Caffeoyl-coenzyme A O-methyltransferase mediates regulation of carbon flux fluctuations during phenylpropenes and lignin biosynthesis in the vegetative organ roots of Asarum sieboldii Miq.

Asarum sieboldii Miq. possesses remarkable medicinal value due to its essential oil enriched with phenylpropenes (e.g., methyleugenol and safrole). Although the biosynthesis of phenylpropenes shares a common pathway with lignin, the regulation mechanisms in carbon flux allocation between them are unclear. This study is the first to genetically verify the carbon flux regulation mechanism in A. sieboldii roots. We regulated the expression of Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT), an essential enzyme in the common pathway, to investigate carbon flux allocation in vegetative organs. Here, the lignin and phenylpropene content fluctuation was analyzed by wet chemistry and GC-MS methods. A bona fide CCoAOMT gene from A. sieboldii was firstly cloned and verified. Preliminary heterologous expression validation in transgenic Arabidopsis thaliana showed that RNAi-induced CCoAOMT down-regulation significantly decreased lignin content by 24% and increased the S/G ratio by 30%; however, AsCCoAOMT over-expression in A. thaliana resulted in a 40% increase in lignin content and a 20% decrease in the S/G ratio when compared to the wild type. Similar trends were noted in homologous transformation in A. sieboldii, although the variations were not conspicuous. Nevertheless, the transgenic A. sieboldii plants displayed substantial differences in the level of phenylpropene compounds methyleugenol and safrole leading to a 168% increase in the methyleugenol/safrole ratio in the over-expression line and a 73% reduction in RNAi-suppression line. These findings suggest that the biosynthesis of phenylpropene constituents methyleugenol and safrole seems to be prioritized over lignin. Furthermore, this study indicated that suppression of AsCCoAOMT resulted in marked root susceptibility to pathogenic fungal disease, implying a significant additional role of CCoAOMT in protecting plant vegetative parts from diseases. Overall, the present study provides important references and suggests that future research should be aimed at elucidating the detailed mechanisms of the carbon flux allocation between phenylpropenes and lignin biosynthesis, as well as the disease resistance competency.

Characteristics and comparative analysis of Mesona chinensis Benth chloroplast genome reveals DNA barcode regions for species identification.

Mesona chinensis Benth (MCB) is an important medicinal and edible plant in Southern China and Southeast Asian countries. Chloroplast (cp) genome is usually used for plant phylogeny, species identification, and chloroplast genetic engineering. To characterize the cp genome and determine the evolutionary position and perform the genetic diversity analysis of MCB, we sequence and characterize the MCB cp genome. The results show that the cp genome of MCB is a single circular molecule with a length of 152,635 bp. It is a typical quadripartite structure, comprising a large single-copy region (LSC, 83,514 bp) and a small single-copy region (SSC, 17,751 bp) separated by two inverted repeat regions (IRs, 51,370 bp). It encodes 129 unique genes, including 84 protein-coding genes (PCGs), 37 transfer RNAs (tRNAs), and 8 ribosomal RNAs (rRNAs). Altogether 127 simple sequence repeats (SSRs) are identified in the MCB cp genome with 86.61% of mononucleotide repeats. Phylogenetic analysis reveals that MCB is most closely related to Ocimum basilicum based on the whole cp genomes. Several highly divergent regions are found, such as trnH_psbA, rps16_trnQ, trnS_trnG, trnE_trnT, psaA_ycf3, rpl32_trnL, ccsA_ndhD, ndhG_ndhI, and rps15_ycf1, which can be proposed for use as DNA barcode regions. Genetic diversity analysis unveils a relatively narrow genetic basis of MCB germplasm resources. Therefore, the innovative breeding of MCB is very urgent and necessary in future research.

Integrated Methylome and Transcriptome Analyses Reveal the Molecular Mechanism by Which DNA Methylation Regulates Kenaf Flowering.

DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) (at concentrations of 0, 100, 200, 400, and 600 µM), and the results showed that pretreatment with 200 µM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 µM 5-azaC (5-azaC200) or with no 5-azaC (control conditions; 5-azaC0). Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 µM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.

5-azacytidine pre-treatment alters DNA methylation levels and induces genes responsive to salt stress in kenaf (Hibiscus cannabinus L.).

Soil salinization is becoming a major threat to the sustainable development of global agriculture. Kenaf is an industrial fiber crop with high tolerance to salt stress and could be used for soil phytoremediation. However, the molecular mechanism of kenaf salt tolerance remains largely unknown. DNA methylation is an important epigenetic modifications phenomena and plays a key role in gene expression regulation under abiotic stress condition. In the present study, the kenaf seedlings were pre-treated or not with 50 µM 5-azacytidine (5-azaC, a DNA methylation inhibitor) and then subjected to different concentrations of NaCl. Results showed that the biomass and antioxidant activities (superoxide dismutase, peroxidase and catalase) of kenaf seedlings pre-treated with 5-azaC were significantly increased, while the contents of superoxide anion (O2-) and malondialdehyde (MDA) were decreased, indicating that 5-azaC pre-treatment could significantly alleviate salt stress injury. Furthermore, the methylation-sensitive amplified polymorphism (MSAP) analysis revealed that DNA methylation level of keanf seedlings pre-treated with 5-azaC significantly decreased. The expression of seven differentially methylated genes responsing to salt stress was significantly changed from real-time fluorescent quantitative (qRT-PCR) analysis. Finally, knocked-down of the l-ascorbate oxidase (L-AAO) gene by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under salt stress. Overall, it was suggested that 5-azaC pre-treatment can significantly improve salt tolerance in kenaf by decreasing ROS content, raising anti-oxidant activities, and regulating DNA methylation and expression of stress-responsive genes.

A comprehensive integrated transcriptome and metabolome analyses to reveal key genes and essential metabolic pathways involved in CMS in kenaf.

KEY MESSAGE: Numbers of critical genes and pathways were found from the levels of transcriptome and metabolome, which were useful information for understanding of kenaf CMS mechanism. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants that leads to the inability to produce or release functional pollen. However, there is lack of comprehensive studies to reveal the molecular basis of CMS occurrence in kenaf. Herein, we performed transcriptome and UPLC-MS-based metabolome analyses in the anthers of a CMS (UG93A) and its maintainer (UG93B) to sort out essential genes and metabolites responding to CMS in kenaf. Transcriptome characterized 7769 differentially expressed genes (DEGs) between these two materials, and pathway enrichment analysis indicated that these DEGs were involved mainly in pentose and glucuronate interconversions, starch and sucrose metabolism, taurine and hypotaurine metabolism. In the metabolome assay, a total of 116 significantly different metabolites (SDMs) were identified between the CMS and its maintainer line, and these SDMs were involved in eight KEGG pathways, including flavone and flavonol biosynthesis, glycerophospholipid metabolism, flavonoid biosynthesis, glycosylphosphatidylinositol-anchor biosynthesi. Integrated analyses of transcriptome and metabolome showed that 50 genes had strong correlation coefficient values (R2 > 0.9) with ten metabolites enriched in six pathways; notably, most genes and metabolites of flavonoid biosynthesis pathways and flavone and flavonol biosynthesis pathways involved in flavonoids biosynthetic pathways were downregulated in CMS compared to those in maintainer. Taken together, the decreased accumulation of flavonoids resulted from the compromised biosynthesis pathways coupled with energy deficiency in the anthers may contribute largely to CMS in UG93A of kenaf.

The complete chloroplast genome sequence of the medicinal plant Sophora tonkinensis.

