Tricin Biosynthesis and Bioengineering

Tricin (3',5'-dimethoxyflavone) is a specialized metabolite which not only confers stress tolerance and involves in defense responses in plants but also represents a promising nutraceutical. Tricin-type metabolites are widely present as soluble tricin O-glycosides and tricin-oligolignols in all grass species examined, but only show patchy occurrences in unrelated lineages in dicots. More strikingly, tricin is a lignin monomer in grasses and several other angiosperm species, representing one of the “non-monolignol” lignin monomers identified in nature. The unique biological functions of tricin especially as a lignin monomer have driven the identification and characterization of tricin biosynthetic enzymes in the past decade. This review summarizes the current understanding of tricin biosynthetic pathway in grasses and tricin-accumulating dicots. The characterized and potential enzymes involved in tricin biosynthesis are highlighted along with discussion on the debatable and uncharacterized steps. Finally, current developments of bioengineering on manipulating tricin biosynthesis toward the generation of functional food as well as modifications of lignin for improving biorefinery applications are summarized.


INTRODUCTION
Flavonoids are a large group of plant-specialized metabolites that are ubiquitous in vascular plants and are also found in non-vascular plant lineages except hornworts (Yonekura-Sakakibara et al., 2019). Structurally, they are featured by a basic diphenylpropane (C6-C3-C6) backbone, which is usually made up of two benzene rings (A-ring and B-ring) and a middle pyrone ring (C-ring; Alseekh et al., 2020). Flavonoids are assigned to different classes according to the oxidation states in the C-rings (Schijlen et al., 2004). At least nine major classes, namely, flavanones, flavones, dihydroflavonols, flavonols, flavan-3-ols, leucoanthocyanidins, anthocyanidins, isoflavones, and aurones, have been described ( Figure 1A; Yang et al., 2018;Nakayama et al., 2019).
Elucidating the biosynthetic pathway for tricin is the pre-requisite for genetic manipulation of soluble and ligninintegrated tricin in different biotechnological applications. Here, we delineate the current understandings on tricin biosynthesis  and discuss the present development and future prospects regarding the biotechnological aspects of engineering the biosynthetic pathway.

TRICIN BIOSYNTHESIS
Early Biosynthesis -The General Phenylpropanoid Pathway Same as other flavonoids, tricin is a downstream metabolite of the general phenylpropanoid pathway (Figure 2) in which ʟ-phenylalanine is first deaminated into cinnamate by phenylalanine ammonia-lyase (PAL; Camm and Towers, 1973;Elkind et al., 1990), followed by cinnamate 4-hydroxylase (C4H)-catalyzed para-hydroxylation of the aromatic ring to form p-coumarate (Russell and Conn, 1967;Russell, 1971;Schilmiller et al., 2009). Afterward, 4-coumarate:coenzymeA ligase (4CL) catalyzes the conversion of p-coumarate into p-coumaroyl-CoA, which serves as the precursor for the biosynthesis of different specialized metabolites, including flavonoids and lignin (Gui et al., 2011;Li et al., 2015). It is long believed that certain 4CL isoforms are specific for flavonoid biosynthesis (Hu et al., 1998;Ehlting et al., 1999;Sun et al., 2013a;Li et al., 2015). An alternative pathway using ʟ-tyrosine as a substrate to produce phenylpropanoids is also present in grasses (Figure 2; Barros et al., 2016). Bifunctional phenylalanine/tyrosine ammonia-lyases (PTAL) in maize and Brachypodium distachyon catalyze the deamination of ʟ-tyrosine to form p-coumarate, while at the same time, these enzymes also harbor PAL activities (Rosler et al., 1997;Barros et al., 2016). PALs and PTALs are highly conserved in grasses, suggesting the co-existence of two parallel pathways for phenylpropanoid production in Poaceae (Barros et al., 2016). In addition, results from feeding experiments using 13 C-labelled ʟ-phenylalanine and ʟ-tyrosine in B. distachyon have suggested that PTAL is likely to be associated with the generation of grass-specific cell-wall-bound p-coumarate units (Barros et al., 2016). It is unknown whether tricin (soluble and lignin-bound) is derived from the PAL and/or PTAL pathway.

