in the lives of trees. In addition to sapwood, it helps provide the mechanical strength necessary to stand upright in a world ruled by gravity, without which trees could not successfully compete in the struggle for light within the forest canopy. Accordingly, many woody plant species also have mechanisms to protect heartwood from rotting fungi and bacteria. Yet, how is this achieved when, being dead (i.e. possessing no living, metabolizing parenchyma cells), heartwood cannot actively respond to attack? As discussed in detail below, this protection involves the infusion of protective compounds (the so-called 'extractives') into pre-lignified sapwood. Conflicting theories have arisen about the initiation and process of heartwood formation (76), which is formed in a manner highly variable between species. It has been proposed, for example, that an increasingly less aerobic environment exists toward the interior of trees, and as this condition progresses, cells die and heartwood forms. A second theory suggests that over time, air enters the tree trunks and that the resulting embolisms cause heartwood formation to begin. A third theory proposes that some heartwood-initiating hormone/substance (77) moves centripetally along the rays from the cambium, and once it reaches a minimal threshold level, heartwood formation occurs. A fourth theory states that it is simply an aging process, where organelles in parenchyma cells become progressively degraded as the distance from the cambium increases (18-21). This particular view is apparently contradicted, however, in species such as maple (Acer), oak (Quercus) and walnut (Juglans), which have larger nuclei in cells near the heart wood/sap wood boundary than in cells in the middle of the sapwood, suggesting that higher metabolic activity actually occurs toward the heartwood/sapwood boundary than towards the periphery ((22), in (76)). Other species such as Pinus radiata show an increase in metabolism at the sapwood/ heartwood boundary that is seasonal (23, 24). The fact that this enhanced metabolism in the transition zone occurs only during a small portion of the year may explain why investigators have not always observed the same phenomenon. Work on other species, such as Nothofagus cunninghamii, Sloanea woollsii and Diospyros pentamera (25), have demonstrated that active parenchyma cells continue to live for years right next to dead, colored cells, that could be mistaken for heartwood cells. In these species, the transition zone is very wide, although it does surround a core of heartwood where all of the cells are dead. More recent work on Garuga pinnata and Ougeinia oojeinensis (26) has also supported this finding. Indeed, it has been cautioned that initiation and mode of heartwood formation may actually differ between given species (i.e. simultaneous initiation has been observed in the ray and axial parenchyma cells in Ougeinia and initiation only in the ray parenchyma cells in Garuga), a point that has rarely been emphasized by the investigators in this field. This would perhaps begin to provide some explanation for the conflicting hypotheses that have plagued heartwood formation investigations for the last half century. Perhaps the most controversial hypothesis for heartwood formation holds that the cells die because they are filled with lethal 'waste' products from external cells (27, 28). That is, heartwood metabolites are putatively transported in sub-toxic concentrations along the rays toward the interior of the plant, where they are then 'disposed of, thus performing a 'detoxication' process. This hypothesis proposes that compounds laid down in the heartwood, such as tannins, (iso)flavonoids, lignans and stilbenes, are simply by-products or 'wastes' of normal cellular metabolism. The authors even went so far as to state that lignin formation itself may be simply a 'detoxication' process to remove the 'excess' phenolic compounds derived through normal cellular respiration (27, 28) and that the role of lignin as a structural component of terrestrial plants may only be of secondary evolutionary significance*. Such an hypothesis has, however, many inconsistencies. For example, careful examination of the heartwood 'waste' products (see next section), reveal that they are most often optically active products of specific biosynthetic pathways (e.g. such as to (-)-plicatic acid 1 in Thuja plicata, see Figure 2) and that the actual metabolites can In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

393

H3CO

CH OH 2

]

"C0 H 2

OH

OCH3


OCH3 1: (-)-Plicatic acid OH

HO"

y

4 R,=R =R =R =:H: (-)-Matairesinol 8 R,=OH, R =R =R =H: (-)-Thujaplicatin a 9 R,=R =R =OH, R =H: (-)-Thujaplicatin b 10 R!=OCH , R =R =R =H: (-)-Thujaplicatin methyl ether a (T.M.E. a) 11 R!=OCH , R =OH, R =R =H: (-)-T.M.E. b 12 R ^ O C H ^ R =R =OH, R =H: (-)-T.M.E. c 13 R R = R = H , R =OH: (-)-7'-Hydroxymatairesinol 2

