Lignin and Its Pathway-Associated Phytoalexins Modulate Plant Defense against Fungi

Fungi infections cause approximately 60–70% yield loss through diseases such as rice blast, powdery mildew, Fusarium rot, downy mildew, etc. Plants naturally respond to these infections by eliciting an array of protective metabolites to confer physical or chemical protection. Among plant metabolites, lignin, a phenolic compound, thickens the middle lamella and the secondary cell walls of plants to curtail fungi infection. The biosynthesis of monolignols (lignin monomers) is regulated by genes whose transcript abundance significantly improves plant defense against fungi. The catalytic activities of lignin biosynthetic enzymes also contribute to the accumulation of other defense compounds. Recent advances focus on modifying the lignin pathway to enhance plant growth and defense against pathogens. This review presents an overview of monolignol regulatory genes and their contributions to fungi immunity, as reported over the last five years. This review expands the frontiers in lignin pathway engineering to enhance plant defense.


Introduction
Plants are relentlessly exposed to pest and pathogen attacks. However, their sessile nature is naturally compensated for by synthesizing stress-responsive metabolites to overcome these attacks. Whereas many ribosome-inactivating proteins are reported to render pathogens proteins inactive to confer immunity in plants [1,2], the cell wall's dynamic and intricate nature provides the first line of defense and environmental cues [3,4]. Several metabolites, including lignin, cellulose, and pectin, contribute to cell wall integrity (CWI) [5]. Lignification, as an integral component of CWI, crucially enhances the two layers of plant innate immunity: pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) and effector-triggered immunity (ETI) [6]. While PTI uses pattern recognition receptors to monitor PAMPs on the cellular surface, ETI relies on nucleotide-binding domain leucine-rich repeat receptors to recognize pathogen effectors inside the cell [7].
The phenylpropanoid pathway is the metabolic hub of plants and produces approximately 8000 metabolites that enhance robust antagonistic and informative interactions between plants and their environments [8]. Recent insights underscore molecular factors regulating phenylpropanoids' metabolism orchestrated by a network of enzyme cascades, including; ligases, oxygenases, transferases, and oxidoreductases [9][10][11]. These enzymes influence the chemical modification of metabolic skeletons through glycosylation, acylation, hydroxylation, and methylation. Therefore, the diversity of phenylpropanoid-derived metabolites depends on them [12].
Lignin production is an off-shoot of the phenylpropanoid pathway. PHENYLALANINE (PAL) is synthesized via the chorismate pathway in plastids and released into the cytosol. It then catalyzes the first of three steps in the general phenylpropanoids pathway. Other regulators of monolignol biosynthesis include C4H, 4CL, the soluble C3H, HCT, CCoAOMTs, COMTs, F5H, CAD, and CCR. Peroxidases and laccases (PRX/LACs) encode monolignol polymerization into intracellular spaces of the cell wall [13][14][15][16]. Knowledge of the regulatory mechanism of monolignol biosynthesis continues to expand. Previously, phydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin were the only known lignin monomers. Recent studies have reported that catechyl (C) and 5-Hydroxy-guaiacyl (5H) monomers contribute to lignin polymerization in some plant species [17][18][19][20]. A total of 11 lignin family enzymes and 24 metabolites are currently associated with the lignin pathway [17][18][19][20]. However, these metabolites are often credited for their direct involvement in plant defense, whereas the enzymes regulating their accumulation remain in the shadows.
Studies have shown that mutant phenotypes of lignin regulators either shut down or severely impair the molecular switches for lignin and other metabolite accumulation. Compromised lignin metabolism affects plant defense against fungi and overall yield output. Therefore, exploiting the potential contribution of lignin and pathway-related metabolites could contribute to plant growth and yield. Over the last five years, many insightful reports have been published regarding lignin biosynthesis and plant defense. Although reviews on phenylpropanoid biosynthesis have recently been published, none has focused on the individual enzymes that regulate lignin formation and their roles in fungal defense. This review provides an overview of lignin pathway enzymes' contributions to defense lignification and other pathway-associated metabolic accumulation.

