Lignin Biosynthesis-related Genes Research Articles

PbAGL7-PbNAC47-PbMYB73 complex coordinately regulates PbC3H1 and PbHCT17 to promote the lignin biosynthesis in stone cells of pear fruit.

Lignification of the cell wall in pear (Pyrus) fruit results in the formation of stone cells, which affects the texture and quality of the fruit. However, it is still unclear that how different transcription factors (TFs) work together to coordinate the synthesis and deposition of lignin. Here, we examined the transcriptome of pear varieties with different stone cell contents and found a key TF (PbAGL7) that can promote the increase of stone cell contents and secondary cell wall thicknesses. In addition, PbAGL7 can facilitate the expression level of lignin biosynthesis-related genes and accelerate the lignin biosynthesis in pear fruit and Arabidopsis. However, PbAGL7 did not directly bind to the promoters of PbC3H1 and PbHCT17 which are crucial genes involved in lignin biosynthesis. On the other hand, yeast two-hybrid (Y2H) library showed that PbNAC47 and PbMYB73 interacted with PbAGL7 in the nucleus. PbNAC47 and PbMYB73 also increased the stone cell and lignin contents, and upregulated the expressions of PbC3H1 and PbHCT17 by binding to the SNBE and AC elements, respectively. Moreover, PbNAC47 also interacted with PbMYB73 to form PbAGL7-PbNAC47-PbMYB73 complex. This complex significantly activated the expression levels of PbC3H1 and PbHCT17 and promoted lignin biosynthesis to form stone cells in pear fruit. Overall, our study provides new insights into the molecular mechanism of TFs that coordinately regulate the stone cell formation in pear fruit and extend our knowledge to understand cell wall lignification in plants.

Ti3C2Tx MXene nanosheets protect Torreya grandis against root rot disease

Root rot is a soil-borne disease primarily caused by fungi. The malady not only decrease the ability of absorbing water and nutrients, but also severely threat agricultural productivity. Recently, a new family member of two-dimensional (2D) transition metal carbide materials, MXene (Ti3C2Tx), has gained much interest as a promising approach to control fungi. However, the efficient use and mechanism of MXene in protecting plant against pathogenic fungus are still rarely reported. Here, the synthesized MXene were first characterized by the atomic force microscopy (AFM), scanning electron microscopy (SEM), dynamic light scattering (DLS), transmission electron microscopy (TEM) and X-ray photoelectron spectra (XPS). MXene application in soil obviously enhanced the root rot disease resistance of T. grandis. Soil microbial community analysis indicated that the abundance of Fusasium genus was decreased by 68.32% after MXene treatment. Further, MXene specially affected the permeability of Fusarium solani via damaging their cell membranes, thereby causing the disintegration and cell death of F. solani. In addition, MXene nanoflakes could transport into roots through T. grandis root air space, which resulted in the accumulation of lignin in roots via enhancing the expression and activities of lignin biosynthesis-related genes in T. grandis roots. Taken together, our finding pioneers comprehensive insights into the antifungal mechanism of MXene against F. solani and the efficiency use of MXene in protecting plant against pathogenic fungus, which will prompt the rapid development of nanotechnology in sustainable forestry.

The MADS-box gene EjAGL15 positively regulates lignin deposition in the flesh of loquat fruit during its storage.

Lignification of fruit flesh is a common physiological disorder that occurs during post-harvest storage, resulting in the deterioration of fruit quality. Lignin deposition in loquat fruit flesh occurs due to chilling injury or senescence, at temperatures around 0°C or 20°C, respectively. Despite extensive research on the molecular mechanisms underlying chilling-induced lignification, the key genes responsible for the lignification process during senescence in loquat fruit remain unknown. MADS-box genes, an evolutionarily conserved transcription factor family, have been suggested to play a role in regulating senescence. However, it is still unclear whether MADS-box genes can regulate the lignin deposition that arises from fruit senescence. Both senescence- and chilling-induced flesh lignification were simulated by applying temperature treatments on loquat fruits. The flesh lignin content during the storage was measured. Transcriptomic, quantitative reverse transcription PCR and correlation analysis were employed to identify key MADS-box genes that may be involved in flesh lignification. The Dual-luciferase assay was utilized to identify the potential interactions between MADS-box members and genes in phenylpropanoid pathway. The lignin content of the flesh samples treated at 20°C or 0°C increased during storage, but at different rates. Results from transcriptome analysis, quantitative reverse transcription PCR, and correlation analysis led us to identify a senescence-specific MADS-box gene, EjAGL15, which correlated positively with the variation in lignin content of loquat fruit. Luciferase assay results confirmed that EjAGL15 activated multiple lignin biosynthesis-related genes. Our findings suggest that EjAGL15 functions as a positive regulator of senescence-induced flesh lignification in loquat fruit.

