Submitted to: Biologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2009
Publication Date: 6/30/2009
Citation: Sullivan, M.L. 2009. Phenylalanine Ammonia Lyase (PAL) Genes in Red Clover: Expression in Whole Plants and in Response to Yeast Fungal Elicitor. Biologia Plantarum. 53(2):301-306. Interpretive Summary: The enzyme, phenylalanine ammonia lyase (PAL), is responsible for the first step in the production of a wide range of natural products based on the phenylpropanoid skeleton. In red clover, o-diphenols, a class of phenylpropanoids, accumulate to high levels and can be oxidized by the enzyme polyphenol oxidase (PPO). When red clover is preserved by ensiling, post-harvest oxidation of o-diphenols by PPO prevents degradation of forage protein to non-protein nitrogen, which is poorly utilized by ruminant animals. To exploit this natural system of protein protection in other forage crops, our group is trying to understand how red clover is able to accumulate the required levels of o-diphenol compounds. This publication reports the isolation and characterization of red clover genes encoding PAL. Red clover has at least four genes encoding PAL, designated PAL1-4. PAL2/3/4 encode nearly identical proteins that are substantially different from PAL1. In some plants, PAL genes are up-regulated (expressed at higher levels) upon infection with a pathogen. To test whether this is true of the red clover PAL genes, we adapted a system utilizing an elicitor prepared from baker’s yeast. Treatment of red clover seedlings with the yeast elicitor resulted in up-regulation of PAL2/3/4 and chalcone synthase (another gene often up-regulated in response to pathogen attack), but not of PAL1 or actin (a gene not expected to be up-regulated in response to pathogen attack). Together, these results suggest PAL1 and PAL2/3/4 play major roles in the biosynthesis of phenylpropanoid compounds under normal conditions, but that PAL2/3/4 are also involved in pathogen defense responses. These results will be useful in understanding o-diphenol and phenylpropanoid biosynthesis in red clover and adapting these pathways for other forages. Additionally, in the course of this study, we have adapted a baker’s yeast fungal elicitor system (previously only used for plant cell suspensions cultures) for use with intact red clover seedlings. This system could greatly facilitate research on fungal diseases and plant-pathogen interaction in red clover.
Technical Abstract: In red clover (Trifolium pratense L.) four unique cDNAs encoding phenylalanine ammonia lyase (PAL, EC 126.96.36.199) were identified (PAL1-4). PAL2-4 encode nearly identical proteins (> 97%) that are only 89% identical to that encoded by PAL1. Under normal growing conditions in young leaves and flowers, PAL1 mRNA accumulates to higher levels than that of PAL2-4, whereas in mature leaves and stems, mRNA levels are similar for PAL1 and PAL2-4. Treatment of red clover seedlings with yeast elicitor (YE) preparation results in an approximately sixfold induction of PAL2-4 transcripts within 1 h of treatment, but only a modest induction (< twofold) of PAL1 transcripts. These results suggest that while both classes of enzymes play a role in biosynthesis of phenylpropanoid compounds under normal growing conditions, PAL2-4 enzymes are also involved in pathogen defense responses.