A Brief Overview of Monolignols Biosynthesis and Lignification
Even though phenylalanine is not a primary precursor to lignin biosynthesis, it initiates the first of three reaction steps to pave the way for lignin production [18,21]. Research advances have discovered eleven enzymes involved in monolignol production and polymerization ( Figure 1) [22]. The functions of each enzyme in the lignin pathway and its defense mechanisms are discussed in this review. In addition, adenosine and cytosine (AC element) enrich DNA motifs to promote lignin synthesis. MYELOBLASTOSIS (MYB) viral oncogene homolog transcription factors possess a rich AC motif that regulates lignin pathway genes, although they do not actively participate in the biosynthesis process. For example, MYB46 and its MYB83 homolog regulate phenylpropanoids and lignin biosynthesis [23,24]. In Arabidopsis thaliana, MYB15 activates PAL, C4H, 4CL, HCT, C3H, COMT, and CAD to enhance lignin accumulation during defense against Pseudomonas syringae DC3000 (AvrRpm1) [25].
Uncompromised pathogens penetrate the apoplast or cytosol through the intercellular voids within the cell wall. Lignification is an essential process that resists the entrance of these pathogens by lignin deposition in the voids via Golgi-mediated vesicles in the cell membrane, as recently proposed [26]. Lignification promotes the chemical alteration of pathogen-secreted cell-wall-degrading enzymes to boost toxin diffusion resistance [6]. Some reports suggest lignification disrupts these pathogen-degrading enzymes and restricts pathogens' mobility in infected cells from infecting new cells [6]. Lignin and callose deposition are also reported to block fungi haustoria from the cell wall [27]. Monolignols biosynthesis and polymerization. The various enzymes leading to monolignol formation are based on current understanding: traditional monolignols (black) and recently discovered monolignols in some plant species (blue). Stage 1: Phenylalanine escapes from the chorismate pathway in the plastid into the cytosol. Stage 2: Enzymatic activities that occur prior to monolignol formation. Stage 3: Monolignols are transported into the apoplast. Stage 4: PRX/LAC encodes monolignol polymerization into lignin. Lignin fills up intercellular voids to enhance cell wall rigidity. Proposed mechanism of monolignol transport: (a) ABC transporters mediate active trafficking of monolignols. (b) Trans-membrane diffusion of monolignols/channels-facilitated membrane transport. (c) ABC transporters channel monolignol glycoside into vacuoles for release at cell death.

PHENYLALANINE AMMONIA-LYASE (PAL)
PAL initiates the general phenylpropanoid pathway reaction by catalyzing the deamination of L-phenylalanine to trans-cinnamic acid and ammonium [16,28]. This process paves the way for several enzymatic activities to produce an enormous array of secondary metabolites, such as lignin, lignan, chlorogenic acid, SA, and stilbene [29]. PAL accumulation is linked to defense mediation against pathogens and pests, even though the mechanism by which they execute these activities is elusive. For example, overexpression (OE) and RNA interference (RNAi) enhanced the expression of soybean GmPAL2.1 against

PHENYLALANINE AMMONIA-LYASE (PAL)
PAL initiates the general phenylpropanoid pathway reaction by catalyzing the deamination of L-phenylalanine to trans-cinnamic acid and ammonium [16,28]. This process paves the way for several enzymatic activities to produce an enormous array of secondary metabolites, such as lignin, lignan, chlorogenic acid, SA, and stilbene [29]. PAL accumulation is linked to defense mediation against pathogens and pests, even though the mechanism by which they execute these activities is elusive. For example, overexpression (OE) and RNA interference (RNAi) enhanced the expression of soybean GmPAL2.1 against Phytophthora sojae infection. The highly expressed PAL induced the accumulation of daidzein, glyceollin, genistein, and salicylic acid (SA) to mediate defense against P. sojae [30]. The rice genome has nine PAL genes. Eight induce resistance against Magnaporthe oryzae infection. In addition, Rhizoctonia solani stimulates quantitative trait loci for resistance in seven OsPAL genes [31].
The Brachypodium distachyon (purple false brome), PAL1, was also identified to induce lignin, SA, cinnamic acid, and fatty acid accumulation in defense against the panicum mosaic virus. However, RNAi-mediated knockdown of BdPAL1 enhances panicum mosaic virus pathogenicity [32]. PAL is also reported to induce lignin and cinnamaldehyde accumulation against P. capsici infection in black pepper and trans-cinnamic acid defense against Xoo [33]. The PAL gene family encodes the production of defense metabolites irrespective of the reaction direction (forward or reverse) and, therefore, are candidate genes for genetic engineering. However, their substratum specificity, catalytic, and protein-wide mechanisms remain elusive, hindering their engineering potential. Table 1 summarizes the current reported role of lignin regulatory genes in fungi immunity in plants.