Early Biosynthesis -Flavonoid Skeleton Formation
The initial biosynthetic steps and enzymes for flavonoid skeleton formation are highly conserved in the plant kingdom. Chalcone synthase (CHS), a prototype in the type III polyketide synthase superfamily, catalyzes sequential condensation of three malonyl-CoAs with p-coumaroyl-CoA to form naringenin chalcone (Figure 2). Chalcone isomerase (CHI)-catalyzed or occasionally spontaneous isomerization further converts naringenin chalcone into naringenin (a flavanone), which is the first flavonoid structure formed in the biosynthetic pathway. Naringenin is the precursor for all other flavonoids, including tricin. It was shown that deficiency of CHSs in maize and rice resulted in depletion in the accumulation of soluble and/ or lignin-integrated tricin (Eloy et al., 2017;Wang et al., 2020a). Although it was not examined previously, CHIs are expected to be involved in tricin biosynthesis based on their conserved catalytic functions in the generation of all classes of flavonoids in plants.

Flavone Nucleus Formation
Using rice (Oryza sativa) as a model system, FNSII was identified to be the primary enzyme generating the flavone nucleus for tricin biosynthesis in grasses ( Figure 5; Lam et al., 2014). Recombinant OsFNSII catalyzes direct conversions of flavanones, i.e., naringenin and eriodictyol, into apigenin and luteolin, respectively, in vitro (Brazier-Hicks and Edwards, 2013;Lam et al., 2014). In addition, over-expression of OsFNSII in Arabidopsis resulted in the accumulation of flavones (apigenin, luteolin, and chrysoeriol) O-glycosides which are normally not present in wild-type plants (Lam et al., 2014). Further analyses of the rice OsFNSII knockout mutant revealed substantial Frontiers in Plant Science | www.frontiersin.org depletion of soluble tricin O-conjugates as well as tricin-lignin in cell walls, demonstrating the direct and predominant involvement of OsFNSII in the generation of both soluble and lignin-integrated tricin in rice (Lam et al., 2014(Lam et al., , 2017. Moreover, the OsFNSII mutant accumulated soluble naringenin but not the other flavanones, e.g., eriodictyol (Lam et al., 2014), and generated altered lignins incorporated with naringenin (Lam et al., 2017), indicating that the in planta substrate of OsFNSII is primarily naringenin.
OsFNSII, or CYP93G1, is a P450 enzyme belonging to the same CYP93G subfamily as OsF2H, or CYP93G2. Using naringenin as a common substrate, OsFNSII and OsF2H are the branch-point enzymes for the biosynthesis of tricin O-conjugates and flavone C-glycosides, respectively (Figures 3, 5).
Phylogenetic analysis revealed that OsFNSII and OsF2H form two separate clades, each containing highly conserved sequences from the grass family ( Figure 6A; Lam et al., 2017). Hence, sub-functionalization of CYP93G members probably preceded lineage divergence within Poaceae, resulting in the widespread distribution of the two classes of flavone-derived metabolites in grasses today. It is noteworthy that grass FNSIIs and F2Hs have a different phylogenetic origin from dicot FNSIIs and F2Hs, all of which exclusively belong to the CYP93B subfamily ( Figure 6A; Kitada et al., 2001;Martens and Mithöfer, 2005;Zhang et al., 2007;Fliegmann et al., 2010;Wu et al., 2016;Zhao et al., 2016;Jiang et al., 2019). Noteworthily, grass species do not contain any CYP93B members and dicots do not have CYP93G members . Functionally, redundant enzymes other than FNSII are likely to be involved in tricin biosynthesis in grasses. For example, the rice OsFNSII mutant still accumulated soluble tricin and other flavones in anthers albeit at reduced levels compared with wild type , while it shows substantial depletion of soluble tricin O-conjugates and tricin-lignin in vegetative tissues (Lam et al., 2014(Lam et al., , 2017. In fact, two rice FNSIs were shown to catalyze the conversion of naringenin into apigenin in vitro Lee et al., 2008b). In addition, maize possesses an FNSI (ZmFNSI-1) which shows in vitro FNS activities and results in the accumulation of flavones when over-expressed in Arabidopsis (Ferreyra et al., 2015;Righini et al., 2019).

