Transport of monolignols and their dimers

mechanism involving Kelch repeat F-box proteins. As glucosylation affects the CA pool, it may also affect PAL activity.

The third hypothesis for the role of MGs is the detoxification of monolignols. As discussed, CA or its degradation products can be harmful to plant cells themselves (I).

Naturally, glucosylation could alleviate these effects. If this was true, it would be interesting to understand why some plants then contain more MGs than the others.

The developing xylem in angiosperm species does not contain monolignol glucosides at the same levels as in gymnosperms (Terashima et al., 2016). The explanation that I will present assumes that the polymerisation sink is important for the monolignol transport. The loss of a polymerisation sink in lac4 lac17 double mutants of Arabidopsis caused a decrease in lignification and an accumulation of phenolics, presumably glucosides, in the vacuole (Perkins et al., 2022). Similarly in the lignin- forming spruce cell culture, impairing the polymerisation sink by scavenging H2O2 by KI, and thus preventing the function of PRXs causes accumulation of some glucosides such as coniferin in the cells (Laitinen et al., 2017). In the developing xylem of Norway spruce, coniferin is present naturally. Is there is imbalance in monolignol production and oxidation in gymnosperms early in the growth season when the MGs are known to accumulate? This could result in an accumulation of monolignol alcohols in the cytosol and thereby force the cell to glycosylate them in order to alleviate the cytotoxicity of free monolignols. The role of MGs is not clear, but identification for their transporter(s) could shed light on this question.

several tree species. As such, the results are also in line with the hypothesis of monolignol diffusion where aglycones do not need a transporter whereas glucosides do (Vermaas et al., 2019; Perkins et al., 2022). However, active transport of CA and pCA have been detected (Miao and Liu, 2010; Alejandro et al., 2012). As pCA content in lignin is much lower than that of CA, the transporter may function in a situation where only minor quantities of pCA are produced, and diffusion would not increase its concentration in the developing CW sufficiently such that a transporter would be needed alongside diffusion, or in a situation where pCA export should follow a localised pattern.

In addition to plant transporters for phenolic aglycones, bacteria have many transporters for lignin-related compounds. For example, Pseudomonas putida has a transporter, PcaK, for 4-hydroxybenzoate and protocatechuate, both degradation products of lignin (Nichols and Harwood, 1997), and Sphingobium sp. strain SYK-6 has an MFS transporter for protocatechuate (Mori et al., 2018). Rhodopseudomonas palustris, a photosynthetic bacterium, has two transporter systems for p-coumarate, caffeate and ferulate: an ABC transporter with a periplasmic binding protein CouPSTU, and a TRAP transporter with a periplasmic binding protein TarP (Salmon et al., 2013).

However, all of the above-mentioned compounds are charged, unlike monolignols, and thus less permeable to membranes based on molecular simulations (Vermaas et al., 2022) which could explain the need of a transporter.

One of the proposed hypotheses was that monolignol dimers could serve as a substrate for a transporter. However, no ATP-dependent transport of pinoresinol, lariciresinol, isolariciresinol, β-O-4 erol (two CAs β-O-4 linked) or pinoresinol monoglucoside could be detected in MF vesicles of Norway spruce developing xylem (II). Pinoresinol did inhibit coniferin transport but was itself not transported. As Vermaas et al. (2019) suggested, these compounds, if formed intracellularly, may diffuse through the membranes.

A surprising effect was seen with MF vesicles prepared from Norway spruce cultured cells: radioactivity accumulated in the vesicles upon incubation together with ¹⁴C-CA in the absence of ATP (Fig 2). Decreasing the temperature hindered, but not completelyinhibited, the accumulation, suggesting that the accumulation was partially dependent on enzymatic activity. This result could be explained by a similar mechanism that Perkins et al. (2022) created artificially with synthetic liposomes

Figure 2. Accumulation of ¹⁴C-CA to MF prepared from A) Norway spruce cultured cells, and B) Norway spruce developing xylem and developing phloem.

with encapsulated LACs. According to this idea, the ¹⁴C-CA diffused into the MF vesicles down the concentration gradient, as there was a system that created a sink inside the vesicles. Oxidising enzymes inside the vesicles would have created ¹⁴C-CA radicals which would have further led to formation of di-, tri-, and oligomers of ¹⁴C-CA.

As bigger oligomers would have been unable to diffuse back out from the vesicles, the net result would have appeared as accumulation of radioactivity. Unfortunately, no comprehensive analyses were conducted on the contents of Norway spruce vesicles after feeding with ¹⁴C-CA. Proteomics analysis did not detect any LACs (nor PRXs) in these vesicles (II), but these enzymes could still be present below detection levels and be capable of oxidising the ¹⁴C-CA, which would lead to polymerisation and accumulation of radioactivity in the vesicles. This result cannot be directly used to

support the theory of diffusion, but I admit that it could be partially explained by the sink-driven diffusion model (Perkins et al., 2022).

There are still variables in monolignol diffusion that need to be considered. The one obstacle between monolignol diffusion into and through a membrane is the composition of the membrane itself. The lipid composition of the PM in lignifying plant cells is not directly known, to my knowledge, and lipid composition affects the diffusion of phenolic compounds through the membrane (Boija et al., 2007). In addition to the lipids, membrane proteins also play a role in membrane permeability as they can decrease the diffusion of smaller compounds such as water and carbon dioxide through the membrane bilayer (Kai and Kaldenhoff, 2014). In addition, monolignols that partition to the membrane will interact with membrane lipids and membrane proteins.

The membrane components should tolerate monolignols passing through them during lignification and the specific effects of monolignols to the membrane remain unknown.

As such, future studies on the lipidomics of lignifying cells, diffusion assays with protein-rich membranes, and studies of the effects of lignin-related phenolics on membrane integrity as well as the function of membrane proteins will be needed to shed light on this system and test the potential role of diffusion in lignification.

Despite all the excellent previous studies on monolignol transport, the complete picture of this process during lignification is still not clear. Most likely, several parallel mechanisms exist, as with many other compounds such as suberin, cutin, and cutan (Xin and Herburger, 2021). There are indications that monomers required to assemble these polymers are transported partly by half-sized ABCG transporters (Panikashvili et al., 2011 and 2010; Yadav et al., 2014; Fabre et al., 2016; Shanmugarajah et al., 2019), and partly by facilitated diffusion with lipid transfer proteins (Deeken et al., 2016; Lee and Suh, 2018; Edqvist et al., 2018; Tafolla-Arellano et al., 2020). In theory, monolignol transport mechanisms could vary between cell types and plant species.

However, the transporter mediated monolignol transport is still a potential mechanism, possibly beside other mechanisms, and my work adds to a growing body of experimental evidence on this topic.