Metabolic engineering and potential functions of proanthocyanidins in poplar

Abstract

Poplar (Populus spp.) is a widely distributed tree genus of significant economic and ecological importance. Poplar trees accumulate proanthocyanidins (PAs) in leaves, roots, and a variety of other tissues. Damage to leaves by insects causes a rapid accumulation of PAs, both at the site of damage and distally in undamaged leaves. This rapid PA accumulation is mediated by the activation of genes encoding enzymes involved in PA synthesis. PAs have been hypothesized to deter insect feeding and reduce the nutritive value of poplar leaf tissue, but experimental evidence supporting a role for PAs as an effective inducible defense against herbivores is lacking. Our recent paper described the identification of a MYB gene that regulates the PA pathway under multiple stress conditions, and we used this gene to constitutively activate the PA pathway in poplar. Here we describe observations that suggest that poplar PAs may have roles besides insect defense, for example, responses to UV light. The PA-modified trees will be a useful tool for analyzing the biological roles of PAs in this important model tree.

Introduction

Proanthocyanidins (PAs), or condensed tannins, are polymeric flavonoids found in a wide variety of plants but are especially common in trees. In herbaceous plants, they are most commonly found in the seed coat where they may help control seed permeability, but also occur in leaves of plants such as sainfoin (Onobrychis viciifolia).¹ In perennials, tannins can be found in roots, bark and leaves, with levels as high as 20% dry weight.² PAs are widely assumed to contribute to plant defense against insect herbivores, an idea derived in part from classic chemical ecology experiments correlating tannin levels in oak leaves with insect herbivory.^3,4 Nevertheless, the evidence for a role of PAs and other tannins in defense is mixed, and recent work is challenging the assumption that these phenolics are necessarily defensive chemicals.^5–7

PAs are upregulated by herbivory and leaf damage in some Populus species, and we first demonstrated that this induction follows transcriptional activation of genes encoding flavonoid pathway enzymes.⁸ We also previously reported that hybrid poplar responds to infection by the compatible fungal biotroph Melampsora medusae with a late upregulation of the PA biosynthetic pathway.⁹ The inducibility of the PA pathway in Populus supports the idea of a role of PAs in poplar defense, and facilitated the identification of the PA regulator MYB134 using expression profiling of genes with similarity to the Arabidopsis TT2 MYB transcription factor. Our recently published report¹⁰ indicates that transgenic P. tremula x tremuloides plants that overexpress the poplar MYB134 gene show strong upregulation of all known flavonoid pathway genes required for PA biosynthesis. Significantly, these plants accumulate up to 50-fold higher PA levels relative to controls, underscoring the importance of the MYB134 gene in regulating poplar PAs.¹⁰

MYB-Overexpressing Transgenic Poplar Plants Show Modified Stress Susceptibility

Under normal greenhouse conditions we observed no phenotypic abnormalities in MYB134-overexpressing poplar (Fig. 1A). However, during a chance outbreak of greenhouse thrips, we noticed more damage to leaves of MYB134 overexpressors than to control (GUS) trees (Fig. 1B). The damage was visible as small holes and tears in leaf blades, which presumably occurred as a result of leaf expansion in areas where the thrips had made small puncture wounds. These observations indicated that the thrips were preferentially feeding on the MYB134 transgenics, suggesting either decreased resistance or increased palatability of this foliage to the pests. This is contrary to the hypothesized defensive role of the PAs; however, the interpretation of these observations is confounded by the unexpected reduction in other phenolic constituents. Specifically, the salicylate-derived phenolic glycoside (PG) concentrations in MYB134 overexpressor leaf tissue are approximately three-fold lower compared to controls. The PGs are known anti-insect defenses of poplar, and previous work has shown that PG consumption negatively impacts lepidopteran larval development and survival.^11,12 Thus it is possible that despite the very high PA levels, the transgenic plants are actually compromised in their defense against herbivores because of reduced PG levels. We are currently investigating this in more detail with insect bioassays and different poplar pests, since the potential impact of PAs for poplar defense is likely to depend on the target insects.

Figure 1.

