Tissue and cellular localization of condensed tannins in poplar roots and potential association with nitrogen uptake.

Condensed tannins are common in vegetative tissues of woody plants, including in roots. In hybrid poplar (Populus tremula x alba; also known as P. x canescens) CT assays indicated they were most concentrated in younger white roots and at the root tip. Furthermore, CT-specific staining of embedded tissue sections demonstrated accumulation in root cap cells and adjacent epidermal cells, as well as a more sporadic presence in cortex cells. In older, brown roots as well as roots with secondary growth (cork zone), CT concentration was significantly lower. The insoluble fraction of CTs was greatest in the cork zone. To determine if CT accumulation correlates with nutrient uptake in poplar roots, a microelectrode ion flux measurement (MIFE™) system was used to measure flux along the root axis. Greatest NH4 + uptake was measured near the root tip, but NO3- and Ca2+ did not vary along the root length. In agreement with earlier work, providing poplars with ample nitrogen led to higher accumulation of CTs across root zones. To test the functional importance of CTs in roots directly, CT-modified transgenic plants could be important tools.

CRISPR/Cas9 Disruption of MYB134 and MYB115 in Transgenic Poplar Leads to Differential Reduction of Proanthocyanidin Synthesis in Roots and Leaves.

Proanthocyanidins (PAs) are common specialized metabolites and particularly abundant in trees and woody plants. In poplar (Populus spp.), PA biosynthesis is stress-induced and regulated by two previously studied transcription factors MYB115 and MYB134. To determine the relative contribution of these regulators to PA biosynthesis, we created single- and double-knockout (KO) mutants for both genes in transgenic poplars using CRISPR/Cas9. Knocking out either MYB134 or MYB115 showed reduced PA accumulation and downregulated flavonoid genes in leaves, but MYB134 disruption had the greatest impact and reduced PAs to 30% of controls. In roots, by contrast, only the MYB134/MYB115 double-KOs showed a significant change in PA concentration. The loss of PAs paralleled the lower expression of PA biosynthesis genes and concentrations of flavan-3-ol PA precursors catechin and epicatechin. Interestingly, salicinoids were also affected in double-KOs, with distinct patterns in roots and shoots. We conclude that the regulatory pathways for PA biosynthesis differ in poplar leaves and roots. The residual PA content in the double-KO plants indicates that other transcription factors must also be involved in control of the PA pathway.

Fungal Community Composition as Affected by Litter Chemistry and Weather during Four Years of Litter Decomposition in Rainshadow Coastal Douglas-Fir Forests.

Climate and litter chemistry are major factors influencing litter decay, a process mediated by microbes, such as fungi, nitrogen-fixing bacteria and ammonia-oxidizing bacteria. Increasing atmospheric CO2 concentrations can decrease nitrogen (N) and increase condensed tannin (CT) content in foliar litter, reducing litter quality and slowing decomposition. We hypothesized that reduced litter quality inhibits microbes and is the mechanism causing decomposition to slow. Litterbags of Douglas-fir needles and poplar leaves with a range of N (0.61-1.57%) and CT (2.1-29.1%) treatment and natural acid unhydrolyzable residue (35.3-41.5%) concentrations were placed along climatic gradients in mature Douglas-fir stands of coastal British Columbia rainshadow forests. The structure (diversity, richness and evenness) and composition of microbial communities were analyzed using DGGE profiles of 18S, NifH-universal and AmoA PCR amplicons in foliar litter after 7, 12, 24 and 43 months of decay. High CT and low N concentrations in leaf litter were associated with changes in microbial community composition, especially fungi. Contrary to our hypothesis, high CT and low N treatments did not inhibit microbial colonization or diversity. The joint effects of air temperature and soil moisture on microbial community composition at our sites were more important than the effects of initial litter chemistry.

CRISPR/Cas9 disruption of UGT71L1 in poplar connects salicinoid and salicylic acid metabolism and alters growth and morphology.

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore effects found only in poplars and willows. Their biosynthesis is poorly understood, but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here, we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knockouts contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.

Factors Affecting Foliar Oregonin and Condensed Tannin in Red Alder (Alnus rubra Bong.): Phytochemicals Implicated In Defense Against Western Tent Caterpillar (Malacosoma californicum Packard).

