Metabolome and Transcriptome Analysis of Sulfur-Induced Kiwifruit Stem Laccase Gene Involved in Syringyl Lignin Synthesis against Bacterial Canker.

Kiwifruit canker is caused by Pseudomonas syringae pv. actinidiae and is one of the most destructive diseases of kiwifruit worldwide. Sulfur can improve the deposit of lignin in kiwifruit stems and induce disease resistance, but the action mechanism at the molecular level remains unclear. This omics-based study revealed that sulfur-induced S lignin synthesis contributes to disease resistance. Histological staining verified sulfur-enhanced total lignin deposition in kiwifruit stems. High-performance liquid chromatography and confocal Raman microscopy showed that sulfur-activated S lignin was mainly deposited in the cell corner. Metabolome and transcriptome analysis revealed that the levels of phenylpropanoid pathway S lignin precursors sinapic acid and sinapyl alcohol were significantly increased and 16 laccase genes were upregulated. Sulfur-induced resistance defense promoted elevated laccase activity by activating the laccase genes, participating in sinapic acid and sinapyl alcohol substance synthesis, and ultimately polymerizing S lignin at cell corner against kiwifruit canker disease.

Modification of lignin composition by ectopic expressing wheat TaF5H1 led to decreased salt tolerance in transgenic Arabidopsis plants.

Lignin is an important cell wall component that provides plants with mechanical support and improved tolerance to pathogen attacks. Previous studies have shown that plants rich in S-lignin content or with a higher S/G ratio always exhibit higher efficiency in the utilization of lignocellulosic biomass. Ferulate 5-hydroxylase, or coniferaldehyde 5-hydroxylase (F5H, or CAld5H), is the critical enzyme in syringyl lignin biosynthesis. Some F5Hs have been characterized in several plant species, e.g., Arabidopsis, rice, and poplar. However, information about F5Hs in wheat remains unclear. In this study, a wheat F5H gene, TaF5H1, together with its native promoter (pTaF5H1), was functionally characterized in transgenic Arabidopsis. Gus staining results showed that TaF5H1 could be expressed predominantly in the highly lignified tissues in transgenic Arabidopsis plants carrying pTaF5H1:Gus. qRT-PCR results showed that TaF5H1 was significantly inhibited by NaCl treatment. Ectopic expression of TaF5H1 driven by pTaF5H1 (i.e., pTaF5H1:TaF5H1) could increase the biomass yield, S-lignin content, and S/G ratio in transgenic Arabidopsis plants, which could also restore the traces of S-lignin in fah1-2, the Arabidopsis F5H mutant, to an even higher level than the wild type (WT), suggesting that TaF5H1 is a critical enzyme in S lignin biosynthesis, and pTaF5H1:TaF5H1 module has potential in the manipulation of S-lignin composition without any compromise on the biomass yield. However, expression of pTaF5H1:TaF5H1 also led to decreased salt tolerance compared with the WT. RNA-seq analysis showed that many stress-responsive genes and genes responsible for the biosynthesis of cell walls were differentially expressed between the seedlings harboring pTaF5H1:TaF5H1 and the WT, hinting that manipulation of the cell wall components targeting F5H may also affect the stress adaptability of the modified plants due to the interference to the cell wall integrity. In summary, this study demonstrated that the wheat pTaF5H1: TaF5H1 cassette has the potential to modulate S-lignin composition without any compromise in biomass yield in future engineering practice. Still, its negative effect on stress adaptability to transgenic plants should also be considered.

Overexpression of EgrIAA20 from Eucalyptus grandis, a Non-Canonical Aux/IAA Gene, Specifically Decouples Lignification of the Different Cell-Types in Arabidopsis Secondary Xylem.

