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.

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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 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.

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.

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: Lignin is the second most abundant natural polymer on Earth and is a major cell wall component in vascular plants. Lignin biosynthesis has three stages: biosynthesis, transport, and polymerization of its precursors. However, there is limited knowledge on lignin precursor transport, especially in monocots. In the present study, we aimed to elucidate the transport mode of lignin monomers in the lignifying tissues of bamboo (Phyllostachys pubescens). The growth manners and lignification processes of bamboo shoots were elucidated, which enabled us to obtain the lignifying tissues reproducibly. Microsomal membrane fractions were prepared from tissues undergoing vigorous lignification to analyze the transport activities of lignin precursors in order to show the ATP-dependent transport of coniferin and p-glucocoumaryl alcohol. The transport activities for both precursors depend on vacuolar type H+-ATPase and a H+ gradient across the membrane, suggesting that the electrochemical potential is the driving force of the transport of both substrates. These findings are similar to the transport properties of these lignin precursors in the differentiating xylem of poplar and Japanese cypress. Our findings suggest that transport of coniferin and p-glucocoumaryl alcohol is mediated by secondary active transporters energized partly by the vacuolar type H+-ATPase, which is common in lignifying tissues. The loading of these lignin precursors into endomembrane compartments may contribute to lignification in vascular plants.

We previously reported the species-specific annual ring formation characteristics of three conifers (slash pine (Pinus elliottii), hinoki (Japanese cypress, Chamaecyparis obtusa) and sugi (Japanese cedar, Cryptomeria japonica)) grown in the same stand over 2 years. We found that the species-specific annual ring formation characteristics affected the inherent difference in wood density among these conifers (slash pine > hinoki > sugi). Plant hormones in cambial-region tissues were believed to affect annual ring formation. However, seasonal variation of the amounts of plant hormones in cambial-region tissues had only been examined in a few tree species. In this study, as the first step to elucidating the role of plant hormones in annual ring formation in conifers, we report the seasonal variations of the auxin (indole acetic acid, IAA) and gibberellin A4 (GA4) levels in cambial-region tissues and their effects on annual ring formation in three conifers (slash pine, hinoki, and sugi) with inherently different wood densities.Sugi (small wood density) had significantly higher levels of IAA and formed more tracheids in the early season than in the late season, although slash pine (large wood density) had higher levels of IAA and formed significantly more tracheids in the late season than in the early season. Hinoki (intermediate wood density) had constant IAA levels and formed a constant number of tracheids throughout the season. There were significant positive correlations between the levels of IAA in cambial-region tissues and the number of tracheids formed during late season in the two conifer species. A close relationship was observed between the seasonal ratio of the IAA levels (late/early season) and wood density. No consistent trend in the change in the level of IAA during the transition from earlywood to latewood formation was recognized among the three conifers. The IAA levels in slash pines were significantly higher than those in sugi and hinoki. The GA4 levels had no significant effect on number of tracheids formed in the three conifers. These results suggest that the species-specific seasonal variation patterns of the IAA levels might lead to the inherent differences in wood density among these three conifers through species-specific characteristics in the formation of annual rings.