Monolignol glucoside transporter (MGT)

the wrong side of this layer. Lignification is said to make the CW somewhat impermeable, although it could permeate some small molecular compounds, as discussed by Yang et al. (2020). However, it is difficult to think that it would permeate all the required monomers with sufficient speed. As discussed by Blokhina et al. (2019), cross-field pits with highly developed plasmodesmata between the ray parenchyma cells and lignifying tracheids, could potentially serve as a route for monolignols. This would mean that monolignols would diffuse a long distance, close to (or even along) membrane structures to enter to the cytoplasm (or the PM) of the lignifying cell. From there they could be transported to SCW. On the other hand, some lignifying cells have only local SCW thickenings where only part of the cell wall becomes lignified. With only local thickenings, the diffusing monolignols from the neighbouring cells have free access to the sites of polymerisation.

Taken together, these results suggest that coniferin is transported by a secondary active transporter that uses a H⁺-gradient. The KM of coniferin transport by PaMGT was 127.4±21.4 μM (II), whereas in hybrid poplar it is 60–80 μM, in Japanese cypress it is 24–26 μM (Tsuyama et al., 2013), and in bamboo it is 32–87 μM (Shimada et al., 2021).

The pCAg transporter in hybrid poplar had a KM closer to that for coniferin in the developing xylem of Norway spruce: 160–260 μM (Tsuyama et al., 2019).

Coniferin transport was not only present in vesicles prepared from developing xylem, but also in vesicles prepared from lignifying cultured cells of Norway spruce. However, the KM value was higher: 463.1±297.0 μM. This difference suggests that Norway spruce may have two coniferin transporters, of which the xylem one has a higher affinity to coniferin (II). Vesicles that were prepared from cultured cells of spruce turned out to be a challenging material, and not all membrane preparations showed coniferin transport. This fact, unfortunately, limited the number of experiments that could be performed with this material, and thus, the inhibitor assays were non-conclusive. It is not clear whether the transport was primarily or secondarily active. If the coniferin transport that was detected was due to the same transporter, PaMGT, a difference in the H⁺-gradient buildup between cultured cells and xylem could also explain the apparently lower KM. Alternatively, PaMGT could be up- or downregulated in each of these tissues by PTMs causing the difference in KM. However, it also remains possible that these two systems have distinct MGTs.

The results of study II, as well as those of Tsuyama et al. (2013, 2019) and Shimada et al. (2021), show that xylem tissues of various species possess H⁺-antiporters involved in MGs transport on vacuolar and/or endosomal membranes. However, the presence of MG transport activity at the vacuolar and/or endosomal membrane of BY-2 cells (II) challenges the idea that MG transport is dedicated for lignin biosynthesis. It may be that a single transporter, here named NtMGT (Nicotiana tabacum MGT) transports both coniferin and pCAg as these substrates inhibit the transport of each other when tested in MF vesicles. BY-2 cells do not produce lignin, and no traces of mono-, di-, or trilignols were detected to be present naturally (I). Still, NtMGT had a higher affinity for coniferin than PaMGT, since its KM was 39.0±0.08 μM. However, this is in the normal range in KM values of tonoplast transporters (Tsuyama et al., 2013). This observation contrasts with the idea that a transporter capable for MG transport should be unique to lignifying cells. It is unlikely, yet not impossible, that non-lignifying BY-2

cells express an active transporter that belongs to the lignin biosynthesis pathway.

These results could be explained by the possibility that the primary in vivo substrate for NtMGT is not MG, but rather some other glycosylated phenolic compound(s). In study II, pinoresinol, but not pinoresinol diglucoside, inhibited coniferin transport in BY-2 cells, similar to PaMGT in spruce xylem. More glucosides should be tested to solve whether PaMGT is more specific than NtMGT. Dima et al. (2015) showed that small, glycosylated lignin oligomers are stored in vacuoles in Arabidopsis leaves and that oligomerisation is likely to occur in the cytosol, followed by glycosylation and transport to vacuoles. We did not specifically search for a wide spectrum of glycosylated compounds in BY-2 cells (I), and it is possible that there are such compounds in these cells that serve as natural substrates for NtMGT. Some studies have been published on BY-2 cell proteomes (e.g. Noguchi et al., 2018; Morel et al., 2006; Baginsky et al., 2004) but, to my knowledge, no studies have reported specifically on the proteomes of tonoplasts in BY-2 cells.