HbNST1 is a positive regulator of the lignin accumulation in strawflower bracts

Strawflower (Helichrysum bracteatum) capitula have papery bracts and thus have the qualities of a naturally dried flower. The involucral bract cells have a secondary cell wall (SCW) of which a crucial component is lignin. Although the constituents of SCWs have been studied extensively in plants, little is known of the mechanism regulating SCW formation, especially lignin biosynthesis in the involucral bracts of strawflower. In this study, a homolog of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, designated HbNST1, was identified as a positive regulator of lignin biosynthesis in strawflower. The transcript level of HbNST1 was the highest in the involucral bracts. Subcellular localization analysis indicated that HbNST1 was localized to the nucleus. Overexpression of HbNST1 in Chrysanthemum indicum promoted the expression of a gene related to lignin biosynthesis, a homolog of cinnamyl alcohol dehydrogenase, designated CiCAD, suggesting that HbNST1 was associated with the accumulation of lignin in the SCW of the involucral bracts. Taken together, the results suggested that HbNST1 positively regulated lignin accumulation in the involucral bracts and mediated the expression of lignin biosynthesis-related genes in strawflower.

Cas9/gRNA-Mediated Mutations in PtrFLA40 and PtrFLA45 Reveal Redundant Roles in Modulating Wood Cell Size and SCW Synthesis in Poplar

Fasciclin-like arabinogalactan proteins (FLAs) play an important role in plant development and adaptation to the environment. However, the roles of FLAs in wood formation remain poorly understood. Here, we identified a total of 50 PtrFLA genes in poplar. They were classified into four groups: A to D, among which group A was the largest group with 28 members clustered into four branches. Most PtrFLAs of group A were dominantly expressed in developing xylem based on microarray and RT-qPCR data. The roles of PtrFLA40 and PtrFLA45 in group A were investigated via the Cas9/gRNA-induced mutation lines. Loss of PtrFLA40 and PtrFLA45 increased stem length and diameter in ptrfla40ptrfla45 double mutants, but not in ptrfla40 or ptrfla45 single mutants. Further, our findings indicated that the ptrfla40ptrfla45 mutants enlarged the cell size of xylem fibers and vessels, suggesting a negative modulation in stem xylem cell size. In addition, wood lignin content in the ptrfla40fla45 mutants was increased by nearly 9%, and the lignin biosynthesis-related genes were significantly up-regulated in the ptrfla40fla45 mutants, in agreement with the increase in wood lignin content. Overall, Cas9/gRNA-mediated mutations in PtrFLA40 and PtrFLA45 reveal redundant roles in modulating wood cell size and secondary cell wall (SCW) synthesis in poplar.

Involvement of PpMYB306 in Pichia guilliermondii-induced peach fruit resistance against Rhizopus stolonifer

Antagonistic yeasts are promising agents for inducing host resistance against pathogens in harvested fruit, but the transcriptional regulatory mechanism involved remains unclear. In our study, Pichia guilliermondii treatment increased the enzyme activities and gene expression of cinnamate 4-hydroxylase (C4H), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) and maintained a higher lignin content in peach fruit after R. stolonifer infection. Transcriptome analysis revealed a total of 17 differentially expressed genes (DEGs) encoding MYB transcription factors (TFs); among them, the gene expression of PpMYB306 was downregulated by P. guilliermondii treatment. In addition, by binding to the promoters of PpC4H, PpCAD1, and PpPOD44, PpMYB306 negatively regulated lignin biosynthesis in peach fruit. Furthermore, transient overexpression of PpMYB306 in peach fruit decreased the expression of PpC4H, PpCAD1, and PpPOD44, reducing the lignin content and thus increasing the lesion diameter associated with soft rot. Taken together, these findings suggested that P. guilliermondii treatment inhibited PpMYB306-mediated the transcriptional repression of lignin biosynthesis-related genes in peach fruit after R. stolonifer infection, which contributed to maintaining a higher lignin content, thereby improving the disease resistance of peach fruit.

