Michael L. Sullivan

Cloning and Characterization of Red Clover Polyphenol Oxidase cDNAs and Expression of Active Protein in Escherichia coli and Transgenic Alfalfa

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%–83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.

Anti-Inflammatory Procyanidins and Triterpenes in 109 Apple Varieties

We evaluated the potential of apple to reduce inflammation. Phenolic compounds and triterpenes were analyzed in 109 apple cultivars. Total phenolics ranged from 29 to 7882 μg g(-1) of fresh weight (FW) in the flesh and from 733 to 4868 μg g(-1) FW in the skin, with flavanols including epicatechin and procyanidins as major components. Ursolic (44.7 to 3522 μg g(-1) FW) and oleanolic (47.2 to 838 μg g(-1) FW) acids dominated the skin triterpene profile. Five chemically contrasting cultivars were fractionated and their immune-modulating activity measured using two cell-based assays targeting key points in the inflammation process. Cultivars exhibiting high contents of procyanidins were the most potent at inhibiting NF-κB while triterpene-rich fractions reduced the promoter activity of the gene of TNFα. This study provides new insights into how apple genetic diversity could be used to alleviate inflammation.

A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent with a Role in Phaselic Acid Biosynthesis

Red clover (Trifolium pratense) leaves accumulate several μmol g−1 fresh weight of phaselic acid [2-O-(caffeoyl)-l-malate]. Postharvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forage crops like alfalfa (Medicago sativa) lack both polyphenol oxidase and o-diphenols, and breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forage crops utilizing this natural system of protein protection. A possible pathway for phaselic acid biosynthesis predicts a hydroxycinnamoyl transferase (HCT) capable of forming caffeoyl and/or p-coumaroyl esters with malate. Genes encoding two distinct HCTs were identified in red clover. HCT1 shares more than 75% amino acid identity with a number of well-characterized shikimate O-HCTs implicated in monolignol biosynthesis. HCT2 shares only 34% amino acid sequence identity with HCT1 and has limited sequence identity to any previously identified HCT. Expression analyses indicate that HCT1 mRNA accumulates to 4-fold higher levels in stems than in leaves, whereas HCT2 mRNA accumulates to 10-fold higher levels in leaves than in stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli indicate that HCT1 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to shikimate but not malate, whereas HCT2 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to malate but not shikimate. Together, these results indicate that HCT1 is involved in monolignol biosynthesis and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.

Immunogold labelling to localize polyphenol oxidase (PPO) during wilting of red clover leaf tissue and the effect of removing cellular matrices on PPO protection of glycerol-based lipid in the rumen

BACKGROUND The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones, which can react with nucleophilic cellular constituents (e.g. proteins) forming protein-phenol complexes that may reduce protein solubility, bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localized PPO in red clover leaf tissue by immunogold labelling and investigated whether red clover lipid was protected in the absence of PPO-induced protein-phenol complexes and plant enzymes (lipases). RESULTS PPO protein was detected to a greater extent (P < 0.001) within the chloroplasts of mesophyll cells in stressed (cut/crushed and wilted for 1 h) than freshly cut leaves for both palisade (61.6 and 25.6 Au label per chloroplast, respectively) and spongy mesophyll cells (94.5 and 40.6 Au label per chloroplast, respectively). Hydrolysis of lipid and C18 polyunsaturated fatty acid biohydrogenation during in vitro batch culture was lower (P < 0.05) for wild-type red clover than for red clover with PPO expression reduced to undetectable levels but only when cellular matrices containing protein-phenol complexes were present. CONCLUSION Damaging of the leaves resulted in over a doubling of PPO detected within mesophyll cells, potentially as a consequence of conversion of the enzyme from latent to active form. PPO reduction of microbial lipolysis was apparent in macerated red clover tissue but not in the absence of the proteinaceous cellular matrix, suggesting that the PPO mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein-phenol complexes.

Proteomic Analysis of Cell Walls of Two Developmental Stages of Alfalfa Stems

Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g., crosslinking) of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible) and basal alfalfa stems (more mature, less digestible) was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 272 proteins in the alfalfa stem cell wall proteome, 153 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicates little overlap in identified proteins among them, which may be largely due to differences in the tissues used as well as differences in experimental approach.

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.

Red Clover HCT2, a Hydroxycinnamoyl-Coenzyme A:Malate Hydroxycinnamoyl Transferase, Plays a Crucial Role in Biosynthesis of Phaselic Acid and Other Hydroxycinnamoyl-Malate Esters in Vivo

In red clover (Trifolium pratense) leaves, phaselic acid (2-O-caffeoyl-l-malate) accumulates to several mmol kg−1 fresh weight and is a crucial component of a natural system that prevents protein breakdown during harvest and storage of this forage crop. Previously, we identified HCT2, a red clover gene encoding a hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferase capable of transferring p-coumaroyl and caffeoyl moieties from their CoA derivatives to malic acid to form the corresponding hydroxycinnamoyl-malate esters in vitro. Here, we carried out a detailed kinetic analysis of the enzyme and examined its in vivo function in red clover via reverse genetics. The kinetic analysis indicates that in vitro, despite similar Km values for the tested hydroxycinnamoyl-CoA derivatives, HCT2 favors transfer to malate of p-coumaroyl and feruloyl moieties over caffeoyl moieties by greater than 5-fold. Reverse reaction (transfer of hydroxycinnamoyl moieties from malate to CoA) by HCT2 was observed with p-coumaroyl-malate but not phaselic acid. Analysis of red clover plants down-regulated for HCT2 expression via RNA interference showed a significant and substantial correlation between HCT2 mRNA levels and phaselic acid accumulation (P < 0.005). In several of the HCT2-silenced plants, phaselic acid and p-coumaroyl-malate levels were reduced to <5% that of wild-type controls. These reductions resulted in easily observable phenotypes including reduced polyphenol oxidase-mediated browning and a reduction in blue epidermal fluorescence under ultraviolet light. These results demonstrate a crucial role for HCT2 in phaselic acid accumulation in red clover and define a previously undescribed pathway for the biosynthesis of hydroxycinnamoyl-malate esters in plants.

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.

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.

Red clover coumarate 3'-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid.

Red clover (Trifolium pratense) leaves accumulate several μmol of phaselic acid [2-O-caffeoyl-l-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases (PPO) prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar PPO activity and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forages utilizing this natural system of protein protection. We have proposed biosynthetic pathways in red clover for phaselic acid that involve a specific hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether the transfer reaction to malate to form phaselic acid involves caffeic acid or p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate. The latter would require a coumarate 3′-hydroxylase (C3′H) capable of hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a cytochrome P450 C3′H (CYP98A44) was identified and its gene cloned from red clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula and Arabidopsis thaliana C3′H proteins that are capable of hydroxylating p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis. CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves. Immune serum raised against CYP98A44 recognizes a membrane-associated protein in red clover stems and leaves and cross-reacts with C3′H proteins from other species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding indicates that in red clover, phaselic acid is likely formed by transfer of a caffeoyl moiety to malic acid, although the existence of a second C3′H capable of hydroxylating p-coumaroyl-malate cannot be definitively ruled out.