More or Less: Recent Advances in Lignin Accumulation and Regulation in Horticultural Crops

Abstract

Lignin is an important secondary metabolite that maintains the mechanical strength of horticultural plants and enhances their ability to respond to external environmental changes such as biotic and abiotic stresses. However, excessive accumulation of lignin can lead to lignification of horticultural products, reducing their taste quality and nutritional value. Therefore, the lignin content of horticultural products needs to be controlled at a reasonable level, and studying and regulating lignin metabolism is very meaningful work. This article focuses on the synthesis, accumulation, and regulation of lignin in horticultural crops in recent years, provides a systematic analysis of its molecular mechanism and application prospects, and sheds insights into the directions that need further research in the future. This article provides an important basis for the regulation of lignin accumulation and lignification in horticultural crops and proposes new ideas for improving the quality of horticultural crops.

1. Introduction

Horticultural plants generally refer to crops with high economic value that are used for human viewing or consumption, mainly including fruit trees, vegetables, flowers, tea, edible fungi, and medicinal plants. The relationship between horticultural plants and humans is extremely close, and we cannot do without these plants in our daily life. Moreover, with the progress of human civilization, many new horticultural plants have been continuously domesticated and cultivated by humans.

On the one hand, lignin can confer on horticultural crops high rigidity, providing plants with resistance to the external environment [1,2]. However, for many horticultural plants, excessive accumulation of lignin can lead to a decrease in the taste, texture, and general quality of horticultural products. Therefore, understanding the accumulation and regulatory mechanisms of lignin in horticultural plants is crucial for improving their resistance and quality.

2. Lignin

Lignin is an important component of the cell wall in vascular plants such as ferns, gymnosperms, and angiosperms. It is mainly deposited in the secondary cell wall of transport tissues, mechanical tissues, and protective tissues [3]. It plays a key role in the evolution of terrestrial vascular plants and has very important biological functions. Lignin is very abundant in nature, second only to cellulose, and its use as a binder and various additives in the chemical industry accounts for its primary economic value [4]. In woody plants, lignin accounts for 25% of the composition, making it the second most abundant organic matter in the world. As the main component of the plant cell wall, lignin is cross-linked with cellulose, hemicellulose, pectin, and other substances [5]. On the one hand, the plant skeleton formed by this cross-linked lignin carbohydrate complex improves the mechanical strength and hardness of plant tissue, increases the penetration ability of cell wall pathogens, and improves the plant’s ability to resist biotic and abiotic stresses [6]. On the other hand, it has a compressive effect and its hydrophobicity makes plant cells less permeable, facilitating the long-distance transportation of water, minerals, and organic matter within the plant body [7].

3. Lignin Biosynthesis and Transcriptional Regulation

3.1. Lignin Biosynthesis

Lignin is synthesized through the phenylpropane pathway and lignin-specific pathway [8]. Phenylalanine goes through deamination, hydroxylation, methylation, and reduction reactions to form three main monomers, coumarin, coniferol, and sinapinol, which are subsequently polymerized to form p-hydroxyphenyl lignin (H lignin), guaiacyl lignin (G lignin), and syringal lignin (S lignin), respectively [9]. The key enzymes involved in this pathway consist of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), p-coumaroyl coumaroyl shikimate/quinate 3′-hydroxylase (C3′H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT), peroxidase (PER), and laccase (LAC) [10].

3.2. Transcriptional Regulation

MYB (v-myb avian myeloblastosis viral oncogene homolog), the main regulatory factor for lignin synthesis, directly induces the expression of most genes. MYB58, MYB63, and MYB85 in Arabidopsis can directly bind to AC elements in the promoter regions of PAL, C4H, 4CL, C3′H, CCoAOMT, CCR, and CAD [11]. And NAC transcription factors are mostly located upstream of the lignin transcription regulation network, indirectly regulating lignin biosynthesis by binding to different MYB transcription factors [12]. For instance, an SG2-type R2R3-MYB transcription factor from chrysanthemum, CmMYB15-like, directly interacts with the AC cis-element in the promoter region of Cm4CL2 and results in lignin accumulation and cell wall thickening [13]. PbrMYB169 from pear is evidently integrated with the AC elements [ACC(T/A)ACC] in the promoter of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2, and LAC18 to regulate lignin deposition and cell wall thickness [14]. PbrMYB24 can also bind to AC elements and MYB binding sites (MBSs) in the promoter of lignin biosynthesis genes to activate their transcription [15]. In apple, transgenic plants overexpressing MdMYB46 presented enhanced secondary cell wall and lignin deposition by directly triggering the promoter of lignin biosynthesis genes [16]. Overexpression of PnMYB2 from Panax notoginsengin resulted in increased lignin accumulation and evidently reinforced thickness of primary and secondary cell walls compared with wild-type plants. Additionally, PnMYB2 could directly interact with the promoter of PnCCoAOMT1, a key lignin biosynthesis gene [17]. In pomelo (Citrus grandis), CgNAC043 is located upstream of CgMYB46 and can simultaneously bind to the promoter of the lignin biosynthesis genes CgCCoAOMT and CgC3H, thereby modulating juice sac granulation [18]. In apple, MdSND1, an NAC transcription factor related to secondary wall development, can activate the expression of MdMYB46/83 to regulate lignin biosynthesis and accumulation [19]. An NAC STONE CELL PROMOTING FACTOR essential for stone cell formation in pear, namely PbrNSC, can bind to the promoters and induce the expression of PbrMYB169, Pbr4CL4, and PbrLAC4 [20].