Blue light exposure intensifies leaf red pigmentation and enhances oxidative stress tolerance in the ornamental bromeliad Neoregelia ‘Fireball’

Neoregelia is a genus of attractive, eye-catching foliage plants in the family Bromeliaceae. Blue light globally regulates multiple biological processes and secondary metabolism in plants. Recently, addition of blue light to the spectrum was reported to intensify pigmentation in some ornamental pot plants. Nevertheless, how blue light influences leaf red pigmentation and oxidative stress tolerance in Neoregelia is unclear. In this study, we performed RNA-seq analysis and a series of assays related to biological, physiological, and phenotypic characteristics. We found that exposure to blue light not only inhibits chlorophyll biosynthesis and accelerates the degradation of chlorophylls, but also up-regulates several flavonoid biosynthesis genes and significantly increases the accumulation of anthocyanins in Neoregelia ‘Fireball’ plants. Ultimately, this response leads to the presence of pure dark red leaves. We also found that blue light enhances oxidative stress tolerance in Neoregelia by promoting the expression of antioxidant-related genes and the accumulation of more proanthocyanidins (PAs). Our results also showed that blue light plays a positive role in lignin biosynthesis by activating the transcription of lignin biosynthesis-related genes in Neoregelia plants exposed to blue light. Furthermore, co-expression network analyses of RNA-seq data indicated that NfWRKY70 may be a key gene involved in the intensified leaf red pigmentation and enhanced oxidative tolerance caused by blue light exposure. Our findings provide novel insights into the regulatory mechanisms underlying blue light-induced leaf red pigmentation and oxidative tolerance, and offer new clues for the coordination between flavonoid and lignin biosynthesis in the plant phenylpropanoid biosynthetic pathway.

Characterization of the ABC Transporter G Subfamily in Pomegranate and Function Analysis of PgrABCG14.

ATP-binding cassette subfamily G (ABCG) proteins play important roles in plant growth and development by transporting metabolites across cell membranes. To date, the genetic characteristics and potential functions of pomegranate ABCG proteins (PgrABCGs) have remained largely unknown. In this study, we found that 47 PgrABCGs were divided into five groups according to a phylogenetic analysis; groups I, II, III, and IV members are half-size proteins, and group V members are full-size proteins. PgrABCG14, PgrABCG21, and PgrABCG47 were highly expressed in the inner seed coat but had very low expression levels in the outer seed coat, and the expression levels of these three PgrABCG genes in the inner seed coats of hard-seeded pomegranate ‘Dabenzi’ were higher than those of soft-seeded pomegranate ‘Tunisia’. In addition, the expression of these three PgrABCG genes was highly correlated with the expression of genes involved in lignin biosynthesis and hormone signaling pathways. The evolution of PgrABCG14 presents a highly similar trend to the origin and evolution of lignin biosynthesis during land plant evolution. Ectopic expression of PgrABCG14 in Arabidopsis promoted plant growth and lignin accumulation compared to wild type plants; meanwhile, the expression levels of lignin biosynthesis-related genes (CAD5, C4H, and Prx71) and cytokinin response marker genes (ARR5 and ARR15) were significantly upregulated in transgenic plants, which suggests the potential role of PgrABCG14 in promoting plant growth and lignin accumulation. Taken together, these findings not only provide insight into the characteristics and evolution of PgrABCGs, but also shed a light on the potential functions of PgrABCGs in seed hardness development.

Comparative Transcriptome Profiling Provides Insights into Plant Salt Tolerance in Watermelon (Citrullus lanatus).

