2024 Volume 12 Pages 281-294
Lignin is an important secondary metabolite in horticultural crops that maintains mechanical strength and enhances the ability to respond to external environmental changes. However, lignin overaccumulation leads to lignification and reduces the taste quality and nutritional value of horticultural crops. Therefore, it is necessary to control the lignin content at a reasonable level in horticultural crops. In recent decades, the biosynthesis and regulation of lignin attracted the widespread attention of researchers, and significant progress has been made in understanding the mechanisms of lignin accumulation. In this review, we summarize the research progress on lignin biosynthesis in horticultural crops. In addition, the effects of different environmental conditions and plant hormones on lignin accumulation were also discussed. This review contributes to a better understanding of the accumulation of lignin in horticultural crops and provides new strategies to enhance the nutritional and commercial value of horticultural products.
Lignin is a primary component of plant cell walls providing structural rigidity and mechanical support in horticultural crops. Lignin forms a complex with cellulose and pectin through interlinking. The lignin-carbohydrate complex builds the plant framework and imparts mechanical strength and hardness to plant tissues, which serves as a physical barrier to enhance the resistance of cell walls against pathogen penetration and improve resistance to abiotic stress [1]. However, in many horticultural crops, overaccumulation of lignin leads to adverse effects such as poor fruit flavor, increased hardness, and bad coloration. These effects lead to a decline in general quality and cause a loss of commodity value. Therefore, understanding the biosynthesis and regulation of lignin is of significant importance in the control of lignin accumulation in horticultural crops. In this study, we reviewed the recent progress on lignin biosynthesis in horticultural crops and its regulation in response to environmental conditions and plant growth regulators. This review contributes to a better understanding of lignin accumulation and provides a basis for improving the quality of horticultural fruit.
In plants, lignin biosynthesis is closely related to sugar metabolism. Fructose 6-phosphate and glucose 6-phosphate participate in both glycolysis and the pentose phosphate pathway, which is upstream in the biosynthesis of lignin (Fig. 1). The intermediates, erythrose 4-phosphate (E-4-P) and phosphoenolpyruvate, enter the shikimate acid pathway to synthesize phenylalanine, which is a well-studied precursor of lignin [2]. In plants, there are three main precursors of lignin monomers: p-coumaroyl alcohol, coniferyl alcohol, and sinapyl alcohol, which are synthesized from phenylalanine (or tyrosine) through the phenylpropanoid pathway. According to the precursors, lignin monomers can be classified as p-hydroxyphenyl lignin (H-unit), guaiacyl lignin (G-unit), and syringyl lignin (S-unit) [1]. As shown in Figure 1, the biosynthesis of lignin undergoes deamination, hydroxylation, methylation, and redox reactions which are catalyzed by a series of enzymes, including phenylalanine ammonialyase (PAL), cinnamic acid 4‐hydroxylase (C4H), 4‐coumarate‐CoA ligase (4CL), coumarate 3‐hydroxylase (C3H), p‐coumaroyl shikimate 3′ hydroxylase (C3′H), hydroxycinnamoyl‐CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), cinnamoyl‐CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), caffeoyl CoA 3‐O‐methyltransferase (CCoAOMT), caffeate/5‐hydroxyferulate 3‐O‐methyltransferase (COMT), and ferulate 5‐hydroxylase (F5H).
2.2 Polymerization of lignin monomersNowadays, although the biosynthesis of lignin monomers has been extensively investigated in various plant species, the research on molecular mechanisms of lignin monomer polymerization is still limited. Lignin monomers are synthesized and transported to cell walls where they are oxidized by laccase (LACs) and peroxidase (PRXs) using O2 and H2O2, respectively, to form lignin monomer radicals (Fig. 1). These radicals then spontaneously couple and polymerize to form the macromolecules of lignin [3, 4]. Previous studies showed that mutations of LACs and PRXs led to a decrease in lignin content and lignin structure changes in Arabidopsis thaliana and Populus trichocarpa [5, 6]. In pomegranate, transcriptomic analysis revealed that two LAC genes, PgLAC37 and PgLAC50, were involved in lignin polymerization, and they were the key candidate genes for the formation of hard seeds [7]. Similarly, in the ornamental plant cleome, silencing the expression of ChLAC8 reduced the lignin content, while overexpression of ChLAC8 significantly increased the lignin content [8]. In addition, Tu et al. [9] found that peroxidase genes (VvPRX4, VvPRX71, and VvPRXN1) were involved in lignin polymerization and drought resistance in grapevine. In citrus leaves, overexpression of the CsPrx25 enhanced lignification, which acted as an apoplastic barrier to Xanthomonas citri subsp. citri infestation [10].
