Intake and digestion of leaves of Quercus calliprinos, Pistacia lentiscus, and Ceratonia siliqua, with and without supplementation of various amounts of polyethylene glycol (PEG), were examined. The tannin contents in these species exerted a substantial negative effect on feed intake and digestion. The effects were species specific and related to tannin content. Once-daily Supplementation with PEG efficiently neutralized the negative effects of tannins. The amount of PEG needed to produce a maximal increase in feed intake was lower than the amount required to produce a maximal increase in digestibility. The intake of digestible crude protein and metabolizable energy was raised in PEGsupplemented animals from a submaintenance level to levels considerably exceeding the maintenance requirement of goats. The response was related to the tannin content of the plant.

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 ( hp-1) and the high pigment-1(w) ( hp-1(w)) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 ( hp-2), high pigment-2(j) ( hp-2(j)) and dark green ( dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1(w) mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A(931)-to-T(931) base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1(w) mutant, on the other hand, a single G(2392)-to-A(2392) transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F(2) population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25

A comprehensive, multi-generation, allele test, carried out in this study, suggests that the tomato mutations dark-green (dg) and high pigment 2(j) (hp-2(j)) are allelic. The hp-2(j) mutant is caused by a mutation in the tomato homolog of the DEETIOLATED1 (DET1) gene, involved in the signal transduction cascade of light perception and morphogenesis. This suggestion is in agreement with the exaggerated photomorphogenic de-etiolation response of homozygous dg mutants grown under modulated light conditions. Sequence analysis of the DET1 gene was carried out in dg mutants representing two different lines, and revealed a single A-to-T base transversion in the second exon of the DET1 gene in comparison with the normal wild-type sequence. This transversion results in a conserved Asparagine(34)-to-Isoleucine(34) amino-acid substitution, and eliminates a recognition site for the AclI restriction endonuclease, present in the wild-type and in the other currently known tomato mutants at the DET1 locus. This polymorphism was used to develop a PCR-based DNA marker, which enables an early genotypic selection for breeding lycopene-rich tomatoes. Using this marker and sequence analysis we demonstrate that an identical base transversion also exists in dg mutants of the cultivar Manapal, in which the natural dg mutation was originally discovered. A linkage analysis, carried out in a F(2) population, shows a very strong linkage association between the DET1 locus of dg mutant plants and the photomorphogenic response of the seedlings, measured as hypocotyl length (12 < LOD Score < 13, R(2) = 51.1%). The results presented in this study strongly support the hypothesis that the tomato dg mutation is a novel allele of the tomato homolog of the DET1 gene.

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Effects were studied on food intake and diet apparent digestibility of giving to goats once daily a basal diet rich in tannin from inclusion of leaves of Quercus calliprinos either supplemented with a high carbohydrate or a high protein food. Also interactions with polyethylene glycol (PEG) were investigated. The results of the present work confirmed our presumption that the content of tannin in some Mediterranean browse is so high that it may negatively affect the utilization of protein in supplementary foods. Neutralizing the tannins with once-daily provision of PEG proved to be an effective means of preventing the negative effect. Providing 10 g/day PEG to goats given Quercus calliprinos leaves ad libitum and supplemented with 300 g/day concentrates containing 160 g crude protein per kg DM increased digestible crude protein intake by 50 g/day. When the concentrate food was given to goats, leaf dry-matter intake decreased significantly (from 664 to 565 g/day) and the goats lost weight rapidly. Therefore, supplementing tannin-rich leaves with concentrate food is recommended only if done in combination with PEG. High protein supplementation increased leaf (from 664 to 844 g/day) and digestible protein intakes (from 4·8 to 92·3 g/day) but a considerable portion of the protein supplementation was wasted due to interaction with tannins. PEG may allow economies in the use of such high-cost foods due to the greater efficiency of protein utilization (digestible crude protein intake increased from 92·3 to 122 g/day) of the supplementary food and to increased intake and protein utilization of the basal leaf diet (from 844 to 1023 g/day).

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A procedure to measure poly(ethylene glycol) (PEG) binding to plant samples is described. The amount of PEG bound to a sample was found to be reproducible with a small coefficient of variation (0.5%). The PEG-binding capacity of plant samples was not affected by predrying of the samples at 90 °C, unlike the colorimetric methods for determination of tannins. Both the PEG-binding and the protein precipitation capacity of the plant samples were found to be useful in predicting the negative effects of tannins on ruminal degradation of the plant material. However, PEG binding to plant samples had an advantage over protein precipitation in samples in which formation of strong tannin−protein complexes results in low extractability of tannins. For this reason, the binding method was found to be preferable to the protein precipitation method in predicting the negative effect of tannins on ruminal degradation of forage plants. Keywords: Poly(ethylene glycol) (PEG); tannin; rumen; digestibility; forage plants; herbivore diet

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