B-Ring Hydroxylations
In the plant kingdom, 3',5'-substituted flavonoids are patchily distributed, because F3'5'Hs, the enzymes responsible for catalyzing 5'-hydroxylation, are only present in isolated plant lineages (Tanaka and Brugliera, 2013). This is in contrast to the ubiquitous nature of flavonoid 3'-hydroxylases (F3'H; exclusively members of the CYP75B subfamily) that gives rise to the prevalence of 3'-substituted flavonoids (Tanaka and Brugliera, 2013). There have been strong interests for the investigation of F3'5'Hs as they are the key enzymes for the generation of delphinidinderived anthocyanins, which confer blue or violet coloration in plant tissues, such as flowers and fruits (Tanaka and Brugliera, 2013). For ornamental purposes, transgenic expression of foreign F3'5'Hs has been employed to engineer novel blue or violet color in roses (Rosa hybrida), chrysanthemums (chrysanthemum morifolium), and carnations (Dianthus caryophyllus), all of which naturally lack delphinidin-derived anthocyanins (Katsumoto et al., 2007;Brugliera et al., 2013;Noda et al., 2013;Tanaka and Brugliera, 2013).
Recently, knockout and knockdown mutant analyses have demonstrated that grass COMT/CAldOMTs are actually bifunctional enzymes required for both tricin and S-lignin biosynthesis (Fornalé et al., 2016;Eudes et al., 2017;Daly et al., 2019;Lam et al., 2019b). Rice and sorghum deficient in COMT/CAldOMT accumulated reduced levels of soluble tricin but increased levels of selgin (mono-methoxylated) and luteolin (non-methoxylated) when compared with wild-type controls (Lam et al., 2015;Eudes et al., 2017). In addition, maize, rice, and sorghum plants deficient in COMT/CAldOMT were depleted in both tricin-lignin and S-lignin (Fornalé et al., 2016;Eudes et al., 2017;Lam et al., 2019b). Apparently, the highly conserved grass COMT/CAldOMT orthologs ( Figure 6C) have likely evolved dual catalytic functions for the two parallel biosynthetic pathways of flavonoids and monolignols, contributing to the widespread occurrence of soluble and lignin-integrated tricin metabolites in the grass family nowadays.

Further O-Conjugations After Tricin Formation
Based on the types of soluble tricin metabolites detected in grasses, O-glycosylations and O-conjugations with monolignols and their acylated derivatives represent the predominant structural modifications of tricin (Dong et al., 2014;Lan et al., 2016a;Eloy et al., 2017;Peng et al., 2017). These modifications occur after the formation of tricin aglycone (Hong et al., 2007;Jiang et al., 2016;Lan et al., 2016a). O-Glycosylations of flavonoids are usually catalyzed by uridine diphosphate (UDP)-dependent glycosyltransferases (UGT; family 1 glycosyltransferases 1; GT1; Ko et al., 2006;Yonekura-Sakakibara and Hanada, 2011;Kim et al., 2015), which utilize UDP sugars as sugar donors . A number of UGTs from rice (Ko et al., 2006(Ko et al., , 2008Hong et al., 2007;Luang et al., 2013;Chen et al., 2014;Peng et al., 2017) and wheat (Shi et al., 2020) are capable of catalyzing the conjugation of sugars, usually glucose, to one or multiple hydroxyl groups of tricin in vitro and/or when over-expressed in transgenic plants. Singlenucleotide polymorphisms (SNPs) in several putative UGTs were also found to be directly associated with the variations of flavone O-glycoside accumulation in different natural cultivars and/or recombinant inbred lines of rice Dong et al., 2014;Peng et al., 2017;Li et al., 2019) and wheat (Shi et al., 2020). The different O-glycosylations could enhance solubility and stability, and might be involved in regulating storage, transport, and detoxification of tricin (Yonekura-Sakakibara and Hanada, 2011).
In addition to sugars, tricin conjugates with monolignols and their derivatives, leading to the formation of soluble tricin-oligolignols along with insoluble tricin-lignin in the cell walls. The soluble tricin-oligolignols in grasses have been found to be either optically active (Wenzig et al., 2005;Xiong et al., 2011) or inactive (racemic; Lan et al., 2016a). The optically active tricin-oligolignols, which have been often referred to as "flavonolignans" (Begum et al., 2010;Chambers et al., 2015;Csupor et al., 2016), may be formed by oxidative radical coupling of tricin with monolignols or their derivatives with the assistance of dirigent proteins, similar to the biosynthesis of lignans (Davin and Lewis, 2003;Umezawa, 2003;Paniagua et al., 2017), in which dirigent proteins serve as auxiliary proteins for guiding the regioselective and stereoselective coupling of phenoxy radicals from monolignols and their analogs. For example, the absolute configuration of a diastereomeric pair of β-O-4 neolignan-type flavonolignans,

