1. General strategy
The constitutive defenses of conifers are ‘generalized’ defenses against a range of organisms that might be expected to try to penetrate the bark during the history of a tree. Constitutive production of a suite of stable defense products of varying properties provides a generalized capacity for resistance to a broad range of organisms. In addition, opportunistic secondary invasions are common, and because they are likely to be a different organism than the original invader it is useful to have multiple and overlapping constitutive defenses present (Fig. 1b,c).
Constitutive defenses are of two basic types: mechanical and chemical. Mechanical defenses are made of structural elements that provide a toughness or thickness to the tissues and inhibit chewing or pulling apart of the bark. They may also include ‘spines’ that can be effective against larger animals – for example, tough, pointed cauline leaves as in monkey puzzle trees. Impregnation of tissues with polymers such as lignins and suberin can add to the mechanical properties and enhance resistance to penetration, degradation and ingestion/chewing, particularly by smaller organisms. Constitutive chemical defenses often occur as pools of stored chemicals (e.g. phenolics, terpenoids and alkaloids) that can be released upon attack. Chemical defenses include antifeedants, toxins, defensive proteins and enzymes, and reservoirs of chemicals such as resins that can flush away, repel or physically entrap small organisms. These defenses are dispersed throughout the various tissues of the bark, which include the periderm, cortex (if the stem is young) and secondary phloem. The secondary xylem can also contain some of these defenses as constitutive products (cf. Pearce, 1996; Fäldt et al., 2003).
A description of the physical and chemical nature of the various constitutive defense structures and their distribution within the bark and among species is important to developing an understanding of defense strategies of conifers. A summary of the major defensive structures of the bark is presented, although this is neither comprehensive nor does it point out all the nuances inherent in the biological diversity of such structures. This description provides the basis for the conceptual framework of integrated defense strategies in bark of conifers.
4. Secondary phloem, the inner defenses
The secondary phloem is a major site of constitutive defense mechanisms. Most conifers share three common constitutive defense components in their secondary phloem: sclerenchyma, calcium oxalate crystals and phenolic bodies, although the relative amount of each varies considerably among species (Hudgins & Franceschi, 2004). A fourth defense that is common in certain taxa is the presence of resin-producing structures such as radial resin ducts (extending into the xylem), axial resin ducts, resin blisters and resin cells. These will be discussed separately, because some conifers do not produce constitutive resin structures in the secondary phloem. The amount and combination of each of these components defines distinct defense strategies and likely different evolutionary pressures.
Bark of all conifer families studied has axial phloem parenchyma cells that are specialized for synthesis and accumulation of phenolic compounds (Fig. 3; Hudgins et al., 2003a; Hudgins et al., 2004). These are referred to as polyphenolic parenchyma cells (PP cells; Franceschi et al., 1998; Krekling et al., 2000). Within their vacuoles, PP cells contain variable amounts of phenolic bodies (Fig. 3) that are thought to serve as antifeedants and as antifungal agents (Beckman, 2000). In addition to their phenolic contents, they also have thickened cell walls, albeit with abundant plasmodesmata that allow for axial and tangential exchange of information, and possibly for defense signaling (Krekling et al., 2000). PP cells are commonly produced as an annual tangential ring, or cylinder, of cells and so can be used to determine age of the region of secondary phloem (Fig. 3a). As the rows of PP cells are ‘displaced’ further away from the cambium through its activity in secondary growth, they become larger and may even divide to produce more PP cells (Krekling et al., 2000). It is significant that PP cells more than 70 yr old in 100-yr-old Norway spruce (Picea abies (L.) Karst.) bark were found to be still living (Krekling et al., 2000). This, along with their presence in all conifer families, attests to their importance to defense reactions in general.
Figure 3. The anatomy and distribution of polyphenolic parenchyma (PP) cells in Picea abies secondary phloem. (a) Transverse section showing annual layers of PP cells separated by sieve cell (S) layers. R, radial ray. Bar, 200 µm. (b) Enlargement of PP cells (radial section) showing the large vacuole (V) filled with phenolics and storage of starch (St) in these living cells. N, nucleus. Bar, 20 µm. (c) Tangential section through a layer of PP cells. Bar, 100 µm. (d) Radial section with files of cylindrical PP cells separated by layers of sieve cells. Bar, 100 µm.