The regulatory network of lignin biosynthesis is complex and diverse, and the regulatory mechanisms of lignin biosynthesis at the transcriptional level are crucial for understanding lignin accumulation in plants. In recent years, several transcriptional factors involved in lignin biosynthesis have been identified in different plant species. NAC transcription factors that are composed of three initials: NAM in Petunia, ATAF in Arabidopsis, and CUC2 in Arabidopsis, are one of the largest families of transcriptional regulators in plants [11]. NACs regulate the lignification in plants and act as the master switch for plant secondary cell wall biosynthesis. Among the NAC family, NAC domain proteins Vascular-related NAC domain (VNDs) and NAC secondary wall thickening promoting factor/Secondary wall-associated NAC domain (NST/SND) are involved in lignin biosynthesis [12]. In Arabidopsis, NST1, NST2, NST6, SND1, VND6, and VND7 were the upstream transcription factors of lignin biosynthesis and capable of activating the entire secondary cell wall biosynthesis. Among them, SND1 and VND7 were not only involved in the biosynthesis of lignin monomers but also regulated lignin polymerization by directly binding to the SNBE element in the promoter region of the AtLAC11 gene [13]. The NAC transcription factors had conservation of gene function in plants, which have been identified in different plant species, such as Eriobotrya japonica, Pyrus pyrifolia, Oryza sativa, Pyrus bretschneideri, Medicago truncatula, and Zea mays (Table 1).
MYB transcription factors, MYB46 and MYB83, which are directly regulated by NACs transcription factor, are the second layer regulators of lignin biosynthesis [21]. MYB46 and MYB83 are central regulators for secondary wall formation and are positively associated with lignin biosynthesis by regulating lignin biosynthetic genes and downstream transcription factors [22]. The key downstream transcription factors directly regulated by MYB46 and MYB83 are mainly from the MYB transcription family (Table 1). Zhou et al. [23] reported that MYB58 and MYB63 are specific transcription activators of lignin biosynthesis. They can activate the promoters of lignin biosynthetic genes by recognizing AC elements, and overexpression of MYB58 and MYB63 caused ectopic accumulation of lignin. In citrus, CsMYB330, which is a homolog of AtMYB58/63, was reported to regulate lignin accumulation through the binding of the Cs4CL1 promoter [24]. Moreover, exogenous ABA treatment enhanced lignin content by promoting the expression of MYB58 and lignin biosynthetic genes in the citrus fruit of pummelo [25]. Different from MYB58 and MYB63, MYB transcription factors MYB3, MYB4, MYB7, MYB32, and MYB75 have been validated as inhibitors of lignin biosynthesis in Arabidopsis. Overexpression of the AtMYB4 gene in tobacco resulted in significant down-regulation of the expression of lignin biosynthetic genes (C4H, 4CL, and CAD), leading to slow plant growth [26]. Repressive MYB transcription factors have also been reported in other horticultural crops, such as CsMYB308 in citrus [24], EjMYB2 in loquat [27], and ZmMYB31 in maize [28].
In addition to NAC and MYB, other transcription factors have been reported to regulate the expression of lignin biosynthetic genes. Zeng et al. [33] reported that EjAP2 interacted with EjMYB1 and EjMYB2 to inhibited chilling temperature-induced lignification in loquat. Besides, EjbHLH1 inhibited the accumulation of lignin in the pulp of loquat by negatively regulating the expression of Ej4CL1 [34]. VvWRKY2, a grapevine transcription factor, regulated lignin biosynthesis by activating the promoter of the C4H gene [35]. In Chinese White Pear, PbBZR1 repressed the promoters of lignin biosynthetic genes, PbCOMT3 and PbHCT6 [36].