Past work shows a significant negative correlation between foliar oregonin concentration and western tent caterpillar (Malacosoma californicum Packard) feeding on red alder (Alnus rubra Bong.). Above an oregonin threshold of 20% leaf dry weight, little feeding by caterpillars is observed. Concentrations of defensive chemicals are influenced by plant genotype, environmental conditions, insect feeding, and the interactions of these factors. Our objective was to measure the effects of nitrogen (N) availability and wounding on foliar oregonin and condensed tannin concentrations in red alder genotypes. One-year-old seedlings from 100 half-sib red alder families were treated with two levels of ammonium nitrate (NH4NO3) for two growing seasons in a common garden. In the second year, leaves from 50 families from the fertilization experiment were used in a bioassay feeding experiment to determine the effects of N fertilization and genotype on WTC damage, and to identify a subset of 20 families with a range of damage to analyze for phytochemical composition. In separate experiments, wound-induction treatments were conducted outdoors and, in a greenhouse using the N treated trees in their third and fourth year, respectively. Foliar condensed tannin, oregonin and N concentrations were measured and ranked among the plant genotypes, and between the two N treatments and two wounding treatments. Results showed that oregonin and condensed tannin concentrations varied among the alder genotypes. Leaf N concentration was negatively correlated with concentration of oregonin. Neither of the measured phenolic compounds responded to wounding. The results suggest that red alder foliar oregonin and condensed tannin are likely constitutive defenses that are largely determined by genotype, and that the negative correlation of defense compounds with plant internal N status holds in this N-fixing tree.

Poplar MYB117 promotes anthocyanin synthesis and enhances flavonoid B-ring hydroxylation by up-regulating the flavonoid 3',5'-hydroxylase gene.

Flavonoids, such as anthocyanins, proanthocyanidins, and flavonols, are widespread plant secondary metabolites and important for plant adaptation to diverse abiotic and biotic stresses. Flavonoids can be variously hydroxylated and decorated; their biological activity is partly dependent on the degree of hydroxylation of the B-ring. Flavonoid biosynthesis is regulated by MYB transcription factors, which have been identified and characterized in a diversity of plants. Here we characterize a new MYB activator, MYB117, in hybrid poplar (Populus tremula×tremuloides). When overexpressed in transgenic poplar plants, MYB117 enhanced anthocyanin accumulation in all tissues. Transcriptome analysis of MYB117-overexpressing poplars confirmed the up-regulation of flavonoid and anthocyanin biosynthesis genes, as well as two flavonoid 3',5'-hydroxylase (F3'5'H) genes. We also identified up-regulated cytochrome b5 genes, required for full activity of F3'5'H . Phytochemical analysis demonstrated a corresponding increase in B-ring hydroxylation of anthocyanins, proanthocyanidins, and flavonols in these transgenics. Similarly, overexpression of F3'5'H1 directly in hybrid poplar also resulted in increased B-ring hydroxylation, but without affecting overall flavonoid content. However, the overexpression of the cytochrome b5 gene in F3'5'H1-overexpressing plants did not further increase B-ring hydroxylation. Our data indicate that MYB117 regulates the biosynthesis of anthocyanins in poplar, but also enhances B-ring hydroxylation by up-regulating F3'5'H1.

Anti-Herbivore Activity of Oregonin, a Diarylheptanoid Found in Leaves and Bark of Red Alder (Alnus rubra).

Plants synthesize a wide range of bioactive secondary metabolites to defend against pests and pathogens. Red alder (Alnus rubra) bark, root, and leaf extract have a long history of use in traditional medicine and hygiene. Diarylheptanoids, especially oregonin ((5S)-1,7-bis(3,4-dihydroxyphenyl)-5-(ß-D-xylopyranosyloxy)-heptan-3-one), have been identified as major bioactive constituents. Diarylheptanoids have become a focus of research following reports of their antioxidant, antifungal, and anti-cancer activities. Recent data suggest that high oregonin concentration is associated with resistance of red alder leaves to western tent caterpillar (Malacosoma californicum) defoliation. Here we test effects of this compound directly on leaf-eating insects. Purified oregonin was examined in insect choice and toxicity tests using lepidopteran caterpillars. The compound exhibited significant anti-feedant activity against cabbage looper (Trichoplusia ni), white-marked tussock moth (Orgyia leucostigma), fall webworm (Hyphantria cunea), and M. californicum at concentrations corresponding to oregonin content of the most resistant alder clones in previous experiments. Toxicity tests were carried out with cabbage looper larvae only, but no contact or ingested toxicity was detected. Our results suggest that oregonin at levels found in red alder leaves early in the growing season may contribute to protecting red alder from leaf-eating insects.