Further studies indicate that bHLH, bZIP, SPL, and other transcription factors are also involved in the transcription regulation of lignin. The atypical bHLH transcription factor CmHLB (HLH PROTEIN INVOLVED IN LIGNIN BIOSYNTHESIS) from chrysanthemum has been confirmed to interact with chrysanthemum KNOTTED ARABIDOPSIS THALIANA7 (CmKNAT7) and has a positive role in stem mechanical strength, cell wall thickness, and lignin content [21]. EjAGL15, a senescence-specific MADS-box gene in loquat, triggered lignin-biosynthesis-related genes to positively modulate the variation in lignin content [22]. CcBLH6, a bell-like homeodomain-containing (BLH) transcription factor from Camellia chekiangoleosa, was consistent with the unique lignification pattern observed during fruit development, and transgenic experiments confirmed the role of CcBLH6 in the control of fruit lignification [23]. The grapevine transcription factor VvWRKY2 showed an ability to bind to the promoter of the VvC4H gene to regulate lignification in response to biotic or abiotic stresses [24]. The LIM transcription factor CaβLIM1a from chickpea (Cicer arietinum) is a transcriptional regulator of CaPAL1, the gatekeeping enzyme of the phenylpropanoid pathway. Its interaction with the nuclear effector PEXEL-like Effector Candidate 25 (ArPEC25) from Ascochyta rabiei would interfere with its DNA-binding ability and result in altered lignin production [25]. An ethylene response factor in apple, MdERF114, can directly interact with the GCC-box in the promoter of MdPEROXIDASE63 (MdPRX63) and activate its transcription, leading to increased lignin accumulation and conferring root resistance to Fusarium solani [26]. The role of EjHSF3, a heat shock factor from loquat, in regulating fruit lignification was realized by activating the promoters of the downstream lignin biosynthesis genes and interacting with the regulator, EjAP2-1 [27]. Overexpression of tomato SlHB8 belonging to the homeodomain-leucine zipper class III transcription factor gene family decreased stem diameter, xylem width, and number of xylem cell layers, accompanied by reduced lignin production [28].

4. Lignin Accumulation

4.1. Growth Developmental Stages

Lignin accumulation is altered in response to developmental stages and can impact plant and product organ development. In pomelo, juice sac granulation and lignin content increased after anthesis with the development of pomelo fruit, and the lignin content near the core was higher than that further away from the core [29]. Similarly, the lignin content showed a continuous increase in winter jujube pericarp during pigmentation [30]. During the growth of Camellia oleifera shell, lignin content increased continuously, leading to a thickened stone cell wall [31]. By contrast, lignin content in storage root gradually decreased during the developmental stages in two sweet potato cultivars [32]. The concentrations of total lignin and its precursors displayed marked decreases during ginger tissue maturation; however, the lignin component was dramatically altered with syringyl lignin mainly enriched in mature rhizomes, contributing to ginger lignification [33]. Lignin content exhibited a decrease in pepper fruits at different stages during growth and maturation, following the same pattern as the lignin precursors [34]. In carrot roots, lignin was mostly deposited in xylem vessels, and with the development of carrot taproots, lignin content continuously decreased, accompanied by a reduced transcript abundance of the lignin biosynthesis genes [35]. Indeed, plant development is a dynamic process that includes changes in structure, phenotype, and internal metabolic substances. Lignin is a secondary metabolite, and its content in plants is largely influenced by other metabolites. In addition, the pattern of lignin content changes in different organs or tissues within the same plant may differ at different developmental stages.

