Prof. Joe Cummins explains why genetically modifying trees and forage crops to reduce their lignin content could make them more susceptible to pests. Other issues related to the GM construct, such as genetic instability, the persistence of antibiotic resistance marker genes in the ecosystem and biosafety in general, have also not been sufficiently considered.
The plant cell is protected by a cell wall that has a structure analogous to reinforced concrete. The cellulose fibrils play the role of steel reinforcing rods, while concrete is represented by lignin. Lignin determines the rigidity, strength and resistance of a plant structure.
When wood fiber is processed to make paper or composite products, lignin must be removed using polluting chemicals and a great deal of energy. Also, the digestibility of animal feed is influenced by lignin content - the greater the lignin content, the poorer the food source. Genetic engineering is now being used to fundamentally modify the lignin of forest trees and animal feed.
Reducing lignin content of fiber and forage leads to greatly reduced costs of preparing fiber and improved digestibility of fodder and forage. However, the advantages of reduced lignin are offset by the disadvantage of plants with reduced lignin, which are more readily attacked by predators such as insects, fungi and bacteria. Indeed, increasing lignin content has been promoted as a defense against pests.
The importance of lignin in disease resistance has been known for well over twenty years [1]. For example, lignification was crucial in reducing predation by spruce bark beetles [2], and lignin in the roots of the date palm played a key role in defense against the fungus Fusarium [3]. It has been suggested that a guaiacyl (a type of lignin subunit) rich lignin was produced as "defence" lignin when Eucalyptus is wounded by a predator [4]. Lignin content of larch species determined the level of heartwood brown-rot decay [5]. Genetic modification of plants to enhance lignin production is covered in United States Patent 5,728,570 [6].
However, Arabidopsis plants modified in the metabolic pathway leading to lignin formation produced abnormal lignin that was associated with severe fungal attacks [7]. Tobacco plants modified to limit production of lignin subunits were susceptible to virulent fungal pathogens, but it was suggested that the precursors of lignin and not lignin that protected plants from pathogens [8]. Genetic modifications for reduced lignin level nevertheless resulted in reduced fitness including increased winter mortality and decreased biomass [9].
It seems clear that plant genetic modification leading to reduced lignin, as proposed for use in pulp and paper or in livestock production, must be fully evaluated for fitness in the environment.
The monomeric structure of lignin influences the properties of the plant material. There are two main types of lignin, quaiacyl lignin and guaiacyl-syringyl. Guaiacyl lignin is characteristic of softwoods, which are resistant to chemical and biological degradation. Guaiacyl-syingyl lignin is typical of hardwoods such as poplar, which are more readily degraded.
Modifying plants with a gene enhancing the proportion of guaiacyl-syringyl lignin therefore provides a lignin more readily degraded by chemicals or enzymes [10]. Reducing lignin content also leads to plants more readily digested with enzymes or chemicals.
Lignin reduction has been achieved using anti-sense genes to limit production of key enzymes on the lignin biosynthesis pathway [11,12]. Multiple genetic transformations of forest trees have been used to enhance production of guaiacyl-syringyl lignin and to limit total lignin production. Four Agrobacterium T-DNA vectors, each with a cauliflower mosaic virus promoter, two of which included anti-sense to limit undesirable enzymes and two with sense constructions to enhance desirable enzymes, were used to simultaneously alter the genome of aspen (Populus tremuloides). This resulted in reduced lignin content of guaiacyl lignin and increased guiaicyl-syringyl proportion in the remaining lignin [13,14].
Even though a potentially desirable end product is created, the multiple transformations (gene stacking) are liable to create chromosome instability leading to translocations, duplications and deletions through homologous recombination during germ cell formation and in somatic tissues (mitotic recombination). Independent studies of transgene integration using T-DNA vectors in aspen showed extensive DNA sequence scrambling at the insertion points [15]. DNA sequence scrambling occurring in the cells during growth is a significant complication in long-lived trees.