Sophora tonkinensis belongs to genus Sophora of the Fabaceae family. It is mainly distributed in the ridge and peak regions of limestone areas in western China and has high medicinal value and important ecological functions. Wild populations of S. tonkinensis are in danger and need urgent conservation. Furthermore, wild S. tonkinensis resources are very limited relative to the needs of the market, and many adulterants are present on the market. Therefore, a method for authenticating S. tonkinensis and its adulterants at the molecular level is needed. Chloroplast genomes are valuable sources of genetic markers for phylogenetic analyses, genetic diversity evaluation, and plant molecular identification. In this study, we report the complete chloroplast genome of S. tonkinensis. The circular complete chloroplast genome was 154,644 bp in length, containing an 85,810 bp long single-copy (LSC) region, an 18,321 bp short single-copy (SSC) region and two inverted repeat (IR) regions of 50,513 bp. The S. tonkinensis chloroplast genome comprised 129 genes, including 83 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The structure, gene order and guanine and cytosine (GC) content of the S. tonkinensis chloroplast genome were similar to those of the Sophora alopecuroides and Sophora flavescens chloroplast genomes. A total of 1,760 simple sequence repeats (SSRs) were identified in the chloroplast genome of S. tonkinensis, and most of them (93.1%) were mononucleotides. Moreover, the identified SSRs were mainly distributed in the LSC region, accounting for 60% of the total number of SSRs, while 316 (18%) and 383 (22%) were located in the SSC and IR regions, respectively. Only one complete copy of the rpl2 gene was present at the LSC/IRB boundary, while another copy was absent from the IRA region because of the incomplete structure caused by IR region expansion and contraction. The phylogenetic analysis placed S. tonkinensis in Papilionoideae, sister to S. flavescens, and the genera Sophora and Ammopiptanthus were closely related. The complete genome sequencing and chloroplast genome comparative analysis of S. tonkinensis and its closely related species presented in this paper will help formulate effective conservation and management strategies as well as molecular identification approaches for this important medicinal plant.

Identification and analysis of RNA editing sites in chloroplast transcripts of kenaf (Hibiscus cannabinus L.).

RNA editing is one of the post-transcriptional modification processes and can lead to changes in sequencing and functioning of corresponding proteins and genetic information. To reveal the composition and characteristic of RNA editing of kenaf chloroplast genome, the RNA editing sites in kenaf chloroplast were predicted and identified using bioinformatics and RT-PCR analysis. The prediction results showed a total of 48 editing sites distributed in 22 genes, all of them were C to U conversion leading to amino acid changes. Further analysis of the position of RNA editing sites revealed that except 11 editing sites located at the first codon base, the other editing sites were found at the second codon base. Then four genes were randomly selected to validate the editing sites. Results showed that it was accurate to study the chloroplast RNA editing sites by bioinformatics method accompanied with cloning sequencing. Furthermore, the protein secondary structure and transmembrane domain of ndhD and atpA that had undergone gene editing also changed after editing. This implied that proteins with structural changes may have an impact on kenaf growth. Meanwhile, the differential editing site was found in chloroplast transcripts in kenaf CMS line and its maintainer line, indicating that chloroplast RNA editing could be associated with kenaf CMS. Therefore, the present study laid a foundation to further reveal the biological functioning of chloroplast RNA editing in CMS and its maintainer lines in kenaf.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L.).

BACKGROUND: Histone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet. RESULTS: In this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf. CONCLUSIONS: We have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

Molecular cloning and subcellular localization of six HDACs and their roles in response to salt and drought stress in kenaf (Hibiscus cannabinus L)

