Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.

No abstract

Lignin is a cell wall component of vascular plants crucial for survival in terrestrial environments. While p -hydroxyphenyl lignin is minor, it is considered to be localised in the outermost part of the cell wall providing strong adhesion between cells, which determines cell shape. Transport of the lignin precursor from the cytosol to the cell wall is critical to regulate temporal and spatial lignin deposition; however, little information on the transport step is available. Here, we report transport activity of p -glucocoumaryl alcohol, a precursor of p -hydroxyphenyl lignin, in a broad-leaved tree (hybrid poplar, Populus sieboldii × P . grandidentata ) and a coniferous tree (Japanese cypress, Chamaecyparis obtusa ). Membrane vesicles of both trees were prepared from differentiating xylem with vigorous lignification and used for transport assays. Several inhibition assays indicated that not ABC transporters but the proton gradient and V-ATPase are involved in p -glucocoumaryl alcohol transport depending on ATP. These results support the hypothesis that p -glucocoumaryl alcohol is loaded into the secretory vesicles and delivered to the cell wall by exocytosis. Furthermore, this transport mechanism was common in both poplar and Japanese cypress, strongly suggesting that p -glucocoumaryl alcohol transport in the differentiating xylem is conserved within woody plants.

Bamboos are among the largest woody grasses and grow very rapidly. Although lignin is a crucial factor for the utilization of bamboo biomass, the lignification mechanism of bamboo shoots is poorly understood. We studied lignification in the bamboo Sinobambusa tootsik during culm development. Elongation growth began in May and ended in late-June, when the lignin content was approximately half that in mature culms. Thioacidolysis analysis indicated that p-hydroxyphenyl units in lignin formed even at late stages of lignification. The syringyl/ guaiacyl ratio varied during culm development. Various lignin precursors were detected in developing culms by liquid chromatography-mass spectrometry. The ferulic acid content decreased from May to June, indicating that ferulic acid was utilized in early stages of cell wall formation. Monolignol glucosides were detected at early stages of lignification, whereas the contents of monolignols, coniferaldehyde, sinapaldehyde, p-coumaric acid, and ferulic acid peaked at later stages of lignification. Therefore, lignin precursors may be supplied differentially during the lignification process. In August, the rate of lignification decreased, although the contents of various lignin precursors peaked, implying that the rate-limiting step in the cessation of lignification in bamboo is transport or polymerization of lignin precursors, rather than their biosynthesis.

Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differentiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii 9 Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differentiating xylem of poplar may be used for the lignification of the xylem.