Trevor M. Fenning

Where will the wood come from? Plantation forests and the role of biotechnology.

Wood is almost as important to humanity as food, and the natural forests from which most of it is harvested from are of enormous environmental value. However, these slow-growing forests are unable to meet current demand, resulting in the loss and degradation of forest. Plantation forests have the potential to supply the bulk of humanitys wood needs on a long-term basis, and so reduce to acceptable limits the harvest pressures on natural forests. However, if they are to be successful, plantation forests must have a far higher yield of timber than their natural counterparts, on much shorter rotation times. To achieve this in reasonable time, biotechnology must be applied to the tree-improvement process, for which large increases in public and private capital investment are needed. However, additional obstacles exist in the form of opposition to plantations, some forest ecocertification schemes, and concerns about aspects of forest biotechnology, especially genetic engineering. It is the intention of this article to explain, in detail, why plantation forests are needed to sustainably meet the worlds demand for wood, why they are not being developed fast enough, and why the application of biotechnology to tree improvement is essential to speeding up this process.

Biosynthesis of the Major Tetrahydroxystilbenes in Spruce, Astringin and Isorhapontin, Proceeds via Resveratrol and Is Enhanced by Fungal Infection

Stilbenes are dibenzyl polyphenolic compounds produced in several unrelated plant families that appear to protect against various biotic and abiotic stresses. Stilbene biosynthesis has been well described in economically important plants, such as grape (Vitis vinifera), peanut (Arachis hypogaea), and pine (Pinus species). However, very little is known about the biosynthesis and ecological role of stilbenes in spruce (Picea), an important gymnosperm tree genus in temperate and boreal forests. To investigate the biosynthesis of stilbenes in spruce, we identified two similar stilbene synthase (STS) genes in Norway spruce (Picea abies), PaSTS1 and PaSTS2, which had orthologs with high sequence identity in sitka (Picea sitchensis) and white (Picea glauca) spruce. Despite the conservation of STS sequences in these three spruce species, they differed substantially from angiosperm STSs. Several types of in vitro and in vivo assays revealed that the P. abies STSs catalyze the condensation of p-coumaroyl-coenzyme A and three molecules of malonyl-coenzyme A to yield the trihydroxystilbene resveratrol but do not directly form the dominant spruce stilbenes, which are tetrahydroxylated. However, in transgenic Norway spruce overexpressing PaSTS1, significantly higher amounts of the tetrahydroxystilbene glycosides, astringin and isorhapontin, were produced. This result suggests that the first step of stilbene biosynthesis in spruce is the formation of resveratrol, which is further modified by hydroxylation, O-methylation, and O-glucosylation to yield astringin and isorhapontin. Inoculating spruce with fungal mycelium increased STS transcript abundance and tetrahydroxystilbene glycoside production. Extracts from STS-overexpressing lines significantly inhibited fungal growth in vitro compared with extracts from control lines, suggesting that spruce stilbenes have a role in antifungal defense.

A common fungal associate of the spruce bark beetle metabolizes the stilbene defenses of Norway spruce

The bark beetle-vectored fungus Ceratocystis polonica degrades stilbenoid defense compounds produced by its conifer host. Norway spruce (Picea abies) forests suffer periodic fatal attacks by the bark beetle Ips typographus and its fungal associate, Ceratocystis polonica. Norway spruce protects itself against fungal and bark beetle invasion by the production of terpenoid resins, but it is unclear whether resins or other defenses are effective against the fungus. We investigated stilbenes, a group of phenolic compounds found in Norway spruce bark with a diaryl-ethene skeleton with known antifungal properties. During C. polonica infection, stilbene biosynthesis was up-regulated, as evidenced by elevated transcript levels of stilbene synthase genes. However, stilbene concentrations actually declined during infection, and this was due to fungal metabolism. C. polonica converted stilbenes to ring-opened, deglycosylated, and dimeric products. Chromatographic separation of C. polonica protein extracts confirmed that these metabolites arose from specific fungal enzyme activities. Comparison of C. polonica strains showed that rapid conversion of host phenolics is associated with higher virulence. C. polonica is so well adapted to its host’s chemical defenses that it is even able to use host phenolic compounds as its sole carbon source.

