Study of spider flower C-lignin reveals two novel monolignol transporters

Although membrane-localized transport proteins can be easily identified in sequence datasets based on homology, their functional characterization remains a significant challenge. A key obstacle lies in the broad substrate specificity observed within transporter protein families, which complicates functional predictions based solely on sequence data (Thomas & Tampé, 2020). Furthermore, the inherent instability and consequent loss of activity of these proteins upon isolation from membranes hinders their purification and in vitro analysis (Hardy et al., 2016). As a result, functional annotations for these proteins often remain incomplete, as exemplified by the limited number of transporters explicitly associated with lignification. The first transporter identified in this context was ABCG29 in Arabidopsis, which mediates the transport of p-coumaryl alcohol (Alejandro et al., 2012). More recently, the coniferyl alcohol transporters ABCG15 in bamboo (Li et al., 2024) and ABCG36 in spruce (Sun et al., 2024) have been characterized, with the latter also facilitating the transport of sinapyl alcohol.

‘The dual capacity of these transporters to mitigate precursor toxicity while modulating lignin composition may indicate that lignification evolved as a detoxification strategy’

To identify novel monolignol transporters, in a paper recently published in New Phytologist, Zhou et al. (2024; doi: 10.1111/nph.20325) refined the research focus by capitalizing on the structurally simple lignin synthesized in the seed coat of spider flower (Cleome hassleriana), an ornamental plant native to South America. This lignin is known as catechyl or C-lignin and is uniquely composed of units derived from caffeyl alcohol (Chen et al., 2012; Fig. 1). By targeting tissues that produce this homopolymeric lignin, the authors hypothesized that genes coding for transporters specific to C-lignin biosynthesis would exhibit heightened expression in the seed coat, enabling their identification against the more complex background of heteropolymeric lignin-related transporters in other tissues. Integration of transcriptomic and proteomic data facilitated the identification of six candidate transporters potentially required for substrate delivery for C-lignin. These candidates underwent functional validation through yeast transport assays to evaluate their role in mediating the transmembrane movement of lignin precursors. Among the candidates, two transporters, ChSTP8 and ChSUC1, exhibited activity with caffeyl alcohol but not with the classical monolignols (i.e. p-coumaryl, coniferyl and sinapyl alcohol). Homology modeling and molecular docking analyses further elucidated the specificity of these transporters, revealing interactions between two conserved amino acid residues and the meta- and para-hydroxyl groups of caffeyl alcohol. The canonical monolignols demonstrated reduced affinity due to structural differences; p-coumaryl alcohol lacks one of the hydroxyl groups, while coniferyl and sinapyl alcohol possess one or two bulkier methoxy groups at these critical positions, resulting in steric hindrance.

Fig. 1

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The study of C-lignin in the seed coat of the spider flower (Cleome hassleriana) reveals novel transporters responsible for translocating caffeyl alcohol from the cytoplasm to the apoplast, where it undergoes polymerization into C-lignin. The transporters are incorporated into a model that combines active transport with passive diffusion to explain the lignification process.

Overexpression of ChPLT3 in an Arabidopsis mutant unable to convert caffeyl alcohol to the G-lignin building block coniferyl alcohol conferred significant resistance to the toxic effects of elevated caffeyl alcohol levels. Notably, a comparable effect was not observed with ChSUC1, a disparity attributable to potential posttranslational modifications of the transporter. Next, both transporters were overexpressed in Medicago truncatula hairy roots in a genetic background promoting the accumulation of caffeyl alcohol at the expense of coniferyl and sinapyl alcohol. Interestingly, the lignin composition in these transgenic hairy root lines displayed an increased proportion of C units derived from caffeyl alcohol. These findings underscore the capacity of the identified transporters to influence lignin biosynthesis by modulating the availability of specific monomeric substrates in the apoplast. A particularly compelling aspect of these experiments is the link between the export of lignin precursors and cellular detoxification mechanisms. The dual capacity of these transporters to mitigate precursor toxicity while modulating lignin composition may indicate that lignification evolved as a detoxification strategy, subsequently repurposed for structural reinforcement and defense functions in plants. This integration of mechanistic and evolutionary perspectives provides a more nuanced understanding of the evolutionary origin of lignin biosynthesis and its overarching biological significance.

