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Title: Phenolic profiles and polyphenol oxidase (PPO) gene expression of red clover (Trifolium pratense) selected for decreased postharvest browning

item Kagan, Isabelle
item Dinkins, Randy
item TAYLOR, NORMAN - University Of Kentucky

Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2016
Publication Date: 7/27/2016
Citation: Kagan, I., Dinkins, R.D., Taylor, N.L. 2016. Phenolic profiles and polyphenol oxidase (PPO) gene expression of red clover (Trifolium pratense) selected for decreased postharvest browning. American Journal of Plant Sciences. 7:1478-1489.

Interpretive Summary: Red clover is a legume eaten by grazing animals. It contains many natural products, known as phenolic compounds that affect grazing animal health. When red clover is being dried in the field for hay, some phenolic compounds interact with an enzyme, commonly known as PPO, to bind to proteins. The proteins are then more available for use by the animal, because they do not break down as quickly during digestion when they are bound to phenolic compounds. This reaction between phenolic compounds and proteins often results in a brown color. Red clover that does not turn brown while drying could provide information on how phenolic compounds and PPO change when no browning occurs. In this study, red clover was bred for browning less while drying. Seed was collected from plants (the "Kenland" red clover variety) that appeared less brown after drying; and from the plants grown from that seed, and selections were made again of plants that browned less after drying. This cycle of planting and selecting was repeated eight times prior to this study. Browning decreased to an intermediate level over those eight cycles of selection. RNA (ribonucleic acid) from leaves from three of these selection cycles was studied to see if the expression of two genes for PPO changed as browning decreased, and no change was found. The phenolic compounds in the original Kenland variety and the eighth selection cycle were measured. Concentrations of two compounds decreased in the eighth selection cycle. One (clovamide) binds to proteins, but it does not need to interact with PPO in order to bind. No changes occurred in the total amounts of formononetin, a phenolic compound that affects reproduction, or in biochanin A, a phenolic compound that prevents loss of protein as ammonia. This results could benefit plant geneticists in understanding that selecting for greener red clover hay leads to a decrease in phenolic compounds that can bind proteins, but expression of two PPO genes, and amounts of some of the phenolic compounds with a role in grazing animal health, are not affected.

Technical Abstract: Red clover (Trifolium pratense L.) is a legume forage abundant in phenolic compounds. It tends to brown when cut for hay, due to oxidation of phenolic compounds catalyzed by polyphenol oxidase (PPO), and subsequent binding to proteins. Selecting for a greener hay may provide information about the relationship of browning, PPO, and phenolics to each other. The red clover Kenland cultivar was selected over eight breeding cycles for decreased browning after being cut and dried 48 h in the field. Expression of PPO1 and PPO3, in Kenland and three of the eight cycles, was compared by real-time quantitative PCR. Phenolic compounds in Kenland and Cycle 8, collected 0, 24, and 48 h after cutting, were quantified by high-performance liquid chromatography (HPLC). Visual browning scores decreased 12% between Kenland and Cycle 8 (P = 0.02). PPO1 and PPO3 gene expression were not affected by selection. Clovamide decreased 26% in Cycle 8 relative to Kenland (P = 0.016). Sissotrin decreased 10% in Cycle 8 (P = 0.043). Total formononetin was unaffected by selection (P=0.63), as was total biochanin A (P=0.45). These results suggest that when selecting clover for decreased postharvest browning, a decrease occurs in phenolic compounds that bind protein independently of PPO. However, PPO1 and PPO3 gene expression, and the major red clover isoflavones, are minimally affected.