3: (-)-Plicatin

3

4

2

OCH^

H3CO

OCH3

0CH3

OH

3

HO

OCH3 OH

3

4

2

3

2

3

3

3

14: (+)-Tsugacetal

1=

3

4

4

3

4

2

4

2

2

Ρ π—Λ

OH

4

Q

HO OH

H CO 3

OCH3

16 R=H,H: (-)-Hinokinin 17 R=0: (-)-7-Oxohinokinin Figure 2a. Selected lignans listed in Table I.



394

H CO

OCH

3

22a (+)-Lariciresinol

3

22b (-)-Lariciresinol

OH

24 R!=H, R = C H : (+)-Isolariciresinol 25 R =R =H: (+)-Isotaxiresinol 26 R!=CH , R =H: (+)-Isotaxiresinol-4-methyl ether 2

1

3

2

3

2

Figure 2b. Selected lignans listed in Table I (cont.).



KhCO

r OCH

27: (+)-Taxiresinol

3

28 R=H: (+)-Syringaresinol 29 R=CH : (+)-Yangambin 3

OR H3CO

H C 3

MeO

30: Ramontoside

H3C0"

γ

"OCH OH

3

H3CO OH

31 R=Xyl: Lyoniside 32 R=Xyl: (-)-Lyoniresinol =(+)-Lyoniresinol 2a2a-0-B-D-xyloside OB-D-xyloside 33 R=H: (+)-Lyoniresinol 34 R=H: (-)-Lyoniresinol 35 R=Glc: (+)-Lyoniresinol 2a-0-6-D-glucoside 36 R=Rhamnose: (+)-Lyoniresinol 2a- O-B-D-rhamnoside Figure 2c. Selected lignans listed in Table I (cont.).


396

H c(r ^

OCH

3

3


OH

37 R=CH OH: rac-Thomasic acid 38 R=COOH: rac-Thomasidioic acid 2

39: Arboreol

40: Isoarboreol

43: (+)-Gummadiol

41: Gmelanone

42: (-)-2'-Bromoisoarboreol

44 R O H , R =H: 46 Dihydro(+)-9-Hydroxysesamin gurnrnadiol 45 R =R =OH: (+)-9,9 -Dihydroxysesamin 1 =

1

2

2

/

Figure 2d. Selected lignans listed in Table I (cont.).



