Methane emission by goats consuming different sources of condensed tannins

Twenty-four yearling Boer × Spanish wethers (7/8 Boer; initial body weight (BW) of 37.5 ± 0.91 kg) were used to assess effects of different condensed tannin (CT) sources on methane (CH₄) emission. Diets were Kobe lespedeza (Lespedeza striata; K), K plus quebracho providing CT at 50 g/kg dry matter (DM) intake (KQ), Sericea lespedeza (Lespedeza cuneata; S), and a 1:1 mixture of K and S (KS). Forages harvested daily were fed at 1.3 times the maintenance metabolizable energy requirement. The experiment was 51 days divided into two phases. In phase A forage diets were fed alone, and in phase B, 25 g/day of polyethylene glycol (PEG) was given mixed with 50 g/day of ground maize grain. Adaptation periods were 28 and 7 days in phases A and B, respectively. After adaptation there were 8 days for feces and urine collections, with gas exchange measured on the last 2 days. Ruminal fluid was collected at the end of the experiment via stomach tube for microbiology assays. The N concentration was 22.8 and 23.6 g/kg DM, in vitro true DM digestibility was 0.698 and 0.648, and the level of CT was 140 and 151 g/kg DM for S and K, respectively. DM intake was similar among treatments in both phases (phase A: 720, 611, 745, and 719 g/day (S.E. = 59.0); phase B: 832, 822, 867, and 880 g/day (S.E. = 55.3) for K, KQ, S, and KS, respectively). N digestibility was affected by treatment in phase A (P<0.05) but not in phase B (phase A: 0.514, 0.492, 0.280, and 0.413 (S.E. = 0.0376); phase B: 0.683, 0.650, 0.638, and 0.662 (S.E. = 0.0203) for K, KQ, S, and KS, respectively). Gross energy digestibility was similar among treatments in phase A (0.475, 0.407, 0.393, and 0.411 (S.E. = 0.0353)) but differed among treatments in phase B (0.449, 0.373, 0.353, and 0.409 for K, KQ, S, and KS, respectively (S.E. = 0.0221)) CH₄ emission was 9.6, 6.8, 10.6, and 8.9 l/day (S.E. = 1.44) in phase A and 19.0, 16.6, 21.8, and 19.2 l/day (S.E. = 1.51) in phase B for K, KQ, S, and KS, respectively (S.E. = 1.25). When data of both phases were pooled, supplementation with PEG in phase B markedly increased (P<0.05) CH₄ emission (9.0 l/day versus 19.1 l/day). In accordance, there was a substantial difference (P<0.05) between phases in in vitro CH₄ emission by ruminal fluid incubated for 3 weeks in a methanogenic medium and with other conditions promoting activity by methanogens (11.5 and 22.9 ml in phases A and B, respectively). Counts of total bacteria and protozoa were similar among treatments in both phases, but values were greater (P<0.05) in phase B versus phase A. In summary, CT from different sources had a disparate influence on N digestion, but similar effects on ruminal microbial CH₄ emission by goats, possibly by altering activity of ruminal methanogenic bacteria though change in actions of other bacteria and/or protozoa may also be involved.     

In sacco rumen disappearance of condensed tannins, fiber, and nitrogen from herbaceous native Texas legumes in goats

Condensed tannins (CT) can play a role in rumen protein and fiber degradability, especially in legumes high in CT. In order to better understand their potential role in ruminant nutrition, three legume species native to Texas, Acacia angustissima var. hirta (prairie acacia) (288.0 g/kg neutral detergent fiber (NDFom), 40.9 g/kg N), Desmodium paniculatum (panicled tick-clover) (479.7 g/kg NDFom, 24.8 g/kg N), and Lespedeza procumbens (trailing bush-clover) (401.0 g/kg NDFom, 21.7 g/kg N) were studied to determine in sacco disappearance rates of key nutritional components compared to that of Medicago sativa (alfalfa) (226.8 g/kg NDFom, 34.6 g/kg N). Herbage was incubated in rumen-cannulated goats fed a basal diet of Sorghum bicolor×S. sudanense (sorghum-Sudan) hay, with disappearance measured at 0, 4, 8, 16, 28, 48 and 96 h. Among the native legumes, the highest CT concentrations were measured in prairie acacia (263 g CT/kg DM foliage) and the lowest (120 g CT/kg DM) in trailing bush-clover. The lowest concentrations of acid detergent fiber (ADFom), NDFom, and sulfuric acid lignin (lignin(sa)) were measured in prairie acacia, the first two fractions being comparable to alfalfa. Proportion remaining was calculated for CT, ADFom, lignin(sa), NDFom, and N for 0, 24 and 48 h of rumen incubation. Disappearance parameters were measured for ADFom, lignin(sa), NDFom and N for the three native legumes and compared to alfalfa. Alfalfa had the highest disappearance of all degradable fractions except lignin(sa). Potential disappearance (PD) fraction for ADFom, lignin(sa) and N were lower for the native legumes versus alfalfa. No differences in N proportion remaining at 24 and 48 h occurred in the native legumes despite differences in protein-bound CT proportion remaining at those same times. Of the native legumes studied, prairie acacia shows the greatest potential for contributing rumen-escape protein, suggesting it may be a candidate for further development as a pasture and rangeland renovation legume.     

