Caffeic Acid O-methyltransferase Research Articles

The maize low-lignin brown midrib3 mutant shows pleiotropic effects on photosynthetic and cell wall metabolisms in response to chilling

Maize (Zea mays L.) is one of the major cereal crops in the world and is highly sensitive to low temperature. Here, changes in photosynthetic and cell wall metabolisms were investigated during a long chilling exposure in inbred line F2 and a low-lignin near-isogenic brown midrib3 mutant (F2bm3), which has a mutation in the caffeic acid O-methyltransferase (COMT) gene. Results revealed that the plant biomass was reduced, and this was more pronounced in F2bm3. Photosynthesis was altered in both lines with distinct changes in photosynthetic pigment content between F2bm3 and F2, indicating an alternative photoprotection mechanism between lines under chilling. Starch remobilization was observed in F2bm3 while concentrations of sucrose, fructose and starch increased in F2, suggesting a reduced sugar partitioning in F2. The cell wall was altered upon chilling, resulting in changes in the composition of glucuronorabinoxylan and a reduced cellulose level in F2. Chilling shifted lignin subunit composition in F2bm3 mutant to a higher proportion of p-hydroxyphenyl (H) units, whereas it resulted in lignin with a higher proportion of syringyl (S) residues in F2. On average, the total cell wall ferulic acid (FA) content increased in both genotypes, with an increase in ether-linked FA in F2bm3, suggesting a greater degree of cross-linking to lignin. The reinforcement of the cell wall with lignin enriched in H-units and a higher concentration in cell-wall-bound FA observed in F2bm3 as a response to chilling, could be a strategy to protect the photosystems.

Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony.

Cut flower quality is severely restrained by stem bending due to low stem strength. Melatonin has been shown to function in many aspects of plant growth and development, yet whether it can enhance stem strength, and the corresponding underlying mechanisms remain unclear. We investigated the role of melatonin in enhancement of stem strength in herbaceous peony (Paeonia lactiflora Pall.) by applying exogenous melatonin and changing endogenous melatonin biosynthesis. Endogenous melatonin content positively correlated with lignin content and stem strength in various P. lactiflora cultivars. Supplementation with exogenous melatonin significantly enhanced stem strength by increasing lignin content and the S/G lignin compositional ratio, up-regulating lignin biosynthetic gene expression. Moreover, overexpression of TRYPTOPHAN DECARBOXYLASE GENE (TDC) responsible for the first committed step of melatonin biosynthesis in tobacco, significantly increased endogenous melatonin, which further increased the S/G ratio and stem strength. In contrast, silencing PlTDC in P. lactiflora decreased endogenous melatonin, the S/G ratio and stem strength. Finally, manipulating the expression of CAFFEIC ACID O-METHYLTRANSFERASE GENE (COMT1), which is involved in both melatonin and lignin biosynthesis, showed even greater effects on melatonin, the S/G ratio and stem strength. Our results suggest that melatonin has a positive regulatory effect on P. lactiflora stem strength.

OsCOMT, encoding a caffeic acid O-methyltransferase in melatonin biosynthesis, increases rice grain yield through dual regulation of leaf senescence and vascular development.

SummaryMelatonin, a natural phytohormone in plants, plays multiple critical roles in plant growth and stress responses. Although melatonin biosynthesis‐related genes have been suggested to possess diverse biological functions, their roles and functional mechanisms in regulating rice grain yield remain largely unexplored. Here, we uncovered the roles of a caffeic acid O‐methyltransferase (OsCOMT) gene in mediating rice grain yield through dual regulation of leaf senescence and vascular development. In vitro and in vivo evidence revealed that OsCOMT is involved in melatonin biosynthesis. Transgenic assays suggested that OsCOMT significantly delays leaf senescence at the grain filling stage by inhibiting degradation of chlorophyll and chloroplast, which, in turn, improves photosynthesis efficiency. In addition, the number and size of vascular bundles in the culms and leaves were significantly increased in the OsCOMT‐overexpressing plants, while decreased in the knockout plants, suggesting that OsCOMT plays a positive role in vascular development of rice. Further evidence indicated that OsCOMT‐mediated vascular development might owe to the crosstalk between melatonin and cytokinin. More importantly, we found that OsCOMT is a positive regulator of grain yield, and overexpression of OsCOMT increase grain yield per plant even in a high‐yield variety background, suggesting that OsCOMT can be used as an important target for enhancing rice yield. Our findings shed novel insights into melatonin‐mediated leaf senescence and vascular development and provide a possible strategy for genetic improvement of rice grain yield.

