The Challenge of Lignin Manipulation: Plant Growth/Development Versus Stem Structural Integrity

There are compelling and long-standing reasons to identify novel ways to more effectively either utilize the lignin biopolymers or manipulate the amounts or forms of carbon allocated to the lignin-forming pathway, for example, to produce more desirable bioproducts in commercially cultivated plant species. Indeed, a number of biotechnological manipulations of both lignin contents and compositions in various plant species have already been carried out; that is, various transgenic/mutant lines have been successfully obtained using standard transformation procedures (see Anterola and Lewis, 2002, for examples and references therein). Generally, though, the effects of drastically reducing lignin contents in both woody and nonwoody vascular plants result in a significant impairment/weakening of the vascular apparatus, for example, collapsed vessels (for a discussion and examples, see Anterola and Lewis, 2002). Such defects potentially lead to severe drawbacks in growing biotechnologically modified plant lines commercially, as this can lead to, for example, premature lodging, weakening of plant stems, and dwarfing during growth/development.

To put the utility of employing mutant and/or genetically modified lines into sharper focus, the following examples should illustrate why this issue deserves attention. It is often overlooked that many lignin mutants have been described over a period spanning nearly a century, particularly the brown midrib mutants (see Anterola and Lewis, 2002). All had significant

FIGURE 13.5 Effects of knocking out Atcad4 and cad5 (cad-c cad-d) in Arabidopsis thaliana (ecotype Wassilewskija). (A) Phenotypical differences between 4-week-old wild type (Ws) and lignin-deficient cad-4 cad-5 double mutant plants. (B) Tensile storage and loss moduli of WT and lignin-deficient (~90%) cad-4 cad-5 double mutant lines. Source: Redrawn from Jourdes et al. (2007). (See Page 24 in Color Section.)

deleterious effects on vascular tissue integrity, and none, to our knowledge, has yet found commercial application. Three of these are COMT, CAD, and cinnamoyl CoA oxidoreductase (CCR) mutants (see Fig. 13.1 for biochemical pathway steps). The first, due to introduction of a 5-hydroxyconiferyl alcohol (22) monomer in lignin, results in brittle stems, which are more susceptible to lodging (Anterola and Lewis, 2002). The CAD double mutation also results in a generally weakened vasculature. The CAD double mutant in A. thaliana has a greatly compromised ability to form monolignols 19, 21, and 23 (by ~90–94%), with only very small amounts of lignin proper being formed (~10% of the natural levels). The resulting stems (of the CAD double mutant) are thus unable to stand upright (Fig. 13.5A) and their tensile modulus is greatly compromised (~50%) when tested in the tension mode (Fig. 13.5B) (Jourdes et al., 2007). Such a phenotype may be a disadvantage for either largescale commercial cultivation, harvesting, or processing due to the weakened vascular apparatus. It is also unknown whether such modifications may also impact/decrease resistance to opportunistic pathogens.

FIGURE 13.6 Effects of mutating CCR in Arabidopsis thaliana. (A) Phenotypical differences between wild type (Ler, left) and irx4 mutant (right) plants. (B) Plot of acetyl bromide lignin determinations in stems at various stages of A. thaliana growth and development. (C) Estimations of H, G, and S monomer amounts released during thioacidolysis of wild type and irx4 extractive-free stem tissues. Source: Redrawn from Patten et al. (2005). (See Page 25 in Color Section.)

CCR mutation in A. thaliana also resulted in a severely dwarfed phenotype and a delayed but coherent lignification program (Fig. 13.6A–C) (Laskar et al., 2006; Patten et al., 2005), whereas in tobacco, it resulted in a compromised vasculature and dwarfing as well (Piquemal et al., 1998). In an analogous manner, 4-coumarate CoA ligase (4CL), PAL, and C4H downregulation resulted in a significant loss of vascular integrity (see Anterola and Lewis, 2002) and/or other effects, such as dwarfing, due (mainly) to reduced lignin levels. Other concerns about deleterious effects on vascular integrity hold also for C3H downregulation (Patten et al., 2007).

These examples underscore the central question as to what extent lignin compositions/contents can actually be manipulated, without introducing structural defects prohibiting field applications of the resulting plant cultivars in, for example, bioethanol/biofuel/bioproduct generation. Another possible concern is that a weaker vascular apparatus may result in plants more susceptible to opportunistic pathogens. In short, it is becoming increasingly evident that a judicious balance must be maintained in growing vascular plants for commercial purposes and in reducing/modifying lignin contents/compositions. However, what flexibility exists in modifying lignin amounts/composition to avoid such adverse growth/developmental effects has not yet been determined.