Tracking Monolignols during Wood Development in Lodgepole Pine 1(W)(OA)

Secondary xylem (wood) formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and then reinforce it with lignin, an amorphous, three-dimensional product of the random radicalcouplingofmonolignols.Theobjectiveofthisstudywastotrackthespatialdistributionofmonolignolsduringdevelopment as they move from symplasm to apoplasm. This was done by feeding [ 3 H]phenylalanine ([ 3 H]Phe) to dissected cambium/ developingwoodfromlodgepolepine(Pinuscontortavarlatifolia)seedlings,allowinguptakeandmetabolism,thenrapidlyfreezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. Parallel experiments showed that radioactivity was incorporated into polymeric lignin and a methanol-soluble pool that was characterized by high-performance liquid chromatography. [ 3 H]Phe was incorporated into expected lignin precursors, such as coniferyl alcohol and p-coumaryl alcohol, as well as pinoresinol. Coniferin, the glucoside of coniferyl alcohol, was detected by high-performance liquid chromatography but was not radioactively labeled. With light microscopy, radiolabeled phenylpropanoids were detected in the rays as well as the tracheids, with the two cell types showing differential sensitivity to inhibitors of protein translation and phenylpropanoid metabolism. Secondary cell walls of developing tracheids were heavily labeled when incubated with [ 3 H]Phe. Insidethecell,cytoplasmwasmoststronglylabeledfollowedbyGolgiandlow-vacuolelabel.InhibitorstudiessuggestthattheGolgi signalcouldbeattributedtoprotein,ratherthanphenylpropanoid,origins.Thesedata,producedwiththebestmicroscopytoolsthat are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols.