A reference assembly for the legume cover crop hairy vetch (Vicia villosa).

Vicia villosa is an incompletely domesticated annual legume of the Fabaceae family native to Europe and Western Asia. V. villosa is widely used as a cover crop and forage due to its ability to withstand harsh winters. Here, we generated a reference-quality genome assembly (Vvill1.0) from low error-rate long-sequence reads to improve the genetic-based trait selection of this species. Our Vvill1.0 assembly includes seven scaffolds corresponding to the seven estimated linkage groups and comprising approximately 68% of the total genome size of 2.03 Gbp. This assembly is expected to be a useful resource for genetically improving this emerging cover crop species and provide useful insights into legume genomics and plant genome evolution.

Near-real time determination of BAHD acyl-coenzyme A transferase reaction rates and kinetic parameters using Ellman's reagent.

BAHD acyl-coenzyme A (CoA) acyltransferases play key roles in a large number of biosynthetic reactions involved in plant specialized metabolism. One approach to measure reaction rates for these enzymes is to quantify the amide or ester reaction products following chromatographic separation of reaction components, an approach that can be labor intensive and time consuming, and complicated by a lack of pure standards. We previously developed and validated an alternative approach using 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to spectrophotometrically monitor reaction progress by the release of free CoA in the reaction. This approach allows near-real time measurement of reaction rates, permitting reaction conditions (buffer, reactant, and enzyme concentrations, etc.) to be changed "on the fly." The ease and rapidity of data collection allows a high density of data points to be collected for determination of kinetic parameters. Here we provide a detailed procedure for using DTNB to measure BAHD acyl-CoA acyltransferase reaction rates, and as an example, use it to determine kinetic parameters for red clover hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase, a BAHD acyl-CoA hydroxycinnamoyltransferase not previously characterized with respect to kinetic parameters. This approach may be more generally applicable to transferases using CoA donors.

Preparation of hydroxycinnamoyl-coenzyme A thioesters using recombinant 4-coumarate:coenzyme A ligase (4CL) for characterization of BAHD hydroxycinnamoyltransferase enzyme activities.

Analyses of the enzymatic activities of hydroxycinnamoyl-coenzyme A (CoA) hydroxycinnamoyltransferases of the BAHD family require hydroxycinnamoyl-CoA thioesters as assay reagents. Here we describe a simple, cost-effective method for preparing p-coumaroyl-, caffeoyl- and feruloyl-CoA thioesters using the Arabidopsis thaliana 4-coumarate:CoA ligase 1 (4CL1) expressed in Escherichia coli. Preparation of the 4CL enzyme, in vitro synthesis of the thioesters, and thioester purification utilizing a C-18 solid phase extraction column are detailed. The hydroxycinnamoyl-CoA thioesters produced are suitable for downstream qualitative and quantitative analyses.

Tools for protein structure prediction and for molecular docking applied to enzyme active site analysis: A case study using a BAHD hydroxycinnamoyltransferase.

Elucidating the structure of an enzyme and how substrates bind to the active site is an important step for understanding its reaction mechanism and function. Nevertheless, the methods available to obtain three-dimensional structures of proteins, such as x-ray crystallography and NMR, can be expensive and time-consuming. Considering this, an alternative is using structural bioinformatic tools to predict the tertiary structure of a protein from its primary sequence, followed by molecular docking of one or more substrates into the enzyme structure model. In the past few years, significant advances have been made in these computational tools, which can give useful information about the active site and enzyme-substrate interactions before the structure can be resolved using physical methods. Here, using common bean (Phaseolus vulgaris) hydroxycinnamoyl-coenzyme A:tetrahydroxyhexanedioic acid hydroxycinnamoyltransferase (HHHT) as an example, we describe methods and workflows for protein structure prediction and molecular docking that can be performed on a personal computer using only open-source tools.

Expression and Variation of the Genes Involved in Rhizobium Nodulation in Red Clover.

Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency, in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency.

Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species.

Relative to other crops, red clover (Trifolium pratense L.) has various favorable traits making it an ideal forage crop. Conventional breeding has improved varieties, but modern genomic methods could accelerate progress and facilitate gene discovery. Existing short-read-based genome assemblies of the ∼420 megabase pair (Mbp) genome are fragmented into >135,000 contigs, with numerous order and orientation errors within scaffolds, probably associated with the plant's biology, which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. Here, we present a high-quality long-read-based assembly of red clover with a more than 500-fold reduction in contigs, improved per-base quality, and increased contig N50 by three orders of magnitude. The 413.5 Mbp assembly is nearly 20% longer than the 350 Mbp short-read assembly, closer to the predicted genome size. We also present quality measures and full-length isoform RNA transcript sequences for assessing accuracy and future genome annotation. The assembly accurately represents the seven main linkage groups in an allogamous (outcrossing), highly heterozygous plant genome.

Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides.

Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play roles in protecting the plant from biotic and abiotic stresses but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5' and 3' rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thioesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase resulted in clovamide accumulation, indicating the host species' pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.

Comparison of Protein Precipitation Ability of Structurally Diverse Procyanidin-Rich Condensed Tannins in Two Buffer Systems.

The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined included Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5% galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages). and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF > LYS ≥ BSA. PP in GVS buffer was 1 to 2.25 times greater than that in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impacts mDP, galloylation, and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature at which GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.

Engineering Alfalfa to Produce 2-O-Caffeoyl-L-Malate (Phaselic Acid) for Preventing Post-harvest Protein Loss via Oxidation by Polyphenol Oxidase.

Many plants accumulate high levels of hydroxycinnamoyl esters and amides in their tissues, presumably to protect against biotic and abiotic stress. Red clover (Trifolium pretense) leaves accumulate high levels [5-15 mmol/kg fresh weight (FW)] of caffeic acid derivatives, including phaselic acid (2-O-caffeoyl-L-malate). Oxidation of caffeoyl-malate by an endogenous polyphenol oxidase (PPO) has been shown to help preserve forage protein after harvest and during storage as silage, which should improve N use efficiency in dairy and other ruminant production systems. The widely grown forage alfalfa lacks both PPO and PPO substrates and experiences substantial loss of protein following harvest. We previously identified a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT, previously called HCT2) responsible for phaselic accumulation in red clover. With the goal of producing PPO-oxidizable compounds in alfalfa to help preserve forage protein, we expressed red clover HMT in alfalfa. Leaves of these alfalfa accumulated mainly p-coumaroyl- and feruloyl-malate (up to 1.26 and 0.25 mmol/kg FW, respectively). Leaves of HMT-expressing alfalfa supertransformed with an RNA interference (RNAi) construct to silence endogenous caffeoyl-CoA acid O-methyltransferase (CCOMT) accumulated high levels of caffeoyl-malate, as well as the p-coumaroyl and feruloyl esters (up to 2.16, 2.08, and 3.13 mmol/kg FW, respectively). Even higher levels of caffeoyl- and p-coumaroyl-malate were seen in stems (up to 8.37 and 3.15 mmol/kg FW, respectively). This level of caffeoyl-malate accumulation was sufficient to inhibit proteolysis in a PPO-dependent manner in in vitro experiments, indicating that the PPO system of post-harvest protein protection can be successfully adapted to alfalfa.

Relationships between Structures of Condensed Tannins from Texas Legumes and Methane Production During In Vitro Rumen Digestion.