4.2. Postharvest Storage

The change in postharvest fruit quality is a complex biological process, and texture is an important aspect of fruit quality which directly affects the storage life, product merchantability, and market competitiveness of horticultural products. Lignification is a common occurrence in fruits and vegetables such as pear, loquat, mangosteen, kiwi and zucchini, and improper storage can lead to a decline in fruit quality and severely limit storage time. Under normal circumstances, postharvest fruits and vegetables will generally experience the process of lignification strengthening. Increased activities of PAL and PER promoted lignin synthesis and accumulation during postharvest pumelo storage, which in turn induced sucrose degradation and an energy deficit [36]. With the extension of shelf life, broccoli florets underwent apparent increases in lignin accumulation, and this effect was reversed by exogenous diacetyl [37]. During cold storage processes, lignification was gradually enhanced in the epidermis of the shoots in water bamboo with the reinforcement of firmness [38]. Compared to the control group, the water bamboo shoot samples subjected to melatonin displayed enhanced lignification during storage [39]. With increasing storage time, two types of snow pea underwent enhanced lignin accumulation, and small snow peas were more susceptible to lignification than large sweet broad peas [40]. Lignin accumulation in samples in both modified polyethylene packages and normal polyethylene packaging gradually increased during storage [41]. Over the period of storage, enhanced respiration may require a significant amount of energy consumption, leading to accelerated senescence and the generation of reactive oxygen species, as well as the strengthened lignification process of horticultural products.

5. Lignin Regulation by External Stimuli

5.1. Environmental Changes

5.1.1. Temperature

Studies have demonstrated that the enhanced accumulation and distribution of lignin within plant tissues can confer resistance to temperature fluctuations. In Chinese cabbage, the lignin content in the samples exposed to low temperatures was apparently higher than that of the control, resulting in the assumption that lignin adjustments are a defense mechanism or response to low temperatures [42]. Similarly, low-temperature-induced enriched accumulation of lignin biosynthesis was seen in postharvest banana and Lei bamboo shoots [43,44]. Just like those under low-temperature conditions, bamboo shoots also underwent lignification when exposed to high temperatures [45]. The primary monolignol contents were substantially increased after heat stress in coffee leaves [46]. These findings again favor the theory that lignin is a polymer that defends against environmental temperature changes.

5.1.2. Drought

Drought stress is one of the most important environmental stresses adversely affecting plant growth and quality. Lignin deposition in plant cells is of great significance for drought adaptability and resistance. It was reported that drought stress in tea plants resulted in a significant increase in lignin content [47]. In tomato, a higher proportion of lignin in the cell walls of leaves was observed due to repeated drought cycles [48]. Drought induced H₂O₂, ABA, and JA accumulation and promoted the expression of CAD genes, contributing to increased lignin synthesis in melon stems [49,50]. Water stress activated the phenylalanine ammonia-lyase genes as well as the primary and secondary metabolism of postharvest carrots, accelerating the lignification process [51].

5.1.3. Salinity

Soil salinization is becoming increasingly severe and is profoundly affecting various aspects of crop growth and development. Plants can adapt to salinity and the resulting injury by modifying the accumulation and distribution of the main cell wall components, especially lignin, to maintain water balance and reduce the transport of harmful ions into the plant. The levels of total and individual phenolic acids in multi-leaf lettuce, such as lignin, gradually increased with increasing concentrations of salt [52]. Similarly, when subjected to salt stress, the monolignol content in coffee leaves was substantially increased as compared to the control [53]. Salt stress might enhance xylem development in tomato root, accompanied by an increased number of lignified cells in vascular bundles as well as a greater deposition of lignin [54]. Increased lignin accumulation under salt stress may enhance the cell-to-cell pathway for water transport and confer reinforced selectivity and decreased ion uptake to protect plant cells exposed to salt stress.

5.1.4. Heavy Metals

Due to the rapid development of industrialization, various heavy metals are constantly added to soil and water, which has become a major global concern as this leads to the loss of agricultural productivity. Heavy metals induce the production of phenolic secondary metabolic pathways and enhance the lignin deposition within cell walls. Heavy metals can damage the cell integrity of the roots, and in severe cases, may lead to cell death in the roots. In the presence of copper (Cu) exposure, lignin deposition of the cell wall in mustard roots was significantly greater than in stress-free control plants [55]. Cu intensively increased PAL activity and lignin accumulation in a dose-dependent manner in copper-treated Matricaria chamomilla, revealing the establishment of a barrier against metal intrusion [56]. The lignin content in lettuce roots treated with cadmium increased by 18% compared with control group [57], whereas increased Zn exposure in growth media evidently reduced the lignin content in the leaves of watermelon seedlings [58].

