First published online October 7, 2004; 10.1105/tpc.104.024406
The Plant Cell 16:3098-3109 (2004)
© 2004 American Society of Plant Biologists
Colocalization of L-Phenylalanine Ammonia-Lyase and Cinnamate 4-Hydroxylase for Metabolic Channeling in Phenylpropanoid Biosynthesis
Lahoucine Achnine,
Elison B. Blancaflor,
Susanne Rasmussen1 and
Richard A. Dixon2
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401
2 To whom correspondence should be addressed. E-mail radixon{at}noble.org; fax 580-224-6692.
Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope- and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.
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