Molecular weight and protein-precipitating ability of condensed tannins from warm-season perennial legumes

The beneficial effects of forages containing condensed tannins (CTs) on ruminants are well documented, but the chemical features of CT that yield benefits have not been defined. Some evaluations of limited numbers of highly purified compounds have resulted in positive correlations between CT molecular weight (M_W) and biological activity, while others have failed to show a correlation. The objectives of this study were to determine if M_W of CT could predict biological activity relative to protein precipitability. M_W of condensed tannin, protein-precipitable phenolics (PPP), and the amount of protein bound (PB) were determined for nine species of warm-season perennial legumes. There was no correlation between PPP or PB and M_W (R² 0.11 and R² 0.02, respectively). However, CT concentration did correlate with PPP and PB (R² 0.81 and R² 0.69, respectively). It was concluded that CT M_W does not explain the variation in protein precipitation by CT from the forage legumes surveyed.     

The condensed tannins of pasture legume species

Condensed tannins have been isolated from legume pasture species and purified by gel chromatography on Sephadex G-50 and LH-20 media. Molecular size di     

Low concentration of condensed tannins from catechu significantly inhibits fatty acid synthase and growth of MCF-7 cells

Tannins exist widely in plants, but because they precipitate proteins, scientists frequently ignore them in search of bioactive components. Catechu, a traditional astringent, is rich in tannins. In this study, we found that condensed tannins from catechu potently inhibited animal fatty acid synthase (FAS). Among them, trimeric condensed tannin showed the most potent inhibition with IC₅₀ of 0.47 μg/ml and it also exhibited strong time-dependent inhibition. Its inhibitory kinetics and reacting sites on FAS were obviously different from the known inhibitors of FAS. Furthermore, condensed tannins were found to suppress the growth of MCF-7 breast cancer cells, and the effect was related to their activity of FAS inhibition. The inhibition of both FAS activity and MCF-7 growth was exhibited by low concentrations of condensed tannins without FAS being precipitated. These results suggest tannins would be a valuable resource of bioactive substances.     

Wound-induced accumulations of condensed tannins in turtlegrass, Thalassia testudinum

Turtlegrass, Thalassia testudinum, produces high concentrations of proanthocyanidins (condensed tannins) which we hypothesized are induced by grazing, as a component of a general wound response. To test this we quantified condensed tannins in a variety of turtlegrass tissues following simulated fish grazing, grazing by the urchin Lytechinus variegatus, and treatment with the natural plant wound hormone jasmonic acid. We observed that simulated fish grazing triggered rapid induction of condensed tannins by an average of 10 mg tannin g⁻¹ dry mass (DM) after 5 days. Condensed tannin accumulations were correlated with a reduction of blade extension rates. Further, we observed that constitutive tannin levels in developing first-rank leaves were strongly correlated with the width of second-rank leaves on each shoot, with an increase of 7.7 mg tannin g⁻¹ tissue dry mass per millimeter blade width on average. We propose that wider source leaves provide additional resources for phenolic biosynthesis. There was no induction of tannins in leaves, meristematic or sheath tissues in response to grazing by the urchin L. variegatus, the presence of potential waterborne cues from nearby grazed plants, or to treatment with 5 mM jasmonic acid. However, urchin grazing did induce tannin production in root/rhizome tissues, where they accumulated to levels 3–4 times higher than in blades (up to 350 mg tannin g⁻¹ tissue dry mass). These results confirm the potential for rapid wound-induced condensed tannin accumulations in T. testudinum. The link between blade widths and the tannin content of new leaves indicates that leaf morphology may be a useful bioindicator for predicting herbivore and disease-resistance in the field.     