Type | Plant species | Transcription factor | Regulation mechanism | References |
---|---|---|---|---|
NAC | Eriobotrya japonica | EjNAC1 | Activates the EjPAL and 4CL promoters, regulates fruit lignification | [14] |
EjNAC3, EjNAC4 | Activates the EjCAD-like promoter, regulates chilling temperature-induced lignification | [15] | ||
Pyrus bretschneideri | PbrNSC | Regulates the expression of Pbr4CL and PbrLAC4, induces lignin biosynthesis in stone cells. | [16] | |
Pyrus pyrifolia | PpNAC187 | Regulates the transcripts of PpCCR and PpCOMT, induces fruit hardening | [17] | |
Punica granatum | PgSND1 | Combines with promoters of PgPAL, Pg4CL, PgF5H, PgCCR and PgCAD | [18] | |
Zea mays | ZmNST3, ZmNST4 | Regulates the transcripts of ZmMYB109/128/149, induces secondary wall deposition | [19] | |
Oryza sativa | OsSWN1 | Regulates the transcripts of the OsLAC gene involved in lignin biosynthesis | [20] | |
MYB | Citrus reticulata | CsMYB330 | Promotes the expression of Cs4CL1, regulates juice sacs granulation | [24] |
CsMYB308 | Inhibits the expression of Cs4CL1, regulates juice sacs granulation | [24] | ||
CsMYB85 | Combines with the promoter of CsMYB330 | [29] | ||
Citrus maxima | CgMYB58 | Activates promoters of CgPAL, Cg4CL, and CgC3H, regulates juice sacs granulation | [25] | |
Eriobotrya japonica | EjMYB1 | Regulates the expression of Ej4CL, regulates pulp lignification | [27] | |
EjMYB2 | Represses promoters of Ej4CL, regulates pulp lignification | [27] | ||
EjMYB8 | Activates promoters of EjPAL and Ej4CL, regulates pulp granulation | [30] | ||
Pyrus bretschneideri | PbrMYB169 | Regulates the expression of lignin biosynthetic genes (4CL, C3H, HCT, CCR, CCOMT, CAD, and LAC) | [31] | |
Malus pumila | MdMYB93 | Regulates the expression of MdPAL, Md4CL, and MdCAD, induces lignin accumulation in the peel | [32] | |
Zea mays | ZmMYB31 | Inhibits the expression of ZmCOMT, combines with the promoter of ZmF5H | [28] | |
Others | Eriobotrya japonica | EjAP2 | Interacts with EjMYB1 and EjMYB2, inhibits pulp lignification | [33] |
EjbHLH1 | Inhibits the expression of Ej4CL1 | [34] | ||
Vitis vinifera | VvWRKY2 | Combines with the promoter of VvC4H | [35] | |
Pyrus bretschneideri | PbBZR1 | Represses the promoters of PbCOMT3 and PbHCT6, inhibits lignin accumulation | [36] |
The color is one of the most important indexes associated with the quality of appearance in horticultural crops. Previous studies found that the accumulation of lignin causes the changes of color in a variety of fruit species [37]. In pear, the overaccumulation of lignin resulted in russet coloration in the exocarp of fruit, which affected consumer preferences. In ‘Xiusu’ pear (russet), lignin content and the expression of lignin biosynthetic genes in the exocarp are significantly increased compared to that in the ‘Dangshansuli’ pear (green) [38]. In addition, bagging treatments could reduce the russet coloration in the exocarp in semi-russet pear. After bagging the pear fruit, the enzyme activities of PAL, C4H, 4CL, CAD, and PRX involved in lignin biosynthesis were inhibited [39]. In ‘Golden Delicious’ apple, transcriptomic analysis revealed that bagging treatment down-regulated the expression of four lignin biosynthetic genes (HCT, CAD, and two PRX) and inhibited russet formation. Moreover, the transcription factor MdLIM11 that repressed PAL gene expression, was involved in the inhibition of lignin biosynthesis and russet coloration in the exocarp [40].
