The law of supply and demand rules monolignol transport

Simple mechanisms are sometimes the hardest to prove. For instance, the Münch hypothesis on how phloem tubes move sugars from source leaves to sink tissues remained just a plausible idea for 86 years until it was experimentally demonstrated in 2016 (Knoblauch et al., 2016). In new work, Mendel Perkins and collaborators (Perkins et al., 2022) tackle another simple long-standing question: how do monolignols, the precursors of lignin, get exported across the plasma membrane to the apoplastic space where lignin polymerization takes place?

Lignin is, after cellulose, the second most abundant biopolymer on the planet and is a potential source of renewable energy and new high-value chemicals. It is hydrophobic and is believed to have been key for the colonization of land by plants, as it rigidifies and impermeabilizes the secondary walls of xylem cells, allowing for long-distance transport of water and taller growth (Renault et al., 2017).

Lignin composition is by definition diverse and highly adaptive: different phenolic precursors vary between species and between tissues, developmental stages, and environmental conditions with each plant, a plasticity that enables finely tuned adaptation (Lourenço et al., 2016). Monolignol precursors need to be transported from the cytoplasm to the cell walls, where laccases and peroxidases radicalize them to induce polymerization. The transport route of monolignols must be highly efficient since lignin accounts for 25% of woody biomass. For years, researchers wondered if the transport of diverse monolignols is mediated by non-specific membrane protein transporters or a more promiscuous mechanism, like simple diffusion, allowed by the kinetics of the powerful monolignol flux.

To date, no transporter has been identified that could account for the diversity of precursors. In 2019, Vermaas et al. (2019) modeled monolignol export and predicted that lignin precursors move across lipid membranes by simple diffusion in the presence of a concentration gradient, but experimental evidence to support this model was missing.

Seeing is believing, and Perkins and colleagues decided to track lignification in xylem vessels of living Arabidopsis roots. Two-photon microscopy allowed them to visually penetrate the tissue at the required depth to observe the autofluorescence associated with lignin and its soluble components. This clever approach avoided the use of specific marker lines that might miss key elements or the static picture provided by staining. To perturb the system, the group tracked changes in lignin polymerization in a laccase mutant. According to the Vermaas model, laccases oxidize soluble monomers that then precipitate to form insoluble lignin, creating a sink that will establish a gradient across the plasma membrane in combination with cytoplasmic monolignol biosynthesis as the source. In the absence of laccases, soluble monomers will accumulate and lignin will be present at lower levels in the wall, a hypothesis that was confirmed in the observed living cells. However, changes in lignification were not observed when the roots were treated with vanadate, an inhibitor of P-type ATPases, such as ABC transporters, suggesting that energy-requiring transporters are not likely the main mechanism by which lignin precursors exit plant cells.

To demonstrate that monolignols can diffuse across membranes, the authors simplified the system to the minimum: they recreated the monolignol gradient by encapsulating fungal laccases in liposomes and incubated these liposomes with the monolignol, coniferyl alcohol, to act as a source (see Figure). When the liposomes contained the polymerization-inducing enzymes, the concentration of coniferyl alcohol decreased in the media while larger phenolic polymers were found inside the vesicles. Furthermore, when imaged with super-resolution microscopy, dots characteristic of lignin autofluorescence were observed inside the liposomes in the absence of transporters.

Figure.

Laccases, contained in liposomes incubated with lignin precursors, are able to polymerize lignin, driving the transport of monomers down a concentration gradient.

These and other experimental data presented by Perkins et al. nicely confirm that monolignols can cross the plasma membrane by simple diffusion and that the gradient generated by partitioning monolignol biosynthesis and consumption in different compartments drives the export. The law of supply and demand indeed rules the lignin market.