5.1.5. Pest and Disease Invasion

Lignin accumulation enhances the hardness and reinforcement of cell walls, provides a solid and strong barrier, and is recognized as the first line of defense against biotic infection. Furthermore, lignin deposition can diffuse pathogens by restricting the access of fungal enzymes and toxins into the host plants and preventing the movement of nutrients from hosts to invaders [2]. Lignification in pear results from plant resistance against diseases caused by Alternaria alternata and Botryosphaeria dothidea, and the PcMYB44-mediated PcmiR397-PcLACs module may be involved in the process of defense-induced lignification [59]. Leaf-spot-infected leaves had increased transcription of lignin biosynthesis genes and enhanced lignin accumulation in an important medicinal plant, Withania somnifera [60]. Exogenous carvacrol enhanced lignin accumulation and delayed cell wall degradation in postharvest pummelo fruit, conferring disease resistance on Diaporthe citri [61]. Alterations in the expression of caffeoyl shikimate esterase (CsCSE1), a key novel enzyme in lignin biosynthesis in cucumber, significantly affected lignin accumulation and plant resistance to Podosphaera xanthii [62]. Enhanced total lignin deposition in kiwifruit stems can induce disease resistance against kiwifruit canker [63]. Early production of lignin might be a key mechanism in repressing the spread of the red leaf blotch disease caused by the fungus Polystigma amygdalinum within the host leaf tissues in almond [64]. Comparison of resistant and susceptible pigeon pea genotypes demonstrated that the genotype resistant against Fusarium wilt possessed higher lignin contents [65]. These findings indicate that lignin accumulation and distribution can be altered to react to pest and disease invasion.

5.1.6. Carbon Dioxide (CO₂)

With the aggravation of climate change, atmospheric CO₂ levels may rise significantly by the end of the 21st century. Elevated CO₂ levels could greatly impact plant growth and development, including changes in phenotype, anatomy, and secondary metabolite accumulation. In the presence of enriched CO₂ levels, there was an upward trend in lignin concentration in celery leaves, and the transcription of most lignin metabolic genes changed in response to elevated CO₂ levels [66]. Similarly, the lignin content in carrot roots under elevated CO₂ conditions was apparently higher than that in the control group [67]. Pea plants grown in an atmosphere enriched with CO₂ showed improved cell wall fortification due to enhanced lignin accumulation, thus efficiently coping with parasite penetration [68]. By contrast, under cold storage, higher CO₂ levels may reduce the lignification process through the inhibition of PAL activity and lignin deposition [69].

5.1.7. Nitric Oxide (NO)

NO acts as a messenger, taking part in various aspects of processes during plant growth and development. Wax apple fruit treated with NO had a lower total lignin content, indicating that NO treatment can alleviate the process of lignification and senescence of wax apple fruit during storage [70]. Cold stress induced increased lignin accumulation and promoted lignification in okra pods and water bamboo shoots, which was reversed with the application of exogenous NO [71,72]. The abundance of mRNA in lignin biosynthesis genes was decreased in postharvest carrots exposed to NO donor sodium nitroprusside (SNP), leading to lower lignin deposition and a lesser degree of taproot lignification [73]. When exposed to the NO scavenger cPTIO, no detectable changes in total lignin content could be detected in sunflower roots, whereas the ratio of G/S lignin was increased, indicating that NO may impact lignin composition [74]. SNP elevated the accumulation of lignin to suppress anthracnose decay in postharvest mango fruit [75]. Similarly, the application of SNP improved the activities of PAL, C4H, CAD, and PER and increased the concentrations of lignin and its monomers, enhancing the hardness of the healing tissue in muskmelon [76].

5.1.8. Melatonin

Melatonin is a kind of biomolecule found in almost all living kingdoms and can regulate many life processes and responses to abiotic and biotic stresses. Recently, melatonin has been demonstrated to be involved in lignin regulation in horticultural crops. Melatonin strengthened the degree of lignification in tea leaves by adjusting the expression of genes involved in lignin synthesis pathway [77]. The melatonin content within plant stems is consistent with lignin content and stem strength in different Paeonia lactiflora cultivars; furthermore, the application of melatonin significantly reinforced stem strength by enhancing lignin production [78]. In the process of preserving horticultural crops postharvest, melatonin can also regulate lignin production to modify senescence, freshness, and disease resistance. Postharvest blueberries exposed to melatonin treatment showed stimulated accumulation of lignin in the fruits and increased disease resistance [79]. However, melatonin has the opposite effect on lignification manipulation during the postharvest storage of asparagus. In postharvest asparagus, the application of melatonin relieved the senescence process by restricting PAL and PER activities and decreasing lignin production, thereby retarding the increase in firmness [80].