Salt stress seriously reduced the yield and quality of watermelon and restricted the sustainable development of the watermelon industry. However, the molecular mechanism of watermelon in response to salt stress is still unclear. In this study, 150 mmol·L−1 NaCl was used to deal with the seedlings of salt-tolerant and salt-sensitive watermelon varieties. Physiological characteristics showed that salt stress significantly reduced the biomass of watermelon seedlings and the accumulation of K+ in roots and leaves and significantly increased the content of Na+, Cl−, and malondialdehyde (MDA). Compared with the salt-sensitive variety, the salt-tolerant variety had higher K+ accumulation, lower Cl−, Cl− accumulation, and MDA content in roots and leaves. Then, RNA-seq was performed on roots and leaves in normal culture and under 150 mmol·L−1 NaCl treatment. A total of 21,069 genes were identified by RNA-seq analysis, of which 1412 were genes encoding transcription factors (TFs). In the comparison groups of roots and leaves, 122 and 123 shared differentially expressed genes (DEGs) were obtained, respectively. Gene ontology (GO) annotation and KEGG enrichment results showed that there were many identical GO terms and KEGG pathways in roots and leaves, especially the pathways that related to sugar or energy (ATP or NADP+/NADPH). In addition, some DEGs related to salt tolerance were identified, such as plant hormone indole-3-acetic acid (IAA) and gibberellin (GA) signal transduction pathway-related genes, K+/Na+/Ca2+-related genes, lignin biosynthesis-related genes, etc. At the same time, we also identified some TFs related to salt tolerance, such as AP2-EREBP, bZIP, bHLH, MYB, NAC, OFP, TCP, and WRKY and found that these TFs had high correlation coefficients with salt tolerance-related genes, indicating that they might have a potential regulatory relationship. Interestingly, one TCP TF (Cla97C09G174040) co-exists both in roots and leaves, and it is speculated that it may be regulated by miR319 to improve the salt tolerance of watermelon.

Lignin biosynthesis regulated by CsCSE1 is required for Cucumis sativus defence to Podosphaera xanthii

Lignin is a complex phenolic compound that can enhance the stiffness, hydrophobicity, and antioxidant capacity of the cell wall; it thus provides a critical barrier against pathogen and insect invaders. Caffeoyl shikimate esterase (CSE) is a key novel enzyme involved in lignin biosynthesis that is associated with genetic improvements in lignocellulosic biomass; however, no research thus far have revealed the role of CSE in resistance to pathogenic stress. CsCSE1 (Cucsa.134370) has previously been shown to highly associated with the response of cucumber to attack by Podosphaera xanthii through RNA sequencing. Here, we detected the exactly role of CsCSE1 in the defence of cucumber to P. xanthii infection. Homologous sequence alignment revealed that CsCSE1 contains two highly conserved lyase domains (GXSXG), suggesting that CsCSE1 possesses CSE activity. Subcellular localization analysis manifested that CsCSE1 was localized to the plasma membrane and endoplasmic reticulum (ER). Functional analysis demonstrated that the transient silencing of CsCSE1 in cucumber dramatically attenuated resistance to P. xanthii, whereas overexpression of CsCSE1 in cucumber markedly increased resistance to P. xanthii. Further investigation of the abundance of lignin in transient transgenic plants revealed that CsCSE1 might actively mediate the disease resistance of cucumber by promoting lignin biosynthesis. CsCSE1 also affects the expression of its downstream lignin biosynthesis-related genes, like CsLAC, CsCOMT, CsCCR, and CsCAD. The results of this study provide targets for the genetic breeding of tolerant cucumber cultivars as well as new insights that could aid the control of plant diseases.

Analysis of lignin metabolism in water bamboo shoots during storage

Water bamboo shoots (WBS) are popular hydrophytic plant in China with a high nutritive value. However, WBS is susceptible to postharvest lignification. The firmness, lignin content, lignin composition, activity of lignin biosynthesis-related enzymes, and expression of lignin biosynthesis-related genes in WBS were determined after postharvest storage at 20 °C or 2 °C for 15 d or 63 d, respectively. The results indicated that lignin present in WBS was composed of G (vanillin and vanillic acid), H (p-hydroxybenzaldehyde and p-hydroxybenzoic acid), and S (syringaldehyde and syringic acid) monomers. During storage, the activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, and peroxidase also increased. Moreover, the upregulated gene expression levels of lignin biosynthesis-related enzymes positively correlated with increasing lignin content, which were the key contributors to the lignification of WBS during storage. We observed that low temperatures delayed lignification by repressing the expression of lignification-related genes and suppressing the activity of lignin biosynthesis-related enzymes.