Download figure to PowerPoint
It is unfortunate that the chemistry of the phenolic components found in PP cells has not been extensively studied. One might expect that different species produce different phenolic components, dependent upon the type of organisms commonly attacking them. There is also some evidence that relative resistance to pathogens might partly be a function of the type of phenolics produced (Brignolas et al., 1995; Bonello et al., 2003). The phenolics deposited are not static structures, as shown by studies of changes in PP cell contents on a seasonal basis (Krekling et al., 2000), and, as will be discussed, PP cells are also responsible for important inducible defense responses. Overall, PP cells represent a very dynamic component of the defense strategy of conifer bark, and because they are the most abundant living cell type in the secondary phloem, the argument can be made that they are the single most important component. The PP cells, along with the ray parenchyma, are also a major site for storage of starch and/or lipids (Fig. 3b; Krekling et al., 2000). In this capacity, they can be seen as a target for beetles and fungi, and constitutive phenolics can be hypothesized to protect the cells themselves as well as prevent fungal penetration towards the cambial zone. In any case, the multiple layers of PP cells between which are the crushed remnants of the sieve cells can be viewed as providing physical and chemical resistance to penetration of the bark (Franceschi et al., 2000).
Sclerenchyma is a mechanical tissue common to all conifer bark, although the amount and type produced varies dramatically among conifer taxa. Sclerenchyma is made of cells with lignified secondary wall thickenings, which can serve as structural elements as well as mechanical defense. They can occur as massive, often irregular-shaped stone cells (sclereids), such as can be seen in many of the Pinaceae (Fig. 4a–d), or as well organized rows of fibers, as in the Taxaceae (Fig. 4e; Hudgins et al., 2004). Stone cells can be present as individual cells or as clusters of cells of variable size (Fig. 4a–d). Their physical toughness can be a deterrent to grazing or bark-boring organisms, particularly if they are abundant (Wainhouse et al., 1990, 1997, 1998; Hudgins et al., 2004).
Figure 4. Sclerenchyma is common in conifer bark but has different forms and distribution patterns. All are transverse sections except (c), which is tangential. (a) Larix occidentalis has fibers (arrows) scattered throughout the secondary phloem. Bar, 100 µm. (b) Picea pungens has large patches of stone cells (SC). Bar, 100 µm. (c) Tangential section of Tsuga heterophylla gives a good representation of the amount of lignified tissue that can be present as stone cells. Bar, 50 µm. (d) Enlargement of a stone cell cluster in Picea abies. The stone cells are generally derived from polyphenolic parenchyma (PP) cells as the phloem ages. Bar, 100 µm. (e) Taxodium sp. showing fibers (arrows) arranged as tangential rows separated by two rows of sieve cells between which is a row of PP cells. Bar, 100 µm.
Download figure to PowerPoint
In contrast to the more or less scattered distribution of stone cells, fibers occur as distinct tangential rows of cells interspersed in a highly ordered organization with rows of PP cells and sieve cells (Fig. 4e). Unlike stone cells, fibers develop from a layer of precursor cells that are distinct from PP cell layers, and they constitute a complete tangential layer of lignified cells disrupted only by radial rays. Full lignification of fibers occurs only after 2–3 yr, so that the two layers of precursor cells closest to the cambium are unlignified or have only partially thickened walls during normal growth (Hudgins et al., 2004). In general, layers of fibers are separated by much fewer layers of other cells (3–5 nonfiber rows) as compared with species with stone cells (7–12 nonstone cell rows between), and appear visually to provide a much more formidable mechanical barrier. In mature secondary phloem, stone cells and fibers are not generally found together. This may be partly due to separate evolution of these mechanical defenses, because stone cells are most abundant in the Pinaceae, whereas fibers are found in other families, and these groups represent a major split in the evolution of conifers (Fig. 5).
Figure 5. The presence or absence of various bark defenses is mapped against a conifer phylogeny. Note that aggressive, tree killing bark beetles are primarily found in the Pinaceae. (a) Distribution of the mechanical defense components, calcium oxalate crystals and sclerenchyma. (b) Distribution of a chemical defense component, resin-producing structures.
Download figure to PowerPoint
The sieve cells themselves may provide some mechanical resistance against penetration. The layers of sieve cells older than 3 or 4 yr, which are dead and no longer participate in assimilate translocation, progressively collapse under the pressure of new cell layers interior to them. They thus form, at least in the Pinaceae where 6–12 layers are produced per year, thick layers of cell wall material.
Calcium oxalate crystals are another potential mechanical defense component that is common in the secondary phloem of conifers. They occur as intracellular deposits in the Pinaceae and extracellular wall deposits in the non-Pinaceae (Fig. 6; Hudgins et al., 2003b), as well as in the periderm of various conifer species (Fig. 2). Although little is known about their formation or function in conifers, the tough physical nature of the crystals and their relative abundance would implicate a role in deterrence of bark-boring or -chewing animals. Because they are chemically inert, they are unlikely to have any effect on fungal attack. Calcium oxalate crystals are common in plants (reviewed by Arnott & Pautard, 1970; Franceschi & Horner, 1980; Webb, 1999; Nakata, 2003; Franceschi & Nakata, 2005), and while a role in bulk calcium regulation has been demonstrated (Franceschi, 1989; Volk et al., 2002; Mazen et al., 2004), they clearly have also evolved secondary functions in defense (Franceschi, 2001; Franceschi & Nakata, 2005). The Pinaceae produce calcium oxalate crystals embedded within the phenolic bodies in vacuoles of modified PP cells, typically as scattered axial strands of crystalliferous cells (Fig. 6c). At maturity, the cell walls of these modified PP cells have a suberin layer and the cells are no longer living, unlike the normal PP cells. In contrast, crystals of the non-Pinaceae are deposited within or along the walls of various cell types of the secondary phloem, depending upon the species (Fig. 6d–f). They are particularly abundant in the Taxodiaceae, where almost all cell walls are heavily encrusted with small crystals, including the walls of fibers. The combination of multiple layers of fibers and heavy incrustation with crystals may provide a particularly potent defense against bark beetles (see Fig. 5a), as proposed by Hudgins et al. (2003b).