Inhibition of miR397 by STTM technology to increase sweetpotato resistance to SPVD

As a critical food crop, sweetpotato (Ipomoea batatas (L.) Lam.) is widely planted all over the world, but it is deeply affected by Sweetpotato Virus Disease (SPVD). The present study utilized short tandem target mimic (STTM) technology to effectively up-regulate the expression of laccase (IbLACs) by successfully inhibiting the expression of miR397. The upstream genes in the lignin synthesis pathway were widely up-regulated by feedback regulation, including phenylalanine ammonialyase (PAL), 4-coumarate-CoAligase (4CL), hydroxycinnamoyl CoA:shikimatetransferase (HTC), caffeicacid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD). Meanwhile, the activities of PAL and LAC increased significantly, finally leading to increased lignin content. Lignin deposition in the cell wall increased the physical defence ability of transgenic sweetpotato plants, reduced the accumulation of SPVD transmitted by Bemisia tabaci (Gennadius), and promoted healthy sweetpotato growth. The results provide new insights for disease resistance breeding and green production of sweetpotato.

Genome-Wide Identification and Characterization of Caffeic Acid O-Methyltransferase Gene Family in Soybean.

Soybean is one of the most important legumes, providing high-quality protein for humans. The caffeic acid O-methyltransferase (COMT) gene has previously been demonstrated to be a critical gene that regulates lignin production in plant cell walls and plays an important function in plant growth and development. However, the COMT gene family has not been studied in soybeans. In this study, 55 COMT family genes in soybean were identified by phylogenetic analysis and divided into two groups, I and II. The analysis of conserved domains showed that all GmCOMTs genes contained Methyltransferase-2 domains. Further prediction of cis-acting elements showed that GmCOMTs genes were associated with growth, light, stress, and hormonal responses. Eventually, based on the genomic data of soybean under different stresses, the results showed that the expression of GmCOMTs genes was different under different stresses, such as salt and drought stress. This study has identified and characterized the COMT gene family in soybean, which provides an important theoretical basis for further research on the biological functions of COMT genes and promotes revealing the role of GmCOMTs genes under stress resistance.

Genome-Wide Identification and Characterization of GhCOMT Gene Family during Fiber Development and Verticillium Wilt Resistance in Cotton

Caffeic acid O-methyltransferases (COMTs) play an essential role in lignin synthesis procession, especially in the plant’s phenylalanine metabolic pathway. The content of COMT genes in cotton and the relationship between their expression patterns have not been studied clearly in cotton. In this study, we have identified 190 COMT genes in cotton, which were classified into three groups (I, II and III), and mapped on the cotton chromosomes. In addition, we found that 135 of the 190 COMT genes result from dispersed duplication (DSD) and whole-genome duplication (WGD), indicating that DSD and WGD were the main forces driving COMT gene expansion. The Ka/Ks analysis showed that GhCOMT43 and GhCOMT41 evolved from GaCOMT27 and GrCOMT14 through positive selection. The results of qRT-PCR showed that GhCOMT13, GhCOMT28, GhCOMT39 and GhCOMT55 were related to lignin content during the cotton fiber development. GhCOMT28, GhCOMT39, GhCOMT55, GhCOMT56 and GhCOMT57 responded to Verticillium Wilt (VW) and maybe related to VW resistance through lignin synthesis. Conclusively, this study found that GhCOMTs were highly expressed in the secondary wall thickening stage and VW. These results provide a clue for studying the functions of GhCOMTs in the development of cotton fiber and VW resistance and could lay a foundation for breeding cotton cultivates with higher quantity and high resistance to VW.

