Michael L Sullivan

Agricultural Research Service, Kerrville, Texas, United States

Are you Michael L Sullivan?

Claim your profile

Publications (16)31.22 Total impact

  • Michael L Sullivan, Jamie L Foster
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. © 2013 Society of Chemical Industry.
    Journal of the Science of Food and Agriculture 01/2013; · 1.76 Impact Factor
  • Source
    Julian C Verdonk, Ronald D Hatfield, Michael L Sullivan
  • Source
    Julian C Verdonk, Ronald D Hatfield, Michael L Sullivan
  • Source
    Julian C Verdonk, Ronald D Hatfield, Michael L Sullivan
  • Julian C. Verdonk, Michael L. Sullivan
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used, however they all have in common the artificial generation of single stranded small ribonucleic acids (RNAs) that are utilized by the endogenous gene silencing machinery of the organism. Artificial microRNAs (amiRNA) can be used to very specifically target genes for silencing because only a short sequence of 21 nucleotides of the gene of interest is used. Gene silencing via amiRNA has been developed for Arabidopsis thaliana (L.) Heynh. and rice using endogenous microRNA (miRNA) precursors and has been shown to also work effectively in other dicot species using the arabidopsis miRNA precursor. Here, we demonstrate that the arabidopsis miR319 precursor can be used to silence genes in the important forage crop species alfalfa (Medicago sativa L.) by silencing the expression of a transgenic beta-glucuronidase (GUSPlus) target gene.
    Botany 12/2012; 91(2):117-122. · 1.23 Impact Factor
  • Michael L Sullivan, Wayne E Zeller
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. Published 2012 by John Wiley & Sons, Ltd.
    Journal of the Science of Food and Agriculture 07/2012; · 1.76 Impact Factor
  • Source
    Julian C Verdonk, Ronald D Hatfield, Michael L Sullivan
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Frontiers in Plant Science 01/2012; 3:279. · 3.60 Impact Factor
  • Source
    Michael L Sullivan, Robert Zarnowski
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Plant physiology 01/2011; 155(3):1060-7. · 6.56 Impact Factor
  • Michael L Sullivan, Robert Zarnowski
    [Show abstract] [Hide abstract]
    ABSTRACT: Red clover (Trifolium pratense) leaves accumulate several mumol 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.
    Planta 11/2009; 231(2):319-28. · 3.35 Impact Factor
  • Source
    George E Schmitz, Michael L Sullivan, Ronald D Hatfield
    [Show abstract] [Hide abstract]
    ABSTRACT: Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.
    Journal of Agricultural and Food Chemistry 02/2008; 56(1):272-80. · 2.91 Impact Factor
  • Source
    Michael L. Sullivan, Ronald D. Hatfield, Deborah A Samac
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: Ensiling forages often leads to degradation of protein to non-protein nitrogen (NPN), which is poorly utilized by ruminants. Postharvest protein degradation is especially high in alfalfa (Medicago sativa L.). In contrast, red clover (Trifolium pratense L.) has up to 90% less protein loss during ensiling due to polyphenol oxidase (PPO) forming o-quinones from endogenous o-diphenols and subsequent binding of o-quinones to cytoplasmic proteins. Here we determined whether an endogenous PPO might be exploited for postharvest protein protection in alfalfa.RESULTS: We isolated an alfalfa PPO gene (MsPPO1) that shares limited sequence identity (70-72%) with red clover PPO genes. MsPPO1 is expressed primarily in flowers and developing seed pods, but not in leaves or stems. Expression of MsPPO1 from a strong constitutive promoter in transgenic alfalfa results in accumulation of PPO transcripts in leaves, but little enzyme activity is detected using a variety of o-diphenol substrates unless assayed in the presence of sodium dodecyl sulfate (SDS). Under this SDS-activated condition, preference of MsPPO1 for tested substrates is catechol >= (-)-epicatechin > caffeic acid. PPO activity in unactivated MsPPO1-alfalfa extracts is sufficient to inhibit proteolysis in the presence of catechol, but not caffeic acid or (-)-epicatechin. Inhibition is less than in extracts of alfalfa expressing the red clover PPO1 gene.CONCLUSION: Endogenous alfalfa PPO, even if expressed in appropriate target tissues, would be less effective at preventing proteolytic losses in ensiled forages than red clover PPO. Published in 2008 by John Wiley & Sons, Ltd.