397 vary substantially between species, thus establishing that no common 'excretion' products are being formed. That is, this particular hypothesis was based on an wholly erroneous and overly simplistic assumption. Instead, heartwood metabolites are not 'wastes' at all, but are instead distinct components of an elaborate defensive arsenal that has evolved to protect these diverse heartwoods from invasion by rotting fungi and bacteria, as well as from other biological challenges. Heartwood metabolites are only significantly laid down, albeit in differing amounts depending on the species, following secondary wall formation and lignification, i.e. their deposition is a post-lignification non-structural mechanism. Moreover, according to Hergert (29), the heartwood 'extractive' precursors are formed and transported along the ray parenchyma cells. They are, perhaps, eventually converted further in the transition zone, at the sapwood/heartwood boundary, into specific heartwood constituents. Thereafter, these metabolites are employed to further seal off bordered pits in the heartwood zone (77, 30) and infuse into adjacent pre-lignified cells. Interestingly, although the precise biochemical events need to be defined, a significant portion of these 'extractive' substances, but not all, become insoluble deposits and can only be removed using 'lignin' dissolution procedures, such as from milled wood lignins (29). It must be emphasized, however, that these heartwood substances, even if partially polymerized in some manner, are not 'abnormal', 'native' or 'secondary' lignins, as previously and erroneously concluded (31). Instead, they are partially polymerized extractives such as polylignans and polymeric (iso)flavonoids. That is, they represent a distinct and unique set of metabolic non-lignin products which are deposited into the lignified secondary xylem tracheids via infusion through the neighboring specialized cells. As Hergert showed many years ago with infrared spectroscopy (29, 32, 33), such heartwood forming substances (then called Braun's native lignins) in western red cedar and western hemlock contain functional groups such as 5-membered lactone rings (32). These cannot directly result from the coupling of monolignols, but instead are derived in these species from lignans, such as a-conidendrin 2, plicatin 3 and matairesinol 4 (see Figure 2), a fact that has been further confirmed by N M R spectroscopic analyses (Hergert, personal communication). Thus, regardless of their chemical composition, the deposition of heartwood characteristic metabolites occurs in a controlled and regulated manner after lignification has ceased and coincides with the death of parenchyma cells in the outer heartwood layers and in the transition zone. In fact, heartwood formation may give an additional perspective to the type of programmed cell death that has become the focus of so much research in recent years. It is also evident that comparable localized 'heartwood-like' forming processes can also occur in regions, for example, surrounding insect attack (and other stresses) where the plant has attempted to seal off the affected zone. Heartwood Formation and the 'Abnormal Lignins' Our knowledge of heartwood formation has not substantially changed over the last 45 years. It should be clear, however, that there is an urgent need to understand both the formation and the precise roles that heartwood constituents play in the physiology of plants, whether solely as defense elements or for additional purposes. Indeed, until their precise biochemical formation and regulation are determined, a definitive understanding of how and why heartwood is formed will not be possible. As Chattaway stated in 1952, 'although various workers have studied heartwood formation intensively, and have even offered explanations for its development in individual genera and species, no one has yet formulated an answer to the fundamental question of what lies behind this uniformity of pattern in the life of so many and such varied trees, so that trees from a very wide variety of families growing in a great range of different habitats produce a stem which contains two types of wood, one an outer layer of relatively pale-colored sapwood, and the other a central core of darker, more durable heartwood' (25). In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


398 She went on to state that 'although the physical changes that give heartwood its characteristics are dependent on the death of the living cells of the sapwood, the cause of heartwood formation must be sought further back in the life of the ray cells, in the agent that stimulates the cells to produce increased quantities of extractives' (emphasis added) (25). Both this summation and its implications hold to this day. It is therefore curious that some scientists would, even to this present time, describe such substances as 'abnormal lignins' in particular woods. Not only do they more closely resemble heartwood metabolites formed by distinct and unique biochemical pathways (discussed below), but they are generated through a distinctive infusion system. Indeed, wood cannot be treated as if it were somehow an homogeneous material with no cell types other than lignified xylem, and with all phenolics being lignin-derived! This is not the case. This chapter will, therefore, not revolve around the initiation or causes of heartwood formation, as such a discussion is premature at this point. This is because we still do not know the precise regulatory control of the biosynthesis of these heartwood compounds. Rather, this discussion will center around what is currently known about the biosynthesis of an important class of heartwood constituents, the lignans, with the belief that an understanding of this biochemical pathway will eventually help lead to the uncovering of the underlying causes of heartwood formation itself. Prior to describing progress to date in defining the unique biochemistry involved in lignan heartwood metabolite accumulation, some commentary on the fundamental differences between heartwood metabolite deposition and lignification is required. Indeed, an appreciation of these fundamental differences is necessary in order to begin to bring a coherent terminology into the field and to eliminate the laxness used in current nomenclature. This is because lignification and heartwood metabolite generating processes (i.e. affording certain 'extractives', 'abnormal lignins' and 'secondary lignins') differ profoundly in several principal ways, namely in their biochemical mechanisms and in their temporal and spatial deposition: 1.

Heartwood metabolite formation, in contrast to lignin synthesis, is first initiated in the center (pith) region of mature woody tissues at some undetermined time. Apparently, comparable substances can also be formed in sapwood in response to biological challenges, such as insect attack. Heartwood metabolites are released from specialized ray parenchyma cells and then further infuse into neighboring cells. Some 48 years ago, Chattaway attempted to graphically illustrate this phenomenon as redrawn in Figure 3 (25). That is, the various heartwood-characteristic substances are released by parenchyma cells through the pit apertures into the lumen of adjacent, dead (lignified) cells and then infuse into neighboring, pre-lignified cells (tracheids). The overall biochemistry involved in their formation and transport is now coming to light, and it is fundamentally distinct from the lignin forming processes.

2.

Heartwood metabolites can vary extensively between species in terms of amount, molecular size and chemical composition, since they can include lignan, (iso)flavonoid, isoprenoid and even alkaloid-derived substances. It is important to note that such compounds can also constitutively accumulate in sapwood, albeit to a much lower extent, presumably due to their lower concentrations in the 'conducting' sapwood ray cells or as a phytoalexin response.

3.

Imperfect characterization of these heartwood/sapwood metabolites, particularly when lignan-derived, has led to vague definitions, such as 'abnormal lignins', 'secondary lignins' and 'Braun's native lignins'. They are not lignins, but instead represent a distinct biochemical class. Indeed, the fact that they can be produced in a range of sizes and can form insoluble deposits, which can only be removed under conditions normally used for lignin removal (e.g. milled wood lignins), in no way permits their classification as lignins.



399

•Ray Parenchyma

Figure 3. Secretion of heartwood constituents by ray parenchyma cells into the lumen of neighboring cells appears to occur through pit apertures. Redrawn from reference (25).



400 4.

In contrast, lignification is a process of individual cell wall maturation. The monolignols, £-/?-coumaryl 5, £-coniferyl 6 and £-sinapyl 7 alcohols, are transported across the plasma membrane in a lignifying cell where they are then polymerized at specific sites (or regions) within the maturing cell wall (see Chapters 15 and 22).

5.

Lignification occurs in distinct and predictable phases of cell wall development, and is the first major transportation of phenolics into the cell wall of the maturing xylem. It occurs in a progressive manner in cells near to the cambial region, and results in secondary wall (xylem) formation during sapwood generation.

6.

Lignins in mature wood tissues that contain little or no heartwood metabolites are essentially colorless; discoloration of both sap- and heartwood is most often due to formation and deposition of non-lignin phenolic metabolites.

7.

Polymerization of monolignols represents the first committed, as well as the final, step in the lignin-forming process in woody plants. On the other hand, heartwood metabolite accumulation, including the generation of appropriate precursors in sapwood parenchymal ray cells, often uses monolignols as the entry point into a distinct biochemical pathway to generate the various lignans.

Selected Elements of the Heartwood Arsenal As mentioned above, various heartwood species contain large and (bio)chemically distinct groups of so-called 'extractives', i.e. non-lignin phenolic compounds that help confer particular properties to specific heartwoods. In this chapter, western red cedar, western hemlock and loblolly pine have been chosen as representatives of heartwood-forming species whose main phenolic heartwood constituents are the lignans. It must be emphasized, however, that the underlying principles of deposition described here are viewed as being applicable to all other classes of heartwood substances in other species, such as those also leading to the tannins, (iso)flavonoids, isoprenoids, tropolones, stilbenes and alkaloids, to name only a few compound classes. Table I and Figure 2 list some of the lignans (compounds 1-4, 8-46) (3-10, 3445) known to date as constituents of the substances deposited in the heartwoods of various gymnosperms and angiosperms. The ones shown represent several subclasses of abundant 8,8'-linked lignans, including the furofuran (28), tetrahydrofuran (22), dibenzylbutane (23), dibenzylbutyrolactone (4, 8), aryltetrahydronaphthalene (1), aryldihydronaphthalene (37), arylnaphthalene (30) and divanillyltetrahydrofuran (15) skeletal types. Four important points about their formation, in terms of the heartwood metabolic arsenal, are discussed below: optical activity, biological activities, magnitude of deposition and biosynthesis.