The digestion of dietary protein bound by condensed tannins in the gastro-intestinal tract of sheep

The digestion of dietary protein bound by condensed tannins (CTs) in ruminants was investigated by determining the extent of dissociation of insoluble ¹²⁵I-BSA + CT complexes administered to abomasally and intestinally fistulated sheep. The extent of dissociation was registered as the true digestibility of iodinated bovine serum albumin (¹²⁵I-BSA). The true digestibility of ¹²⁵I-BSA originally bound to Leucaena pallida CT (0.721) was lower (P<0.05) than that of ¹²⁵I-BSA originally bound to L. leucocephala CT (0.880) between the abomasum and terminal ileum. These results indicate that differences in the ability of CT to inhibit ¹²⁵I-BSA digestion in vivo matched the relative abilities of the same CT to bind BSA in vitro, indicating that the in vitro BSA-binding assay for ranking CT behaviour was biologically relevant in vivo. Furthermore, the true digestibility of CT-bound ¹²⁵I-BSA between the mouth and faeces permitted the prediction of the quantitative contribution that CT-bound dietary proteins make to improved nitrogen supply to the small intestines.     

Developing a conceptual model of possible benefits of condensed tannins for ruminant production

Enteric methane (CH₄) emissions from ruminants have compelled a wide range of research initiatives to identify environmental abatement opportunities. However, although such mitigations can theoretically be attained with feed additives and feeding strategies, the limited empirical evidence on plant extracts used as feed additives does not support extensive or long-term reductions. Nevertheless, their strategic use (i.e. alone or combined in a simultaneous or consecutive use) may provide not only acceptable CH₄ abatement levels, but also relevant effects on animal physiology and productivity. Condensed tannins (CT) represent a range of polyphenolic compounds of flavan-3-ol units present in some forage species that can also be added to prepared diets. Methods to determine CT, or their conjugated metabolites, are not simple. Although there are limitations and uncertainties about the methods to be applied, CT are thought to reduce CH₄ production (1) indirectly by binding to the dietary fibre and/or reducing the rumen digestion and digestibility of the fibre and (2) directly by inhibiting the growth of rumen methanogens. On the basis of their role in livestock nutrition, CT influence the digestion of protein in the rumen because of their affinity for proteins (e.g. oxidative coupling and H bonding at neutral pH) that causes the CT-protein complex to be insoluble in the rumen; and dissociate in the abomasum at pH 2.5 to 3.0 for proteolysis and absorption in the small intestine. CT may also reduce gastro-intestinal parasite burdens and improve reproductive performance, foetal development, immune system response, hormone serum concentrations, wool production and lactation. The objectives of this paper are to discuss some of the beneficial and detrimental effects of CT on ruminant production systems and to develop a conceptual model to illustrate these metabolic relationships in terms of systemic physiology using earlier investigations with the CT-containing legume Lotus corniculatus. Our conceptual model indicated four complex and long-lasting relationships (digestive, toxicological, physiological and morphological) that can alter the normal biology of the animal. These relationships are interdependent, integrative, and sometimes, complementary to each other. This conceptual model can be used to develop mechanistic models to improve the understanding of the interaction between CT and the ruminants as well as to guide research initiatives of the impact of polyphenol-rich foods on human health.     

Growth and cashmere production by Spanish goats consuming ad libitum diets differing in protein and energy levels

