Lignin Monomer Synthesis Research Articles

MYB168 and WRKY20 transcription factors synergistically regulate lignin monomer synthesis during potato tuber wound healing.

Lignin is a critical component of the closing layer of the potato (Solanum tuberosum L.) tuber during healing; however, the molecular mechanism of its formation remains poorly understood. To elucidate the molecular mechanism of tuber healing, we screened the genes encoding transcription factors that regulate lignin synthesis(StMYB24/49/105/144/168, StWRKY19/20/22/23/34) and the key genes involved in lignin monomer synthesis (PHENYLALANINE AMMONIA LYASE 5 (StPAL5) and CINNAMYL ALCOHOL DEHYDROGENASE 14 (StCAD14)) for induced expression after wounding using transcriptome data. DLR, Y1H, EMSA, and ChIP-qPCR assays revealed that StMYB168 could bind directly to the StPAL5 and StCAD14 promoters to activate their expression and that StWRKY20 enhanced this regulation with a synergistic effect. Y2H, BiFC, and Co-IP assays showed that StMYB168 interacted with StWRKY20 to form a MYB-WRKY complex. Furthermore, transient overexpression of StMYB168 and StWRKY20 in Nicotiana benthamiana leaves upregulated the expression of NbPAL and NbCAD10 and promoted lignin accumulation in the leaves. In addition, overexpression of StWRKY20 and StMYB168 together resulted in higher expression levels of NbPAL and NbCAD10 and higher levels of lignin monomer and total lignin. In contrast, silencing of StMYB168 and StWRKY20 in potato significantly reduced the lignin content of wounded tubers. In conclusion, StMYB168 and StWRKY20 are important regulators of lignin biosynthesis in potato tubers during healing and can positively regulate lignin biosynthesis by forming a complex. The elucidation of this regulatory module provides information on the regulatory mechanism of lignin monomer synthesis in wounded tubers at the transcriptional level.

A Preliminary Study on the Identification of Genes Involved in Lignification in the Endocarp of Bared-Nut Walnut (Juglans regia L.) in Xinjiang, China

This study focused on the “Xinlu” walnut and explored the molecular regulatory mechanism of lignin synthesis in the endocarp, aiming to explain the formation of bared-nut walnuts through morphological, metabolomic, and transcriptomic techniques. It was found that the synthesis of lignin, cellulose, p-coumaryl alcohol, and sinapyl alcohol was severely inhibited in the pulpy-hue (PUH) of the endocarp. We obtained 14 modules (gene sets) significantly correlated with the lignification factor (LIG) and 1548 hub genes. Additionally, we identified a MEplum3 module involved in endocarp lignin synthesis, primarily participating in phenylalanine biosynthesis and the lignin biosynthetic process. Meanwhile, we constructed a gene co-expression network for the MEplum3 module and identified a key hub gene for lignin synthesis—JrCAD10. Among the different tissues of “Xinlu”, the expression level of JrCAD10 in the scleritic-hue (SCH) was significantly higher than in other tissues, with a relative copy number (RCN) of 3.2. However, JrCAD10 expression was severely suppressed in the PUH. The suppression of JrCAD10 expression led to the inhibition of lignin monomer synthesis, which further resulted in inhibited lignin synthesis, thus forming the bared-nut walnut. Our findings provide new insights into understanding the regulation of lignin synthesis and offer a possible explanation for the formation of bared-nut walnuts.

Tartary Buckwheat (Fagopyrum tataricum) NAC Transcription Factors FtNAC16 Negatively Regulates of Pod Cracking and Salinity Tolerant in Arabidopsis.