Flavan-3-ols in Norway spruce: Biosynthesis, accumulation and function in response to attack by the bark beetle-associated fungus Ceratocystis polonica

Monomeric and polymeric flavan-3-ols are antifungal defense compounds in Norway spruce (Picea abies). Proanthocyanidins (PAs) are common polyphenolic polymers of plants found in foliage, fruit, bark, roots, rhizomes, and seed coats that consist of flavan-3-ol units such as 2,3-trans-(+)-catechin and 2,3-cis-(–)-epicatechin. Although the biosynthesis of flavan-3-ols has been studied in angiosperms, little is known about their biosynthesis and ecological roles in gymnosperms. In this study, the genes encoding leucoanthocyanidin reductase, a branch point enzyme involved in the biosynthesis of 2,3-trans-(+)-flavan-3-ols, were identified and functionally characterized in Norway spruce (Picea abies), the most widespread and economically important conifer in Europe. In addition, the accumulation of flavan-3-ols and PAs was investigated in Norway spruce saplings after wounding or inoculation with the fungal pathogen Ceratocystis polonica, which is vectored by bark beetles (Ips typographus) and is usually present during fatal beetle attacks. Monomeric and dimeric flavan-3-ols were analyzed by reverse-phase high-pressure liquid chromatography, while the size and subunit composition of larger PAs were characterized using a novel acid hydrolysis method and normal phase chromatography. Only flavan-3-ol monomers with 2,3-trans stereochemistry were detected in spruce bark; dimeric and larger PAs contained flavan-3-ols with both 2,3-trans and 2,3-cis stereochemistry. Levels of monomers as well as PAs with a higher degree of polymerization increased dramatically in spruce bark after infection by C. polonica. In accordance with their role in the biosynthesis of 2,3-trans-(+)-flavan-3-ols, transcript abundance of Norway spruce LEUCOANTHOCYANIDIN REDUCTASE genes also increased significantly during fungal infection. Bioassays with C. polonica revealed that the levels of 2,3-trans-(+)-catechin and PAs that are produced in the tree in response to fungal infection inhibit C. polonica growth and can therefore be considered chemical defense compounds.

Ri-plasmid mediated transformation and regeneration of Ulmus procera (English Elm)

Infection of Ulmus procera (English elm) cloneSR4 internodal stem explants with Agrobacteriumtumefaciens C58 c1 pRiA4b resulted in callusdevelopment and extensive hairy root production. Shoots which regenerated from hairy roots, followingan extended culture period, were dwarf in stature,with reduced apical dominance and wrinkled leaves whencompared with wild type U. procera SR4. Shootswere rooted successfully and plants with extensiveroot systems have been transferred to soil. Thetransgenic status of regenerants was confirmed by PCRanalysis and DNA sequencing of pRiA4b TL- and TR- DNArolA (329 bp) and agropine synthase (490 bp)primed amplimers, which were 100% homologous to theexpected sequences. No vir D1 primed PCRproducts were obtained, indicating that the Agrobacterium was successfully removed. Thepotential of Ri plasmid mediated transformation forinducing altered elm xylem structure, restrictedspread of the Dutch elm disease fungus and inphytoremediation is discussed.

An elm EST database for identifying leaf beetle egg-induced defense genes

BackgroundPlants can defend themselves against herbivorous insects prior to the onset of larval feeding by responding to the eggs laid on their leaves. In the European field elm (Ulmus minor), egg laying by the elm leaf beetle ( Xanthogaleruca luteola) activates the emission of volatiles that attract specialised egg parasitoids, which in turn kill the eggs. Little is known about the transcriptional changes that insect eggs trigger in plants and how such indirect defense mechanisms are orchestrated in the context of other biological processes.ResultsHere we present the first large scale study of egg-induced changes in the transcriptional profile of a tree. Five cDNA libraries were generated from leaves of (i) untreated control elms, and elms treated with (ii) egg laying and feeding by elm leaf beetles, (iii) feeding, (iv) artificial transfer of egg clutches, and (v) methyl jasmonate. A total of 361,196 ESTs expressed sequence tags (ESTs) were identified which clustered into 52,823 unique transcripts (Unitrans) and were stored in a database with a public web interface. Among the analyzed Unitrans, 73% could be annotated by homology to known genes in the UniProt (Plant) database, particularly to those from Vitis, Ricinus, Populus and Arabidopsis. Comparative in silico analysis among the different treatments revealed differences in Gene Ontology term abundances. Defense- and stress-related gene transcripts were present in high abundance in leaves after herbivore egg laying, but transcripts involved in photosynthesis showed decreased abundance. Many pathogen-related genes and genes involved in phytohormone signaling were expressed, indicative of jasmonic acid biosynthesis and activation of jasmonic acid responsive genes. Cross-comparisons between different libraries based on expression profiles allowed the identification of genes with a potential relevance in egg-induced defenses, as well as other biological processes, including signal transduction, transport and primary metabolism.ConclusionHere we present a dataset for a large-scale study of the mechanisms of plant defense against insect eggs in a co-evolved, natural ecological plant–insect system. The EST database analysis provided here is a first step in elucidating the transcriptional responses of elm to elm leaf beetle infestation, and adds further to our knowledge on insect egg-induced transcriptomic changes in plants. The sequences identified in our comparative analysis give many hints about novel defense mechanisms directed towards eggs.