Impact of melatonin application on lignification in water bamboo shoot during storage.

Melatonin, a crucial bioactive molecule, involved in several physiological processes in plants. This study investigated the effects of melatonin (MT) treatment on lignification, including firmness, lignin, lignified-enzyme activities, the expression patterns of genes encoding corresponding enzymes and transcription factors in water bamboo shoot during storage for 8days. MT treatment decreased the firmness and content of lignin. It inhibited the degradation of total phenols and ascorbic acid and delayed the lignin biosynthesis, via reducing the activities of phenylalanine ammonia-lyse cinnamyl alcohol dehydrogenase and peroxidase, as well as lignin biosynthesis-related genes expression levels. Transcription factors of ZlNAC1, ZlNAC2, ZlNAC3 and ZlNAC4 from NAC family and ZlMYB1 and ZlMYB2 from MYB family were increased in water bamboo shoot after harvest and MT-treated markedly reduced their expression. Therefore, our findings supply a fundamental understanding of MT treatment suppression of lignification and establish a foundation for further research on transcriptional regulation.

Identification and Functional Analysis of the CgNAC043 Gene Involved in Lignin Synthesis from Citrusgrandis "San Hong".

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo (Citrus grandis). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043, that is involved in the regulation of lignin biosynthesis in Citrus grandis, which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of ‘San hong’ as the material, the staining for lignin with HCl−phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46, which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043, was transformed into the mesocarp of ‘San hong’ by Agrobacterium-mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation.

GhODO1, an R2R3-type MYB transcription factor, positively regulates cotton resistance to Verticillium dahliae via the lignin biosynthesis and jasmonic acid signaling pathway

In plants, MYB transcription factors play diverse roles in growth, development, and response to abiotic and biotic stresses. However, the signaling processes of these transcription factors in defense against pathogen attacks remain largely unknown. This study isolated a novel R2R3-type MYB transcription factor GhODO1 from cotton (Gossypium hirsutum) and functionally characterized its positive role in tolerance to Verticillium dahliae. GhODO1 was induced by V. dahliae and jasmonic acid (JA) and transient expression of fused GhODO1-GFP in onion epidermal cells showed that GhODO1 protein was localized in the cell nucleus. Knockdown of GhODO1 significantly reduced the resistance of cotton to V. dahliae, whereas GhODO1 ectopic overexpression in Arabidopsis conferred enhanced resistance to V. dahliae. Lignin deposition was significantly decreased in GhODO1-silenced cotton plants after V. dahliae inoculation and mock treatment. The expression levels of genes and activities of enzymes involved in lignin biosynthesis were reduced in GhODO1-silenced cotton plants compared to the TRV:00. Yeast one-hybrid assays revealed that GhODO1 protein interacts with the promoters of lignin biosynthesis-related genes Gh4CL1 and GhCAD3, directly activates their expression, and enhances total lignin accumulation. Moreover, GhODO1 silencing compromised JA-mediated defense signaling and JA accumulation. These results show that GhODO1 is involved in cotton resistance to V. dahliae by involving the lignin biosynthesis and the JA signaling pathway.

Overexpression of pPLAIIIγ in Arabidopsis Reduced Xylem Lignification of Stem by Regulating Peroxidases.

Patatin-related phospholipases A (pPLAs) are a group of plant-specific acyl lipid hydrolases that share less homology with phospholipases than that observed in other organisms. Out of the three known subfamilies (pPLAI, pPLAII, and pPLAIII), the pPLAIII member of genes is particularly known for modifying the cell wall structure, resulting in less lignin content. Overexpression of pPLAIIIα and ginseng-derived PgpPLAIIIβ in Arabidopsis and hybrid poplar was reported to reduce the lignin content. Lignin is a complex racemic phenolic heteropolymer that forms the key structural material supporting most of the tissues in plants and plays an important role in the adaptive strategies of vascular plants. However, lignin exerts a negative impact on the utilization of plant biomass in the paper and pulp industry, forage digestibility, textile industry, and production of biofuel. Therefore, the overexpression of pPLAIIIγ in Arabidopsis was analyzed in this study. This overexpression led to the formation of dwarf plants with altered anisotropic growth and reduced lignification of the stem. Transcript levels of lignin biosynthesis-related genes, as well as lignin-specific transcription factors, decreased. Peroxidase-mediated oxidation of monolignols occurs in the final stage of lignin polymerization. Two secondary cell wall-specific peroxidases were downregulated following lowered H2O2 levels, which suggests a functional role of peroxidase in the reduction of lignification by pPLAIIIγ when overexpressed in Arabidopsis.

