Condensed tannins as antioxidants that protect poplar against oxidative stress from drought and UV-B.

Condensed tannins (CTs, proanthocyanidins) are widespread polymeric flavan-3-ols known for their ability to bind proteins. In poplar (Populus spp.), leaf condensed tannins are induced by both biotic and abiotic stresses, suggesting diverse biological functions. Here we demonstrate the ability of CTs to function as physiological antioxidants, preventing oxidative and cellular damage in response to drought and UV-B irradiation. Chlorophyll fluorescence was used to monitor photosystem II performance, and both hydrogen peroxide and malondialdehyde content was assayed as a measure of oxidative damage. Transgenic MYB-overexpressing poplar (Populus tremula × P. tremuloides) with high CT content showed reduced photosystem damage and lower hydrogen peroxide and malondialdehyde content after drought and UV-B stress. This antioxidant effect of CT was observed using two different poplar MYB CT regulators, in multiple independent lines and different genetic backgrounds. Additionally, low-CT MYB134-RNAi transgenic poplars showed enhanced susceptibility to drought-induced oxidative stress. UV-B radiation had different impacts than drought on chlorophyll fluorescence, but all high-CT poplar lines displayed reduced sensitivity to both stresses. Our data indicate that CTs are significant defences against oxidative stress. The broad distribution of CTs in forest systems that are exposed to diverse abiotic stresses suggests that these compounds have wider functional roles than previously realized.

Discovery of salicyl benzoate UDP-glycosyltransferase, a central enzyme in poplar salicinoid phenolic glycoside biosynthesis.

The salicinoids are anti-herbivore phenolic glycosides unique to the Salicaceae (Populus and Salix). They consist of a salicyl alcohol glucoside core, which is usually further acylated with benzoic, cinnamic or phenolic acids. While salicinoid structures are well known, their biosynthesis remains enigmatic. Recently, two enzymes from poplar, salicyl alcohol benzoyl transferase and benzyl alcohol benzoyl transferase, were shown to catalyze the production of salicyl benzoate, a predicted potential intermediate in salicinoid biosynthesis. Here, we used transcriptomics and co-expression analysis with these two genes to identify two UDP-glucose-dependent glycosyltransferases (UGT71L1 and UGT78M1) as candidate enzymes in this pathway. Both recombinant enzymes accepted only salicyl benzoate, salicylaldehyde and 2-hydroxycinnamic acid as glucose acceptors. Knocking out the UGT71L1 gene by CRISPR/Cas9 in poplar hairy root cultures led to the complete loss of salicortin, tremulacin and tremuloidin, and a partial reduction of salicin content. This demonstrated that UGT71L1 is required for synthesis of the major salicinoids, and suggested that an additional route can lead to salicin. CRISPR/Cas9 knockouts for UGT78M1 were not successful, and its in vivo role thus remains to be determined. Although it has a similar substrate preference and predicted structure as UGT71L1, it appears not to contribute to the synthesis of salicortin, tremulacin and tremuloidin, at least in roots. The demonstration of UGT71L1 as an enzyme of salicinoid biosynthesis will open up new avenues for the elucidation of this pathway.

Tannins in plant-herbivore interactions.

Tannins are the most abundant secondary metabolites made by plants, commonly ranging from 5% to 10% dry weight of tree leaves. Tannins can defend leaves against insect herbivores by deterrence and/or toxicity. Contrary to early theories, tannins have no effect on protein digestion in insect herbivores. By contrast, in vertebrate herbivores tannins can decrease protein digestion. Tannins are especially prone to oxidize in insects with high pH guts, forming semiquinone radicals and quinones, as well as other reactive oxygen species. Tannin toxicity in insects is thought to result from the production of high levels of reactive oxygen species. Tannin structure has an important effect on biochemical activity. Ellagitannins oxidize much more readily than do gallotannins, which are more oxidatively active than most condensed tannins. The ability of insects to tolerate ingested tannins comes from a variety of biochemical and physical defenses in their guts, including surfactants, high pH, antioxidants, and a protective peritrophic envelope that lines the midgut. Most work on the ecological roles of tannins has been correlative, e.g., searching for negative associations between tannins and insect performance. A greater emphasis on manipulative experiments that control tannin levels is required to make further progress on the defensive functions of tannins. Recent advances in the use of molecular methods has permitted the production of tannin-overproducing transgenic plants and a better understanding of tannin biosynthetic pathways. Many research areas remain in need of further work, including the effects of different tannin types on different types of insects (e.g., caterpillars, grasshoppers, sap-sucking insects).