Thirty-six Spanish goat wethers (196 ± 12.9 days of age and 17.5 ± 4.60 kg BW at experiment initiation) from a herd previously selected for cashmere growth were used to determine effects and interactions of ad libitum consumption of diets differing in concentrations of CP (10% and 15%) and ME (2.00, 2.35 and 2.70 Mcal/kg; DM basis) on growth and cashmere fiber production in an 84-day, fall-season experiment. DM intake was greater (P < 0.05) for diets with 15% vs. 10% CP (736 vs. 655 g/day) but similar among ME concentrations (P > 0.10). ADG was greater (P < 0.02) for 15% vs. 10% CP (79 vs. 53 g/day) and increased linearly (P < 0.10) as dietary ME level increased (58, 62 and 78 g/day for 2.00, 2.35 and 2.70 Mcal/kg ME, respectively). Total fleece weight at the end of the experiment was similar (P > 0.10) between CP levels but increased linearly (P < 0.03) as ME concentration in the diet increased (197, 239 and 227 g/day). Guard hair weight was not affected by dietary treatments (P > 0.10); cashmere length was similar among treatments (P > 0.10); and cashmere fiber diameter was not altered by dietary ME level (P > 0.10) but was greater (P < 0.05) for 15% vs. 10% CP (16.92 vs. 16.06 μm). Cashmere weight was influenced by an interaction between CP and ME levels (P < 0.05); cashmere fiber weight with 10% CP was 92, 82 and 95 g, and with 15% CP was 63, 115 and 99 g for 2.00, 2.35 and 2.70 Mcal ME/kg diets, respectively (SE = 10.2). However, the ranking of treatment means for initial cashmere fiber weight was similar to that at the end of the experiment, although differences at the beginning were non-significant and of lesser magnitude than at the end. In conclusion, further research with influences of concentrations of CP and ME in diets consumed ad libitum on cashmere fiber growth of growing US Spanish wether goats is required, and animal numbers and allocation to treatments deserve careful consideration in studies of cashmere fiber production.     

Mixing sainfoin and lucerne to improve the feed value of legumes fed to sheep by the effect of condensed tannins

The aim of this study was to investigate whether the use of sainfoin-based condensed tannins (CT) enhances feed value when given with tannin-free legumes (lucerne) to sheep. The experiments were conducted with fresh sainfoin and lucerne harvested at two stages (vegetative stage as compared with early flowering) in the first growth cycle. Fresh sainfoin and lucerne forages were combined in ratios of 100 : 0, 75 : 25, 25 : 75 and 0 : 100 (denoted S100, S75, S25 and S0, respectively). Voluntary intake, organic matter digestibility (OMD) and nitrogen (N) retention were measured in sheep fed the different sainfoin and lucerne mixtures. Loss of dry matter (DM) and N from polyester bags suspended in the rumen, abomasum and small intestine (SI) was also measured using rumen-fistulated sheep and intestinally fistulated sheep. The CT content in sainfoin (S100) decreased with increasing percentage of lucerne in the mixture (mean value from 58 g/kg DM for S100 to 18 g/kg DM for S25) and with growth stage (S100: 64 to 52 g/kg DM). OMD did not differ between different sainfoin/lucerne mixture ratios. Sainfoin and lucerne had an associative effect (significant quadratic contrast) on voluntary intake, N intake, total-tract N digestibility, N in faeces and urine (g/g N intake) and N retained (g/g N intake). Compared with lucerne mixtures (S0 and S25), high-sainfoin-content mixtures (S100 and S75) increased the in situ estimates of forage N escaping from the rumen (from 0.162, 0.188 for S0 and S25 to 0.257, 0.287 for S75 and S100) but decreased forage N intestinal digestibility (from 0.496, 0.446 for S0 and S25 to 0.469, 0.335 for S75 and S100). The amount of forage N disappearing from the bags in the SI (per g forage N) was the highest for high-sainfoin mixtures (from 0.082, 0.108 for S100 and S75 to 0.056, 0.058 for S25 and S0, P < 0.001). Rumen juice total N (tN) and ammonia N (NH3-N) values were the lowest in the high-sainfoin diet (mean tN 0.166 mg/g in S100 as compared with 0.514 mg/g in S0; mean NH3-N 0.104 mg/g in S100 as compared with 0.333 mg/g in S0, P < 0.001).     

Relationship between intake of tannin-containing tropical tree forage,PEG supplementation,and salivary haze development in hair sheep and goats

The objective of this study was to estimate the relationship between tannin binding salivary protein (TBSP) and condensed tannins (CT) intake in hair sheep and creole goats. Foliage was obtained from trees with different levels of CT content; animals were offered foliage ad libitum, with or without polyethylene glycol (PEG). Saliva haze development (SHD) was evaluated as evidence for TBSP. PEG consumption did not affect dry matter intake (DMI) (P > 0.05). Lignin (R = −0.714, P < 0.001) and Crude Protein (CP) (R = 0.622, P < 0.001) contents had a stronger association with DMI than CT (R = 0.622, P < 0.011) in sheep; no significant association was found in goats. The positive relationship between tannin intake and SHD (P < 0.05) was not confirmed after PEG supplementation in sheep (P > 0.09), but remained significant for goats (P < 0.01), except for those fed Lysiloma latisiliquum (P = 0.07). Foliage lignin or CP contents are better predictors of foliage intake than CT. Sheep and goats fed with tropical tree forages containing different levels of tannins exhibited differences in intake behavior; moreover, individual variations in TBSP expression helps explaining foliage DMI.     