The thick and hard fruit shell of Fagopyrum tataricum (F. tataricum) represents a processing bottleneck. At the same time, soil salinization is one of the main problems faced by modern agricultural production. Bioinformatic analysis indicated that the F. tataricum transcription factor FtNAC16 could regulate the hull cracking of F. tataricum, and the function of this transcription factor was verified by genetic transformation of Arabidopsis thaliana (A. thaliana). Phenotypic observations of the wild-type (WT), OE-FtNAC16, nst1/3 and nst1/3-FtNAC16 plant lines confirmed that FtNAC16 negatively regulated pod cracking by downregulating lignin synthesis. Under salt stress, several physiological indicators (POD, GSH, Pro and MDA) were measured, A. thaliana leaves were stained with NBT (Nitroblue Tetrazolium) and DAB (3,3’-diaminobenzidine), and all genes encoding enzymes in the lignin synthesis pathway were analyzed. These experiments confirmed that FtNAC16 increased plant sensitivity by reducing the lignin content or changing the proportions of the lignin monomer. The results of this study may help to elucidate the possible association between changes in lignin monomer synthesis and salt stress and may also contribute to fully understanding the effects of FtNAC16 on plant growth and development, particularly regarding fruit pod cracking and environmental adaptability. In future studies, it may be useful to obtain suitable cracking varieties and salt-tolerant crops through molecular breeding.

Drought-induced ABA, H2O2 and JA positively regulate CmCAD genes and lignin synthesis in melon stems

BackgroundCinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems.ResultsHere we studied the effects of abscisic acid (ABA), hydrogen peroxide (H2O2) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H2O2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H2O2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H2O2 are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H2O2 and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H2O2 to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS.ConclusionsCmCADs but CmCAD4 are positively regulated by ABA, H2O2 and JA under drought stress and participate in lignin synthesis.

Physiological, Biochemical, and Transcriptomic Responses of Neolamarckia cadamba to Aluminum Stress.

Aluminum is the most abundant metal of the Earth’s crust accounting for 7% of its mass, and release of toxic Al3+ in acid soils restricts plant growth. Neolamarckia cadamba, a fast-growing tree, only grows in tropical regions with acidic soils. In this study, N. cadamba was treated with high concentrations of aluminum under acidic condition (pH 4.5) to study its physiological, biochemical, and molecular response mechanisms against high aluminum stress. High aluminum concentration resulted in significant inhibition of root growth with time in N. cadamba. The concentration of Al3+ ions in the root tip increased significantly and the distribution of absorbed Al3+ was observed in the root tip after Al stress. Meanwhile, the concentration of Ca, Mg, Mn, and Fe was significantly decreased, but P concentration increased. Aluminum stress increased activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase from micrococcus lysodeiktic (CAT), and peroxidase (POD) in the root tip, while the content of MDA was decreased. Transcriptome analysis showed 37,478 differential expression genes (DEGs) and 4096 GOs terms significantly associated with treatments. The expression of genes regulating aluminum transport and abscisic acid synthesis was significantly upregulated; however, the genes involved in auxin synthesis were downregulated. Of note, the transcripts of several key enzymes affecting lignin monomer synthesis in phenylalanine pathway were upregulated. Our results shed light on the physiological and molecular mechanisms of aluminum stress tolerance in N. cadamba.

Correlation Analysis of Lignin Accumulation and Expression of Key Genes Involved in Lignin Biosynthesis of Ramie (Boehmeria nivea).

The phloem of the stem of ramie (Boehmeria nivea) is an important source of natural fiber for the textile industry. However, the lignin content in the phloem affects the quality of ramie phloem fiber. In this study, the lignin content and related key gene expression levels were analyzed in the phloem and xylem at different developmental periods. The results showed that the relative expression levels of lignin synthesis-related key genes in the xylem and phloem of the stem gradually decreased from the fast-growing period to the late maturation period, but the corresponding lignin content increased significantly. However, the relative expression levels of a few genes were the highest during the maturation period. During all three periods, the lignin content in ramie stems was positively correlated with the expression of genes, including PAL, C4H and 4CL1 in the phenylpropanoid pathway, F5H and CCoAOMT in the lignin-specific synthetic pathway, and CAD in the downstream pathway of lignin synthesis, but the lignin content was negatively correlated with the expression of genes including 4CL3 in the phenylpropanoid pathway and UDP-GT in the shunt pathway of lignin monomer synthesis. The ramie 4CL3 recombinant protein prefers cinnamic acid as a substrate during catalysis, and it negatively regulates lignin synthesis. It is speculated that ramie 4CL3 is mainly involved in the synthesis of ramie flavonoid compounds, and that 4CL1 is mainly involved in lignin synthesis.