CINNAMATE 4-HYDROXYLASE (C4H)
The C4H is a member of the CYP73A class of P450-associated monooxygenase family proteins that encodes the hydroxylation of p-coumaric acid from cinnamic acid. C4H activities promote cell wall lignification and biosynthesis of other plant defense metabolites [34][35][36]. The soybean C4H1 gene is highly responsive to pathogens and encodes defense lignification against P. sojae. Whereas the gmc4h1-mutant plants are highly susceptible to P. sojae, the OE-GmC4H1 lines in N. benthamiana significantly accumulated lignin for immunity induction [34]. C4H1, C4H2, and C4H3 expression vary from tissue to tissue in Pyrus bretschneideri (pear plant) [35]. Transcripts of C4H1 and C4H3 defensively accumulate lignin and robust cell walls in Arabidopsis plants overexpressing these genes [35]. A related study reports that OsC4H complements pathogenesis and antioxidant-related genes to activate defense against pests [36]. Pathway perturbations can also externally or internally influence biological functions, such as metabolic changes [37]. A reprogrammed phenylpropanoid pathway by piperonylic acid (PA)-mediated inhibition of C4H triggers systematic resistance against a broad spectrum of pathogens [38]. The C4H-inhibited Solanum lycopersicum (tomato) increased flavonoid production with enhanced immune signaling, cell wall modification, phenolic compounds, and SA accumulation [38]. Elicitor proteins and transcription factors have also been reported to activate C4H defense against fungi (Table 1).

4-COUMARATE-COA -COENZYME A LIGASE (4CL)
The 4CL protein distributes the flux among different metabolic pathways. It is the precursor for downstream biosynthesis of other metabolites, such as stilbenes and flavonoids, and also encodes the esterification of p-coumaroyl CoA to p-coumaric acid for lignin production [9]. A Fraxinus mandshurica OE-4CL2 in tobacco plants enhanced lignin accumulation but inhibited hemicellulose production. This resulted in a 250 % increase in coniferyl alcohol levels, fortifying cell wall and xylem cell layer thickness. Overexpression lines in soybean significantly induced resistance against P. sojae by accumulating daidzein, genistein, and glyceollins. The Fm4CL2 ortholog from Dryopteris fragrans (Df4CL2), transformed into tobacco via an Agrobacterium tumefacient-mediated system, increased lignin and flavonoids concentration, further suggesting 4CL could play a crucial role in cell-wallmediated defense [39][40][41].
Transcription factors activate the expression of phenylpropanoid genes. The peach WRKY70 activates 4CL and PAL promoters to elevate total phenolics, flavonoids, and lignin biosynthesis against a rot initiation fungus, Rhizopus stolonifer [42]. WRKY, MYB, and bHLH transcription factors can also switch on lignin biosynthetic genes (4CL, PAL) in Pinus strobus (eastern white pine) after perceiving nematode (Bursaphelencus xylophilus)inflicted injuries [43]. The high expression of 4CL and PAL induces stilbenoids, pinosylvin monomethyl, and monoethyl ethers elicitation to mediate plant defense [43]. In related findings involving Botrytis cinerea (gray mold) infection in blueberry fruits, methyl jasmonate (MeJA) treatment restrained the decaying success of gray mold in the fruits through 4CL-, C4H-, and PAL-induced production of NO, H 2 O 2 , phenolic, and flavonoid [44]. A virus-induced gene silencing (VIGS) of 4CL30 in cotton compromised lignin and flavonoid accumulation but increased caffeic and ferulic acid levels to confer immunity against Verticilia dahlia [67]. The central position of flux distribution showed that 4CL is an essential enzyme in downstream defense modulation (Table 1) and could play a critical role in lignin pathway engineering.