Previous studies showed that a series of purified condensed tannins (CTs) from warm-season perennial legumes exhibited high variability in their modulation of methane production during in vitro rumen digestion. The molecular weight differences between these CTs did not provide correlation with either the in vitro CH4 production or the ability to precipitate bovine serum albumin. In an effort to delineate other structure-activity relationships from these methane abatement experiments, the structures of purified CTs from these legumes were assessed with a combination of methanolysis, quantitative thiolysis, ¹H-13C HSQC NMR spectroscopy and ultrahigh-resolution MALDI-TOF MS. The composition of these CTs is very diverse: procyanidin/prodelphinidin (PC/PD) ratios ranged from 98/2 to 2/98; cis/trans ratios ranged from 98/2 to 34/66; mean degrees of polymerization ranged from 6 to 39; and % galloylation ranged from 0 to 75%. No strong correlation was observed between methane production and the protein precipitation capabilities of the CT towards three different proteins (BSA, lysozyme, and alfalfa leaf protein) at ruminal pH. However, a strong non-linear correlation was observed for the inhibition of methane production versus the antioxidant activity in plant sample containing typical PC- and PD-type CTs. The modulation of methane production could not be correlated to the CT structure (PC/PD or cis/trans ratios and extent of galloylation). The most active plant in methane abatement was Acacia angustissima, which contained CT, presenting an unusual challenge as it was resistant to standard thiolytic degradation conditions and exhibited an atypical set of cross-peak signals in the 2D NMR. The MALDI analysis supported a 5-deoxy flavan-3-ol-based structure for the CT from this plant.

Spectrophotometric determination of reaction rates and kinetic parameters of a BAHD acyltransferase using DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid]).

Hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferases are BAHD family acyltransferases that transfer hydroxycinnamoyl moieties from a CoA-thioester to an acceptor amine or alcohol to form an N-hydroxycinnamoyl amide or O-hydroxycinnamoyl ester, respectively, with the concomitant release of free CoA. One approach to measure reaction rates for these enzymes is to quantify the hydroxycinnamoyl amide or ester reaction product following chromatographic separation of reaction components. This approach can be labor-intensive and time-consuming. As an alternative, we examined the use of 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to spectrophotometrically quantify, in real time, the release of free CoA during the transferase reaction. Using a hydroxycinnamoyl-CoA:l-DOPA hydroxycinnamoyl transferase as a model, we show that DTNB has little to no effect on the transferase reaction and can be used to provide a good estimate of hydroxycinnamoyl amide formation, thus allowing for the quick and easy collection of reaction rate data and determination of transferase kinetic parameters. This approach should be applicable to a wide range of hydroxycinnamoyl-CoA and other BAHD acyltransferases.

Identification of bean hydroxycinnamoyl-CoA:tetrahydroxyhexanedioate hydroxycinnamoyl transferase (HHHT): use of transgenic alfalfa to determine acceptor substrate specificity.

MAIN CONCLUSION: Transgenic alfalfa ( Medicago sativa L.) provides a useful reverse genetics platform to elucidate acceptor substrate specificity for uncharacterized BAHD family hydroxycinnamoyl-CoA hydroxycinnamoyl transferases. Tissues of many plant species accumulate hydroxycinnamoyl derivatives, often esters, thought to serve in protection against biotic and abiotic stresses. In many cases, these specialized metabolites are produced by BAHD family hydroxycinnamoyl-CoA hydroxycinnamoyl transferases (HCTs). Bean (Phaseolus vulgaris) leaves contain both hydroxycinnamoyl-malate esters and an HCT activity capable of making them. In seeking to identify this HCT from bean, we identified a gene whose predicted protein showed a high degree of sequence similarity (75%) to the Trifolium pratense (red clover) enzyme that carries out this reaction. The encoded bean protein, however, failed to carry out the malate transfer reaction when expressed in Escherichia coli. Expression of the gene in alfalfa (Medicago sativa) resulted in accumulation of several new hydroxycinnamates not present in nontransformed alfalfa, many of which corresponded to phenolics present in bean. Using accurate mass and UV absorption spectral data, we identified the acceptor substrate for this HCT as tetrahydroxyhexanedioic acids and demonstrated this predicted transferase activity with the E. coli-expressed protein. This finding adds to the growing number of BAHD family HCTs that have been characterized with respect to substrate specificity. Such data, combined with primary sequence and protein structural data will allow for a better understanding of the structure/function relationships of these enzymes and may eventually aid the rational design of such enzymes for altered substrate specificities. Additionally, expression of HCTs of unknown substrate specificity in alfalfa and characterization of the resulting accumulated novel metabolites could be a useful approach to characterizing putative BAHD HCT enzymes.

