Downregulation of Cinnamoyl CoA Reductase Affects Lignin and Phenolic Acids Biosynthesis in Salvia miltiorrhiza Bunge

The biosynthesis of salvianolic acid B shares the phenylpropanoid pathway with lignin, and cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is a specific enzyme in the lignin pathway. In this study, a CCR gene (SmCCR1) from Salvia miltiorrhiza Bunge was cloned using DNA walking technology (GenBank ID: JF798634). The full-length SmCCR1 is 2,489?bp long and consists of four introns and five exons encoding a polypeptide of 324 amino acid residues. Sequence alignment revealed that SmCCR1 shares 83?% identity with CCR sequences reported in Camellia oleifera and other plant species. Expression pattern analysis indicated that expression of SmCCR1 can be induced by exposure to Xanthomonas campestris pv. Campestris or methyl jasmonate. To demonstrate its functioning, we selected a 296-bp fragment and established an RNA interference construct that was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants exhibited dwarfing phenotypes, and both syringyl and guaiacyl lignin monomers were decreased more than 60?%. In contrast, biosynthesis of phenolic acids??danshensu, rosmarinic acid, and salvianolic acid B??was strongly induced by 2.03-, 1.41-, and 1.45-fold, respectively, in the roots of transgenic plants from line CCR-10. Consistent with these phytochemical changes, downregulation of SmCCR1 also affected the expression of related genes in the phenolics and lignin biosynthetic pathways. Our results also provide potential opportunities for engineering danshensu and salvianolic acid B production in S. miltiorrhiza.     

Convergent Evolution of Syringyl Lignin Biosynthesis via Distinct Pathways in the Lycophyte Selaginella and Flowering Plants

Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago. Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C3′H and F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and suggest a new way to genetically reconstruct lignin biosynthesis in higher plants.     

O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.     

Biochemical characterization of a putative wheat caffeic acid O-methyltransferase

A wheat (Triticum aestivum L., near isogenic line of Hamlet) O-methyltransferase (OMT) was previously reported as a putative caffeic acid OMT (TaCOMT1), involved in lignin biosynthesis, based on its high sequence similarity with a number of graminaceous COMTs. The fact that the putative TaCOMT1 exhibits a significantly high sequence homology to another recently characterized wheat flavone-specific OMT (TaOMT2), and that molecular modeling studies indicated several conserved amino acid residues involved in substrate binding and catalysis of both proteins, prompted an investigation of its appropriate substrate specificity. We report here that TaCOMT1 exhibits highest preference for the flavone tricetin, and lowest activity with the lignin precursors, caffeic acid/5-hydroxyferulic acid as the methyl acceptor molecules, indicating that it is not involved in lignin biosynthesis. We recommend its reannotation to a flavone-specific TaOMT1 that is distinct from TaOMT2.     

Integrated Metabolo-Proteomic Approach to Decipher the Mechanisms by Which Wheat QTL (Fhb1) Contributes to Resistance against Fusarium graminearum

Background

Resistance in plants to pathogen attack can be qualitative or quantitative. For the latter, hundreds of quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. Integrated non-target metabolomics and proteomics, using high resolution hybrid mass spectrometry, were applied to identify the mechanisms of resistance governed by the fusarium head blight resistance locus, Fhb1, in the near isogenic lines derived from wheat genotype Nyubai.

Findings

The metabolomic and proteomic profiles were compared between the near isogenic lines (NIL) with resistant and susceptible alleles of Fhb1 upon F. graminearum or mock-inoculation. The resistance-related metabolites and proteins identified were mapped to metabolic pathways. Metabolites of the shunt phenylpropanoid pathway such as hydroxycinnamic acid amides, phenolic glucosides and flavonoids were induced only in the resistant NIL, or induced at higher abundances in resistant than in susceptible NIL, following pathogen inoculation. The identities of these metabolites were confirmed, with fragmentation patterns, using the high resolution LC-LTQ-Orbitrap. Concurrently, the enzymes of phenylpropanoid biosynthesis such as cinnamyl alcohol dehydrogenase, caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, flavonoid O-methyltransferase, agmatine coumaroyltransferase and peroxidase were also up-regulated. Increased cell wall thickening due to deposition of hydroxycinnamic acid amides and flavonoids was confirmed by histo-chemical localization of the metabolites using confocal microscopy.

Conclusion

The present study demonstrates that the resistance in Fhb1 derived from the wheat genotype Nyubai is mainly associated with cell wall thickening due to deposition of hydroxycinnamic acid amides, phenolic glucosides and flavonoids, but not with the conversion of deoxynivalenol to less toxic deoxynivalenol 3-O-glucoside.     

Syncytium gene expression in Glycine max[PI 88788] roots undergoing a resistant reaction to the parasitic nematode Heterodera glycines

The plant parasitic nematode, Heterodera glycines is the major pathogen of Glycine max (soybean). H. glycines accomplish parasitism by creating a nurse cell known as the syncytium from which it feeds. The syncytium undergoes two developmental phases. The first is a parasitism phase where feeding sites are selected, initiating the development of the syncytium. During this earlier phase (1–4 days post infection), syncytia undergoing resistant and susceptible reactions appear the same. The second phase is when the resistance response becomes evident (between 4 and 6 dpi) and is completed by 9 dpi. Analysis of the resistant reaction of G. max genotype PI 88788 (G. max_{[PI 88788]}) to H. glycines population NL1-RHg/HG-type 7 (H. glycines_{[NL1-RHg/HG-type 7]}) is accomplished by laser microdissection of syncytia at 3, 6 and 9 dpi. Comparative analyses are made to pericycle and their neighboring cells isolated from mock-inoculated roots. These analyses reveal induced levels of the jasmonic acid biosynthesis and 13-lipoxygenase pathways. Direct comparative analyses were also made of syncytia at 6 days post infection to those at 3 dpi (base line). The comparative analyses were done to identify localized gene expression that characterizes the resistance phase of the resistant reaction. The most highly induced pathways include components of jasmonic acid biosynthesis, 13-lipoxygenase pathway, S-adenosyl methionine pathway, phenylpropanoid biosynthesis, suberin biosynthesis, adenosylmethionine biosynthesis, ethylene biosynthesis from methionine, flavonoid biosynthesis and the methionine salvage pathway. In comparative analyses of 9 dpi to 6 dpi (base line), these pathways, along with coumarin biosynthesis, cellulose biosynthesis and homogalacturonan degradation are induced. The experiments presented here strongly implicate the jasmonic acid defense pathway as a factor involved in the localized resistant reaction of G. max_{[PI 88788]} to H. glycines_{[NL1-RHg/HG-type 7]}.     

Differentially expressed proteins during an incompatible interaction between common bean and the fungus Pseudocercospora griseola

The common bean (Phaseolus vulgaris L.) is the main source of protein and an important source of minerals in several countries around the world. Angular leaf spot, caused by the fungus Pseudocercospora griseola, is one of the major diseases of the common bean. In this work, we used two-dimensional gel electrophoresis and mass spectrometry to analyze alterations in the proteome of common bean leaves challenged with an incompatible race of P. griseola. Twenty-three differentially expressed proteins were detected in leaves of cultivar AND 277 collected at 12, 24 and 48 h after inoculation. The proteins were digested with trypsin and submitted to MALDI-TOF/TOF and MicrOTOF-Q electrospray mass spectrometry. Nineteen of them were identified upon MS/MS fragmentation. Most of these proteins are involved with amino acid metabolism, terpenoid metabolism, phenylpropanoid biosynthesis, antioxidant systems, vitamin and cofactor metabolism, plant–pathogen interaction, carbohydrate metabolism, photosynthesis, or genetic information processing, showing that the interaction in this pathosystem affects different genes from various metabolic pathways and processes.     

Indole Glucosinolate Biosynthesis Limits Phenylpropanoid Accumulation in Arabidopsis thaliana

Plants produce an array of metabolites (including lignin monomers and soluble UV-protective metabolites) from phenylalanine through the phenylpropanoid biosynthetic pathway. A subset of plants, including many related to Arabidopsis thaliana, synthesizes glucosinolates, nitrogen- and sulfur-containing secondary metabolites that serve as components of a plant defense system that deters herbivores and pathogens. Here, we report that the Arabidopsis thaliana reduced epidermal fluorescence5 (ref5-1) mutant, identified in a screen for plants with defects in soluble phenylpropanoid accumulation, has a missense mutation in CYP83B1 and displays defects in glucosinolate biosynthesis and in phenylpropanoid accumulation. CYP79B2 and CYP79B3 are responsible for the production of the CYP83B1 substrate indole-3-acetaldoxime (IAOx), and we found that the phenylpropanoid content of cyp79b2 cyp79b3 and ref5-1 cyp79b2 cyp79b3 plants is increased compared with the wild type. These data suggest that levels of IAOx or a subsequent metabolite negatively influence phenylpropanoid accumulation in ref5 and more importantly that this crosstalk is relevant in the wild type. Additional biochemical and genetic evidence indicates that this inhibition impacts the early steps of the phenylpropanoid biosynthetic pathway and restoration of phenylpropanoid accumulation in a ref5-1 med5a/b triple mutant suggests that the function of the Mediator complex is required for the crosstalk.     

Caffeoyl coenzyme A O-methyltransferase and lignin biosynthesis.

Lignin, a complex phenylpropanoid compound, is polymerized from the monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These three monolignols differ only by the 3- and 5-methoxyl groups. Therefore, enzymatic reactions controlling the methylations of the 3- and 5-hydroxyls of monolignol precursors are critical to determine the lignin composition. Recent biochemical and transgenic studies have indicated that the methylation pathways in monolignol biosynthesis are much more complicated than we have previously envisioned. It has been demonstrated that caffeoyl CoA O-methyltransferase plays an essential role in the synthesis of guaiacyl lignin units as well as in the supply of substrates for the synthesis of syringyl lignin units. Caffeic acid O-methyltransferase has been found to essentially control the biosynthesis of syringyl lignin units. These new findings have greatly enriched our knowledge on the methylation pathways in monolignol biosynthesis.     

Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii

Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts.     

Soybean root growth inhibition and lignification induced by p-coumaric acid

The effects of 0.25–2 mM p-coumaric acid, a phenylpropanoid metabolite with recognized allelopathic properties, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) activities, soluble and cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H₂O₂) level and lignin content and its monomeric composition in soybean (Glycine max (L.) Merr.) roots. At ≥0.25 mM, exogenously supplied p-coumaric acid induced premature cessation of root growth, increased POD activity and lignin content and decreased the H₂O₂ content. At ≥0.5 mM, the allelochemical decreased the cell viability and PAL activity. When applied jointly with PIP (an inhibitor of the cinnamate 4-hydroxylase, C4H), 1 mM p-coumaric acid increased lignin content. In contrast, the application of MDCA (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with p-coumaric acid did not increase lignin content. The lignin monomeric composition of p-coumaric acid-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and guaiacyl (G) units. Taken together, these results suggest that p-coumaric acid's mode of action is entry via the phenylpropanoid pathway, resulting in an increase of H and G lignin monomers that solidify the cell wall and restrict soybean root growth.