BACKGROUND: Histone acetylation is an important epigenetic modification that regulates gene activity in response to stress. Histone acetylation levels are reversibly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The imperative roles of HDACs in gene transcription, transcriptional regulation, growth and responses to stressful environment have been widely investigated in Arabidopsis. However, data regarding HDACs in kenaf crop has not been disclosed yet. RESULTS: In this study, six HDACs genes (HcHDA2, HcHDA6, HcHDA8, HcHDA9, HcHDA19, and HcSRT2) were isolated and characterized. Phylogenetic tree revealed that these HcHDACs shared high degree of sequence homology with those of Gossypium arboreum. Subcellular localization analysis showed that GFP-tagged HcHDA2 and HcHDA8 were predominantly localized in the nucleus, HcHDA6 and HcHDA19 in nucleus and cytosol. The HcHDA9 was found in both nucleus and plasma membranes. Real-time quantitative PCR showed that the six HcHDACs genes were expressed with distinct expression patterns across plant tissues. Furthermore, we determined differential accumulation of HcHDACs transcripts under salt and drought treatments, indicating that these enzymes may participate in the biological process under stress in kenaf. Finally, we showed that the levels of histone H3 and H4 acetylation were modulated by salt and drought stress in kenaf. CONCLUSIONS: We have isolated and characterized six HDACs genes from kenaf. These data showed that HDACs are imperative players for growth and development as well abiotic stress responses in kenaf.

Complete sequence of kenaf (Hibiscus cannabinus) mitochondrial genome and comparative analysis with the mitochondrial genomes of other plants.

Plant mitochondrial (mt) genomes are species specific due to the vast of foreign DNA migration and frequent recombination of repeated sequences. Sequencing of the mt genome of kenaf (Hibiscus cannabinus) is essential for elucidating its evolutionary characteristics. In the present study, single-molecule real-time sequencing technology (SMRT) was used to sequence the complete mt genome of kenaf. Results showed that the complete kenaf mt genome was 569,915 bp long and consisted of 62 genes, including 36 protein-coding, 3 rRNA and 23 tRNA genes. Twenty-five introns were found among nine of the 36 protein-coding genes, and five introns were trans-spliced. A comparative analysis with other plant mt genomes showed that four syntenic gene clusters were conserved in all plant mtDNAs. Fifteen chloroplast-derived fragments were strongly associated with mt genes, including the intact sequences of the chloroplast genes psaA, ndhB and rps7. According to the plant mt genome evolution analysis, some ribosomal protein genes and succinate dehydrogenase genes were frequently lost during the evolution of angiosperms. Our data suggest that the kenaf mt genome retained evolutionarily conserved characteristics. Overall, the complete sequencing of the kenaf mt genome provides additional information and enhances our better understanding of mt genomic evolution across angiosperms.

Analysis of chloroplast differences in leaves of rice isonuclear alloplasmic lines.

The chloroplast being an important organelle of plant cells could possibly be associated with plant cytoplasmic male sterility (CMS). To better understand the correlation between (CMS) and chloroplast, we presented a comprehensive analysis based on the changes of photosynthetic parameters, chloroplasts ultrastructure, soluble sugar and starch content, the relative expression of sugar and starch metabolism genes, and chloroplast genome in rice isonuclear alloplasmic CMS lines at the flowering stage. Leaf gas exchange parameters did not affect by CMS lines (M2BS and M2A), although intercellular CO2 concentration (C i) was influenced in both M2BS and M2A. Ultrastructural observation results indicated that many starch granules were observed in the chloroplast of CMS lines, especially bigger size in M2BS, while few ones in M2B. Only the chloroplasts of M2A contained some additional number of lipoids compared with those of the other two lines (M2B and M2BS). Soluble sugar and starch contents in CMS lines (M2BS and M2A) were significantly higher than those in maintainer line (M2B) (p < 0.01). The relative expression of sugar and starch metabolism genes indicated the imbalance of starch and sugar synthesis and decomposition may lead to accumulation of starch granules and demonstrated the presence of cytoplasmic effects. Moreover, chloroplast genome sequencing results showed similarity in both CMS lines, which revealed different single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) models compared with their maintainer line. Those models were located in psbD, rpoC2, rpl33, psbB, ndhA, ndhH, and intergenic regions. These findings, aligned with the possible association of CMS characteristics with cpDNA and genetically close relationship among both CMS lines, may contribute for future research.