Protein Precipitation Behavior of Condensed Tannins from Lotus pedunculatus and Trifolium repens with Different Mean Degrees of Polymerization.

The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited high-purity profiles by 1D/2D NMR and purities >90% (determined by thiolysis). At pH 6.5, large CTs with a mean degree of polymerization (mDP) of ∼18 exhibited similar protein precipitation behaviors and were significantly more effective than medium CTs (mDP ∼9). Medium CTs exhibited similar capacities to precipitate ALF or BSA, but showed small but significant differences in their capacity to precipitate LYS. All CTs precipitated ALF more effectively than BSA or LYS. Aggregation of CT-protein complexes likely aided precipitation of ALF and BSA, but not LYS. This study, one of the first to use CTs of confirmed high purity, demonstrates that the mDP of CTs influences protein precipitation efficacy.

Clover, red (Trifolium pratense).

Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express ß-glucuronidase (GUS) reporter genes as well as for hairpin RNA-mediated silencing of endogenous genes for polyphenol oxidase and a transferase crucial in phaselic acid accumulation.

Perennial peanut (Arachis glabrata Benth.) leaves contain hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase activity and accumulate hydroxycinnamoyl-tartaric acid esters.

Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg(-1) crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.

Beyond brown: polyphenol oxidases as enzymes of plant specialized metabolism.

Most cloned and/or characterized plant polyphenol oxidases (PPOs) have catechol oxidase activity (i.e., they oxidize o-diphenols to o-quinones) and are localized or predicted to be localized to plastids. As a class, they have broad substrate specificity and are associated with browning of produce and other plant materials. Because PPOs are often induced by wounding or pathogen attack, they are most generally believed to play important roles in plant defense responses. However, a few well-characterized PPOs appear to have very specific roles in the biosynthesis of specialized metabolites via both tyrosinase (monophenol oxidase) and catechol oxidase activities. Here we detail a few examples of these and explore the possibility that there may be many more "biosynthetic" PPOs.

Polyphenol oxidase affects normal nodule development in red clover (Trifolium pratense L.).

Polyphenol oxidase (PPO) may have multiple functions in tissues depending on its cellular or tissue localization. Here we use PPO RNAi transformants of red clover (Trifolium pratense) to determine the role PPO plays in normal development of plants, and especially in N2-fixing nodules. In red clover, PPO was not essential for either growth or nodule production, or for nodule function in plants grown under optimal, N-free conditions. However, absence of PPO resulted in a more reduced environment in all tissues, as measured by redox potential, and caused subtle developmental changes in nodules. Leaves and, to a lesser extent nodules, lacking PPO tended to accumulate phenolic compounds. A comparison of nodules of two representative contrasting clones by microscopy revealed that nodules lacking PPO were morphologically and anatomically subtly altered, and that phenolics accumulated in different cells and tissues. Developing nodules lacking PPO were longer, and there were more cell layers within the squashed cell layer (SCL), but the walls of these cells were less thickened and the cells were less squashed. Within the N2-fixing zone, bacteroids appeared more granular and were less tightly packed together, and were similar to developmentally compromised bacteroids elicited by catalase mutant rhizobia reported elsewhere.

Gene expression patterns, localization, and substrates of polyphenol oxidase in red clover ( Trifolium pratense L.).