Proteomic analysis of the key mechanism of exocarp russet pigmentation of semi-russet pear under rainwater condition

This study aimed to explore the mechanism of exocarp russet pigmentation of semi-russet ‘Cuiguan’ sand pears at the proteome level following rainwater. Exocarp russeting was characterized in semi-russet sand pears cultivated in the rain shelter and control (rainwater) groups via microscopy and ultra-microscope observations, followed by determination of lignin content and enzyme activities. The differentially expressed proteins between different cultivation conditions were analyzed using the isobaric tags for relative or absolute quantitation (iTRAQ) technology, followed by confirmation by real-time quantitative polymerase chain reaction (qPCR). The rust color of the outer pericarp of ‘cuiguan’ was rapidly accumulated on 60–80 days after full bloom (DAFB) in both two groups. Under the shelter condition, most stratum corneum cracks were shallow and had less suberization. The lignin content as well as the enzyme activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), catechol-O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR) and peroxidase (POD) in exocarp from the rain shelter group were all lower than that in control group. Additionally, 309 differentially expressed proteins were identified in the rain shelter group, and eight down-regulated proteins associated with lignin monomer synthesis were identified, such as PAL (E9JWF1), CCR1 (F4Y9E7), CAD (U3PGC0), two peroxidases (K9URQ0 and M5W3Y0, respectively), two COMT (S5TPC3 and Q09K02, respectively) and omega-hydroxypalmitate O-feruloyl transferase (HHT1) (M5VMR7), and their expression was confirmed by qPCR. Moreover, many defense proteins were significantly down-regulated. These findings suggest that the rain shelter treatment may affect phenylalanine lignin monomer synthesis and subsequent defense metabolism by regulating corresponding proteins, thus decreasing exocarp russet accumulation in semi-russet pear. This study provides a further understanding of pear russeting and plant defense mechanisms.

EjHAT1 Participates in Heat Alleviation of Loquat Fruit Lignification by Suppressing the Promoter Activity of Key Lignin Monomer Synthesis Gene EjCAD5.

Texture attributes such as firmness and lignification are important for fruit quality. Lignification has been widely studied in model plants and energy crops, but fruit lignification has rarely been investigated, despite having an adverse effect on fruit quality and consumer preference. Chilling-induced loquat fruit lignification that occurs after harvest can be alleviated by heat treatment (HT) applied prior to low temperature storage. Enzyme activity assay showed that HT treatment could retard the low temperature-induced increase in cinnamyl alcohol dehydrogenase (CAD) activity. Transcript analysis and substrate activity assays of recombinant CAD proteins highlighted the key role of EjCAD5 in chilling-induced lignin biosynthesis. A novel homeobox-leucine zipper protein ( EjHAT1) was identified as a negative regulator of EjCAD5. Therefore, the effect of HT treatment on lignification may be partially due to the suppression of the EjCAD5 promoter activity by EjHAT1.