Efficient purification of the diarylheptanoid oregonin from red alder (Alnus rubra) leaves and bark combining aqueous extraction, spray drying and flash-chromatography.

INTRODUCTION: The diarylheptanoid xyloside oregonin ((5S)-1,7-bis(3,4-dihydroxyphenyl)-5-(ß-d-xylopyranosyloxy)-heptan-3-one) has significant medicinal potential and is found at high concentration in leaves and bark of red alder (Alnus rubra). OBJECTIVES: To establish inexpensive and easily scaled methods for the extraction and purification of oregonin from timber by-products. METHODS: We developed a method combining aqueous extraction with spray drying of red alder extract into a powder, thus reducing the need for organic solvents used in traditional Soxhlet extraction or in solvent partitioning. Flash chromatography was utilised to purify oregonin from crude spray-dried alder extract. RESULTS: Crude spray-dried alder extract was comprised of an average of 9% of the diarylheptanoid compound oregonin. Less than 10% thermal degradation of oregonin was observed using extraction temperatures between 25°C and 50°C, followed by spray drying. The structure of purified oregonin was validated using high-performance liquid chromatography (HPLC), mass spectrometry (MS), ultraviolet spectroscopy (UV), and nuclear magnetic resonance (NMR). CONCLUSION: The developed method was robust, repeatable, and yielded purified oregonin of greater than > 95% purity (average of 95.8%). Our analysis represents the most complete NMR characterisation of oregonin reported to date.

MYB134-RNAi poplar plants show reduced tannin synthesis in leaves but not roots, and increased susceptibility to oxidative stress.

The importance of the poplar MYB134 gene in controlling condensed tannin (CT) biosynthesis was tested by suppressing its expression using RNA interference (RNAi). MYB134-RNAi plants grew normally but showed reduced accumulation of stress-induced CTs in leaves. RNA-seq analysis indicated that flavonoid- and CT-related genes, as well as additional CT regulators, were strongly and specifically down-regulated by MYB134 suppression. This confirmed that the primary MYB134 target is the leaf flavonoid and CT pathway. Root CT accumulation was not impacted by MYB suppression, suggesting that additional CT regulators are active in roots and emphasizing the complexity of the regulation of CTs in poplar. To test the effect of CT down-regulation on oxidative stress resistance, leaves of MYB134-RNAi and control plants were exposed to the reactive oxygen species generator methyl viologen. MYB134-RNAi leaves sustained significantly more photosystem II damage, as seen in reduced chlorophyll fluorescence, compared with wild-type leaves. MYB134-RNAi leaves also contained more hydrogen peroxide, a reactive oxygen species, compared with the wild type. Our data thus corroborate the hypothesis that CT can act as an antioxidant in vivo and protect against oxidative stress. Overall, MYB134 was shown to be a central player in the regulation of CT synthesis in leaves.

The Occurrence of Sulfated Salicinoids in Poplar and Their Formation by Sulfotransferase1.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

Condensed tannins are inducible antioxidants and protect hybrid poplar against oxidative stress.

Condensed tannins (CTs) have been studied extensively as potential defenses against pests and pathogens, and for their beneficial effects on human health. They are known to possess high in vitro antioxidant capacity, but whether they can function as in planta antioxidants for protection against oxidative stress has not been previously tested. Here, we show that stress induction of CTs in poplar (Populus) is matched closely by an increase in antioxidant activity under both high light and nitrogen deficiency. We also investigate the effects of CTs as in vivo antioxidants directly, using transgenic poplar plants which overexpress poplar MYB transcription factors that regulate the CT pathway. These transgenics have 50-fold higher CT concentrations than controls, and and also have dramatically higher antioxidant activity. High-CT and control poplar leaves were exposed to methyl viologen for 24 h. Chlorophyll fluorescence was used to measure maximum quantum efficiency of photosystem II photochemistry (Fv/Fm), and leaf discs were stained with 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) to assess hydrogen peroxide and superoxide levels. After methyl viologen exposure, high-CT transgenics retained higher Fv/Fm ratios and accumulated less hydrogen peroxide and superoxide than the controls. Our findings indicate that high-CT concentrations protect poplar against methyl viologen-induced oxidative stress and suggest a broader function of CTs than previously supposed.

MYB Repressors as Regulators of Phenylpropanoid Metabolism in Plants.