Structure basis of the caffeic acid O-methyltransferase from Ligusiticum chuanxiong to understand its selective mechanism

Caffeic acid O-methyltransferase from Ligusticum chuanxiong (LcCOMT) showed strict regiospecificity despite a relative degree of preference. Compared with caffeic acid, methyl caffeate was the preferential substrate by its low Km and high Kcat. In this study, we obtained the SAM binary (1.80 Å) and SAH binary (1.95 Å) complex LcCOMT crystal structures, and established the ternary complex structure with methyl caffeate by molecular docking. The active site of LcCOMT included phenolic substrate pocket, SAM/SAH ligand pocket and conserved catalytic residues as well. The regiospecificity of LcCOMT that permitted only 3-hydroxyl group to be methylated arise from the interactions between the active site and the phenyl ring. However, the propanoid tail governed the relative preference of LcCOMT. The ester group in methyl caffeate stabilized the anionic intermediate caused by His268-Asp269 pair, whereas caffeic acid was unable to stabilize the anionic intermediate due to the adjacent carboxylate anion in the propanoid tail. Ser183 residue formed an additional hydrogen bond with SAH and its role was identified by S183A mutation. Ile318 residue might be a potential site for determination of substrate preference, and its mutation led to the change of tertiary conformation. The results supported the selective mechanism of LcCOMT.

Construction of an Artificial Biosynthetic Pathway for Zingerone Production in Escherichia coli Using Benzalacetone Synthase from Piper methysticum.

Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger (Zingiber officinale). In this study, it was confirmed that a type III polyketide synthase (PKS) gene (pmpks) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase (optal), cinnamate-4-hydroxlase (sam5), caffeic acid O-methyltransferase (com), 4CL (4cl2nt), BAS (pmpks), and BAR (rzs1), in Escherichia coli. Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.

H2S aids osmotic stress resistance by S-sulfhydration of melatonin production-related enzymes in Arabidopsis thaliana

Hydrogen sulfide closed Arabidopsis thaliana stomata by increasing the transcription of melatonin-producing enzymes and the post-translational modification levels to combat osmotic stress. Hydrogen sulfide (H2S) and melatonin (MEL) reportedly have similar functions in many aspects of plant growth, development and stress response. They regulate stomatal movement and enhance drought resistance. However, their physiological relationship is not well understood. Here, their crosstalk involved in osmotic stress resistance in Arabidopsis thaliana was studied. Exogenous H2S and MEL closed stomata under normal or osmotic stress conditions and increased the relative water contents of plants under osmotic stress conditions. At the same time, exogenous H2S and MEL responded to osmotic stress by increasing the content of proline and soluble sugar, and reducing malondialdehyde (MDA) content and relative conductivity. Using mutants in the MEL-associated production of serotonin N-acetyltransferase (snat), caffeic acid O-methyltransferase (comt1) and N-acetylserotonin methyltransferase (asmt), we determined that H2S was partially dependent on MEL to close stomata. Additionally, the overexpression of ASMT promoted stomatal closure. Exogenous H2S increased the transcription levels of SNAT, ASMT and COMT1. Furthermore, exogenous H2S treatments increased the endogenous MEL content significantly. At the post-translational level, H2S sulfhydrated the SNAT and ASMT, but not COMT1, enzymes associated with MEL production. Thus, H2S appeared to promote stomatal closure in response to osmotic stress by increasing the transcription levels of MEL synthesis-related genes and the sulfhydryl modification of the encoded enzymes. These results increased our understanding of H2S and MEL functions and interactions under osmotic stress conditions.

The role of watermelon caffeic acid O-methyltransferase (ClCOMT1) in melatonin biosynthesis and abiotic stress tolerance