MYB134 overexpressor P. tremula x tremuloides (clone INRA 353-38) trees exhibit a distinct damage phenotype after a greenhouse thrips outbreak. (A) Photograph of typical GUS control leaf after the outbreak. (B) Photograph of typical MYB134 overexpressor 353-38 leaf after thrips outbreak showing leaf damage (arrow). (C) Percentage of control and MYB134 overexpressor trees with leaves showing signs of thrips damage after infestation (GUS, n = 24 trees; MYB134-OE, n = 20 trees; all trees 8 weeks old).

Alternatively, PA accumulation following herbivore damage may reflect other functions, or a broader response to stress in poplar leaves. Our recent report showed that abiotic stresses, in particular UV-B exposure, cause a strong transcriptional activation of the PA biosynthetic pathway and a significant increase in leaf PA concentrations in hybrid aspen plants.¹⁰ Genes for biosynthesis of flavonols, common UV screens in plants, were also induced, indicating that the UV-B treatment imposed was appropriate for eliciting a UV-B stress response. PA synthesis in response to UV-B radiation has been reported only for a few species, for example birch.¹³ Therefore we repeated this UV induction experiment with P. trichocarpa clone Nisqually 1, the genotype for which the complete genome sequence is available. Both wound- and UV-B-inducible PA accumulation were demonstrated with this genotype as well (Fig. 2). The enhancement of PA accumulation as a result of UV-B exposure confirms our original observation, and suggests that the induction of PAs in poplars by UV-B may have some functional importance.

Figure 2.

Stress-induced accumulation of PAs in leaves of Populus trichocarpa (clone Nisqually 1). (A) PA levels in control and plier-wounded leaves 7 days after treatment. (B) PA levels in control and UV-B-exposed poplar leaves 7 days after treatment. UV-B exposure was for 8 h day-1 in a growth chamber, as described in Mellway et al.¹⁰ PAs were quantified using the acid-butanol method.¹⁸ Bars indicate means of four trees per treatment, with error bars indicating the SE of mean. Asterisks indicate significant differences using Student's t test (*p < 0.05).

The accumulation of PAs in UV-B-exposed leaves may be a direct response to elevated UV-B, or the result of secondary effects, for example, increased levels of reactive oxygen molecules. As an initial step in evaluating such a stress-protective role, six-week-old MYB134-overexpressing and control saplings were exposed to UV-B irradiation, and growth monitored over a two-week period. Under normal greenhouse growth conditions, MYB134 overexpressors and controls exhibited no significant difference in height or other growth parameters.¹⁰ However, after two weeks of elevated UV-B irradiation, MYB134 overexpressors had a significantly (Student's t test, p < 0.05) greater height increase than control plants (30.1 ± 1.5 and 23.9 ± 2.1 cm, respectively). This suggests that the elevated PA levels may help to protect the leaves against the damaging effects associated with elevated UV-B radiation. Again, the minor secondary alterations to phenolic metabolism in leaves of MYB134-overexpressing trees confound the interpretation of the results, since these transgenics had an approximately two-fold increase in total flavonol glycoside concentrations relative to controls. Flavonols are known to be potent UV-B protective molecules, although their levels here were low compared to the PAs. Additional analyses will need to be conducted to detangle the roles of different phenolic metabolites in resistance to UV-B or other stresses.

Conclusions

Oxidative stress is a common component of many different biotic and abiotic stress conditions, including excess light, pathogen infection and possibly mechanical damage.^14,15 Under some conditions, PAs may act as antioxidants^5,16 and it has been suggested that oxidative stress protection may be an important function of many plant phenolics.¹⁷ Our results with leaf PAs in poplar are consistent with this idea. PAs are likely multifunctional and may contribute to plant health in multiple ways. We note that investigations into potential functions of PAs need to take into account their chemical and structural diversity.⁷ Metabolic engineering of the PA pathway in Populus will facilitate experiments to further define the ecological roles of these widespread plant chemicals.

Addendum to: Mellway RD, Tran LT, Prouse MB, Campbell MM, Constabel CP. The wound-, pathogen- and UV-B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar. Plant Physiol. 2009;150:924–941. doi: 10.1104/pp.109.139071.