HYDROXYCINNAMOYL TRANSFERASE (HCT)
The HCT distributes the mass flux among C-, G-, 5H, and S-lignin. It also forms p-coumaroyl shikimic acid from p-coumaroyl CoA and then reversely encodes caffeoyl shikimate conversion to caffeoyl CoA [68,69]. However, the latter process is being questioned for possible redundancy. In O. sativa, the negative regulation of cell death elicitation mediated by the APIP5 transcriptional factor that binds to OsPHCT4 is mitigated by APIP5-RNAi [70]. This process frees up the activation of tryptamine HCTs (OsTBT1 and OsTBT2) and tyramine HCTs (OsTHT1 and OsTHT2) to enhance immunity against M. oryzae through lignin and phenolamide accumulation [70]. Populus trichocarpa WRKY transcription factor regulates HCT2 to mediate defense against Sphaerulina musiva [71], while MYB15 turns on monolignol synthetic genes, including HCT, for lignin-mediated ETI [25]. Populus tomentosa Carr PtoHCT1 also relies on caffeoyl-CoA and shikimic acid substrates to synthesize caffeoyl shikimate. PoptrHCT1 and -2 from Populus trichocarpa, a close relative of P. tomentosa, contribute to plant defense [20]. Similar investigations involving HCT defense against fungi have been reported (Table 1).

CAFFEOYL SHIKIMATE ESTERASE (CSE)
CSE catalyzes the direct conversion of caffeoyl shikimate to caffeate acid. The reverse catalytic activity of HCT in converting caffeoyl shikimate to caffeoyl-CoA has raised controversy upon CSE discovery [72], suggesting this process could be redundant in the lignin pathway. Even though there is no established consensus, available reports suggest CSE could be more efficient than HCT in lignin biosynthesis [73]. A few recent reports have elucidated the function of CSE in lignin production. However, no distinct bioassay demonstrating the defense function of this enzyme in vitro via the higher lignin content has been reported in the last five years. CSE from a hybrid Populus significantly encodes lignin accumulation [74]. Moreover, its OE-PbCSE1 lines in pea fruits increased lignin content in the stem [75], while its mutant lines decreased lignin production [76].
Sugar cane ShMYB78 regulates suberin accumulation by activating COMT and ketoacyl-CoA synthase (ShKCS20) [82]. Suberin is a vital metabolite that provides a physical barrier against pathogens, water loss, and wound healing and could spike interest in possible engineering attempts [82]. CRISPR-Cas9-mediated editing of StCCoAOMT in Russet Burbank potato induces suberin and lignin elicitation to resist P. infestans [83]. In addition, the bread wheat plant lignin-induced cell wall thickening was enhanced by TaCCoAOMT for Fusarium head blight resistance [84].

CINNAMOYL COA REDUCTASE (CCR)
CCR encodes the formation of hydroxycinnamaldehydes from hydroxycinnamoyl-CoA, the first committed step in monolignol production. Loss of CCR function in angiosperm inhibits lignin accumulation and increases susceptibility to pathogens [89]. B. nepus CCR1 gene participates in H-and G-lignin synthesis and vascular systems formation, while the BnCCR2 encodes S-lignin production. OE-BnCCR (1 and 2) phenotypes delayed flowering time and resulted in poor leaf and vascular system development [89]. BnCCR1 and BnCCR2 increased glucosinolate (GLSs) concentration [89], which could remedy chemical defense against fungi diseases through hormone signaling and pathogen perception [90][91][92].

CINNAMYL ALCOHOL DEHYDROGENASE (CAD)
CAD encodes the NADPH-dependent reduction of various hydroxy-cinnamaldehydes to their respective monolignol alcohols [93]. Rice CAD2 transcript abundantly accumulates in young seedlings and confers cell-wall-mediated immunity against Xanthomonas oryzae pv. oryzae (Xoo) [93]. Cell wall fortification has been explored to control Sclerotinia sclerotiorum. BnCAD5 and F5H induce rapid accumulation of S-lignin against S. sclerotiorum infection [26]. A comparative transcriptional analysis in Manduca sexta (stem-boring herbivore), Trichobaris mucorea (stem borer)-attacked, and healthy wild tobacco Nicotiana attenuata implicated CAD activity for enhanced lignin deposition in parenchymal cells and pith of the insect-attacked plants. However, cad mutants restored the stem-boring ability of the herbivores without inhibiting growth. Ethylene and jasmonate were subsequently identified to signal pith lignification [94].
Trichoderma harzianum is a plant fungicide used for foliar application, seeds, and soil treatment to control fungi pathogens. The commercial fungicide 3Tac is developed from T. harzianum to control Botrytis, Fusarium, and Penicillium spp. Studies have shown that T. harzianum induces immunity in S. lycopersicum L (tomato) against RKN, Meloidogyne incognita through increased expression of CAD, PAL, C4H, and CCOMT for lignin, flavonoids, and phenols accumulation against M. incognita [95]. The transformation of another CAD2 gene from Pyrus pyrifolia (pear) into a tomato plant via an Agrobacterium-mediated system defensibly accumulated lignin in leaves, stems, and fruits [96].