Transcriptomic Characterization of the Effects of Selenium on Maize Seedling Growth

Selenium (Se) is a trace mineral element in soils that can be beneficial to plants in small amounts. Although maize is among the most economically important crops, there are few reports on the effects of Se on maize seedling growth at the molecular level. In this study, the growth of maize seedlings treated with different concentrations of Na2SeO3 was investigated, and the physiological characteristics were measured. Compared with the control, a low Se concentration promoted seedling growth, whereas a high Se concentration inhibited it. To illustrate the transcriptional effects of Se on maize seedling growth, samples from control plants and those treated with low or high concentrations of Se were subjected to RNA sequencing. The differentially expressed gene (DEG) analysis revealed that there were 239 upregulated and 106 downregulated genes in the low Se treatment groups, while there were 845 upregulated and 1,686 downregulated DEGs in the high Se treatment groups. Both the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analyses showed a low concentration of the Se-stimulated expression of “DNA replication” and “glutathione (GSH) metabolism”-related genes. A high concentration of Se repressed the expression of auxin signal transduction and lignin biosynthesis-related genes. The real-time quantitative reverse transcription PCR (qRT-PCR) results showed that in the low Se treatment, “auxin signal transduction,” “DNA replication,” and lignin biosynthesis-related genes were upregulated 1.4- to 57.68-fold compared to the control, while, in the high Se concentration treatment, auxin signal transduction and lignin biosynthesis-related genes were downregulated 1.6- to 16.23-fold compared to the control. Based on these transcriptional differences and qRT-PCR validation, it was found that a low dosage of Se may promote maize seedling growth but becomes inhibitory to growth at higher concentrations. This study lays a foundation for the mechanisms underlying the effects of Se on maize seedling growth.

A Cotton Lignin Biosynthesis Gene, GhLAC4, Fine-Tuned by ghr-miR397 Modulates Plant Resistance Against Verticillium dahliae.

Plant lignin is a component of the cell wall, and plays important roles in the transport potential of water and mineral nutrition and plant defence against biotic stresses. Therefore, it is necessary to identify lignin biosynthesis-related genes and dissect their functions and underlying mechanisms. Here, we characterised a cotton LAC, GhLAC4, which participates in lignin biosynthesis and plant resistance against Verticillium dahliae. According to degradome sequencing and GUS reporter analysis, ghr-miR397 was identified to directedly cleave the GhLAC4 transcript through base complementary. GhLAC4 knockdown and ghr-miR397 overexpression significantly reduced basal lignin content compared to the control, whereas ghr-miR397 silencing significantly increased basal lignin levels. Based on staining patterns and GC/MS analysis, GhLAC4 acted in G-lignin biosynthesis. Under V. dahliae infection, we found that G-lignin content in ghr-miR397-knockdowned plants significantly increased, compared to these plants under the mock treatment, while G-lignin contents in GhLAC4-silenced plants and ghr-miR397-overexpressed plants treated with pathogen were comparable with these plants treated with mock, indicating that GhLAC4 participates in defence-induced G-lignin biosynthesis in the cell wall. Knockdown of ghr-miR397 in plants inoculated with V. dahliae promoted lignin accumulation and increased plant resistance. The overexpression of ghr-miR397 and knockdown of GhLAC4 reduced lignin content and showed higher susceptibility of plants to the fungal infection compared to the control. The extract-free stems of ghr-miR397-knockdowned plants lost significantly less weight when treated with commercial cellulase and V. dahliae secretion compared to the control, while the stems of ghr-miR397-overexpressed and GhLAC4-silenced plants showed significantly higher loss of weight. These results suggest that lignin protects plant cell walls from degradation mediated by cellulase or fungal secretions. In summary, the ghr-miR397-GhLAC4 module regulates both basal lignin and defence-induced lignin biosynthesis and increases plant resistance against infection by V. dahliae.