In microsomal vesicles prepared from poplar xylem, pCAg transport is inhibited by coniferin and by its aglycone form, pCA (Tsuyama et al., 2019), but neither syringin nor sucrose were inhibitory. On the other hand, poplar microsomes do not transport

L-coniferin but only D-coniferin, which is the natural form (Maeda et al., 2019), suggesting that the poplar transporter is quite specific for its substrate. However, determining the substrate specificities of MGTs may not necessarily help uncover the role of MGs and the MGTs.

Is MGT then a monolignol transporter? There is an underlying question of whether MGs are an integral part of lignin biosynthesis or not. Thus, it is useful to discuss the role of MGs in lignifying tissue. Perhaps MGs 1) are the transported form of monolignols, 2) regulate monolignol biosynthesis pathway, or 3) detoxifiy monolignols? To address the first hypothesis, Tsuyama et al. (2013) suggested that coniferin is routed to the SCW via endosomal vesicles that are loaded by a secondarily active coniferin transporter. This would be roughly similar to the transport of anthocyanins to vacuoles. It has been shown that anthocyanins are loaded into small vesicles by MATE transporters, and the vesicles are then transported to vacuoles (Gomez et al., 2011; Zhao, 2015). However, a different type of anthocyanin transport exists that involves glutathione-S-transferases (GST). Additionally, secretory vesicles are known to play an important role in SCW formation: woody tissues of hybrid poplar

showed changes in structural polysaccharides, but also in the levels of small phenolic compounds when Secretory Carrier-Associated Membrane Protein3 (SCAMP3) was silenced using an RNAi construct (Obudulu et al., 2018). This observation indicates that membrane trafficking can readily influence phenolic metabolism. Even further, a mechanism similar to the one detected by Jeon et al. (2022) where lignin monomers were transported via autophagic vesicles during pathogen attack would fit nicely to the hypothesis of MGs serving as lignin monomers if they were loaded into vesicles by MGTs and then exported to the SCW. On the other hand, the accumulation of MGs in vacuoles could be involved in lignification as discussed in the introduction. During PCD, the tonoplast and PM disintegrate liberating the MGs if they do indeed accumulate in vacuoles. Deglucosylation of MGs by β-glucosidases would then cause a sudden wave of monolignols in the SCW. In Norway spruce, the lignin structure in the final SCW layer, S3, is relatively rich in dibenzodioxocin structure (Kukkola et al., 2004). It is possible that this structure forms due to changes in monolignol concentrations (Brunow et al., 1998). However, there is no specific data on the lignification of the S3 layer and whether vacuolar burst is correlated in timing in Norway spruce, meaning this mechanism is highly speculative.

The second hypothesis for the role of MGs involves feedback inhibition. Feedback inhibition is common in many pathways. For example, in Arabidopsis, flavonols are glycosylated and transported to the apoplast to prevent inhibition of PAL and chalcone synthase (Yin et al., 2012). Various monolignol biosynthetic enzymes are also inhibited by several pathway intermediates as shown by a large-scale study in poplar (P.

trichocarpa) (Wang et al., 2014). CAD, the last enzyme of the pathway, can catalyse a reverse reaction and use CA as a substrate to form coniferylaldehyde. In vitro, coniferin noncompetitively inhibits the reverse reaction of CAD in loblolly pine (Pinus taeda) (O’Malley et al., 1992). Whether coniferin does this in planta, and with the concentrations normally present in the cell, remains unclear. Glucosylation of CA may hinder the reverse reaction of CAD by reducing the CA pool. On the contrary, based on the feeding of labelled CA and coniferylaldehyde to ginkgo stems, it has been shown that MGs could be converted into monolignol aldehyde glucosides, and then hydrolysed and converted back into alcohol forms by CAD to serve as lignin precursors (Tsuji et al., 2005). Guan et al. (2022) suggested that CA can function as a signalling molecule to regulate the phenylpropanoid pathway by affecting PAL activity via a

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.