Polyphenol oxidase (PPO) genes and their corresponding enzyme activities occur in many plants; natural PPO substrates and enzyme/substrate localization are less well characterized. Leaf and root PPO activities in Arabidopsis and five legumes were compared with those of high-PPO red clover ( Trifolium pratense L.). Red clover PPO enzyme activity decreased leaves > stem > nodules > peduncle = petiole > embryo; PPO1 and PPO4 genes were expressed early in leaf emergence, whereas PPO4 and PPO5 predominated in mature leaves. PPO1 was expressed in embryos and nodules. PPO substrates, phaselic acid and clovamide, were detected in leaves, and clovamide was detected in nodules. Phaselic acid and clovamide, along with caffeic and chlorogenic acids, were suitable substrates for PPO1, PPO4, and PPO5 genes expressed in alfalfa ( Medicago sativa L.) leaves. PPO enzyme presence and activity were colocalized in leaves and nodules by cytochemistry. Substrates and PPO activity were localized in developing squashed cell layer of nodules, suggesting PPO may have a developmental role in nodules.

Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and PPO substrates that can reduce post-harvest proteolysis.

BACKGROUND: Studies of perennial peanut (Arachis glabrata Benth.) suggest its hay and haylage have greater levels of rumen undegraded protein (RUP) than other legume forages such as alfalfa (Medicago sativa L.). Greater RUP can result in more efficient nitrogen utilization by ruminant animals with positive economic and environmental effects. We sought to determine whether, like red clover (Trifolium pretense L.), perennial peanut contains polyphenol oxidase (PPO) and PPO substrates that might be responsible for increased RUP. RESULTS: Perennial peanut extracts contain immunologically detectible PPO protein and high levels of PPO activity (>100 nkatal mg(-1) protein). Addition of caffeic acid (PPO substrate) to perennial peanut extracts depleted of endogenous substrates reduced proteolysis by 90%. Addition of phenolics prepared from perennial peanut leaves to extracts of either transgenic PPO-expressing or control (non-expressing) alfalfa showed peanut phenolics could reduce proteolysis >70% in a PPO-dependent manner. Two abundant likely PPO substrates are present in perennial peanut leaves including caftaric acid. CONCLUSIONS: Perennial peanut contains PPO and PPO substrates that together are capable of inhibiting post-harvest proteolysis, suggesting a possible mechanism for increased RUP in this forage. Research related to optimizing the PPO system in other forage crops will likely be applicable to perennial peanut.

Efficacy of various naturally occurring caffeic acid derivatives in preventing post-harvest protein losses in forages.

BACKGROUND: In red clover, oxidation of endogenous o-diphenols by polyphenol oxidase (PPO) inhibits post-harvest proteolyis. This system is transferable to alfalfa by providing PPO (via a transgene) and o-diphenol PPO substrates (via exogenous application). To exploit the PPO system for protein protection, it would be advantageous to produce PPO substrates in alfalfa, which lacks them. We assessed the extent of PPO-mediated proteolytic inhibition by phenolic compounds, especially those whose biosynthesis could be engineered into alfalfa. RESULTS: Tested compounds included o-diphenols (caffeic acid, phaselic acid, chlorogenic acid, clovamide) and monophenols (p-coumaric acid, p-coumaroyl-malic acid). In the presence of PPO, 2 mmol o-diphenol g⁻¹ protein reduced 24 h proteolysis 68-87% (P < 0.001) and as little as 0.25 mmol g⁻¹ protein still decreased 24 h proteolysis 43-60% (P < 0.001). At high concentrations, clovamide inhibited 24 h proteolysis 50% (P < 0.001) in the absence of PPO, likely due to non-PPO oxidation. Monophenol p-coumaric acid did not inhibit 24 h proteolyis, although high levels of its malate ester did exhibit PPO- and oxygen-independent inhibition (37%, P < 0.001). CONCLUSIONS: For PPO-mediated proteolytic inhibition, pathways for both phaselic acid and chlorogenic acid may be good targets for engineering into alfalfa. Clovamide may be useful for inhibiting proteolysis without PPO.