In addition, lignin is closely associated with the accumulation of carotenoids. In ‘Harumi’ mandarin fruit, the juice sacs of large fruit that accumulated high level of lignin were in pale yellow color. The carotenoids contents in the juice sacs of large fruit were significantly lower than that in the small fruit, in which lower lignin was accumulated [41]. Shi et al. [25] found that exogenous application of ABA up-regulated the expression of lignin biosynthetic genes and enhanced lignin content in the juice sacs, which led to the fading of orange color in the citrus juice sacs. A similar phenomenon was also observed in the carrot, in which the increase of lignin content led to reduce carotenoid accumulation [42]. To date, however, the molecular mechanisms on crosstalk between lignin and carotenoid biosynthesis still remain unclarified in horticultural crops.
3.2 TextureThe cell wall is a key determinant of the texture in horticultural crops, and its properties influence the mechanical deformation and disruption of plant tissues during the chewing process. Lignin interacts with other cell wall components (cellulose and pectin), and recent research on the developmental mechanisms of lignified cells at the single-cell level revealed that lignin and cellulose progressively filled lignified cells during cell development [43]. The overaccumulation of lignin increases the mechanical strength and hardness of cell walls, which led to lignification in fruit. In citrus fruit, the rapid increase of lignin content led to occurrence of granulation in the juice sacs of orange, pumelo, and mandarin [44], and as a result the juice sacs became hardening, colorless, and flavor loss. In addition, lignin is the main component of stone cells, and it substantially influence pear fruit texture. In pear, the number of stone cells in the pulp was positively correlated with lignin content [45]. In addition, lignin content was significantly associated with flesh firmness in loquat, and the increase in lignin contents led to the postharvest deterioration of texture in loquat [43].
3.3 SugarIn plants, lignin biosynthesis is closely related to sugar metabolism. Glucose serves as the carbon source for lignin biosynthesis. Acerbo et al. [46] reported that D-glucose labeled with C14 was infused into a spruce twig, and radioactive lignin was detected in the cambium, suggesting that D-glucose was the primary organic source of lignin. In citrus, the increase of lignin in large fruit during the granulation process was accompanied by the reduction of fructose and glucose [41]. In peach fruit, sugar accumulation, particularly glucose, directly influences lignin biosynthesis and lignin deposition occurs after the peak of sugar accumulation [47]. To date, the effect of lignin on sugar metabolism has been investigated only in a few horticultural crops, and the molecular mechanisms of sugar consumption during lignin biosynthesis are not yet completely understood.
3.4 Postharvest diseasesLignin accumulation is recognized as the first line of defense against biotic infection because it enhances the mechanical strength of plant cell walls or forms suberization tissues. The accumulation of lignin limits the entry of pathogens and toxins into the plant and preventing the movement of nutrients from the host to the invader [48]. The expression of lignin biosynthetic genes and the rate of lignin accumulation in soft rot-resistant mutant sr cabbage were significantly higher than that in the wild type after pectobacterium carotovorum ssp. carotovorum (Pcc) infiltration, suggesting that lignin accumulation plays a favorable role in resistance to Pcc [49]. Lignification of fruits, such as pear, apple, and kiwifruit, results from plant resistance against diseases caused by Alternaria alternata and Botryosphaeria dothidea [50]. Moreover, enhanced accumulation of lignin in kiwifruit stems induces resistance to canker disease [51]. At the transcriptional level, Zhu et al. [52] found that GhODO1, a R2R3-type MYB transcription factor, positively regulated the resistance to Verticillium dahliae in cotton by activating lignin biosynthesis and the jasmonic acid signaling pathway. In addition, MYB44 regulated lignin biosynthesis and improved the resistance to fungi in pear [50].
Various methods have been recently investigated to improve disease resistance in horticultural crops to increase lignin accumulation. In cherry tomato fruit, melatonin treatment effectively induced lignification and improved postharvest fruit resistance to gray mold [53]. In pummelo fruit, postharvest treatment of carvacrol enhanced lignin content, delayed cell wall degradation, and conferred disease resistance to Diaporthe citri [54]. Wang et al. [55] reported that preharvest spraying of sodium nitroprusside significantly improved wound healing in postharvest reticulated melons, and lignin content and the activities of enzymes involved in lignin biosynthesis were increased. In addition, methionine, carbon monoxide, and arginine were also found to be effective in activating lignin biosynthesis and enhancing the disease resistance of postharvest jujube fruit against black spot rot and Alternaria rot [56].