5.1.9. Other Bioactive Regulators

Kiwifruit is prone to lignification induced by chilling stress, and this process can be alleviated by hydrogen-rich water [81]. A chitosan coating suppressed postharvest juice sac granulation and lignification in pummelo fruit by inhibiting the activities and transcript levels of lignin synthesis genes [82]. Cucumber seeds primed with exogenous chitosan induced enhanced resistance against cucumber powdery mildew disease in cucumber seedlings, which was well correlated with lignin production [83]. Exogenous γ-aminobutyric acid promoted the expression of lignin-related genes and increased the accumulation of lignin, thereby conferring on apple seedlings resistance to long-term drought stress [84]. Glucose acts not only as a structural substance but also a signaling molecule, playing an important regulatory role in plant growth and development. The application of exogenous glucose markedly improved the accumulation of lignin in pear calli [85]. Exogenous silicon reinforced stem strength by enhancing lignin accumulation, particularly G lignin and S lignin, and secondary cell wall thickness in herbaceous peony [86].

5.2. Hormonal Stimuli

5.2.1. Gibberellin

It is reported that gibberellin can greatly impact lignification in horticultural crops by regulating lignin accumulation and secondary growth. Gibberellin can induce lignin accumulation and enlarged secondary xylem in carrot root, accompanied by increased transcript levels of lignin-related genes [87,88]. Similar results were achieved for sweet potato roots exposed to gibberellin [89]. In table grape, gibberellic acid promotes the expression of the lignin-related genes and results in alterations in cell wall composition and pedicel structure, leading to an increase in berry drop [90]. However, gibberellin was demonstrated to have an opposite effect on lignification and secondary growth by directly regulating the activity of a basic peroxidase isoenzyme, an enzyme involved in lignin biosynthesis [91]. These results indicate that gibberellin may have a different impact on lignin accumulation depending on the plant species, organ type, and developmental stage.

5.2.2. Brassinosteroid

In garlic, brassinosteroid-mediated lignin accumulation may be largely responsible for adaption to salt stress [92]. BR triggered the transcription of PAL, 4CL, and CAD genes and lignin-related enzyme activities and activated the synthesis of lignin precursors, thereby accelerating the wound healing of potato tubers [93]. The expression of PAL, 4CL, CCR, and CCoAOMT was significantly increased after exogenous brassinosteroid application in watermelon. Furthermore, compared with Zn stress alone, brassinosteroid treatment evidently enhanced the activities of PAL, 4CL, POD, and lignin content, indicating a potential role of brassinosteroid-mediated lignin accumulation in heavy metal tolerance [94]. Similarly, 24-epibrassinolide treatment alleviated the adverse effects induced by salinity stress and Phytophthora melonis damping-off in cucumber seedlings by influencing cell wall reassembly and lignin-related genes [95,96]. Brassinosteroid is also involved in methane-mediated adventitious root development in marigold, altering the levels of lignin and other cell wall components [97]. Therefore, it can be concluded that the role of brassinosteroid in abiotic and biotic stress resistance may be related to its participation in lignin metabolism.

5.2.3. Ethylene

The application of ethephon, an ethylene-releasing compound, increased lignin content and delayed stem bending of snapdragon cultivars, whereas plants exposed to silver thiosulfate (an ethylene action inhibitor) exhibited a higher bending rate compared with the control [98,99]. Fruits of Fragaria chiloensis subjected to ethephon at the large green developmental stage displayed stimulated lignin biosynthesis and increased mRNA abundance of FcPOD27 [100]. Exogenous ethylene resulted in the accumulation of cellulose and lignin in tomato, indicating that ethylene treatment also brought about changes in cell wall composition [101]. Ethylene significantly stimulated the expression of lignin-related genes and strengthened lignification in common beans during storage, whereas 1-methylcyclopropene, an ethylene inhibitor, had the opposite effect [102]. By contrast, 1-methylcyclopropene treatment accelerated lignin accumulation and delayed cellulose degradation during papaya fruit ripening [103].