Identify of Fast-Growing Related Genes Especially in Height Growth by Combining QTL Analysis and Transcriptome in Salix matsudana (Koidz).

The study on the fast-growing traits of trees, mainly valued by tree height (TH) and diameter at breast height (DBH), is of great significance to promote the development of the forest industry. Quantitative trait locus (QTL) mapping based on high-density genetic maps is an efficient approach to identify genetic regions for fast-growing traits. In our study, a high-density genetic map for the F1 population was constructed. The genetic map had a total size of 5,484.07 centimorgan (cM), containing 5,956 single nucleotide polymorphisms (SNPs) based on Specific Length Amplified Fragment sequencing. Six fast-growing related stable QTL were identified on six chromosomes, and five stable QTL were identified by a principal component analysis (PCA). By combining the RNA-seq analysis for the two parents and two progenies with the qRT-PCR analysis, four candidate genes, annotated as DnaJ, 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1), Caffeic acid 3-O-methyltransferase 1 (COMT1), and Dirigent protein 6 (DIR6), that may regulate height growth were identified. Several lignin biosynthesis-related genes that may take part in height growth were detected. In addition, 21 hotspots in this population were found. The results of this study will provide an important foundation for further studies on the molecular and genetic regulation of TH and DBH.

Lignin provides mechanical support to herbaceous peony (Paeonia lactiflora Pall.) stems

Stem bending caused by mechanical failure is a major constraint for high-quality herbaceous peony (Paeonia lactiflora Pall.) cut flowers, but little is known about the underlying factors. In this study, two P. lactiflora cultivars, Xixia Yingxue (bending) and Hong Feng (upright), were used to investigate differences in stem bending. The results showed that the stem mechanical strength of Hong Feng was significantly higher than that of Xixia Yingxue, and the thickening of the secondary cell wall and the number of thickened secondary cell wall layers in Hong Feng were significantly higher than those in Xixia Yingxue. Moreover, compared with Xixia Yingxue, Hong Feng showed greater lignification of the cell wall and lignin deposition in the cell walls of the sclerenchyma, vascular bundle sheath and duct. All three types of lignin monomers were detected. The S-lignin, G-lignin, and total lignin contents and the activities of several lignin biosynthesis-related enzymes were higher in Hong Feng than in the other cultivar, and the S-lignin content was closely correlated with stem mechanical strength. In addition, 113,974 full-length isoforms with an average read length of 2106 bp were obtained from the full-length transcriptome of P. lactiflora stems, and differential expression analysis was performed based on the comparative transcriptomes of these two cultivars. Ten lignin biosynthesis-related genes, including 26 members that were closely associated with lignin content, were identified, and multiple upregulated and downregulated transcription factors were found to positively or negatively regulate lignin biosynthesis. Consequently, lignin was shown to provide mechanical support to P. lactiflora stems, providing useful information for understanding the formation of P. lactiflora stem strength.

Integrative analysis of wood biomass and developing xylem transcriptome provide insights into mechanisms of lignin biosynthesis in wood formation of Pinus massoniana

Lignin is an important renewable energy source as an excellent new battery fuel and ideal substitutes for the petrochemical industry. However, the molecular mechanism underlying lignin biosynthesis in wood formation of P. massoniana remains unexplored. Thus, an integrative analysis of wood biomass and the developing xylem transcriptome was performed to identify genes involved in lignin biosynthesis. A total of 1624 differentially expressed genes (DEGs) were identified, consisting of 797 upregulated and 827 downregulated genes (MaxG vs MinG). Additionally, 122 candidate genes and 17 DEGs were successfully annotated to the lignin biosynthesis pathway. All upregulated MYB and NAC genes were regulators of secondary cell wall formation. Moreover, the qRT-PCR analyses shown that 9 lignin biosynthesis-related genes and 7 transcription factor-encoding genes were upregulated (MaxG vs MinG), which indicated that the downregulation of lignin biosynthesis-related genes might be the possible causes of growth retardation and dwarf phenotype in some P. massoniana individuals. The identification of lignin biosynthesis-related genes can provide valuable genetic basis and resource for further researches on molecular mechanisms of lignin biosynthesis and contribute to the future investigations of bioengineering and synthetic biology to regulate lignin content in wood formation for the pulp and wood utilization industry.