Lignin genetic engineering is promoted as a promising strategy to improve fiber production but the drawbacks of anti-sense manipulation and transgene stability are not seriously dealt with. Trees genetically modified to produce low lignin are called "super" trees [16] with little consideration of pest resistance and genetic stability. Field and pulping performance of transgenic poplars with altered lignin was evaluated to be superior by the developers of the poplar and abnormal pest damage was not found [17]. However, the pest damage studies were cursory and not compared with experimental controls, but with norms reported by government agencies.
The antibiotic resistance markers from the leaves of transgenic aspen have been studied for their persistence in the soil. The field study showed that the marker DNA of the aspen leaves persisted for as much as four months in the soil [18]. The persistence of antibiotic resistance genes in the forest ecosystem is likely to impact not only soil microbes, but human and animal inhabitants of the forest as well.
Lignin content increases as crops age or are stressed. Animal feed rich in lignin is poorly digestible and considered to be of low quality. Grass, alfalfa or maize with reduced lignin or lignin with increased guaiacyl-syringyl proportion (readily digested) may provide a large economic benefit in animal production, provided that the genetic modifications do not result in susceptibility to predatory insects, fungi and bacteria and do not compromise food or feed safety (for example, fungus food contamination may lead to pollution of food with toxins, causing liver damage and cancer).
The main technique used to produce lignin modifications is anti-sense genes designed to reduce one or another enzyme level on the pathway to lignin production. Maize with improved forage quality was produced by down-regulating the enzyme O-methyl transferase to limit lignin production [19]. Tall fescue pasture grass with improved forage digestibility was produced using an anti-sense gene for the lignin precursor enzyme cinnamyl alcohol dehydrogenase [20]. Alfalfa down-regulated for lignin enzyme caffeoyl coenzyme A 3-O-methyl transferase produced plants with increased guaiacyl-syringyl lignin proportions leading to improved rumen digestibility [21,22].
There is little question that the forage and fodder with reduced lignin and lignin with improved composition are more desirable food sources for grazing animals. However, the downside of lignin manipulation - greater disease susceptibility - was not thoroughly considered by developers of crops with modified lignin. The developers seem to ignore safety issues while they promote the modified crops.
Furthermore, smooth brome grass clones selected using conventional breeding showed that reduced lignin was associated with severe rust fungus disease [23]. Alfalfa selected for forage quality (including reduced lignin) had reduced vigour but was not expected to affect levels of disease resistance [24]. Sudan grass selected for brown- midrib trait (an indicator of reduced lignin) experienced severe yield reductions and environmental sensitivity, particularly during cooler growing seasons [25].
Lignin modification of trees and forage crops has been a focus of research in genetic engineering. But lignin provides both fundamental structural features and resistance to animal and microbial pests. Lignin enhancement that leads to greater forage or tree pulp quality also leads to susceptibility to disease, while lignin enhancement that leads to great disease resistance makes forage less digestible and tree pulp more expensive to process.
The economic consequences of effective lignin modification could be tremendous, but producing forests and rangelands highly susceptible to insects, fungi and bacteria would lead to economic and environmental disaster. The low lignin trait is comparable to a loss in immune functions comparable to AIDS in mammals. The chemical corporations might well welcome a huge increase in pesticides to fight disease in forests and pastures. Nevertheless, the best strategy is to proceed prudently and honestly evaluate the consequences of far reaching genetic engineering experiments.
Note added by editor: Another consideration is ecological. Wood, with its naturally high lignin content, generally takes a long time to decay and recycle in the ecosystem, probably for good reasons. It is a long-term energy store complementing the shorter-term energy storage depots, which enables the ecosystem to function most efficiently and effectively (see "Why are organisms so complex? A lesson in sustainability", SiS 21). Slow-decaying wood is also a major carbon sink. Reducing its lignin content to enhance degradation will end up returning carbon dioxide too rapidly to the atmosphere, thereby exacerbating climate change (see "Why Gaia needs rainforests" SiS 20).
Article first published 05/06/04
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