5.2.4. Auxin

Exogenous treatment with 200 μM NAA, a synthetic auxin, decreased lignin accumulation in pear fruit stone cells and also dramatically reduced the transcript level of PbrNSC for lignin and cellulose biosynthesis [104]. In cucumber, high auxin levels resulting from amino acid permease 2 mutation accumulated in the roots, accompanied by increased concentrations of lignin [105]. Auxin-like 2,4-dichlorophenoxyacetic acid can slow senescence in postharvest citrus by modifying lignin-biosynthesis-related genes and lignin contents in fruit peels [106]. Similar results were also observed in pummel exposed to indole-3-acetic acid (IAA) [107].

5.2.5. Other Hormones

Increased abscisic acid (ABA) and jasmonic acid (JA) levels induced by water stress positively regulated the expression levels and activity of CmCAD and lignin accumulation in melon stems [49]. ABA treatment triggered CgMYB58 expression and regulated the transcription of downstream lignin-related genes, affecting juice sac granulation and lignin accumulation in pummel [107]. Lignin concentrations in kiwifruit plants treated with methyl jasmonate (MJ) or salicylic acid (SA) were reduced by at least 20% as compared to the control group [108]. Loquat fruits are vulnerable to chilling stress, flesh firmness, and lignin accumulation resulting from postharvest storage at low temperatures. Pretreatment with MeJA can relieve this low-temperature-induced lignification [109]. By contrast, exogenous MeJA can enhance the activity of PAL, 4CL, CAD, and PER enzymes and upregulate the expression of related genes and lignin accumulation to inhibit fungal decay in kiwifruit [110].

6. Lignin Regulation at the Molecular Level

6.1. Different Layers Contributing to Lignin Accumulation

6.1.1. mRNA and Abundance Alterations Identified by Transcriptome

Transcriptome remains a common method of identifying genes and their expression patterns according to different conditions and requirements. Based on the RNA seq data generated from collected pulp samples, a co-expression network of structural genes and MYB, NAC, and WRKY family transcription factors for lignin synthesis in pear was constructed based on weighted gene co-expression network analysis (WGCNA) [111]. In response to citrus blight stress, genes involved in lignin biosynthesis were upregulated, leading to the hypothesis that citrus blight may lead to root lignification [112]. With the aid of de novo transcriptome sequencing, 66 generated unigenes and 10 candidate genes were found to be involved in lignin biosynthesis in radish [113]. Differential expression of lignin-related genes was observed in cucumber inbred lines differing in downy mildew resistance, leading to altered lignin accumulation in the two lines [114]. Shading treatments induced reduced lignin accumulation in asparagus, and 37 differentially expressed genes were found to be related to lignin metabolism via transcriptome profiling [115].

6.1.2. MicroRNA (miRNA)

miRNAs are a type of non-coding RNA with regulatory functions in various plant processes, being approximately 20–25 nucleotides in length. Blocking IbmiR319a in transgenic sweet potato plants resulted in increased brittleness and reduced lignin content [116]. Transient overexpression of miR7125 or repression of its target, CCR, in apple fruit decreased lignin deposition under light-induced conditions [117]. In chickpea, overexpressing CamiR397 decreased the transcript levels of its targets, LAC4 and LAC17L and lignin accumulation in the root xylem and reduced xylem wall thickness [118]. A similar role of miR397 was observed during stone cell development in pear fruit [119]. With small RNA sequencing, Bm-miR172c-5p from Bacopa monnieri was found to cleave the F5H gene involved in the lignin biosynthesis pathway; furthermore, transgenic plants harboring Bm-miR172c-5p showed suppressed F5H expression and reduced lignification [120]. With the extension of Cd or alkali treatment, the lignin content in potato roots gradually increased; miR4243-x and the novel miRNA novel-m3483-5p were demonstrated to interfere with shikimate O-hydroxycinnamoyltransferase and cinnamic alcohol dehydrogenase (CAD), respectively, in the lignin biosynthesis pathway [121,122]. In summary, most recent studies discovered that miRNA mainly acts on structural genes in lignin synthesis, but there are a few reports on lignin-related regulatory factors controlled by miRNA.

