Monolignol toxicity

ether, dihydro-coniferyl alcohol, acetovanillone, ferulic acid, and lariciresinol. In the cell culture, these same compounds could be detected in the medium, but CA, guaiacylglycerol-β-coniferyl ether, dihydro-coniferyl alcohol, lariciresinol, and vanillin decreased or disappeared entirely during the 8-days of culturing. In contrast, ferulic acid accumulated and iso-hydroxymatairesinol appeared. In addition, the cells metabolised CA and its degradation products. The same compounds, as well as their glucosides, were detected in the cells.

Based on the results of this study, I suggest that CA or its degradation products are toxic to plant cells and that cells glycosylate these compounds to decrease their toxicity.

This hypothesis is also in line with the observation that Arabidopsis plants are dwarfed, have ectopic lignin formation, and increased monolignol glucoside levels when the phenylpropanoid- and monolignol pathways were induced by overexpressing transcription factors MYB58 and MYB63 (Zhou et al., 2009; Perkins et al., 2020).

Increasing polymerisation (and thus decreasing the concentration of the end products and pathway intermediates) by overexpressing LAC4 and LAC7 rescues the growth phenotype in these plants (Perkins et al., 2020). The results of study I are also in line with more recent results by Guan et al. (2022) which showed that CA is toxic to Arabidopsis seedlings.

Multiple other phenolic compounds show toxic effects in plants and bacteria.

Externally added pCA is toxic to Arabidopsis seedlings and yeast cells and this toxicity can be decreased by directly transporting pCA through the PM by ABCG29 (Alejandro et al., 2012). This data indicates that the toxicity of pCA is a function of pCA itself, and not a function of its degradation products. In petunia (Petunia hybrida), treatment with benzaldehyde (between 3–30 mM) for 3 h caused membrane disruption in a dose- dependent manner (Adebesin et al., 2017). More volatile secondary metabolites (such as phenylpropanoids and benzenoids) accumulated after down-regulation of their transporter, PhABCG1, leading to disruption of the membrane integrity in the cell. As monolignols and lignin-like compounds can diffuse through membranes (Vermaas et al., 2019), a possibility remains that some of these compounds alter membrane integrity. Phenolic aldehydes are known to be toxic to microbes (Kunjapur and Prather, 2015) and the effects are often said to occur at the level of the membranes. Vanillin is an aldehyde that is also detected in BY-2 cells after CA supplementation (I). Vanillin is toxic to different bacteria, and the effect is largely caused by its dissipation of ion

gradients and inhibition of respiration (Fitzgerald et al., 2004). Eugenol (~5 mM) and cinnamaldehyde (>30 mM) are also toxic to bacteria but the level of effect varies between bacterial species (Gill and Holley, 2004).

Phenolic compounds are sometimes discussed to cause ion leakage through membranes by serving as ion-carriers. Results by Hossain et al., (2021) support this hypothesis as caffeic acid, caffeic acid methyl ester, and 3,4-dihydroxybenzoic acid increase the membrane permeability to Na⁺. The exact effect of each individual phenolic molecule depends on the chemical nature of the molecule and on the properties of the bilayer and its components. Pinoresinol, but not, for example, CA or pCA, inhibits coniferin transport (II). Pinoresinol could be a direct inhibitor of coniferin transporter by binding to the substrate-binding site or another accessible region of the transporter. On the other hand, transporters have a hydrophobic region(s) that interact with membrane lipids. Hypothetically, pinoresinol present in the membrane could alter the protein–lipid interactions and cause a general inhibition of multiple transporters. The interactions of membrane proteins with surrounding lipids can be affected by hydrophobic compounds (Sikkema et al., 1995). Thus, V- ATPase that is responsible for creating the H⁺ gradient that coniferin transporter uses (II, see below) could also be inhibited, and the decrease of H⁺ gradient could cause an inhibition of coniferin transport. On the other hand, an effect on the H⁺ -gradient by altering the ion permeability of the membrane (Hossain et al., 2021) would surely inhibit coniferin-H⁺ antiport. Pinoresinol was also detected in the BY-2 cells and in the culture medium after CA supplementation and, in the light of this discussion, could potentially contribute to the growth-hindering effects seen in Study I.

CA, one of the major monolignols, is toxic to plant cells (I) and both lignifying and neighbouring cells produce and export it, as has been reported in Norway spruce (Blokhina et al., 2019). However, only lignifying cells die through PCD. A sink in the SCW seems to be important for monolignol transport (Perkins et al., 2022) and this would help to decrease the monolignol concentration in the cytoplasm. But is the sink in the SCW of TEs sufficient to drive transport out from the neighbouring cells that remain alive? Or do they need an additional mechanism to detoxify monolignols or their degradation products? It is easy to think of a polymerisation sink in the middle lamella where the lignification initiates but how about after this layer has become highly lignified? From the perspective of neighbouring cells, polymerisation occurs on

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.