Phenolics and condensed tannins of high altitude Pteridium arachnoideum in relation to sunlight exposure,elevation, and rain regime

Non-adapted plants growing in high altitude such as bracken fern Pteridium arachnoideum are exposed to environmental extremes that may induce a chemical adaptive response. Here we show that there is a non-uniform distribution of low (LMP) and high molecular weight (HMP) phenolics in the frond parts of P. arachnoideum growing at high elevation. LMP–HMP levels were measured in sun-exposed (E) and self shaded (SS) pinnae between 2100 and 3190 m in the tropical Andes, during dry and rainy seasons. While there was no difference in E vs. SS contents of LMP at 2100 m, E accumulated greater LMP–HMP concentrations relative to SS as altitude increased. This difference was increased during the dry season. Linear correlations between the position of each pinnae relative to the ground level and LMP–HMP occurred along a 2570–3190 m transect. Water restriction in the dry season also caused increase of LMP and HMP. We conclude that excess UV-B radiation and water availability are important modelers of the non-adapted plant acclimation response to stress in tropical high mountain habitats.     

The relationship between heart rate and energy expenditure in Alpine, Angora, Boer and Spanish goat wethers consuming different quality diets at level of intake near maintenance or fasting

Six Alpine (AL; 38.4 ± 3.0 kg), Angora (AN; 23.1 ± 2.7 kg), Boer (BO; 40.8 ± 4.5 kg) and Spanish (SP; 33.6 ± 2.2 kg) wethers (1.5 yr of age) were used to determine the effects of time of the day and potential interactions between time, genotype and diet quality on energy expenditure (EE), heart rate (HR) and EE:HR when fed near maintenance and fasting. The experiment consisted of four simultaneous crossovers, with 21 d for adaptation before measures. Diets were 60% concentrate (CON: 15% CP) and ground alfalfa hay (FOR: 23% CP), offered in two meals at 8:00 and 16:00 h. Energy expenditure was determined from O₂ consumption and production of CO₂ and CH₄ over 2-day periods in fed and fasting states (total 4-day fasting period). Fasting EE was higher during the day than night, with values generally highest at 16:00–17:00 h. Animal within breed affected EE, HR and EE:HR (P < 0.05). The diurnal pattern in EE varied with diet (P < 0.05), although total daily EE was not different between diets. Before the morning meal, there were a number of hours during which EE was greater for CON than for FOR. However, at both meals the rise in EE was considerably greater for FOR versus CON, lasting for 3–4 h. The same general pattern in HR was observed, although the period of time when there was a dietary difference after the afternoon meal was shorter. For both fed and fasted goats, EE:HR differed among hours of the day (P < 0.05). EE:HR tended (P < 0.09) to differ between diets (5.99 and 6.21 for CON and FOR, respectively) and to be affected (P < 0.09) by an interaction between breed and diet (AL: 5.84 and 6.38; AN: 5.91 and 5.73; BO: 6.05 and 6.58; and SP: 6.17 and 6.15 kJ/(kg BW^0.75 × day):heart beats/min) for CON and FOR, respectively. In conclusion, for use of HR to predict EE by goats, it appears desirable to determine the ratio of EE:HR with a diet similar to that consumed during prediction and over an extended period of time.     

Effect of molecular weight of condensed tannins from warm-season perennial legumes on ruminal methane production in vitro