Besides facilitating the identification of relevant transporters, the focus on low-complexity lignin, also provided novel insights into the molecular mechanisms regulating lignin biosynthesis in a tissue-specific context. Although the C-lignin polymer itself is made of caffeyl alcohol building blocks only, the apoplast intriguingly harbors a substantial pool of coniferyl alcohol that remains unincorporated into the C-lignin matrix (Tobimatsu et al., 2013). The authors leveraged this observation to develop a model that elucidates the deposition of C-lignin, integrating both the diffusion and transporter hypotheses. According to this model, caffeyl alcohol diffuses into the apoplast, where it undergoes polymerization into C-lignin. This polymerization process is proposed to suppress the incorporation of coniferyl alcohol into the lignin polymer, thereby creating a distinct compositional profile of the lignin structure. When the rate of polymerization in the apoplast is insufficient to sustain passive diffusion at the early stage of C-lignin polymerization, the model further posits that the cytotoxic caffeyl alcohol monomers are actively transported across the plasma membrane by the identified transporters. This dynamic interplay between transporter-mediated processes and passive diffusion underscores a finely tuned mechanism that ensures efficient lignin precursor delivery under varying physiological conditions.

Building upon this study, there are unprecedented opportunities to identify novel transporters involved in lignification, even in nonmodel plant species. With the advances in sequencing technologies, these are no longer constrained by limitations in accuracy or accessibility. The true bottleneck for this approach is the access to plants or tissues that exhibit a low-complexity lignin fingerprint. A phylogenetic analysis conducted by the authors revealed no clear evolutionary relationship between species capable of synthesizing C-lignin. However, it is crucial to acknowledge that current investigations into C-lignin are fragmented. The observation that C-lignin deposition occurs exclusively in specific tissues and within tightly regulated temporal windows (seed coats 12 d after pollination in the case of C. hassleriana) highlights the challenge linked to its detection and supports the assertion that the absence of detected C-lignin does not unequivocally confirm its absence in a given species. The present study underscores the importance of analyzing lignin composition across a broad spectrum of plant tissues, encompassing diverse species and developmental stages. Such a holistic approach to studying lignin diversity and deposition dynamics may serve as a foundation for uncovering novel insights into lignin polymerization processes and their functional roles.

In addition to the emphasis placed on the transport of caffeyl alcohol, the negative results reported in this study are equally significant and offer valuable insights into the underlying mechanisms of monolignol transport. The inability of the transporters to recognize or transport certain monolignols sheds light on the structural features that are critical for transporter activity. By identifying the specific molecular characteristics that distinguish transportable substrates from nontransportable ones, this work contributes to a deeper understanding of the substrate specificity of these transport systems. Such information is essential for elucidating the structural requirements that govern transporter–substrate interactions, including aspects such as functional groups and steric configuration. Moreover, the findings present a compelling strategy for further exploration. The docking information paves the way to screen and possibly predict which other transporters might have the capacity to transport specific monolignols. Given the typically large size of these transporter families, this computational approach could significantly streamline the identification of potential transporters by narrowing down the pool of candidates before experimental validation. The incorporation of AI and machine learning could further enhance this process by analyzing vast datasets to identify patterns and predict transporter–substrate interactions with greater accuracy and efficiency providing a framework for future studies aimed at characterizing the molecular determinants of substrate recognition and transport efficiency in lignification.

This work not only advances fundamental biological understanding but also aligns with broader goals of sustainable agriculture and the bioeconomy. For instance, by translocating potentially toxic phenylpropanoids to the apoplast, precise regulation of monolignol transport can mitigate the metabolic trade-offs between lignin engineering and plant growth. Moreover, modulating the expression or efficiency of the transporters provides a pathway to reduce lignin content or alter its structure, thereby enhancing the digestibility of lignocellulosic biomass for biofuel production while preserving the structural integrity essential for plant robustness. The focus on C-lignin in this study is particularly significant, as this lignin polymer is distinguished by its linear structure and well-defined monomer product distribution upon depolymerization, providing important advantages for valorization (Li et al., 2023).

While the characterization of the two novel monolignol transporters has significantly advanced our understanding of lignification, it opens new avenues for further exploration. For instance, from an economic perspective, it is key to understand how the identified transporters can be leveraged to design lignin tailored to specific needs. From a fundamental perspective, a key area for further investigation is the molecular mechanism supporting the presented model explaining why available coniferyl alcohol is excluded from incorporation into the C-lignin polymer. Additionally, the proposed model's relationship to vesicle-transport theory remains unclear and warrants further exploration. The research also triggers the question of how many additional transporters are involved in the translocation of both canonical and noncanonical lignin monomers across membranes, as well as the factors that determine their specificity for particular monolignols. Addressing these questions will not only deepen our understanding of lignification but also open new avenues for practical applications in agriculture and bioengineering.