Analysis of CmCADs and three lignifying enzymes in oriental melon (‘CaiHong7’) seedlings in response to three abiotic stresses

Abstract Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195), a key enzyme in lignin monomer synthesis pathway, is responsible for the conversion of cinnamyl aldehydes to cinnamyl alcohols. Our previous work identified five CAD genes in the melon genome and named CmCAD1 to CmCAD5, respectively. The further promoter analysis of CmCADs revealed elements involved in stress response and signaling pathway. However, what abiotic stresses the genes respond to remain unclear. Thus three abiotic stresses were conducted on oriental melon seedlings (‘CaiHong7’) as drought, salt and wound. In our study, lignin deposition was significantly promoted in stems and roots, and stem sections showed deeper staining with phloroglucinol-HCl under the three abiotic stresses than the control, indicating a higher lignin accumulation promoted by abiotic stress. Activities of CAD and peroxidase (POD; EC 1.11.1.7), as well as phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), were generally induced to significant levels by the abiotic stresses, implying that lignin synthesis pathway may be activated. CmCAD2 was significantly up-regulated in stems and roots under salt treatment but was most strongly induced in whole seedlings under drought stress. Also, CmCAD2 was significantly induced by wound treatment as well as the other CmCADs. However, the other four CmCAD genes exhibited tissue-specific inductions under drought and salt stresses. Expression analysis suggested that CmCAD2 might be a major gene in lignin deposition responding to the abiotic stresses.

Monolignol biosynthesis is associated with resistance to Sclerotinia sclerotiorum in Camelina sativa.

The ascomycete Sclerotinia sclerotiorum is a necrotrophic plant pathogen with an extremely broad host range. It causes stem rot in Camelina sativa, a crucifer with great potential as an alternative oilseed crop. Lignification is a common phenomenon in the expression of resistance against necrotrophs, but the molecular mechanisms underlying this defence response are poorly understood. We present histochemical, gene expression and biochemical data investigating the role of monolignols in the resistance of C. sativa to S. sclerotiorum. Comparative studies with resistant and susceptible lines of C. sativa revealed substantial differences in constitutive transcript levels and gene regulation patterns for members of the gene family encoding cinnamoyl-CoA reductase (CCR), the first enzyme specifically committed to the synthesis of lignin monomers. These differences were associated with anatomical and metabolic factors. While the induction of CsCCR2 expression after inoculation with S. sclerotiorum was associated with the deposition of lignin mainly derived from guaiacyl monomers, high constitutive levels of CsCCR4 paralleled a high syringyl lignin content in healthy stems of resistant plants. The results provide evidence that plant cell wall strengthening plays a role in the resistance of C. sativa to S. sclerotiorum, and that both constitutive and inducible defence mechanisms contribute to reduced symptom development in resistant germplasm. This study provides the first characterization of quantitative resistance in C. sativa to S. sclerotiorum.

Early detection of wood quality in Hevea brasiliensis (Rubber wood) using cinnamyl alcohol dehydrogenase (CAD) activity and lignification

Lignins are phenolic polymers of the cell wall and cinnamyl alcohol dehydrogenase (CAD) is the key enzyme involved in the synthesis of lignin monomers. A study was conducted in stems of 18 wild Hevea germplasm accessions and five popular clones to localize and correlate CAD activity and lignification at various stages of xylogenesis through tissue printing and in situ localization as well as quantitative image analysis techniques. Simultaneously, quantification of lignin produced was also done by classical histochemistry and gravimetric estimation. CAD activity was maximum during the early stages of stem development and minimum during the mature stage of xylogenesis whereas the pattern of lignification showed a reverse trend indicating that as xylogenesis increased, CAD activity decreased. The quantity of lignin produced was also increased in association with progress of secondary thickening. The percentage of lignin at different developmental stages of the stem was highly significant. The percentage of lignin ranged from 21.04–27.4 % in wild Hevea germplasm and 20.01–23.00 % in popular clones. Of the 18 wild accessions screened, nine showed significantly higher percentage of lignin over five popular clones. The present study revealed that CAD activity is involved in the lignification process in Hevea stem and its activity is restricted in the mature xylem. The CAD activity and lignification pattern in the juvenile growth phase can be used as reliable tools for the early detection of wood quality in Heveabrasiliensis.

