Persistence of the soil seed bank requires both dormancy and resistance to seed decay organisms. However, there is little or no information evaluating biochemical responses of dormant weed seeds to pathogens. Wild oat caryopses were incubated with four pathogenic fungal isolates to evaluate the response of the pathogen defense enzyme, polyphenol oxidase (PPO). Caryopsis PPO activity was induced by three Fusarium spp. isolates previously obtained from whole seeds incubated in the field whereas caryopsis PPO activity was decreased by a Pythium isolate. Fusarium avenaceum isolate F.a.1 caused the greatest PPO induction and was studied in more detail. When whole wild oat seeds were incubated on F.a.1, PPO activity was induced in seeds, hulls (lemma and palea), and caryopses. Incubation of whole seeds on F.a.1 gradually induced caryopsis PPO activity over an 8-d period, whereas incubation of caryopses on F.a.1 over a 4-d period caused a greater and more rapid induction of PPO activity. Very little PPO activity could be leached from untreated caryopses, but nearly all of the induced PPO activity in F.a.1-treated caryopses was readily leached when incubated in buffer. In Western blots, both untreated and F.a.1-treated leachates contained a ∼57-kilodalton (kD) protein, putatively the mature and relatively inactive form of PPO. However, lower molecular weight antigenic proteins between ∼52 and ∼25 kD were strongly induced in F.a.1-treated caryopses, with this induction being correlated with the increase in PPO activity. We hypothesize that dormant weed seeds possess biochemical defenses against pathogens and, more specifically, that proteolysis in the presence of fungal pathogens may release an activated form of PPO from the surface of wild oat caryopses and hulls.

Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO’s lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid encapsulation in protein–phenol complexes would lead to an effective protection of lipids against ruminal biohydrogenation. From several studies it has also become apparent that the degree of PPO activation could influence the mode and degree of protection. In conclusion, this review demonstrates that protein-bound phenols and encapsulation in protein–phenol complexes, induced by PPO-mediated diphenol oxidation, could be of interest when aiming to protect lipids against pre-ruminal and ruminal degradation.

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol–protein complexes.

Introduction. Wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is the most important disease of banana worldwide, causing severe yield losses in commercial and local consumption cultivars. Chemical control is currently the most used method to manage the Fusarium wilt of banana, although it is toxic, expensive and dangerous. Therefore, control through genetic improvement is widely encouraged. Hence, breeding was carried out to develop banana hybrids with putative resistance. Materials and methods. Hybridization of identified resistant diploids with commercial triploids and tetraploids was carried out; it resulted in development of 22 hybrids with improved agronomic characters. In a second step, screening of these hybrids for resistance to Foc race 1 was attempted using the double cup method of challenge inoculation with the pathogen in the roots of the hybrids at optimum level. Host responses of the susceptible and resistant hybrids were examined under greenhouse conditions through biochemical and isozyme analysis. Results. Six hybrids among the 22 hybrids with improved agronomic characters were selected based on their yield and they were evaluated for resistance. Three of them were resistant to Foc and the others were susceptible. The resistance mechanism involving the relation of enzymes such as peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine lyase (PAL), and biochemicals such as phenols, proline and lignin showed relatively higher activity in resistant hybrids and parents than susceptible ones. Isozyme analysis of PO and PPO carried out using selected resistant and susceptible hybrids revealed the induction of specific isoforms in the resistant hybrids upon challenge inoculation. Conclusion. Three hybrids were identified as potential hybrids with good yield and resistance to Foc race 1.

Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products. In this study, wheat PPO sequences (mRNA) were searched/BLASTed in the NCBI database and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into two clusters (I and II) and that three genes (‘i’) of cluster II seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNPs) were detected between two haplotypes of the PPO gene on chromosome 2D. Eighty of these were found in the coding region (coding (c) SNPs) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers (STS-H) were designed at some non-synonymous cSNPs sites and were used to investigate the correlations between allelic variants and PPO activity of seeds – a total of 130 common wheat varieties were evaluated in 2 years. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of a single dominance molecular marker, the multiplex polymerase chain reaction (PCR) system of STS-H and STS01 markers was also studied, based on the complementary between them.