3.5 Physiological disordersPlant physiological disorders that are caused by non-pathological conditions, such as light, weather, water, and nutrients, affect the functioning of plant systems. In horticultural crops, lignin metabolism can cause or respond to physiological disorders. Previous studies showed that overaccumulation of lignin often occurred in citrus juice sacs, which led to the physiological disorders of a ‘gritty’ texture and dryness of the juice sacs. In addition, the rapid accumulation of lignin depletes nutrients in the juice sacs and disrupts physiological metabolism in the fruit [44]. Prolonged treatment of papaya fruit with 1-methylcyclopropene (1-MCP) led to a flesh softening disorder known as ‘rubbery’. The flesh cannot soften normally and exhibits an elasticity similar to rubber. This phenomenon is associated with elevated lignin and the content of cellulose in the flesh [57]. In addition, lignification is sensitive to low temperatures in many fruits, such as loquat, pear, mangosteen, zucchini, and kiwifruit [50, 58]. Improperly low-temperature treatment led to quality deterioration, increased lignin content and cell wall strength, and severely restricted storage life of fruit.
In addition to the transcriptional regulation of lignin biosynthesis, lignin accumulation in horticultural crops is also deeply influenced by environmental conditions, storage methods, biologics, and plant hormones. According to the existing reports, several pre- and postharvest factors have been shown to affect the change of lignin accumulation (Fig. 2). Different methods can significantly change lignin accumulation patterns and improve the storage quality of horticultural crops.
In general, low temperature is the main factor that induces fruit lignification. Cai et al. [58] reported that loquat was a cold-sensitive fruit, and prone to chilling injury during cold storage. Low temperature treatment up-regulated the expression of lignin biosynthetic genes in loquat fruit, and as a result, lignin was overaccumulation and the juiciness of the flesh was reduced. In citrus fruit, the lignification in navel oranges was mainly caused by low temperatures in winter. CsMYB15 binds to the promoter of lignin biosynthetic gene Cs4CL2, was activated by cold stress, and ultimately led to lignification of the juice sacs in navel oranges [59]. In contrast to low temperatures, heat treatment can inhibit lignin accumulation and improve fruit quality. Postharvest heat treatment significantly suppressed the expression of lignin biosynthetic gene EjNAC1 and inhibited lignification in loquat fruit after low-temperature storage [60]. Zhang et al. [61] found that spraying with 42 °C hot water effectively suppressed the activities of enzymes PAL, C4H, and POD and reduced the lignin content in the juice sacs of ‘Guanximiyou’.
4.1.2 Storage atmosphere controlControlled atmosphere (CA) and modified atmosphere (MA) are two effective postharvest technologies that are used to control the gas composition of the storage environment in agriculture. The postharvest storage atmosphere control reduces the degree of lignification and improves quality in horticultural crops. In the flesh of cherimoya fruit, a high CO2 atmosphere (20%) inhibited the accumulation of lignin and enhanced the activity of the PAL enzyme at chilling temperature [62]. MA packaging (2% O2, 5% CO2, and 93% N2) significantly reduced the lignification of bamboo shoots by suppressing the formation of cellulose and lignin [63]. In green asparagus, it was found that 60% and 80% high-O2 MA packaging could effectively slow the rate of increase in lignin levels during the storage time, which kept the tender texture of asparagus spears [64].
4.2 Biochemical regulation 4.2.1 TemperatureIn recent years, increasing novel biologics have been applied in horticultural crops during postharvest storage. Among these, chitosan, which can regulate lignin biosynthetic enzyme activity, has been widely used to maintain postharvest fruit quality. Chitosan, derived mainly from plants, animals, and microorganisms, is the only alkaline polysaccharide among natural polysaccharides. Preharvest spraying of chitosan (0.1%) increased the activities of key enzymes related to lignin biosynthesis and lignin monomer levels in melon wounds. In addition, the spraying of chitosan increased POD activity and H2O2 content, which led the lignin monomers to polymerize and lignin deposited on the wounds [65]. In addition to chitosan, other novel biologics, such as oligochitosan, hinokitiol, epsilon-polylysine, and methyl thujate, were also effective to induce lignin accumulation and inhibit spore germination, germ tube elongation, and mycelial growth of fungal phytopathogens in horticultural crops [66, 67].