Sequence analysis and functional characterization of the promoter of the Picea glauca Cinnamyl Alcohol Dehydrogenase gene in transgenic white spruce plants

The enzyme Cinnamyl Alcohol Dehydrogenase (CAD) catalyses the last step of lignin monomer synthesis, and is considered as a molecular marker of cell wall lignification in different plants species. Here, we report the isolation and analysis of 5' flanking genomic DNA regions upstream to the CAD gene, from two conifers, i.e. white spruce (Picea glauca (Moench) Voss) and loblolly pine (Pinus taeda L.). Sequence comparisons with available CAD gene promoters from angiosperms highlighted the conservation of cis-elements matching MYB, WRKY and bHLH binding sites. Functional characterization of the P. glauca CAD promoter used P. glauca seedlings stably transformed with a DNA fragment of 1,163 base pairs (PgCAD) fused to the beta-glucuronidase (GUS) gene. Histochemical observations of different vegetative organs of the transgenic trees showed that this sequence was sufficient to drive GUS expression in lignifying tissues, and more specifically in differentiating xylem cells. Quantitative RT-PCR experiments also indicated that the native CAD gene was preferentially expressed in differentiating xylem both in stems and roots. In addition, GUS expression driven by the PgCAD promoter was wound-inducible which was consistent with the accumulation of CAD mRNA in response to jasmonate application and mechanical wounding. The spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood.

Light, the circadian clock, and sugar perception in the control of lignin biosynthesis

Experiments were undertaken to investigate some of the mechanisms that may function to regulate lignin biosynthesis (lignification) in Arabidopsis thaliana. Northern blot analyses revealed that several genes encoding enzymes involved in the synthesis of lignin monomers displayed significant changes in transcript abundance over a diurnal cycle. Northern blot analysis also suggested that some of the changes in diurnal transcript abundance were likely to be attributable to circadian regulation, whereas others were likely to be attributable to light perception. Comparison of circadian changes in transcript abundance of lignin biosynthetic genes between wild-type plants and the sex1 mutant, which is impaired in starch turnover, suggested that carbon availability related to starch turnover might determine the capacity to synthesize lignins. This hypothesis was supported by the observation that the sex1 mutant accumulated fewer lignins than wild-type plants. Consistent with the relationship between carbon availability and lignin accumulation, analysis of dark-grown wild-type A. thaliana seedlings uncovered a role for sugars in the regulation of lignin biosynthesis. Analysis of lignin accumulation, as determined by qualitative changes in phloroglucinol staining, suggested that metabolizable sugars positively influence the abundance of lignins. Transcriptome analysis supports the hypothesis that sugars are not merely a source of carbon skeletons for lignification, but they also function as a signal to enhance the capacity to synthesize lignins.

Cloning and characterization of a ryegrass ( Lolium perenne) gene encoding cinnamoyl-CoA reductase (CCR)

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyzes the reduction of cinnamoyl acid-CoA esters into their corresponding aldehydes, the first step in the biosynthesis of lignin. The synthesis of lignin monomers is strictly associated with the activity of this enzyme which occurs in significant concentrations in lignin producing tissues. A 1207 bp cDNA ( LpCCR) encoding a polypeptide of 344 amino acids with a predicted molecular mass of 37.4 kDa was isolated from a ryegrass ( Lolium perenne) stem cDNA library. The identity of the LpCCR was established by comparison of the deduced polypeptide sequence with other isolated plant CCR enzymes. A motif, NWYCY, representing a putative CCR active site is conserved in the encoded LpCCR amino acid sequence. The encoded polypeptide exhibits sequence similarity to CCRs from different plants, the highest identities (87 and 86%, respectively) being to CCRs from Festuca arundicinea and Hordeum vulgare. Phylogenetic analysis shows that LpCCR and CCR from other monocotyledons form a group distinct from dicot CCRs. Genomic Southern blot hybridization demonstrated that LpCCR probably is represented as a single-copy gene in the ryegrass genome. The isolation of a single genomic CCR clone ( gLpCCR) from a ryegrass leaf library further supports this observation. The gLpCCR contains four introns, the same number found in the Eucalyptus gunnii gCCR. Computer analysis of a 1333 bp 5′-flanking region of gLpCCR suggests the presence of binding sites for a MYB transcription factor and a binding site for a P-box factor of the Dof class transcription factors. In agreement with the involvement of CCR in lignification, the ryegrass CCR mRNA was detected in stem tissue only. The isolation and characterization of gLpCCR provides tools for genetically modifying plants with reduced lignin content thereby increasing forage digestibility.