Tricin-Lignin Formation
Tricin is incorporated into lignin polymers in grass cell walls by radical coupling (Lan et al., 2015), essentially the same way lignification takes place solely with monolignols (coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol) in typical non-grass vascular plants (i.e., gymnosperms, dicots, and non-grass monocots). The compatibility of tricin with radical coupling was demonstrated by biomimetic oxidations of tricin with monolignols using peroxidase/hydrogen peroxide and silver (I) oxide as oxidants (Lan et al., 2015). Tricin was found to cross-couple to monolignols exclusively via the 4'-O-β-coupling mode ( Figure 7B; del Río et al., 2012;Lan et al., 2015), probably because the radical from the 4'-hydroxyl group of tricin is more stabilized than the other possible radicals as supported by a density functional theory study . Thus, in plant cell walls, it is expected that tricin is first oxidized by phenol oxidases, presumably laccases (LAC) and/or peroxidases (PRX; Figure 7A; Tobimatsu and Schuetz, 2019), and then coupled with monolignol radicals or acylated monolignol radicals to form tricin-(4'-O-β)-linked phenylpropane units in the lignin polymers ( Figure 7B). As tricin is unable to undergo dehydrodimerization, and it does not cross-couple directly with growing lignin polymers, tricin predominantly incorporates into the starting ends of the final lignin polymer chains (Lan et al., 2015). Thus, tricin is expected to serve as a nucleation site for lignification (Lan et al., 2015;Berstis et al., 2021).
Lignin-integrated tricin content in grasses was estimated to be around 0.5-7 mg/g whole cell wall or 2-33 mg/g lignin by thioacidolysis (Lan et al., 2016b). These contents are several folds higher than extractable tricin content (Lan et al., 2016b), suggesting that the majority of tricin synthesized in grasses is incorporated into lignin polymers in cell walls.
Meanwhile, tricin-lignin is only detected in leaves of alfalfa, albeit at much lower quantity than those in grasses (Lan et al., 2016b). Intriguingly, although tricin is restricted to certain dicot lineages, its flavone precursors, including apigenin, luteolin, and/or chrysoeriol, are widely distributed in non-tricinaccumulating dicots (Harborne, 1974). Hence, the occurrences of tricin derivatives are probably resulting from independent and convergent recruitment of novel enzyme activities in those isolated tricin-accumulating dicot lineages.
The Medicago-unique CYP75B enzymes required for tricin biosynthesis are distinct from the grass A3'H/C5'Hs with regard to their catalytic properties and phylogenetic origins (Lam et al., 2015;Lui et al., 2020). For example, the 5'-hydroxylase activity is restricted to chrysoeriol for the grass enzymes but is extended to other 3'-methoxylated flavonoids for the Medicago enzymes.
Interestingly, the Thr-to-Gly substitution in the substrate recognition site 6 domain is critical for these Medicago enzymes to catalyze the 5'-hydroxylation reactions (Lui et al., 2020). On the other hand, the equivalent position is replaced by a Leu residue in the grass A3'H/C5'Hs (Lui et al., 2020), but it is unknown whether this could account for their more specific substrate preference for 5'-hydroxylation. Meanwhile, the Medicagounique CYP enzymes have likely acquired the novel 5'-hydroxylase activities through neofunctionalization of redundant CYP75B F3'Hs following the divergence of the Medicago genus from other lineages in the legume family (Lui et al., 2020). Convergent evolution of CYP75B F3'5'H had also occurred independently in several Asteraceae lineages for the generation of delphinidinderived blue/violet pigments (Seitz et al., 2006(Seitz et al., , 2015. By sharp contrast, A3'H/C5'Hs are highly conserved amongst grasses, consistent with prevalence of tricin in the grass family (Lam et al., 2019a). It would be intriguing to decipher the enzymology and evolution of B-ring hydroxylations for tricin biosynthesis in other isolated dicot lineages.

