Functional characterization of caffeic acid O-methyltransferase in internode lignification of switchgrass(Panicum virgatum)

Caffeic acid O-methyltransferase(COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1,was isolated from switchgrass(Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde(CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.

Natural sources,refined extraction,biosynthesis,metabolism,and bioactivities of dietary polymethoxyflavones(PMFs)

Polymethoxyflavones(PMFs)are a type of uncommon dietary flavonoids,characterized by more than one methoxy group,which exist in limited plant species,like Citrus species and Kaempferia parviflora.In addition,different PMFs,such as nobiletin,sinensetin,tangeretin,and casticin,have been isolated from these natural sources.PMFs have received increasing attention due to their multiple bioactivities,such as antioxidant,anti-inflammatory,anti-cancer,metabolic regulatory,immunoregulatory,neuroprotective,and skin protective effects.These bioactivities of PMFs should be associated with the regulation of critical molecular targets and the interaction with gut microbiota.In order to provide a comprehensive and updated review of PMFs,their natural sources,refined extraction,biosynthesis,metabolism,and bioactivities are summarised and discussed,with the emphasis on the molecular mechanisms of PMFs on regulating different chronic diseases.Overall,PMFs may be promising flavonoids to the forefront of nutraceuticals for the prevention and/or treatment of certain human chronic diseases.

Revealing the role of CCoAOMT1: fine-tuning bHLH transcription factors for optimal anther development

The tapetum,a crucial innermost layer encompassing male reproductive cells within the anther wall,plays a pivotal role in normal pollen development.The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1,a gatekeeper TF in the tapetum.Nevertheless,the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown.In this study,we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089,probably through their K259 site.Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089.Intriguingly,elevated CCoAOMT1 expression resulted in defective pollen development,mirroring the phenotype observed in bhlh010 bhlh089 mutants.Moreover,our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1.Importantly,transgenic plants with the probHLH089::bHLH089^(K259A)construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants.Collectively,these findings emphasize the need to maintain balanced TF homeostasis for male fertility.They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development.

Methylation in hangtaimycin biosynthesis and its antibacterial activities

About two-thirds of small molecule drugs contain methyl group and it plays a very important role in the drug development.So,methyltransferases catalyzing the methylation have always attracted great attention.Hangtaimycin(HTM)is a potent hepatoprotective agent.Previous study showed that its biosynthetic gene cluster contained three methyltransferase domains,but their characteristics in HTM biosynthetic pathway has not been revealed.In this study,we clarified multi-methylations in HTM biosynthesis in vivo.It showed that the two S-adenosylmethionine-dependent methyltransferases(SAM-MTs)of HtmA2(-module 6)-MT domain and HtmB2(-module 18)-MT domain are responsible for the installation of methyl group at C-45 and N-12,respectively,whereas the FK506 methyltransferase(FKMT)type O-methyltransferase of HtmB1(-module 16)-MT domain take care of the methylation at O-21 of HTM.We also reported the antibacterial activities of HTM in this study,and found that it showed activities against M.luteus,B.thuringiensis and A.baumannii with MIC of 4μg/mL,4μg/mL,and 64μg/mL,respectively.

一个新的忽地笑O-甲基转移酶基因的克隆与原核表达

采用RACE技术克隆得到一个新的忽地笑O-甲基转移酶(OMT)基因,命名为LaOMT。LaOMT的cDNA序列为1 379 bp,开放阅读框1 077 bp,预测编码蛋白包含358个氨基酸。序列比对分析发现,LaOMT编码的蛋白序列具有依赖于S-腺苷-L-甲硫氨酸(SAM)的高度保守的结构域,与葡萄、大豆等其他植物OMTs蛋白的相似性为50%左右。将LaOMT基因连接到原核表达载体pET28a上,转化大肠杆菌Rosetta(DE3)的SDS-PAGE电泳结果显示,重组蛋白受异丙基硫代半乳糖苷(IPTG)诱导表达,分子量大小约为45 kDa,与已报导的其他植物的OMTs蛋白大小基本一致。此外,半定量RT-PCR分析结果表明,LaOMT在忽地笑的叶、鳞茎、根和花中均有表达,其中花中的表达量最低。