Melatonin is a pleiotropic signaling molecule that regulates plant growth and responses to various abiotic stresses. The last step of melatonin synthesis in plants can be catalyzed by caffeic acid O-methyltransferase (COMT), a multifunctional enzyme reported to have N-acetylserotonin O-methyltransferase (ASMT) activity; however, the ASMT activity of COMT has not yet been characterized in nonmodel plants such as watermelon (Citrullus lanatus). Here, a total of 16 putative O-methyltransferase (ClOMT) genes were identified in watermelon. Among them, ClOMT03 (Cla97C07G144540) was considered a potential COMT gene (renamed ClCOMT1) based on its high identities (60.00–74.93%) to known COMT genes involved in melatonin biosynthesis, expression in almost all tissues, and upregulation under abiotic stresses. The ClCOMT1 protein was localized in the cytoplasm. Overexpression of ClCOMT1 significantly increased melatonin contents, while ClCOMT1 knockout using the CRISPR/Cas-9 system decreased melatonin contents in watermelon calli. These results suggest that ClCOMT1 plays an essential role in melatonin biosynthesis in watermelon. In addition, ClCOMT1 expression in watermelon was upregulated by cold, drought, and salt stress, accompanied by increases in melatonin contents. Overexpression of ClCOMT1 enhanced transgenic Arabidopsis tolerance against such abiotic stresses, indicating that ClCOMT1 is a positive regulator of plant tolerance to abiotic stresses.

Melatonin synthesis genes N-acetylserotonin methyltransferases evolved into caffeic acid O-methyltransferases and both assisted in plant terrestrialization.

Terrestrialization is one of the most momentous events in the history of plant life, which leads to the subsequent evolution of plant diversity. The transition species, in this process, had to acquire a range of adaptive mechanisms to cope with the harsh features of terrestrial environments compared to that of aquatic habitat. As an ancient antioxidant, a leading regulator of ROS signaling or homeostasis, and a presumed plant master regulator, melatonin likely assisted plants transition to land and their adaption to terrestrial ecosystems. N-acetylserotonin methyltransferases (ASMT) and caffeic acid O-methyltransferases (COMT), both in the O-methyltransferase (OMT) family, catalyze the core O-methylation reaction in melatonin biosynthesis. How these two enzymes with close relevance evolved in plant evolutionary history and whether they participated in plant terrestrialization remains unknown. Using combined phylogenetic evidence and protein structure analysis, it is revealed that COMT likely evolved from ASMT by gene duplication and subsequent divergence. Newly emergent COMT gained a significantly higher ASMT activity to produce greater amounts of melatonin for immobile plants to acclimate to the stressful land environments after evolving from the more environmentally-stable aquatic conditions. The COMT genes possess more conserved substrate-binding sites at the amino acid level and more open protein conformation compared to ASMT, and getting a new function to catalyze the lignin biosynthesis. This development directly contributed to the dominance of vascular plants among the Earth's flora and prompted plant colonization of land. Thus, ASMT, together with its descendant COMT, might play key roles in plant transition to land. The current study provides new insights into plant terrestrialization with gene duplication contributing to this process along with well-known horizontal gene transfer.

UV-B treatment enhances phenolic acids accumulation and antioxidant capacity of barley seedlings

The effects of ultraviolet B (UV-B, 280–315 nm) on phenolic acids content, key enzymes activity and antioxidant capacity as well as the related gene expressions of barley seedlings were investigated in the present study. Results showed that UV-B treatment increased phenolics content including vanillic acid, p-coumaric acid, ferulic acid and erucic acid in barley seedlings, significantly. The mRNA levels and activities of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), p-coumaric acid 3-hdroxylase (C3H), caffeic acid O-methyltransferase (COMT) and ferulic acid 5-hydroxylase (F5H) involved in phenolic acid synthesis increased under UV-B treatment as well. In addition, UV-B radiation enhanced the antioxidant enzymes activity and the gene expressions of barley seedlings, including superoxide dismutase (SOD), peroxidase (POD) and glutathione reductase (GR), and improved 2,2-diazo-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging ability. In conclusion, UV-B treatment led to the upper-regulation of related enzymes activity and gene expression, resulting in the accumulation of phenolic acids in barley seedlings, thus contributing to the enhancement of antioxidant system.

