Abstract
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 radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm. This was done by feeding [(3)H]phenylalanine ([(3)H]Phe) to dissected cambium/developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, then rapidly freezing 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. Inside the cell, cytoplasm was most strongly labeled followed by Golgi and low-vacuole label. Inhibitor studies suggest that the Golgi signal could be attributed to protein, rather than phenylpropanoid, origins. These data, produced with the best microscopy tools that are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols.
Highlights
Secondary xylem formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols
The abundance of coniferin led to the hypothesis that, first, coniferin is exported during xylem development, coniferin b-glucosidases in the secondary cell wall cleave off the Glc, generating coniferyl alcohol for lignification in the apoplast (Freudenberg, 1959)
In order to interpret autoradiography results showing where [3H]Phe and its derivatives are found in developing wood, it was necessary to determine the biochemical fate of the [3H]Phe
Summary
Secondary xylem (wood) formation in gymnosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm This was done by feeding [3H]phenylalanine ([3H]Phe) to dissected cambium/ developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, rapidly freezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. The abundance of coniferin led to the hypothesis that, first, coniferin is exported during xylem development, coniferin b-glucosidases in the secondary cell wall cleave off the Glc, generating coniferyl alcohol for lignification in the apoplast (Freudenberg, 1959). During secondary cell wall deposition in lodgepole pine (Pinus contorta var latifolia), the protoplasts of the developing tracheids have prominent Golgi with grape-like clusters of vesicles being shed from the transGolgi network (Samuels et al, 2002). This work led to the following question: do these abundant Golgi vesicles carry monolignols to the developing secondary cell wall?
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