34 35 10 34, 36 36 36 36 35 37 6, 9, 10 6-8, 10

Y Y Y Y

Y Y Y Y

Cupressaceae Pinaceae Cupressaceae Cupressaceae Cupressaceae Pinaceae Pinaceae Pinaceae Cupressaceae Cupressaceae Cupressaceae

Western Hemlock Norway Spruce Western Red Cedar Western Hemlock Mountain Hemlock Balsam Fir Grand Fir Norway Spruce Western Red Cedar Western Red Cedar

Tsuga heterophylla Picea abies

Thuja plicata

Tsuga heterophylla Tsuga mertensiana Abies amabilis Abies grandis Picea abies Calocedrus formosana

Thuja plicata

2 (-)-cc-conidendrin

3 (-)-plicatin

4 (-)-matairesinol

10-12 (-)-thujaplicatin methyl ethers Thuja plicata

35 38 37 37 37

Y Y Y Y Y

Pinaceae Cupressaceae Cupressaceae Cupressaceae Cupressaceae

Norway Spruce Taiwan hemlock

Picea abies

Tsuga chinensis var. formosana

Calocedrus formosana



151iovil

14 (+)-tsugacetal

16 (-)-hinokinin

17 (-)-7-oxohinokinin

18 (-)-savinin

Continued on next page.

34 35 Y Y Cupressaceae Pinaceae

Western Hemlock Norway Spruce

Tsuga heterophylla Picea abies

13 (-)-7'-hydroxymatairesinol

8,9 (-)-thujaplicatin a & b

3-5, 10

Y

Reference

Reductase Derived?

Cupressaceae

Family

Western Red Cedar

Common Name

Thuja plicata

Species

1 (-)-plicatic acid

Compound

Table I. Lignans found in heartwood



37 37

Y Y

Cupressaceae Cupressaceae

40 39 39 40 39 39 40 41 41 42 43

Y Y Y Y Y N N Y Y

Taxaceae Taxaceae Taxaceae Taxaceae Taxaceae Magnoliaceae Magnoliaceae Flacourtiaceae Violales Fabaceae

Common yew Japanese yew Common yew Japanese yew Common yew Japanese yew Common yew Japanese yew Common yew Yellow poplar Yellow poplar

Taxas baccata Taxus cuspidata

Taxus baccata Taxus cuspidata



Taxus baccata

Liriodendron tulipifera

Liriodendron tulipifera

Flacourtia ramontchi

Quercus petraea

23a (-)-secoisolariciresinol

2 4 (+)-isolariciresinol

2 5 (H-)-isotaxiresinol

2 6 (+)-isotaxiresinol-4methyl ether

27 (+)-taxiresinol

2 8 (+)-syringaresinol

2 9 (+)-yangambin

3 0 ramontoside

31 lyoniside =(+)-lyoniresinol 2a- = known conversions = probable conversions — = potential conversions — = pathway demonstrated in Forsythia Thuja

and

Dirigent Τι Protein

species.

X.OH Ù

^"

JÛ

Gmelina,

(y)~*^

...Or

arborea

OHfV h

in Gmelina

3

ΗΟ^γ

arborea 40: Isoarboreol

Η

-

0

Ό

^

H\0—V"OH

0

47a: (+)-Pinoresinol !

1

4

ό

3

;

(+)-Gummadiol

OCH3

(+)-Pinoresinol/ j (+)-Lariciresinol Reductase

NADPH

Reductase

Gmelina

NADPH

HO

-

I

ΗΙί—ί'ΐΟΗ

OCH3 22a: (+)-Lariciresinol 42: (-)-2'-Bromo-isoarboreol

46: (+)-Dihydrogummadiol

(+)-Pinoresinol/ (+)-Lariciresinol Reductase

.OH

-OCH3

NADPH H CO 3

v

H^CO^

latairesinol 17: (-)-7-Oxohinokinin

V

in Tsuga heterophylla

H,CO

^

0

Η

Thuja

3: (-)-Plicatin

plicata

| i

H„CO

v

H CO. 3

OCH

3

OH 2: (-)-a-Conidendrin

Figure 4.

OCH, 13: (-)-7'-Hydroxy matairesinol

HO^Y OCH3 OH 9: (-)-Thujaplicatin b

Proposed biosynthetic pathway to various heartwood lignans.