Combining protein and metabolic engineering strategies for biosynthesis of melatonin in Escherichia coli

BackgroundMelatonin has attracted substantial attention because of its excellent prospects for both medical applications and crop improvement. The microbial production of melatonin is a safer and more promising alternative to chemical synthesis approaches. Researchers have failed to produce high yields of melatonin in common heterologous hosts due to either the insolubility or low enzyme activity of proteins encoded by gene clusters related to melatonin biosynthesis.ResultsHere, a combinatorial gene pathway for melatonin production was successfully established in Escherichia coli by combining the physostigmine biosynthetic genes from Streptomyces albulus and gene encoding phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris and caffeic acid 3-O-methyltransferase (COMT) from Oryza sativa. A threefold improvement of melatonin production was achieved by balancing the expression of heterologous proteins and adding 3% glycerol. Further protein engineering and metabolic engineering were conducted to improve the conversion of N-acetylserotonin (NAS) to melatonin. Construction of COMT variant containing C303F and V321T mutations increased the production of melatonin by fivefold. Moreover, the deletion of speD gene increased the supply of S-adenosylmethionine (SAM), an indispensable cofactor of COMT, which doubled the yield of melatonin. In the final engineered strain EcMEL8, the production of NAS and melatonin reached 879.38 ± 71.42 mg/L and 136.17 ± 1.33 mg/L in a shake flask. Finally, in a 2-L bioreactor, EcMEL8 produced 1.06 ± 0.07 g/L NAS and 0.65 ± 0.11 g/L melatonin with tryptophan supplementation.ConclusionsThis study established a novel combinatorial pathway for melatonin biosynthesis in E. coli and provided alternative strategies for improvement of melatonin production.

Pathogen and drought stress affect cell wall and phytohormone signaling to shape host responses in a sorghum COMT bmr12 mutant

BackgroundAs effects of global climate change intensify, the interaction of biotic and abiotic stresses increasingly threatens current agricultural practices. The secondary cell wall is a vanguard of resistance to these stresses. Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot) cause internal damage to the stalks of the drought tolerant C4 grass, sorghum (Sorghum bicolor (L.) Moench), resulting in reduced transpiration, reduced photosynthesis, and increased lodging, severely reducing yields. Drought can magnify these losses. Two null alleles in monolignol biosynthesis of sorghum (brown midrib 6-ref, bmr6-ref; cinnamyl alcohol dehydrogenase, CAD; and bmr12-ref; caffeic acid O-methyltransferase, COMT) were used to investigate the interaction of water limitation with F. thapsinum or M. phaseolina infection.ResultsThe bmr12 plants inoculated with either of these pathogens had increased levels of salicylic acid (SA) and jasmonic acid (JA) across both watering conditions and significantly reduced lesion sizes under water limitation compared to adequate watering, which suggested that drought may prime induction of pathogen resistance. RNA-Seq analysis revealed coexpressed genes associated with pathogen infection. The defense response included phytohormone signal transduction pathways, primary and secondary cell wall biosynthetic genes, and genes encoding components of the spliceosome and proteasome.ConclusionAlterations in the composition of the secondary cell wall affect immunity by influencing phenolic composition and phytohormone signaling, leading to the action of defense pathways. Some of these pathways appear to be activated or enhanced by drought. Secondary metabolite biosynthesis and modification in SA and JA signal transduction may be involved in priming a stronger defense response in water-limited bmr12 plants.

Integrative iTRAQ-based proteomic and transcriptomic analysis reveals the accumulation patterns of key metabolites associated with oil quality during seed ripening of Camellia oleifera