6.1.3. Protein

Proteomics, as a key technology for exploring various life changes, has become one of the core components of life science research in the post-genomic era due to its ability to explain the molecular mechanisms of various physiological processes at the protein level through dynamic protein expression differences. Key enzymes involved in the lignin pathway, such as PAL, POD, 4CL, and CCoAMT1, were significantly induced in BcGs1-triggered disease resistance in tomato leaves [123]. The regulatory network for the biosynthesis of lignin, a principal component of stone cells, was reconstructed with generated pear proteome data [124]. The differential resistance to the parasitic weed Orobanche cumana in sunflower cultivars is partly attributed to altered defense-related proteins involved in the biosynthesis of lignin [125]. With the isobaric tag for relative and absolute quantitation (iTRAQ), PER47, LAC4, and β-glucosidase 15 (BGLU15) were considered to ultimately affect lignin biosynthesis and stone cell formation in pear pollinated with different varieties of pollen [126]. In the presence of UV-B stress or Botrytis cinerea infection, most of the proteins involved in the lignin pathway were substantially increased in Morus alba, indicating their significant role in the responses to abiotic and biotic stress [127].

6.1.4. Metabolic

Metabolomics is a method that allows for studying the internal metabolites and their changes in organisms. Its main research subjects include lipids, sugars, alkaloids, flavonoids, amino acids, and other substances. Qualitative and quantitative analysis of metabolites in organisms can reveal the types, quantities, and alterations of metabolites under specific conditions. In peach, lignin composition and accumulation affect fruit texture and quality, and metabolite and transcriptome analysis revealed that peach cultivars with different levels of lignin accumulation may be attributed to differential expression of Pp4CL2, Pp4CL3, and PpCOMT2 [128]. Many metabolites derived from the phenylpropanoid pathway contributing to cell wall formation and the lignin production of stone cells were observed near pear fruit core [129]. As a result of Cu exposure, Citrus grandis leaves accumulated more lignin-related metabolites to cope with the stress induced by Cu [130].

6.2. Molecular Modules and Networks within and beyond NAC-MYB Layers

To date, most molecular modules and networks for lignin regulation have been found to be coupled with NAC-MYB layers. That is, MYB transcription factors directly bind to the AC elements in the promoters of lignin biosynthesis genes, with NAC transcription factors upstream of MYBs. For instance, CsMYB15 directly interacted with the Cs4CL2 promoter and induced its transcription, thereby leading to high lignin accumulation in citrus [131]. In recent years, with the deepening of research on lignin regulation, it has been found that there are other mechanisms that regulate lignin metabolism. EjERF39 could directly bind to the DRE element in the promoter of Ej4CL and activate its expression level, ultimately promoting low-temperature-induced lignification in postharvest loquat fruit [132]. Grapevine VlbZIP30 directly interacted with the G-box cis-element in the promoter of the lignin biosynthetic gene VvPRX N1 to regulate lignin deposition [133].

7. Application Fields

7.1. Abiotic Resistance

Abiotic stresses refer to the negative impact of abiotic factors on organisms in a specific environment, including physical and chemical factors in the environment, such as drought, salt stress, and extreme temperature. With the deterioration of environmental conditions, abiotic stresses pose important challenges to the growth and development of horticultural crops, as well as their yield and quality. Some research has found that the modulation of lignin levels and deposition can effectively cope with or adapt to abiotic stresses. Overexpression of a caffeoyl-CoA O-methyltransferase gene, CCoAOMT, from Paeonia ostia resulted in markedly higher lignin levels and conferred drought stress tolerance on transgenic plants [134]. Transgenic plants hosting AgNAC1, an NAC transcription factor from celery, modified lignin contents and improved salt tolerance [135]. MdMYB46 could induce lignin deposition and confer salt and osmotic stress tolerance on apple by directly binding to the promoter of genes involved in lignin biosynthesis and stress signal transduction pathways [16]. Increased monolignol contents and/or increased S/G ratios were determined in transgenic sweet potato plants hosting the IbCAD1 gene and might enhance the antioxidation capacity against low-temperature stress and pathogen attack [136].

7.2. Biotic Resistance

Lignin accumulation is a useful and reliable defense indicator for biotic resistance, and some outstanding attempts to study it have been made in horticultural crops via lignin engineering. Overexpression of an MYB transcription factor, CmMYB19, from chrysanthemum enhanced lignin accumulation and impeded the reproduction of aphids on the host [137]. LrNAC35, an NAC member from Lilium regale, could directly activate the expression of lignin-related genes in petunia (Petunia hybrida) and contributed to reduced susceptibility to cucumber mosaic virus and tobacco mosaic virus attack by increasing lignin deposition in the cell walls [138]. Inhibition of microRNA397b in Malus hupehensis promoted MhLAC7 expression and resulted in elevated lignin accumulation, conferring increased tolerance to Botryosphaeria dothidea infection [139].