B-Ring O-Methylations
The enzymes responsible for the 3'-and 5'-O-methylation reactions remain elusive for tricin biosynthesis in dicots. It is possible that they are also COMT/CAldOMT enzymes, as in the case for the grass bifunctional OMTs. In fact, Arabidopsis knockout mutant analyses demonstrated the dual roles of COMT/CAldOMT in the production of monolignols and flavonoids (Do et al., 2007;Tohge et al., 2007;Nakatsubo et al., 2008). However, there is no tricin accumulation in Arabidopsis, presumably due to the absence of F3'5'H enzymes. Meanwhile, the expression of an endogenous COMT gene is upregulated in transgenic alfalfa over-expressing the gene encoding N-acetylserotonin O-methyltransferase (MsASMT1), which catalyzes the final step in melatonin biosynthesis (Cen et al., 2020). In addition to increased melatonin formation, the transgenic alfalfa plants produced elevated amounts of various soluble chrysoeriol-and tricin-derived metabolites (Cen et al., 2020), which might be resulting from increased COMT activities. However, FgCOMT1 isolated from the tricin-accumulating legume fenugreek (Trigonella foenum-graecum; Kuwabara et al., 2003) could O-methylate 5-hydroxyferulic acid but not quercetin (a 3'-hydroxylated flavonol) or tricetin in vitro (Qin et al., 2012). Over-expression of FgCOMT1 in Arabidopsis atomt1 knockout mutant only partially restored the accumulation of sinapoyl aldehyde and sinapic acid (intermediates of the monolignol biosynthetic pathway) but not isorhamnetin (a 3'-methoxylated flavonol; Qin et al., 2012 which are mainly consisting of endosperms, are poor in phytochemicals and minerals (Awika, 2011). Their consumption as staple food in developing countries is associated with micronutrient malnutrition due to the lack of dietary diversity (Bhullar and Gruissem, 2013). To overcome this problem, biofortification through metabolic engineering has been pursued to introduce different phytochemicals and minerals in endosperms of cereal grains (Bhullar and Gruissem, 2013;Saltzman et al., 2013). As a prime example, golden rice engineered with the β-carotene biosynthetic pathway in endosperm was developed to combat vitamin A deficiency (Ye et al., 2000;Paine et al., 2005;Owens, 2018). Following the success of golden rice, cereal crops that accumulate high contents of iron, zinc, and various carotenoids in the edible endosperm have been developed using genetic engineering (Wirth et al., 2009;Johnson et al., 2011;Saltzman et al., 2013;Blancquaert et al., 2015;Singh et al., 2017;Zhu et al., 2018). Recently, transgenic rice with endosperms fortified with flavonoids, anthocyanins, or stilbenoids was also successfully engineered (Baek et al., 2013;Ogo et al., 2013;Zhu et al., 2017), representing potential functional staple food containing different health-beneficial phenolics. Although tricin and its derivatives have been characterized with many different health-promoting properties (Cai et al., 2004;Duarte-Almeida et al., 2007;Yazawa et al., 2011;Murayama et al., 2012;Jung et al., 2014Jung et al., , 2015Lee et al., 2015;Shalini et al., 2016), they are rarely present in human diets. Tricin is abundant in vegetative tissues of grasses but is not present in cereal endosperm due to the absence of expression of genes required for tricin biosynthesis (Ogo et al., 2013). Primary dietary sources of tricin include whole cereal grains such as rice, wheat, oat, and barley, in which small amounts of tricin are preserved in the bran (pericarp, testa, aleurone, and embryo; Poulev et al., 2018Poulev et al., , 2019, as well as some grass-derived food products, such as sugarcane juice (Duarte-Almeida et al., 2007) and barley leaf powders .
Functional food crops that are fortified with tricin could be generated by engineering the entire biosynthetic pathway in edible tissues. Previously, transgenic rice seeds that accumulate tricin were generated by expression of genes from multiple species encoding rice PAL, rice CHS, parsley FNSI, soybean FNSII, blue viola F3'5'H, and rice COMT/CAldOMT (Ogo et al., 2013). Recent establishment of the endogenous biosynthetic pathways in grasses (Lam et al., 2014(Lam et al., , 2015(Lam et al., , 2019a and Medicago legumes (Lui et al., 2020) as well as further elucidation of the regulatory mechanism should facilitate more effective metabolic engineering in plants or edible tissues that do not naturally produce tricin-type metabolites.