Camellia oleifera (C. oleifera) is one of the four major woody oil-bearing crops in the world and has relatively high ecological, economic, and medicinal value. Its seeds undergo a series of complex physiological and biochemical changes during ripening, which is mainly manifested as the accumulation and transformation of certain metabolites closely related to oil quality, especially flavonoids and fatty acids. To obtain new insights into the underlying molecular mechanisms, a parallel analysis of the transcriptome and proteome profiles of C. oleifera seeds at different maturity levels was conducted using RNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) complemented with gas chromatography-mass spectrometry (GC-MS) data. A total of 16,530 transcripts and 1228 proteins were recognized with significant differential abundances in pairwise comparisons of samples at various developmental stages. Among these, 317 were coexpressed with a poor correlation, and most were involved in metabolic processes, including fatty acid metabolism, α-linolenic acid metabolism, and glutathione metabolism. In addition, the content of total flavonoids decreased gradually with seed maturity, and the levels of fatty acids generally peaked at the fat accumulation stage; these results basically agreed with the regulation patterns of genes or proteins in the corresponding pathways. The expression levels of proteins annotated as upstream candidates of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) as well as their cognate transcripts were positively correlated with the variation in the flavonoid content, while shikimate O-hydroxycinnamoyltransferase (HCT)-encoding genes had the opposite pattern. The increase in the abundance of proteins and mRNAs corresponding to alcohol dehydrogenase (ADH) was associated with a reduction in linoleic acid synthesis. Using weighted gene coexpression network analysis (WGCNA), we further identified six unique modules related to flavonoid, oil, and fatty acid anabolism that contained hub genes or proteins similar to transcription factors (TFs), such as MADS intervening keratin-like and C-terminal (MIKC_MADS), type-B authentic response regulator (ARR-B), and basic helix-loop-helix (bHLH). Finally, based on the known metabolic pathways and WGCNA combined with the correlation analysis, five coexpressed transcripts and proteins composed of cinnamyl-alcohol dehydrogenases (CADs), caffeic acid 3-O-methyltransferase (COMT), flavonol synthase (FLS), and 4-coumarate: CoA ligase (4CL) were screened out. With this exploratory multiomics dataset, our results presented a dynamic picture regarding the maturation process of C. oleifera seeds on Hainan Island, not only revealing the temporal specific expression of key candidate genes and proteins but also providing a scientific basis for the genetic improvement of this tree species.

Changes in phenotype and gene expression under lead stress revealed key genetic responses to lead tolerance in Medicago sativa L.

Lead (Pb) is a serious heavy metal soil pollutant. It can be absorbed and accumulated by plant roots and impact plant growth. Medicago sativa L. (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Little is known about the mechanism by which alfalfa responds to Pb stress at the molecular level. In this study, three alfalfa genotypes (a lead-resistant type (LR), a lead-sensitive type (LS) and an intermediate type (IN)) with contrasting abilities to resist lead were exposed to different durations of Pb treatment. Next-generation sequencing (NGS)-based RNA-seq technology was employed to characterize the root transcriptomes of three genotypes of alfalfa and identify differentially expressed genes (DEGs) during Pb stress. Genotypes LR and LS displayed different mechanisms of tolerance. In LR, the accumulation of more resistant substances was induced by the upregulation of sucrose synthase, glucan endo-1,3-beta-glucosidase, beta-amylase 3, probable trehalose-phosphate phosphatase J, 6-phosphofructo-2-kinase delta-1-pyrroline-5-carboxylate synthase (P5CS) and δ-ornithine aminotransferase (δ-OAT). In addition, flavin monooxygenase (YUCCA), 4-coumarate:CoA ligase-like protein (4CL), cinnamoyl-CoA reductase-like protein (CCR), ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) were upregulated, leading to root development in a short time under Pb stress. Further study of the expression levels of metal transport-related genes, such as NRAMP (metal transporter), MATE (multidrug and toxin extrusion), HIPPs (heavy metal-associated isoprenylated plant proteins), MTP (metal tolerance protein), and ABC transporter, suggested that these genes were differentially expressed after lead treatment in the three alfalfa genotypes. Our research provides useful information for further studies on the molecular mechanism of Pb resistance in Medicago sativa L.

De novo transcriptome assembly of Angelica dahurica and characterization of coumarin biosynthesis pathway genes.

Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav (A. dahurica) is a famous Chinese herb known for the production of coumarins, important secondary metabolites with wide-ranging pharmacological activities. In particular, the methoxylated coumarins like those produced by A. dahurica are known for their anti-inflammatory, anti-cancer, and anti-oxidant pharmacological effects. However, the molecular mechanism of coumarin biosynthesis in A. dahurica has not been studied. Such investigation could help scientists harness the biosynthesis potential of methoxylated coumarins. Here we present, three transcriptomes corresponding to leaf, root, and stem tissues of A. dahurica. A total of 114,310 unigenes with an average length of 1118bp were de novo assembled, and 81,404 (71.21%) of those unigenes were annotated. Then, 181 unigenes encoding the seven key enzymes involved were identified, for which COMT (Caffeic acid 3-O-methyltransferase) was spatially used in a phylogenetic analysis, and some of these key enzyme genes were verified by qRT-PCR. Differentially expressed genes and root-specific-expressed genes were identified, by comparing genes' profile activity between roots and other tissues. Furthermore, multiple genes encoding key enzymes or transcription factors related to coumarin biosynthesis were identified and analyzed. This study is the first to report comprehensive gene information of A. dahurica at the transcriptional level, and to distinguish candidate genes related to its biosynthesis of coumarin, thus laying a foundation for this pathway's further exploration in A. dahurica.

The Cysteine-Rich Peptide Snakin-2 Negatively Regulates Tubers Sprouting through Modulating Lignin Biosynthesis and H2O2 Accumulation in Potato.

Potato tuber dormancy is critical for the post-harvest quality. Snakin/Gibberellic Acid Stimulated in Arabidopsis (GASA) family genes are involved in the plants’ defense against pathogens and in growth and development, but the effect of Snakin-2 (SN2) on tuber dormancy and sprouting is largely unknown. In this study, a transgenic approach was applied to manipulate the expression level of SN2 in tubers, and it demonstrated that StSN2 significantly controlled tuber sprouting, and silencing StSN2 resulted in a release of dormancy and overexpressing tubers showed a longer dormant period than that of the control. Further analyses revealed that the decrease expression level accelerated skin cracking and water loss. Metabolite analyses revealed that StSN2 significantly down-regulated the accumulation of lignin precursors in the periderm, and the change of lignin content was documented, a finding which was consistent with the precursors’ level. Subsequently, proteomics found that cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT) and peroxidase (Prx), the key proteins for lignin synthesis, were significantly up-regulated in silencing lines, and gene expression and enzyme activity analyses also supported this effect. Interestingly, we found that StSN2 physically interacts with three peroxidases catalyzing the oxidation and polymerization of lignin. In addition, SN2 altered the hydrogen peroxide (H2O2) content and the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that StSN2 negatively regulates lignin biosynthesis and H2O2 accumulation, and ultimately inhibits the sprouting of potato tubers.

Phytotoxic property of metabolites isolated from Garcinia gardneriana

Garcinia gardneriana is a medicinal tree species used in Brazil in the treatment of hepatitis and gastritis. This use is attributed to phenolic compounds, mainly 7-epiclusianone, guttiferone-A and fukugetin, which present several proven biological activities. However, to the best of our knowledge, no study on the phytotoxic activity of G. gardneriana extracts has been conducted until now. This research proposed to isolate and quantify by high-performance liquid chromatography (HPLC) the major compounds from G. gardneriana seed extracts in ethyl acetate and to evaluate their phytotoxic activities. The natural products 7-epiclusianone, guttiferone-A and fukugetin were quantified at concentrations varying from 0.46 to 1.13 mg mL−1 and were isolated with yields ranging from 7% to 23% (w/w). The phytotoxic assay indicated that the crude extract showed no action on the dry matter of Sorghum bicolor plants, but the isolated compounds fukugetin and 7-epiclusianone had moderate activity. On the other hand, guttiferone-A displayed a greater herbicide activity than glyphosate, a positive control, even in almost three times lower concentrations, causing severe intoxication in the plants. This work is the first report on this activity by the extract of G. gardneriana and its isolated compounds. Besides that, guttiferone-A showed up as a scaffold for the development of new agrochemicals. Complementing these findings, computational studies suggested that this benzophenone can interact effectively with transferase enzymes type, in special caffeic acid O-methyltransferase from S. bicolor (PDB code: 4PGH), indicating a possible mechanism of action in this plant.

RNAi-Based Gene Silencing in Sugarcane for Production of Biofuel.

RNA interference (RNAi) is an innate cellular mechanism triggered by a double-stranded RNA (dsRNA) molecule causing selective inhibition of gene expression. Here, we demonstrated the RNAi technology for gene silencing in sugarcane for biofuel production. This chapter describes an efficient model system that established to target the caffeic acid O-methyltransferase (COMT) gene and the RNAi construct is designed and delivered through Agrobacterium mediated stable sugarcane transformation. Also, the approach for an analysis of resulting putative transgenic plants for a targeted RNAi mediated gene silencing is described.