The objectives of the present study were to determine if the molecular weight of condensed tannins (CT) from warm-season perennial legumes affects the biological activity of CT relative to suppression of methane (CH₄) production by ruminants, and to identify potential North American native forage plants to use for mitigation of enteric CH₄ emission. Eight North American native warm-season perennial legumes were evaluated: Leucaena retusa Benth. (littleleaf leadtree), Desmanthus illinoensis (Michx.) MacMill. Ex B.L. Rob. & Fernald (Illinois bundleflower), Lespedeza stuevei Nutt. (tall lespedeza), Mimosa strigillosa Torr. & A. Gray (powderpuff), Neptunia lutea (Leavenworth) Benth. (yellow puff), two ecotypes of Acacia angustissima var. hirta (Nutt.) B.L. Rob (prairie acacia), and Desmodium paniculatum (L.) DC. var. paniculatum (panicledleaf ticktrefoil). Two introduced legumes were also included: Arachis glabrata Benth. (rhizoma peanut) and Lespedeza cuneata (Dum. Cours.) G. Don (sericea lespedeza). Forages were fermented with cattle rumen fluid for 48 h anaerobically using an in vitro gas production technique. D. paniculatum, L. stuevei, and M. strigillosa were high in CT, ranging from 11.7 to 12.5%. D. illinoensis, L. cuneata, N. lutea, and two ecotypes of A. angustissima var. hirta had less CT (P < 0.05), ranging from 8.1 to 8.9%, whereas L. retusa and A. glabrata had the least CT (P < 0.05), measuring 3.2 and 0.5%, respectively. Weight-average molecular weight (M_W) of CT ranged from 1483 Da for L. cuneata to 552 Da for L. stuevei. In vitro CH₄ production was greatest for L. retusa and A. glabrata at 40.7 mg/g DM and 38.2 mg/g DM, respectively. The least amount of in vitro CH₄ was produced by fermentation of two ecotypes of A. angustissima var. hirta, which measured 0.8 and 0.6 mg/g DM, respectively. In vitro CH₄ production regressed on CT M_W resulted in a R² of 0.0009 (P = 0.80), strongly suggesting that CT M_W does not explain the biological activity of in vitro CH₄ production by the forage legumes surveyed. Five of the seven North American native warm-season perennial legumes have promise for use in ruminant diets for the purpose of CH₄ emission mitigation.     

Effects of condensed tannins on behavior and performance of a specialist aphid on aspen

Genes involved in plant defences against herbivores and pathogens are often highly polymorphic. This is a putative sign that balancing selection may have operated reciprocally on the hosts and their herbivores. Spatial and temporal variations (for example, in soil nutrients and the plants'' ontogenetic development) may also modulate resistance traits, and thus selection pressures, but have been largely overlooked in theories of plant defences. Important elements of defences in Populus tremula (hereafter aspen) are phenolic compounds, including condensed tannins (CTs). Concentrations of CTs vary considerably with both variations in external factors and time, but they are also believed to provide genotype‐dependent resistance, mainly against chewing herbivores and pathogens. However, evidence of their contributions to resistance is sparse. Detailed studies of co‐evolved plant–herbivore associations could provide valuable insights into these contributions. Therefore, we examined correlations between CT levels in aspen leaves and both the feeding behavior and reproduction of the specialist aspen leaf aphid (Chaitophorus tremulae) in varied conditions. We found that xylem sap intake and probing difficulties were higher on genotypes with high‐CT concentrations. However, aphids engaged in more nonprobing activities on low‐CT genotypes, indicating that CTs were not the only defence traits involved. Thus, high‐CT genotypes were not necessarily more resistant than low‐CT genotypes, but aphid reproduction was generally negatively correlated with local CT accumulation. Genotype‐specific resistance ranking also depended on the experimental conditions. These results support the hypothesis that growth conditions may affect selection pressures mediated by aphids in accordance with balancing selection theory.     

Feeny revisited: condensed tannins as anti-herbivore defences in leaf-chewing herbivore communities of Quercus