Bioengineering for Biorefinery
Grasses show great potential as a source of lignocellulosic biomass. A large amount of lignocellulose is produced annually as agricultural residues from worldwide cultivation of grass grain crops, including maize, wheat, rice, barley, and sorghum, as well as grass sugar crops, such as sugarcane and sweet sorghum. In addition, grass energy crops, such as Miscanthus, Erianthus, switchgrass, and bamboo, which show notably high biomass productivity, are attractive lignocellulose feedstocks for various biorefinery applications (Tye et al., 2016;Bhatia et al., 2017;Umezawa, 2018;Umezawa et al., 2020). Because of the prominent impacts of lignin on the usability of lignocellulose in both polysaccharide-and lignin-oriented biorefinery applications, bioengineering approaches to control lignin content and structure in grass cell walls have been actively investigated (Umezawa, 2018(Umezawa, , 2020Halpin, 2019;Coomey et al., 2020). However, due to our limited knowledge regarding the biological functions and physicochemical properties of tricin-lignin, it is still uncertain how tricin-lignin influences the usability of grass biomass. Thus far, not much has been examined on the effects of manipulating tricin biosynthesis on the utilization properties of grass biomass for different biorefinery applications.
As tricin could serve as a nucleation site for lignification, reducing the content of tricin used for lignification may result in reduction of lignin content and biomass recalcitrance, which may in turn improve the production of fermentable sugars from biomass in the polysaccharide-oriented biorefinery processes (Halpin, 2019). Indeed, tricin-depleted rice mutants deficient in FNSII (Lam et al., 2017) or A3'H/C5'H (Lam et al., 2017(Lam et al., , 2019a displayed reduced lignin content and improved cell wall digestibility. In contrast, however, tricin-depleted maize mutant deficient in CHS showed increased lignin level and reduced cell wall digestibility in leaves albeit no alteration in either lignin content or cell wall digestibility in stems (Eloy et al., 2017). The altered lignin content in the CHS-deficient maize leaf cell walls was attributed at least partially to the consequence of the increased carbon flux toward the branching monolignol biosynthesis pathway upon the blockage of the entry of the flavonoid pathway where CHS plays the major role (Eloy et al., 2017). These studies on tricin-depleted grass mutants implicated that disrupting tricin biosynthetic genes not only impedes the formation of tricin-lignin but also affects the formation of the core lignin polymer units derived from monolignols, although the mechanisms underlying this phenomenon remain unclear. Further manipulations of different tricin biosynthetic genes in different grass species are imperative to determine the precise relationships between tricin, lignin content and composition, and cell wall digestibility in tricindepleted grasses.
On the other hand, increasing the levels of tricin serving as initiation sites for lignin polymerization would theoretically reduce the molecular weight of the lignin polymers, which may potentially improve the efficiency of lignin deconstruction in the polysaccharide-oriented biorefinery processes (Berstis et al., 2021). A recent computational study determined that the bond strengths of the 4'-O-β linkages between the tricinand monolignol-derived lignin polymer units are comparable to the major β-O-4 linkages connecting the internal monolignolderived lignin polymer units, suggesting that introduction of more tricin units in lignin polymers is unlikely to increase the energy for lignin depolymerization (Berstis et al., 2021). Nonetheless, whether such tricin bioengineering strategy to attenuate lignin molecular weight and depolymerization efficiency requires further exploration.
Meanwhile, grass crops bioengineered toward high tricinlignin content could bring benefits in the lignin-oriented biorefinery approaches by amplifying the supply of tricin or tricin-derived aromatic chemicals. It has been estimated that large quantity of tricin could be released from grass lignins (Ralph, 2020;del Río et al., 2020). However, challenges ahead include developing technologies for efficient extraction and isolation of tricin from grass lignins to meet the stringent purity specifications as well as industrializing the production with maximized cost effectiveness and minimized environmental impacts. As the most abundant aromatic polymers on Earth, lignin has a great potential to serve as starting materials for sustainable production of bulk or functionalized aromatic chemicals (Ragauskas et al., 2014;Rinaldi et al., 2016;Umezawa et al., 2020). Accordingly, chemical and biochemical approaches to depolymerize lignin into useful low molecular weight aromatic compounds have been extensively pursued (Schutyser et al., 2018;Sun et al., 2018;Renders et al., 2019;Abu-Omar et al., 2021). As these studies have mostly focused on the conversions of the major monolignol-derived phenylpropane units in lignin, the consequences of lignin-integrated tricin units in various catalytic and bio-catalytic lignin depolymerization strategies remain an intriguing subject for further investigations.

AUTHOR CONTRIBUTIONS
PL, YT, and CL wrote the manuscript with help from all the other authors. All authors contributed to the article and approved the submitted version.