    Journal of the Science of Food and Agriculture 01/2008; · 1.76 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants with subsequent increases in the efficiency of N utilization and the level of beneficial polyunsaturated fatty acids in their products (meat and milk). It has also been reported that red clover feeding alters the rumen microbial population compared to grass feeding. This study investigated whether the observed shifts in the microbial population of the rumen when ruminants are fed red clover silage (RC) as opposed to grass silage (G) represented an adaptation by the micro-organisms to increase the utilization of PPO-protected protein and glycerol-based lipid.RESULTS: The experiment consisted of two periods where ruminally fistulated dairy cows were offered either RC or G for 2 weeks, followed by collection of rumen fluid, which was then used in in vitro incubations to investigate lipolysis and proteolysis over time in plant material derived from red clover plants with either wild type PPO expression (PPO+) or PPO expression reduced to undetectable levels by gene silencing (PPO-). Proteolysis and lipolysis (P < 0.05) were lower after 24 h of incubation in the PPO+ treatment than the PPO- treatment irrespective of rumen fluid. Biohydrogenation of C18 polyunsaturated fatty acids was also lower on the PPO+ treatment than the PPO- treatment, with no effect of rumen fluid.CONCLUSION: These results suggest that microbial changes to red clover feeding did not result in an increased ability of the micro-organisms in the present study to utilize either PPO-protected protein or glycerol-based lipid.
    Journal of the Science of Food and Agriculture 01/2008; · 1.76 Impact Factor
  • Source
    Michael L. Sullivan, Ronald D. Hatfield
    [Show abstract] [Hide abstract]
    ABSTRACT: Many forages experience significant proteolytic losses when preserved by ensiling. Such losses in alfalfa (Medicago sativa L.) are especially high, with degradation of 44 to 87% of the forage protein to nonprotein N (NPN). In contrast, red clover (Trifolium pratense L.) has up to 90% less proteolysis during ensiling. Here we demonstrate that the combination of polyphenol oxidase (PPO) and o-diphenol PPO substrates, both abundantly present in red clover, is responsible for postharvest proteolytic inhibition in this forage crop. Proteolysis in red clover leaf extracts increased nearly fivefold when endogenous o- diphenols were removed by gel filtration but returned to starting levels by adding back an exogenous o-diphenol. Proteolysis in leaf extracts of red clover plants silenced for PPO expression was dramatically in- creased compared to control plants. Leaf extracts of transgenic alfalfa expressingaredcloverPPOgeneshowedanearlyfivefoldo-diphenol- dependent decrease in proteolysis compared to those of control alfalfa. We also demonstrate that PPO levels 10- to 20-fold lower than those typically found in red clover are sufficient for proteolytic inhibition, that as little as 0.25 mmol o-diphenol mg 21 protein has a substantial impact on proteolysis, that a wide variety of o-diphenols are functional substrates in proteolytic inhibition, and that proteolysis is reduced for PPO-expressing alfalfa in small-scale ensiling experiments. Together, these results indicate that PPO and o-diphenols can be an effective treatment to prevent protein loss in ensiled forage crops.
    Crop Science - CROP SCI. 01/2006; 46(2).
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Plant physiology 11/2004; 136(2):3234-44. · 6.56 Impact Factor
  • Source
    Michael L. Sullivan
  • Source
    Michael L. Sullivan

Publication Stats

116 Citations
31.22 Total Impact Points

Institutions

  • 2012
    • Agricultural Research Service
      Kerrville, Texas, United States
  • 2009–2011
    • United States Department of Agriculture
      • Agricultural Research Service (ARS)
      Washington, D. C., DC, United States
  • 2004
    • University of Wisconsin, Madison
      • Agricultural Research Service
      Madison, MS, United States