ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

Abstract

Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

Fig. 1.

Preparation and characterization of plasma membrane (PM) vesicles and the transport of monolignols by inside-out plasma membrane vesicles. (A) Protein gel-blot analysis of membrane fractions by probing with antibodies against Arabidopsis plasma membrane H⁺-ATPase (PM-H⁺-ATPase), vacuolar H⁺-pyrophosphatase (V-PPase, tonoplast marker), and endoplasmic reticulum-binding protein (Bip, ER marker). (B) H⁺-ATPase activities of different membrane preparations in the presence or absence of MgATP. The results are from three replicates. (C) Portions of HPLC traces showing the recovered coniferyl alcohol from the inverted vesicles in the uptake assay in the presence (a) and absence (b) of ATP, the presence of sodium vanadate and ATP (c), and the absence of phenolic substrate (d). (D) Time course of the transport of coniferyl alcohol into the inside-out vesicles. The results are the mean and SD of two replicates. (E and F) The kinetics of transport of coniferyl alcohol into Arabidopsis inside-out plasma membrane vesicles for coniferyl alcohol (E) and MgATP (F) concentrations. The data are plotted by a nonlinear regression analysis fit to the Michaelis–Menten equation. The insets in E and F show Lineweaver–Burk plots. The data are the means and SD of two or three replicates.

Fig. 2.

Inhibition of ATP-dependent uptake of plasma membrane vesicles to coniferyl alcohol by inhibitors of membrane transporters. Inside-out vesicles were incubated with 100 μM coniferyl alcohol, to which we added different inhibitors at varied concentrations as described in Materials and Methods. The average transport activity under MgATP and monolignol only was set to 100%. The data are the means and SD of three replicates. *Significant changes at P < 0.01 and **significant changes at P < 0.05, compared with the control, under Student's t test.

Fig. 3.

Preparation of vacuolar membranes and vacuolar sequestration assay. (A) Protein gel-blot analysis of selected fractions probed with antibodies against V-PPase, Bip, and plasma membrane H⁺-ATPase. (B) Portions of HPLC traces showing recovered coniferyl alcohol 4-O-glucoside (coniferin) from the vacuolar vesicles in the sequestering assay in the presence (a) and absence (b) of ATP, the presence of sodium vanadate and ATP (c), and the presence of gramicidin D and ATP (d). (C) Effects of the membrane transporter inhibitors sodium vanadate (1 mM) and gramicidin D (5 μM) on the ATP-dependent uptake of coniferin by vacuolar vesicles. Data were from three replicates. *Significant changes at P < 0.01 under t test. (D and E) The kinetics of transport of coniferin into vacuolar vesicles for coniferin (D) and MgATP (E) concentrations. The data are from two or three replicates and are plotted by nonlinear regression analysis fit to the Michaelis–Menten equation. Insets show Lineweaver–Burk plots.