7.3. Fruit Development and Ripening

Lignin accumulation is essentially required for fruit development and can affect fruit quality formation to a great extent. Ripening Inducing Factor (FaRIF), an NAC transcription factor highly expressed in strawberry receptacles, can influence lignin deposition and accumulation to regulate fruit ripening and quality formation [140]. PpMYB36, encoding a pear MYB transcription factor, was demonstrated to contribute to lignin accumulation and russet coloration in pear fruit [141]. PbrSAUR13, a member of the small auxin-up RNA gene family in Pyrus bretschneideri, enhanced lignin synthesis and stone cell accumulation in the fruit, whereas PbrSAUR52 had the opposite effect [142]. CsMYB330 can recognize and bind AC elements in the Cs4CL1 promoter to positively regulate fruit juice sac lignification, whereas CsMYB308 might be a transcriptional repressor [143].

7.4. Lodging Resistance and Stem Strength

Stem lodging is a critical problem adversely influencing crop productivity throughout the world. It involves stem breaking and bending, a state in which the plant cannot return to its vertical stance anymore. It is widely recognized that stem lodging interferes with crop photosynthesis and ultimately impacts yield and quality formation. It has been demonstrated that lignin deposition enhances plant cell walls, provides mechanical strength, and is closely related to resistance to lodging. The role of lignin in stem strength and lodging resistance was extensively investigated in herbaceous peony [144,145]. Ca treatment could increase lignin accumulation and reinforce the mechanical strength in peony stems, whereas ethyl glycol tetraacetic acid (EGTA), an effective Ca²⁺ chelator, had a negative role [146,147]. PlMYB83 could directly activate the promoter of PlMYB43, PlCOMT2, and PlLAC4 to manipulate lignin production, secondary wall thickness, and stem strength [148].

7.5. Postharvest Storage Regulation

Over the period of postharvest storage, horticultural products are usually prone to low-temperature-induced stress, ultimately resulting in morphological changes, lignin accumulation, and reduced quality. A feasible and effective way to protect postharvest storage products can be achieved through modulating lignin accumulation and distribution. On the one hand, lignin production can be decreased to maintain fruit quality when exogenous active substances are applied. On the other hand, worsening fruit appearance and integrity resulting from oxidative stress can be alleviated by enhancing or engineering lignin synthesis. Exogenous L-cysteine and γ-aminobutyric acid could alleviate the degree of lignification and delay senescence to promote the quality of postharvest loquat fruit [149]. Tuberous roots of transgenic sweet potato lines overexpressing IbLfp, encoding a lignin-forming peroxidase, showed improved tolerance to cold storage during low-temperature storage due to enhanced lignin accumulation [150].

8. Prospects

Lignin is an important secondary metabolite and one of the main components making up plant cell walls. Lignin can provide mechanical support for horticultural crops and the ability to respond to external environmental changes. At the same time, lignin is an important dietary fiber, being widely present in horticultural crops such as vegetables and fruits, and has an important regulatory effect on human health. However, the excessive presence of lignin can affect the texture, taste, and quality of horticultural products. Therefore, it is necessary to maintain the lignin content of horticultural crops within a certain range. At present, most research on the quality of horticultural plants mainly focuses on nutritional quality indicators such as vitamins, carotenoids, and anthocyanins, and there is a lack of research on other nutrient substances. The pathway of lignin synthesis in most horticultural crops is not clear enough, and most of the mined transcription regulatory factors are based on the NAC-MYB regulatory network. There are still few reports on other regulatory factors modulating the distribution and accumulation of lignin in horticultural plants, and the functions of these lignin-related genes still need to be further verified through biotechnology, molecular technology, and biochemical technology.

Excessive lignin can affect the taste and quality of crops, while insufficient lignin can lead to poor crop resistance, reduced yield, and even death. Studies in ornamental crops and non-edible organs have shown that lignin plays an important role in plant lodging resistance, stress resistance, and disease resistance. However, for other edible horticultural crops, excessive accumulation of lignin can lead to lignification and a decreased quality of horticultural products, resulting in irreversible economic losses. Therefore, it is necessary to reasonably regulate lignin metabolism and improve the quality of horticultural crops. This can be approached from the following aspects: firstly, research on lignin strengthening in ornamental plants can be carried out to reinforce stem strength, enhancing their ability to resist against lodging, biotic and abiotic stresses, and diseases; secondly, through the regulation of lignin metabolism in specific organs by exogenous active substances, the production of horticultural products with a low lignin content can be achieved; thirdly, lignin synthesis and regulatory factors specifically expressed in horticultural products can be explored, and horticultural germplasm resources with low lignification could be created through gene editing and other technologies.