Abstract. 1. Community level oak–tannin–insect patterns have been largely unexplored since Paul Feeny's ground‐breaking research. Two hypotheses were tested for Quercus velutina and Q. alba in the Missouri Ozarks: abundance and richness of leaf‐chewing herbivores are negatively correlated with foliar condensed tannin concentrations and variation in condensed tannin concentrations explains variation in herbivore community structure. 2. In 2001, foliar condensed tannins in the understorey and canopy of these two oak species were quantified simultaneously with censuses of herbivores in May, during leaf expansion, and in June and August, when leaves were fully expanded. Thirty‐eight of the 134 species encountered had densities sufficient to be analysed individually (n = 10). Of those, Acronicta increta (Noctuidae) and Attelabus sp. (Curculionidae), both oak specialists, were negatively correlated with condensed tannins in the canopy of Q. alba. One additional specialist, Chionodes pereyra (Gelechiidae), was marginally negatively correlated with condensed tannins in the understorey of Q. velutina. Understorey species richness of May Q. velutina herbivores was negatively correlated with condensed tannins, as were total canopy insect density and species richness of August herbivores on Q. alba. 3. Principal component analysis (PCA) of insect abundances indicated that understorey and canopy Q. velutina and Q. alba had different communities of leaf‐chewing insects. Furthermore, condensed tannin levels contributed significantly to variation in PCA scores for Q. velutina, explaining 25% of the total variation. 4. Overall, these results indicate that specialists were more likely than generalists both to correlate negatively with condensed tannins and to occur in lower tannin habitats; abundance and richness of both early and late season fauna correlated negatively with tannins; and species were more likely to correlate negatively with condensed tannins when feeding on Q. alba than on Q. velutina and when feeding in the canopy than in the understorey. Future studies of tannin–insect interactions should manipulate leaf quality in combination with manipulations of other factors that likely influence community structure.     

The tannosome is an organelle forming condensed tannins in the chlorophyllous organs of Tracheophyta

Background and Aims

Condensed tannins (also called proanthocyanidins) are widespread polymers of catechins and are essential for the defence mechanisms of vascular plants (Tracheophyta). A large body of evidence argues for the synthesis of monomeric epicatechin on the cytosolic face of the endoplasmic reticulum and its transport to the vacuole, although the site of its polymerization into tannins remains to be elucidated. The aim of the study was to re-examine the cellular frame of tannin polymerization in various representatives of the Tracheophyta.

Methods

Light microscopy epifluorescence, confocal microscopy, transmission electron microscopy (TEM), chemical analysis of tannins following cell fractionation, and immunocytochemistry were used as independent methods on tannin-rich samples from various organs from Cycadophyta, Ginkgophyta, Equisetophyta, Pteridophyta, Coniferophyta and Magnoliophyta. Tissues were fixed in a caffeine–glutaraldehyde mixture and examined by TEM. Other fresh samples were incubated with primary antibodies against proteins from both chloroplastic envelopes and a thylakoidal chlorophyll-carrying protein; they were also incubated with gelatin–Oregon Green, a fluorescent marker of condensed tannins. Coupled spectral analyses of chlorophyll and tannins were carried out by confocal microscopy on fresh tissues and tannin-rich accretions obtained through cell fractionation; chemical analyses of tannins and chlorophylls were also performed on the accretions.

Key Results and Conclusions

The presence of the three different chloroplast membranes inside vacuolar accretions that constitute the typical form of tannin storage in vascular plants was established in fresh tissues as well as in purified organelles, using several independent methods. Tannins are polymerized in a new chloroplast-derived organelle, the tannosome. These are formed by pearling of the thylakoids into 30 nm spheres, which are then encapsulated in a tannosome shuttle formed by budding from the chloroplast and bound by a membrane resulting from the fusion of both chloroplast envelopes. The shuttle conveys numerous tannosomes through the cytoplasm towards the vacuole in which it is then incorporated by invagination of the tonoplast. Finally, shuttles bound by a portion of tonoplast aggregate into tannin accretions which are stored in the vacuole. Polymerization of tannins occurs inside the tannosome regardless of the compartment being crossed. A complete sequence of events apparently valid in all studied Tracheophyta is described.     

A comparison of two strategies to modify the hydroxylation of condensed tannin polymers in Lotus corniculatus L

A full-length sense Antirrhinum majus dihydroflavonol reductase (DFR) sequence was introduced into birdsfoot trefoil (Lotus corniculatus L.) in experiments aimed at modifying condensed tannin content and polymer hydroxylation in a predictable manner. Analysis of transgenic plants indicated lines that showed enhanced tannin content in leaf and stem tissues. In contrast to previous data from root cultures, levels of propelargonidin units were not markedly elevated in lines with enhanced tannin content. RT-PCR analysis of four selected lines indicated a correlation between enhanced tannin content and expression of the introduced DFR transgene. Using a contrasting approach we introduced a flavonoid 3'5' hydroxylase (F3'5'H) sequence derived from Eustoma grandiflorum into Lotus root cultures. Expression of the transgene was associated with increased levels of condensed tannins and in this case there was also no alteration in polymer hydroxylation. These results suggest that additional mechanisms may exist that control the hydroxylation state of condensed tannins in this model species.