The phenylpropanoid pathway gives rise to lignin, flavonoids, and other metabolites and is regulated by MYB transcription factors. Many R2R3-MYB transcriptional activators are known, but the prevalence of MYB repressors has only recently become recognized. This review article summarizes recent progress on function and mechanism of these MYB repressors. The characterized phenylpropanoid R2R3-MYB repressors comprise two phylogenetic clades that act on the lignin and general phenylpropanoid genes, or the flavonoid genes, respectively; anthocyanin R3-MYB repressors form a separate clade. While some flavonoid MYBs repressors can bind basic-helix-loop-helix factors and disrupt the MBW complex, for the lignin repressor MYBs interactions with promoter cis-elements have been demonstrated. The role of the conserved repression motifs that define the MYB repressors is not yet known, however.

Two R2R3-MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar.

The phenylpropanoid pathway leads to the production of many important plant secondary metabolites including lignin, chlorogenic acids, flavonoids, and phenolic glycosides. Early studies have demonstrated that flavonoid biosynthesis is transcriptionally regulated, often by a MYB, bHLH, and WDR transcription factor complex. In poplar, several R2R3 MYB transcription factors are known to be involved in flavonoid biosynthesis. Previous work determined that poplar MYB134 and MYB115 are major activators of the proanthocyanidin pathway, and also induce the expression of repressor-like MYB transcription factors. Here we characterize two new repressor MYBs, poplar MYB165 and MYB194, paralogs which comprise a subgroup of R2R3-MYBs distinct from previously reported poplar repressors. Both MYB165 and MYB194 repressed the activation of flavonoid promoters by MYB134 in transient activation assays, and both interacted with a co-expressed bHLH transcription factor, bHLH131, in yeast two-hybrid assays. Overexpression of MYB165 and MYB194 in hybrid poplar resulted in greatly reduced accumulation of several phenylpropanoids including anthocyanins, proanthocyanidins, phenolic glycosides, and hydroxycinnamic acid esters. Transcriptome analysis of MYB165- and MYB194-overexpressing poplars confirmed repression of many phenylpropanoid enzyme genes. In addition, other MYB genes as well as several shikimate pathway enzyme genes were downregulated by MYB165-overexpression. By contrast, leaf aromatic amino acid concentrations were greater in MYB165-overexpressing poplars. Our findings indicate that MYB165 is a major repressor of the flavonoid and phenylpropanoid pathway in poplar, and may also affect the shikimate pathway. The coordinated action of repressor and activator MYBs could be important for the fine tuning of proanthocyanidin biosynthesis during development or following stress.

Molecular Controls of Proanthocyanidin Synthesis and Structure: Prospects for Genetic Engineering in Crop Plants.

Proanthocyanidins (PAs) are widespread oligomeric and polymeric flavan-3-ols with significant benefits to human and animal health. As products of the general flavonoid pathway, the biosynthesis of the flavan-3-ols is well-understood and the major enzyme-encoding genes that determine PA structure have been identified. However, the mechanism of PA polymerization remains unknown. The most important transcription factors regulating PA biosynthesis are the MYB factors, potent tools for enhancing PA biosynthesis in plants. In some species, simple overexpression of these transcription factors has led to spectacular successes in upregulating PA synthesis. However, targeted metabolic engineering of the PA structure has not yet been achieved.

Phytochemical analysis of salal berry (Gaultheria shallon Pursh.), a traditionally-consumed fruit from western North America with exceptionally high proanthocyanidin content.

Salal (Gaultheria shallon Pursh.) is a wild perennial shrub of the Ericaceae and common in coastal forests of western North America, and its berries were an important traditional food for First Nations in British Columbia. Salal berries were investigated for phytochemical content and antioxidant capacity over the course of fruit development. The proanthocyanidin content was extremely high in young berries (280.7 mg/g dry wt) but dropped during development to 52.8 mg/g dry wt. By contrast, anthocyanins accumulated only at the late berry stages. Total antioxidant capacity, as measured by the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method, reflected both proanthocyanidin and anthocyanin content, and in mature berries reached 36 mmol Trolox equivalents/100 g dry wt. More detailed phytochemical analysis determined that delphinidin 3-O-galactoside is the dominant anthocyanin, and that the berries are also rich in procyanidins, including procyanidin A2 which has been implicated in anti-adhesion activity for uropathogenic E. coli. Proanthocyanidins were 60% prodelphinidin, and overall concentrations were higher than reported for many Vaccinium species including blueberry, lingonberry, and cranberry. Overall, the phenolic profile of salal berries indicates that these fruit contain a diversity of health-promoting phenolics.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection.

Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(-)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula × Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

An improved butanol-HCl assay for quantification of water-soluble, acetone:methanol-soluble, and insoluble proanthocyanidins (condensed tannins).

BACKGROUND: Condensed tannins (CT) are the most abundant secondary metabolite of land plants and can vary in abundance and structure according to tissue type, species, genotype, age, and environmental conditions. Recent improvements to the butanol-HCl assay have separately helped quantification of soluble and insoluble CTs, but have not yet been applied jointly. Our objectives were to combine previous assay improvements to allow for quantitative comparisons of different condensed tannin forms and to test protocols for analyses of condensed tannins in vegetative plant tissues. We also tested if the improved butanol-HCl assay can be used to quantify water-soluble forms of condensed tannins. RESULTS: Including ~50% acetone in both extraction solvents and final assay reagents greatly improved the extraction and quantification of soluble, insoluble and total condensed tannins. The acetone-based method also extended the linear portion of standard integration curves allowing for more accurate quantification of samples with a broader range of condensed tannin concentrations. Estimates of tannin concentrations determined using the protocol without acetone were lower, but correlated with values from acetone-based methods. With the improved assay, quantification of condensed tannins in water-soluble forms was highly replicable. The relative abundance of condensed tannins in soluble and insoluble forms differed substantially between tissue types. CONCLUSIONS: The quantification of condensed tannins using the butanol-HCl assay was improved by adding acetone to both extraction and reagent solutions. These improvements will facilitate the quantification of total condensed tannin in tissues containing a range of concentrations, as well as to determine the amount in water-soluble, acetone:MeOH-soluble and insoluble forms. Accurate determination of these three condensed tannin forms is essential for careful investigations of their potentially different physiological and ecological functions.

Poplar MYB115 and MYB134 Transcription Factors Regulate Proanthocyanidin Synthesis and Structure.

The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a high-proanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 3',5'-hydroxylases and cytochrome b5 Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.

Influence of Genotype, Environment, and Gypsy Moth Herbivory on Local and Systemic Chemical Defenses in Trembling Aspen (Populus tremuloides).

Numerous studies have explored the impacts of intraspecific genetic variation and environment on the induction of plant chemical defenses by herbivory. Relatively few, however, have considered how those factors affect within-plant distribution of induced defenses. This work examined the impacts of plant genotype and soil nutrients on the local and systemic phytochemical responses of trembling aspen (Populus tremuloides) to defoliation by gypsy moth (Lymantria dispar). We deployed larvae onto foliage on individual tree branches for 15 days and then measured chemistry in leaves from: 1) branches receiving damage, 2) undamaged branches of insect-damaged trees, and 3) branches of undamaged control trees. The relationship between post-herbivory phytochemical variation and insect performance also was examined. Plant genotype, soil nutrients, and damage all influenced phytochemistry, with genotype and soil nutrients being stronger determinants than damage. Generally, insect damage decreased foliar nitrogen, increased levels of salicinoids and condensed tannins, but had little effect on levels of a Kunitz trypsin inhibitor, TI3. The largest damage-mediated tannin increases occurred in leaves on branches receiving damage, whereas the largest salicinoid increases occurred in leaves of adjacent, undamaged branches. Foliar nitrogen and the salicinoid tremulacin had the strongest positive and negative relationships, respectively, with insect growth. Overall, plant genetics and environment concomitantly influenced both local and systemic phytochemical responses to herbivory. These findings suggest that herbivory can contribute to phytochemical heterogeneity in aspen foliage, which may in turn influence future patterns of herbivory and nutrient cycling over larger spatial scales.

Functional characterization of two acyltransferases from Populus trichocarpa capable of synthesizing benzyl benzoate and salicyl benzoate, potential intermediates in salicinoid phenolic glycoside biosynthesis.

Salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Although their biosynthetic pathway is not known, recent work has suggested that benzyl benzoate may be a potential biosynthetic intermediate. Two candidate genes, named PtACT47 and PtACT49, encoding BAHD-type acyl transferases were identified and are predicted to produce such benzylated secondary metabolites. Herein described are the cDNA cloning, heterologous expression and in vitro functional characterization of these two BAHD acyltransferases. Recombinant PtACT47 exhibited low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM(-1) s(-1)) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA: salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, including benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM(-1) s(-1)) and lowest Km (55.3 µM) were observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA: benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters, such as hexenyl benzoate, at trace levels. Although the activities demonstrated are consistent with roles in salicinoid biosynthesis, direct tests of this hypothesis using transgenic poplar must still be performed.