407


complex mixtures may result in the developed heartwood via the infusion process previously discussed. In any event, as far as the lignans are concerned, pinoresinol 47, lariciresinol 22, secoisolariciresinol 23 and matairesinol 4 appear to be general precursors for a wide array of 8,8Minked lignans (Figure 4). Indeed, as described below, all of the data obtained to date with western red cedar and western hemlock point to the fact that the biochemical transformations leading to heartwood formation are carried out by very specific enzymes, with strict stereochemical specificities for their substrates. Stereoselective Entry into the Heartwood Lignan Pathway: Dirigent Proteins. In initial investigations in lignan biosynthesis using Forsythia intermedia, it was found that the entry point into lignan formation occurred via coupling of two ^-coniferyl alcohol 6 molecules to give (+)-pinoresinol 47a. From this observation, the discovery was made that in order to control this regio- and stereospecificity, a coupling agent, identified as a 78 kDa dirigent protein, was necessary (52, 53). This finding has since been extended to various plant systems, including western red cedar and western hemlock. The 78 kDa dirigent protein apparently only serves to bind and orient the substrate molecules and has no detectable active (oxidative capacity) site (53). As discussed in Chapter 22, stereoselective coupling occurs only when auxiliary oxidative capacity is provided through addition of either a non-specific oxidase or a free radical initiator. Thus, the means to achieve stereoselective coupling leading to optical activity differs markedly from that currently viewed to produce lignins, where only non-specific coupling has to this point been implicated. The effect of the 78 kDa protein on stereoselective coupling of £-coniferyl alcohol 6 is shown in Figure 5. The 78 kDa protein, which apparently exists as a homotrimer of 27 kDa subunits, was first cloned from a Forsythia intermedia stem cDNA library. The resulting cDNA encoded an 18 kDa secreted protein containing several potential N-glycosylation sites, with sugar moieties accounting for the difference in native versus cloned polypeptide size. This cDNA was transferred to an eukaryotic expression system, which employs protein production by a Spodoptera frugiperda (fall army worm) cell line following infection with the baculovirus strain containing the cDNA of interest, and subsequently shown to produce functional recombinant dirigent protein. Thus, reactions of Forsythia laccase with £-coniferyl alcohol 6 in the presence of the recombinant dirigent protein afforded only (+)-pinoresinol 47a formation (see Figure 5). Therefore, the protein conferring regio- and stereospecificity in the first step of 8,8'-linked lignan biosynthesis has unambiguously been cloned. This cDNA was then used as a probe to screen western red cedar and western hemlock cDNA libraries, resulting in the cloning of several homologs (see Chapter 22). Current work is underway to heterologously express these and verify that the isolated putative dirigent protein clones are functional. However, their presence strongly suggests that the pathway is also present in western red cedar and western hemlock. Significantly, sequence analysis of all of the cloned dirigent protein cDNAs revealed no homology to any other protein of known function (manuscript in preparation). The identification and cloning of the dirigent protein is an unprecedented finding, since not only does it represent the first example of stereoselective bimolecular freeradical coupling in nature, but it is manifested in a manner that could not have been predicted based on existing chemical, biochemical or molecular precedents. Moreover, since different plant species clearly catalyze distinct coupling modes (i.e. to give lignans with linkages other than 8,80, it is envisaged that this is also directed by comparable dirigent proteins. Put in another way, a new class of proteins has apparently been discovered. Stereospecific Reduction of Heartwood Lignan Precursors. The first enzyme identified in the lignan pathway was initially isolated from F. intermedia, i.e. the bifunctional reductase that converts (+)-pinoresinol 47a into (+)-lariciresinol 22a, and (+)-lariciresinol 22a into (-)-secoisolariciresinol 23a, and trivially called In Lignin and Lignan Biosynthesis; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


OCH

3

3

oxidase/ oxidant

,OH

HO

47: (±)-Pinoresinols

bCH

3

49: (±)-Dehydrodiconiferyl alcohols

Hd

OH

6: £-Coniferyl alcohol

O H ^ "

^OCH

,OH

3

50: erythro/threo(±)-Guaiacylglycerol 8-

Abnormal Lignins - American Chemical Society

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