Wood (secondary xylem) formation is regulated by auxin, which plays a pivotal role as an integrator of developmental and environmental cues. However, our current knowledge of auxin-signaling during wood formation is incomplete. Our previous genome-wide analysis of Aux/IAAs in Eucalyptus grandis showed the presence of the non-canonical paralog member EgrIAA20 that is preferentially expressed in cambium. We analyzed its cellular localization using a GFP fusion protein and its transcriptional activity using transactivation assays, and demonstrated its nuclear localization and strong auxin response repressor activity. In addition, we functionally tested the role of EgrIAA20 by constitutive overexpression in Arabidopsis to investigate for phenotypic changes in secondary xylem formation. Transgenic Arabidopsis plants overexpressing EgrIAA20 were smaller and displayed impaired development of secondary fibers, but not of other wood cell types. The inhibition in fiber development specifically affected their cell wall lignification. We performed yeast-two-hybrid assays to identify EgrIAA20 protein partners during wood formation in Eucalyptus, and identified EgrIAA9A, whose ortholog PtoIAA9 in poplar is also known to be involved in wood formation. Altogether, we showed that EgrIAA20 is an important auxin signaling component specifically involved in controlling the lignification of wood fibers.

Engineering Pseudomonas putida for improved utilization of syringyl aromatics.

Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.

Lignin Distribution on Cell Wall Micro-Morphological Regions of Fibre in Developmental Phyllostachys pubescens Culms.

Bamboo is a natural fibre reinforced composite with excellent performance which is, to a certain extent, an alternative to the shortage of wood resources. The heterogeneous distribution and molecular structure of lignin is one of the factors that determines its performance, and it is the key and most difficult component in the basic research into the chemistry of bamboo and in bamboo processing and utilization. In this study, the distribution of lignin components and lignin content in micro-morphological regions were measured in semi-quantitative level by age and radial location by means of visible-light microspectrophotometry (VLMS) coupled with the Wiesner and Maule reaction. There as guaiacyl lignin and syringyl lignin in the cell wall of the fibre. Lignin content of the secondary cell wall and cell corner increased at about 10 days, reached a maximum at 1 year, and then decreased gradually. From 17 days to 4 years, the lignin content of the secondary cell wall in the outer part of bamboo is higher than that in the middle part (which is, in turn, higher than that in the inner part of the bamboo). VLSM results of the micro-morphological regions showed that bamboo lignification developed by aging. Guaiacyl and syringl lignin units can be found in the cell wall of the fibre, parenchyma, and vessel. There was a difference in lignin content among different ages, different radial location, and different micro-morphological regions of the cell wall. The fibre walls were rich in guaiacyl lignin in the early stage of lignification and rich in syringyl units in the later stage of lignification. The guaiacyl and syringyl lignin deposition of bamboo green was earlier than that of the middle part of bamboo culm, and that of the middle part of bamboo culm was earlier than that of bamboo yellow. The single molecule lignin content of the thin layer is higher than that of thick layers, while the primary wall is higher than the secondary cell wall, showing that lignin deposition is consistent with the rules of cell wall formation. The obtained cytological information is helpful to understand the origin of the anisotropic, physical, mechanical, chemical, and machining properties of bamboo.

Bioconversion of syringyl lignin into malic acid by Burkholderia sp. ISTR5.

Syringyl monomeric units are the most common intermediates encountered during hardwood lignin degradation. In the present study, efficient utilization of syringaldehyde (SAld), syringic acid (SAc) by Burkholderia sp. ISTR5 (R5) has been shown. The proteogenomic analysis of Burkholderia sp. ISTR5 was done to understand the enzymes involved in the degradation of syringaldehyde and syringic acid. Various proteins such as aldehyde dehydrogenase, laccase, and oxidoreductases were highly upregulated during growth on syringaldehyde and syringic acid. R5 completely transformed both the substrates SAld and SAc to other hydrocarbons in 48 h and 24 h, respectively. Moreover, bioconversion of syringyl lignins followed an unusual pathway and accumulated a considerable amount of industrially valuable chemical malic acid in the reaction titer. This study shows the robust chassis of R5 to cope with the aromatic aldehydic stress and simultaneous bioconversion into valuable products for an efficient biorefinery.