Figure 6. The distribution of calcium oxalate crystals in various conifer species. Panels (c–f) are scanning electron microscope back-scattered electron (BSE) images where crystals appear as bright particles. (a) Young Pinus sp. stem in transverse section. C, cortex; P, phloem; X, xylem. Bar, 250 µm, (b) The same Pinus section viewed with crossed polarizing filters, with crystals showing up as bright spots in the phloem. Bar, 250 µm. (c) A crystal parenchyma cell in Cedrus libani with four prismatic crystals (arrows). Bar, 5 µm. (d) Transverse section of Taxus brevifolia phloem. Crystals appear as white spots along the walls of the sieve elements and ray parenchyma. Bar, 50 µm. (e) Tangential section of Juniperus scopulonum phloem. Crystals are abundant along the walls of almost all cell types. Bar, 50 µm. (f) Radial section of Chamaecyparis nootkatensis. Crystals are abundant along the walls of most cell types. Bar, 50 µm.
Download figure to PowerPoint
A major constitutive defense of particular importance to the Pinaceae is resin-producing and -storing structures. These include radial resin ducts derived from radial rays, axial resin ducts or canals, resin blisters and resin cells (Fig. 7a–c). The ducts and blisters have a lining of plastid-enriched epithelial cells (Fig. 7d) that synthesize terpenoid resins and secrete them into an extracellular lumen where they accumulate under pressure. Resin cells accumulate resin internally under pressure and expand into fairly large structures (Fig. 7c). Upon damage by wounding or by an invading organism, the pressurized resin is released and can repel or flush the organism out of the bark, entrap the organism in sticky resin or otherwise kill the invader due to the toxic nature of the resin. Resin flow from radial phloem ducts can be enhanced by their connection to constitutive or induced axial resin ducts in the xylem (Christiansen et al., 1999a; Nagy et al., 2000). When the volatile components of the released resin evaporate, the nonvolatile components will crystallize to sterilize and seal the wounded region effectively. Whereas resin-producing structures are found in all Pinaceae, they do not occur constitutively in the secondary phloem of some other conifer taxa (Hudgins et al., 2003a, 2004). It is interesting to note that those species that lack constitutive phloem resin structures are the same species that have tangential fiber rows and high amounts of calcium oxalate crystals (see Fig. 5b). Although resin is considered to be a defense against bark-boring insects, and although a correlation between resin duct number and resistance to pine weevil has been shown (Alfaro et al., 1997), it is clear that other strategies have evolved that can work equally or more effectively (Hudgins et al., 2004). The chemistry and biochemistry of conifer resins has been extensively reviewed (Gershenzon & Croteau, 1991; Bohlmann et al., 1998; Phillips & Croteau, 1999, and others) and so is not covered here.
Figure 7. Resin-producing structures found in conifer secondary phloem. (a) Radial resin duct in Pseudotsuga menziesii. Bar, 100 µm. (b) Axial resin duct in Thuja plicata. Bar, 100 µm. (c) Resin cells in Abies grandis. Bar, 50 µm. (d) Epithelial cells (E) of a resin duct from Picea abies. Bar, 10 µm.
Download figure to PowerPoint
The role of radial ray cells in constitutive defense has not been determined. These cells in conifers tend to have thin walls, and generally lack accumulation of polyphenolics. However, in a few species that have been examined, they have an abundance of the enzyme phenylalanine ammonia lyase (PAL), a key enzyme in the phenylpropanoid pathway, and it has been speculated that they may be involved in production of soluble phenolics that can be secreted (Franceschi et al., 1998; Hudgins et al., 2004). Because they represent the main radial pathway for movement of information in the bark, ray parenchyma likely function in signal sensing and transduction at some level (Hudgins & Franceschi, 2004). Recent work has shown that they contain 1-aminocyclopropane-1-carboxycylic acid oxidase, a key enzyme in ethylene biosynthesis, and that they may be involved in ethylene-mediated responses to attack (Hudgins & Franceschi, 2004). However, the ray parenchyma also provides a nutrient-rich radial infection route for pathogenic fungi, and therefore rays may need their own defense mechanisms.