PEROXIDASES and LACCASES (PRX and LACs)
Plant cell wall lignification is catalyzed by class III peroxidase (PRX) and laccase (LACs) enzymes [97,98] for defense modulation and breakdown of hydrogen peroxides in the cytosol and chloroplast [99]. An apoplast CsPRX25 protein in Citrus sinensis induces cell wall lignification to mediate defense against pathogens [100]. Blossom-end rot also induces ROS, H 2 O 2 , and lignin accumulation. According to Reitz & Mitcham, enhanced expression of PRXs in blossom-end rot-infected tomatoes participates in defense lignification [101]. In addition, two PRX genes (VlPRX21 and VlPRX35) in the grapevine are involved in transresveratrol conversion to δ-vinifera and could be essential genes for δ-viniferin engineering for enhanced fungal defense in plants [102]. Histochemical analysis showed the localization of lignin in the xylem cell wall was linked to DcPRX30, DcPRX32, and DcPRX62 activities in the taproot epidermal zones of carrots, leading defense lignification [103].

Phytoalexins Associated with the Lignin Pathway Enzymes
Apart from lignin being the final product and most crucial metabolite in this pathway, other antifungal defense metabolites accumulate along the same path ( Figure 2). Current advances link coumarin accumulation to the catalytic activities leading to p-coumaryl CoA formation. Therefore, PAL, 4CL, and HCT play a role in coumarin biosynthesis. The feruloyl-CoA formation from the p-coumaryl CoA precursor forms the committed step for coumarin accumulation with the involvement of the CCoAOMT enzyme. Moreover, ironassisted hydroxylation of cinnamate, p-coumarate, caffeate, and ferulate also accumulates simple coumarins. Umbelliferone, esculetin, and scopoletin are simple coumarins whose biosynthesis follows this route [106][107][108]. Coumarins have generally been reported as plant microbiome regulators, principally regulating three crucial activities: nutrient improvement, pathogen inhibition, and abiotic stress tolerance [107,109,110].
Stilbenes are also phenolic phytoalexins whose accumulation is also associated with the lignin pathway regulators. They are unique for their C 6 -C 2 -C 6 carbon skeleton [111]. PAL, C4H, and 4CL activities in the phenylpropanoid pathway leading to p-coumaroyl-CoA formation, as elaborated in Figures 1 and 2, generate an active intermediate for trans-resveratrol production. Finally, stilbene synthase (STS) catalyzes the conversion of p-coumaryl-CoA to the stilbene skeleton by initially converting p-coumaroyl-CoA and a three-unit malonyl-CoA to trans-resveratrol. STS also converts cinnamoyl-CoA to transpinosylvin. Moreover, resveratrol-O-methyl transferase is enhanced by VvMYB14 and VvMYB15 for stilbene production [112][113][114]. The defense involvement of stilbene against fungi and viral diseases are recently reported [115][116][117].
Furthermore, caffeic acid is the precursor to ferulic acid. Both share the same route from the phenylalanine precursor through the 4-hydroxycinnamic acid precursor leading to the formation of caffeic acid. Caffeic acid subsequently becomes the precursor to ferulic acid biosynthesis in plants, regulated by COMT enzymes in the lignin pathway. As well as lignin and lignan biosynthesis intermediates, caffeic, ferulic, and dihydro ferulic acids are lignocellulose compounds. They induce cell wall stiffness by crosslinking with lignin and other polysaccharides [118,119]. PAL, C4H, and 4CL chronologically catalyze the formation of the coumaroyl CoA precursor for downstream biosynthesis of daidzein and genistein through the initial enzymatic activities of chalcone synthase (CHS). Daidzein and genistein accumulation is induced by fungi, bacterial, and viral infections [120,121].  Figure 1. The proposed monolignol transport mechanisms (a-c) are also the same as in Figure 1.