Lignin Composition and Timing of Cell Wall Lignification Are Involved in Brassica napus Resistance to Stem Rot Caused by Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) is an economically and globally significant disease in oilseed rape (Brassica napus) caused by the necrotrophic ascomycete Sclerotinia sclerotiorum. This study explored the role of cell wall reinforcement by lignin as a relevant factor for effective plant defense against attack by this pathogen. Expression of key genes in the phenylpropanoid pathway and the induced synthesis of lignin in infected stem tissues were investigated in a study comparing a susceptible ('Loras') and a moderately resistant cultivar ('Zhongyou 821' [ZY821]). Data revealed an earlier and more rapid defense activation in ZY821 through upregulation of transcript levels of genes related to key steps in the phenylpropanoid pathway associated with increased lignin deposition in the resistant B. napus genotype. Expression level of BnCAD5, encoding a cinnamyl alcohol dehydrogenase, responsible for conversion of monolignol to lignin, was more rapidly upregulated in ZY821 than 'Loras'. The similar expression pattern of BnCAD5 and the gene BnF5H, encoding for ferulate-5-hydroxylase, which catalyzes the synthesis of syringyl (S) lignin precursors, suggests that BnCAD5 is involved in S lignin formation. Histological observations confirmed these results, showing an earlier increase of S lignin deposition in the infected resistant genotype. Deposition of guaiacyl lignin was detected in both genotypes and is thus considered a component of basal, cultivar-independent defense response of B. napus to stem rot. The results indicate the importance of cell wall modification for quantitative stem rot resistance by responses in the phenylpropanoid metabolism generating distinct lignin types on different temporal scales.

Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor.

Ferulate 5-hydroxylase (F5H) of the monolignol pathway catalyzes the hydroxylation of coniferyl alcohol, coniferaldehyde and ferulic acid to produce 5-hydroxyconiferyl moieties, which lead to the formation of sinapic acid and syringyl (S) lignin monomers. In contrast, guaiacyl (G) lignin, the other major type of lignin monomer, is derived from polymerization of coniferyl alcohol. In this study, the effects of manipulating S-lignin biosynthesis in sorghum (Sorghum bicolor) were evaluated. Overexpression of sorghum F5H (SbF5H), under the control of the CaMV 35S promoter, increased both S-lignin levels and the ratio of S/G lignin, while plant growth and development remained relatively unaffected. Maüle staining of stalk and leaf midrib sections from SbF5H overexpression lines indicated that the lignin composition was altered. Ectopic expression of SbF5H did not affect the gene expression of other monolignol pathway genes. In addition, brown midrib 12-ref (bmr12-ref), a nonsense mutation in the sorghum caffeic acid O-methyltransferase (COMT) was combined with 35S::SbF5H through cross-pollination to examine effects on lignin synthesis. The stover composition from bmr12 35S::SbF5H plants more closely resembled bmr12 stover than 35S::SbF5H or wild-type (WT) stover; S-lignin and total lignin concentrations were decreased relative to WT or 35S::SbF5H. Likewise, expression of upstream monolignol biosynthetic genes was increased in both bmr12 and bmr12 35S::SbF5H relative to WT or 35S::SbF5H. Overall, these results indicated that overexpression of SbF5H did not compensate for the loss of COMT activity. KEY MESSAGE: Overexpression of F5H in sorghum increases S-lignin without increasing total lignin content or affecting plant growth, but it cannot compensate for the loss of COMT activity in monolignol synthesis.

Analytical Py-GC/MS of Genetically Modified Poplar for the Increased Production of Bio-aromatics.