Engineering of a water-soluble plant cytochrome P450, CYP73A1, and NMR-based orientation of natural and alternate substrates in the active site.

CYP73A1 catalyzes cinnamic acid hydroxylation, a reaction essential for the synthesis of lignin monomers and most phenolic compounds in higher plants. The native CYP73A1, initially isolated from Jerusalem artichoke (Helianthus tuberosus), was engineered to simplify purification from recombinant yeast and improve solublity and stability in the absence of detergent by replacing the hydrophobic N terminus with the peptitergent amphipathic sequence PD1. Optimized expression and purification procedures yielded 4 mg engineered CYP73A1 L(-1) yeast culture. This water-soluble enzyme was suitable for 1H-nuclear magnetic resonance (NMR) investigation of substrate positioning in the active site. The metabolism and interaction with the enzyme of cinnamate and four analogs were compared by UV-visible and 1H-NMR analysis. It was shown that trans-3-thienylacrylic acid, trans-2-thienylacrylic acid, and 4-vinylbenzoic acid are good ligands and substrates, whereas trans-4-fluorocinnamate is a competitive inhibitor. Paramagnetic relaxation effects of CYP73A1-Fe(III) on the 1H-NMR spectra of cinnamate and analogs indicate that their average initial orientation in the active site is parallel to the heme. Initial orientation and distances of ring protons to the iron do not explain the selective hydroxylation of cinnamate in the 4-position or the formation of single products from the thienyl compounds. Position adjustments are thus likely to occur during the later steps of the catalytic cycle.

Lignification and cinnamyl alcohol dehydrogenase activity in developing stems of tomato and poplar: a spatial and kinetic study through tissue 2

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in the synthesis of lignin monomers. Tissue prints were used to localize CAD activity in tomato and poplar tissues. In parallel, lignin deposition was estimated by classical histochemical tests. Image analysis was also used to quantify the amount of CAD activity and the extent of lignification throughout the development of stems. Tomato stems showed a limited lignification. CAD activity, restricted to the xylem, reached a maximum during the early stages of development and showed a patched pattern on the xylem ring. In contrast, CAD activity was much stronger in poplar stems and closely associated with all lignified areas: starting with localized areas within the xylem and finally completely covering the xylem ring. The enzyme showed a significant activity in primary and secondary xylem, and both lignin deposition and CAD activity increased as the plant matured. These results show that CAD activity, which is important for lignification, can easily be detected in plant tissues by tissue prints. They also reveal that tomato (herbaceous stems) and poplar (woody stems) dramatically differ in the developmental pattern of CAD within their lignified tissues.

Specific Inhibition of Lignification in Bryonia dioica

Rubbing-induced lignification in Bryonia dioica internodes is significantly impaired by N(o-hydroxyphenyl) and N(o-aminophenyl) sulfinamoyl tertiobutyl acetate, specific inhibitors of cinnamyl alcohol dehydrogenase, an enzyme which is strictly associated with lignin monomer synthesis. Along with the reduction of lignification, these inhibitors counteract the inhibition of elongation due to rubbing. These results indicate that lignification participates in the thigmomorphogenetic growth response of Bryonia dioica internodes. In a general way, the data point to the causal role of lignification in the limitation of plant growth.