Smelling the tree and the forest: elm background odours affect egg parasitoid orientation to herbivore induced terpenoids

Parasitic insects use herbivore induced plant volatiles as signals for host location. However, their responses to these volatiles in the background of natural habitat odours need further evaluation for developing successful biological control strategies. Field elms (Ulmus minor Miller (Ulmaceae)) release a blend of volatiles in response to oviposition of the elm leaf beetle, Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), a major urban and forest pest in the USA and Australia. This induced blend attracts the beneficial egg parasitoid Oomyzus gallerucae Fonscolombe (Hymenoptera: Eulophidae). Our olfactory assays showed that an odorous background of non-attractive host plant volatiles from feeding damaged elms or (Z)-3-hexenyl acetate masks the attractive effect of the host-induced (E)-β-caryophyllene to O. gallerucae. Quantitative GC–MS analyses revealed decreased concentrations of (Z)-3-hexenyl acetate accompanied by highly increased concentrations of sesquiterpenes in oviposition and feeding treated elms compared to undamaged elms. This finding hints to how the parasitoid might distinguish between different odorous backgrounds. It is corroborated by the outcome of our field study in natural elm stands, where the egg parasitoid parasitized more host egg masses due to an artificially induced blend of elm terpenoids.

Elm defence against herbivores and pathogens: morphological, chemical and molecular regulation aspects

Elms (Ulmus spp.) have long been appreciated for their environmental tolerance, landscape and ornamental value, and the quality of their wood. Although elm trees are extremely hardy against abiotic stresses such as wind and pollution, they are susceptible to attacks of biotic stressors. Over 100 phytopathogens and invertebrate pests are associated with elms: fungi, bacteria and insects like beetles and moths, and to a lesser extent aphids, mites, viruses and nematodes. While the biology of the pathogen and insect vector of the Dutch elm disease has been intensively studied, less attention has been paid so far to the defence mechanisms of elms to other biotic stressors. This review highlights knowledge of direct and indirect elm defences against biotic stressors focusing on morphological, chemical and gene regulation aspects. First, we report how morphological defence mechanisms via barrier formation and vessel occlusion prevent colonisation and spread of wood- and bark-inhabiting fungi and bacteria. Second, we outline how secondary metabolites such as terpenoids (volatile terpenoids, mansonones and triterpenoids) and phenolics (lignans, coumarins, flavonoids) in leaves and bark are involved in constitutive and induced chemical defence mechanisms of elms. Third, we address knowledge on how the molecular regulation of elm defence is orchestrated through the interaction of a huge variety of stress- and defence-related genes. We conclude by pointing to the gaps of knowledge on the chemical and molecular mechanisms of elm defence against pest insects and diseases. An in-depth understanding of defence mechanisms of elms will support the development of sustainable integrated management of pests and diseases attacking elms.

Genetic manipulation with elms.

Genetic manipulation by transformation is a powerful alternative to conventional elm breeding programs, allowing the rapid introduction of small numbers of desirable genes into elite genotypes without disrupting their better genetic features. To achieve this, systems for the micropropagation and regeneration of English elm (Ulmus procera), American elm (Ulmus americana,), and wych elm (Ulmus glabra) have been established using Driver & Kuniyuki Walnut based media. Following extensive testing, protocols for the genetic modification of elms have been developed using Agrobacterium tumefaciens, A. rhizogenes and biolistics. Transformed regenerant elms have been produced using wild type A. tumefaciens and A. rhizogenes. For the first time, genetically modified regenerant elms have been obtained using antibiotic resistance selectable markers, beta-glucuronidase and putative disease resistance genes, with binary vectors and disarmed Agrobacterium strains. Jellyfish green fluorescent protein has successfully been used as a reporter gene in particle inflow gun studies. The potential value of this effective elm transformation and regeneration system for the control of Dutch elm disease is discussed.