4.2.2 Plant hormonesPlant hormones play a crucial role in regulating lignin accumulation in horticultural crops. Nowadays, plant hormones, such as ethylene and gibberellin (GA), have been widely used in preharvest and postharvest to manipulate lignin accumulation in fruit and vegetables. Ethylene is a key factor regulating lignin accumulation during the maturation of horticultural crops. A current report showed that ethylene treatment inhibited anthocyanin biosynthesis but increased lignin content in strawberry at the immature stage [68]. During postharvest storage, ethylene treatment significantly promoted the accumulation of lignin and PAL enzyme activity in the pulp of loquat. In contrast, 1-MCP, an inhibitor of ethylene reception, reduced lignin content and PAL enzyme activity in loquat pulp [69]. In addition, Kumarihami et al. [70] found that the preharvest dipping treatment of chitosan affected endogenous ethylene biosynthesis and lignin biosynthetic genes, which was effective in maintaining the firmness and prolonging the shelf life of kiwifruit during postharvest storage.
GAs have been reported to have an important effect on lignification in horticultural crops. Wang et al. [71] found that GA treatment induced lignin accumulation in carrot roots by up-regulating the expression of lignin biosynthetic genes. However, GA was found to have negative effects on the lignification in Zinnia elegans by directly regulating the activity of basic peroxidase isoenzyme [72]. A similar phenomenon was also observed in citrus and pears. The postharvest treatment of GA significantly inhibited the accumulation of lignin and suppressed granulation in the juice sacs of citrus fruit [73]. GA application down-regulated the expression level of lignin biosynthetic genes and reduced the accumulation of sclereid in the pulp of pear during ripening [74]. These results indicate that GA may have different effects on lignin accumulation depending on the plant species, organ type, and developmental stage.
In addition to ethylene and GA, other plant hormones have been found to regulate lignin accumulation in horticultural crops. Cao et al. [75] and Xu et al. [76] reported that plant hormones, MeJA and auxin, effectively inhibited lignin accumulation in loquat and pear. The MeJA-treated loquat fruit reduced fruit hardness and internal browning after chilling temperature storage, and PAL and CAD enzyme activities were significantly lower in the MeJA-treated loquat fruit than those of untreated fruit. In pear, exogenous treatment of NAA, a synthetic auxin, reduced lignin accumulation in stone cells and significantly decreased the expression of the lignin biosynthetic gene PbrNSC. Strigolactone and ABA, which are two plant hormones derived from the cleavage of carotenoids, are the positive regulators of lignin accumulation in horticultural crops. In strawberry fruit, exogenous strigolactone significantly increased the activity of lignin biosynthetic enzymes and promoted the accumulation of antioxidants and lignin [77]. In pummelo, exogenous ABA treatment enhanced lignin content by promoting the expression levels of MYB58 and lignin biosynthetic genes, PAL1, PAL2, 4CL1, and C3H [25].
lignin is an important index that associated with the quality of horticultural crops. Overaccumulation of lignin impairs the texture and taste, and causes dramatic decline in the quality of horticultural crops. In contrast, insufficient accumulation of lignin leads to poor stress resistance to stresses and causes abnormal phenotypes even plant death. Therefore, it is necessary to maintain the level of lignin within an optimum range in horticultural crops. To date, although multiple genes involved in lignin biosynthesis have been reported, there are still many gaps in our understanding of the transcriptional regulation of lignin accumulation in horticultural crops. For example, the molecular mechanisms by which lignin monomers polymerize into lignin macromolecules are not clear, and it is not understood whether other flavonoid substances are involved in the polymerization process. In addition, most of the identified transcription factors regulating lignin accumulation were based on the NAC-MYB regulatory network. There are few reports on other transcription factors regulating lignin accumulation in horticultural crops, and research on constructing multi-gene regulatory networks and other gene interaction regulatory networks is limited. The functions of most lignin biosynthetic genes still need to be further verified through biotechnology, molecular technology, and biochemical techniques. Therefore, in future research, isolating new genes related to lignin biosynthesis and modification, and exploring the key regulatory factors of lignin accumulation are of great significance in horticultural crops. These will not only contribute to a new understanding of lignin biosynthesis, but also provide effective and precise strategies for controlling lignin accumulation in horticultural crops.