Genetic engineering is a powerful tool to steer bio-oil composition towards the production of speciality chemicals such as guaiacols, syringols, phenols, and vanillin through well-defined biomass feedstocks. Our previous work demonstrated the effects of lignin biosynthesis gene modification on the pyrolysis vapour compositions obtained from wood derived from greenhouse-grown poplars. In this study, field-grown poplars downregulated in the genes encoding CINNAMYL ALCOHOL DEHYDROGENASE (CAD), CAFFEIC ACID O-METHYLTRANSFERASE (COMT) and CAFFEOYL-CoA O-METHYLTRANSFERASE (CCoAOMT), and their corresponding wild type were pyrolysed in a Py-GC/MS. This work aims at capturing the effects of downregulation of the three enzymes on bio-oil composition using principal component analysis (PCA). 3,5-methoxytoluene, vanillin, coniferyl alcohol, 4-vinyl guaiacol, syringol, syringaldehyde, and guaiacol are the determining factors in the PCA analysis that are the substantially affected by COMT, CAD and CCoAOMT enzyme downregulation. COMT and CAD downregulated transgenic lines proved to be statistically different from the wild type because of a substantial difference in S and G lignin units. The sCAD line lead to a significant drop (nearly 51%) in S-lignin derived compounds, while CCoAOMT downregulation affected the least (7-11%). Further, removal of extractives via pretreatment enhanced the statistical differences among the CAD transgenic lines and its wild type. On the other hand, COMT downregulation caused 2-fold reduction in S-derived compounds compared to G-derived compounds. This study manifests the applicability of PCA analysis in tracking the biological changes in biomass (poplar in this case) and their effects on pyrolysis-oil compositions.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Molecular cloning of two novel peroxidases and their response to salt stress and salicylic acid in the living fossil Ginkgo biloba.

BACKGROUND AND AIMS: Peroxidase isoenzymes play diverse roles in plant physiology, such as lignification and defence against pathogens. The actions and regulation of many peroxidases are not known with much accuracy. A number of studies have reported direct involvement of peroxidase isoenzymes in the oxidation of monolignols, which constitutes the last step in the lignin biosynthesis pathway. However, most of the available data concern only peroxidases and lignins from angiosperms. This study describes the molecular cloning of two novel peroxidases from the 'living fossil' Ginkgo biloba and their regulation by salt stress and salicylic acid. METHODS: Suspension cell cultures were used to purify peroxidases and to obtain the cDNAs. Treatments with salicylic acid and sodium chloride were performed and peroxidase activity and gene expression were monitored. KEY RESULTS: A novel peroxidase was purified, which preferentially used p-hydroxycinnamyl alcohols as substrates and was able to form dehydrogenation polymers in vitro from coniferyl and sinapyl alcohols. Two peroxidase full-length cDNAs, GbPrx09 and GbPrx10, were cloned. Both peroxidases showed high similarity to other basic peroxidases with a putative role in cell wall lignification. Both GbPrx09 and GbPrx10 were expressed in leaves and stems of the plant. Sodium chloride enhanced the gene expression of GbPrx09 but repressed GbPrx10, whereas salicylic acid strongly repressed both GbPrx09 and GbPrx10. CONCLUSIONS: Taken together, the data suggest the participation of GbPrx09 and GbPrx10 in the developmental lignification programme of the cell wall. Both peroxidases possess the structural characteristics necessary for sinapyl alcohol oxidation. Moreover, GbPrx09 is also involved in lignification induced by salt stress, while salicylic acid-mediated lignification is not a result of GbPrx09 and GbPrx10 enzymatic activity.

Parallels in lignin biosynthesis: A study in Selaginella moellendorffii reveals convergence across 400 million years of evolution.

A hallmark of vascular plants is the development of a complex water-conducting system, which is physically reinforced by the heterogeneous aromatic polymer lignin. Syringyl lignin, a major building block of lignin, is often thought to be uniquely characteristic of angiosperms; however, it was demonstrated over fifty years ago that that syringyl lignin is found in another group of plants, known as the lycophytes, the ancestors of which diverged from all the other vascular plant lineages 400 million years ago.1 To determine the biochemical basis for this common biosynthetic ability, we isolated and characterized cytochrome P450-dependent monooxygenases (P450s) from the lycophyte Selaginella moellendorffii and compared them to the enzyme that is required for syringyl lignin synthesis in angiosperms. Our results showed that one of these P450s encodes an enzyme that is functionally analogous to but phylogenetically independent from its angiosperm counterpart. Here, we discuss the evolution of lignin biosynthesis in vascular plants and the role of Selaginella moellendorffii in plant comparative biology and genomics.