Furthermore, caffeic acid is the precursor to ferulic acid. Both share the same route from the phenylalanine precursor through the 4-hydroxycinnamic acid precursor leading to the formation of caffeic acid. Caffeic acid subsequently becomes the precursor to ferulic acid biosynthesis in plants, regulated by COMT enzymes in the lignin pathway. As well as lignin and lignan biosynthesis intermediates, caffeic, ferulic, and dihydro ferulic acids are lignocellulose compounds. They induce cell wall stiffness by crosslinking with lignin and other polysaccharides [118,119]. PAL, C4H, and 4CL chronologically catalyze the formation of the coumaroyl CoA precursor for downstream biosynthesis of daidzein and genistein through the initial enzymatic activities of chalcone synthase (CHS). Daidzein and genistein accumulation is induced by fungi, bacterial, and viral infections [120,121]. Steps 1, 2, 3, and 4 are the same as in Figure 1. The proposed monolignol transport mechanisms (a-c) are also the same as in Figure 1.
Lignans are vital physiological, developmental, and ecological plant metabolites. They are formed by coupling reactions of monolignols and defend against herbivores and pathogens [122]. Plants' dirigent protein crucially regulates the initial coupling reactions that form lignans. PINORESINOL-LARICIRESINOL REDUCTASES (PLR) then encode successive reduction reactions to form lariciresinol and secoisolariciresinol from pinoresinol. A soybean dirigent protein (GmDIR22) was identified to regulate coniferyl alcohol coupling into lignan (+)-pinoresinol to restrict P. sojae hyphal growth. An enhanced concentration of yatein was detected in the roots and leaves of mycorrhizal plants in conferring resistance against B. cinerea infections [123]. The chemical structures of lignin pathway-associated phytoalexins shown in Figure 3.
Lignans are vital physiological, developmental, and ecological plant metabolites. They are formed by coupling reactions of monolignols and defend against herbivores and pathogens [122]. Plants' dirigent protein crucially regulates the initial coupling reactions that form lignans. PINORESINOL-LARICIRESINOL REDUCTASES (PLR) then encode successive reduction reactions to form lariciresinol and secoisolariciresinol from pinoresinol. A soybean dirigent protein (GmDIR22) was identified to regulate coniferyl alcohol coupling into lignan (+)-pinoresinol to restrict P. sojae hyphal growth. An enhanced concentration of yatein was detected in the roots and leaves of mycorrhizal plants in conferring resistance against B. cinerea infections [123]. The chemical structures of lignin pathway-associated phytoalexins shown in Figure 3.

Missing Links in the Lignin Research, Prospects, and Conclusions
The lignin pathway is a crucial vehicle for plant information and communication interactions with their environment and a source of bioactive compounds for plant defense. As a result, a thorough understanding of the pathway enzymes and their interactions will contribute significantly to the beneficial exploits of fungi defense tradeoffs. Enormous literature on key genes regulating lignin biosynthesis and their activities abound. This review dissected a plethora of them, including some defense metabolites that accumulate along the lignin pathway. Engineering these candidate genes in food crops could promote disease resistance to enhance crop yield. However, there are several unanswered questions on lignin metabolism that could facilitate its engineering processes. The shikimate pathway involves seven enzymatic steps to form folates and aromatic amino acids in plants, including phenylalanine. This process exclusively occurs in the plastid, and shikimate provides the required substrate for phenylalanine formation. The mechanism involved in shikimate transition into the cytosol for lignin biosynthesis is currently unknown. In addition, CSE directly converts caffeoyl shikimate to caffeic acid, a shorter route to monolignol biosynthesis. It is also unclear if this process renders the HCT role in reverse reaction redundant. Further identifying the most efficient route between the two could enhance lignin genetic manipulations to address pathogen defense. More lignin monomers are identified in some plant species. Intriguingly, current reports only focused on the dimerization and polymerization reactions that form lignan and lignin, respectively, but the key functions of the individual monolignols relative to plant defense are unknown. In a nutshell, addressing these gaps will improve the attempts of lignin pathway engineering to enhance plant defense against fungi.