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Conservation and management of medicinally important plants are among the necessary tasks all over the world. The genus Dracocephalum (Lamiaceae) contains about 186 perennials, or annual herb species that have been used for their medicinal values in different parts of the world as an antihyperlipidemic, analgesic, antimicrobial, antioxidant, as well as anticancer medicine. Producing detailed data on the genetic structure of these species and their response against climate change and human landscape manipulation can be very important for conservation purposes. Therefore, the present study was performed on six geographical populations of two species in the Dracocephalum genus, namely, Dracocephalum kotschyi, and Dracocephalum oligadenium, as well as their inter-specific hybrid population. We carried out, population genetic study, landscape genetics, species modeling, and genetic cline analyses on these plants. We present here, new findings on the genetic structure of these populations, and provide data on both geographical and genetic clines, as well as morphological clines. We also identified genetic loci that are potentially adaptive to the geographical spatial features and genocide conditions. Different species distribution modeling (SDM) methods, used in this work revealed that bioclimatic variables related to the temperature and moisture, play an important role in Dracocephalum population's geographical distribution within IRAN and that due to the presence of some potentially adaptive genetic loci in the studied plants, they can survive well enough by the year 2050 and under climate change. The findings can be used for the protection of these medicinally important plant., (© 2024. The Author(s).)

Menthone-type monoterpenes are the main active ingredients of Schizonepeta tenuifolia Briq. Previous studies have indicated that light intensity influences the synthesis of menthone-type monoterpenes in S. tenuifolia, but the mechanism remains unclear. WRKY transcription factors play a crucial role in plant metabolism, yet their regulatory mechanisms in S. tenuifolia are not well understood. In this study, transcriptome data of S. tenuifolia leaves under different light intensities were analyzed, identifying 57 candidate transcription factors that influence monoterpene synthesis. Among these, 7 members of the StWRKY gene family were identified and mapped onto chromosomes using bioinformatics methods. The physicochemical properties of the proteins encoded by these StWRKY genes, their gene structures, and cis-acting elements were also studied. Comparative genomics and phylogenetic analyses revealed that Sch000013479 is closely related to AaWRKY1, AtWRKY41, and AtWRKY53, and it was designated as StWRKY1. Upon silencing and overexpressing the StWRKY1 transcription factor in S. tenuifolia leaves, changes in the expression of key genes in the menthone-type monoterpene synthesis pathway were observed. Specifically, when StWRKY1 was effectively silenced, the content of (-)-pulegone significantly decreased. These results enhance our understanding of the impact of StWRKYs on monoterpene synthesis in S. tenuifolia and lay the groundwork for further exploration of the regulatory mechanisms involved in the biosynthesis of menthone-type monoterpenes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Obtaining a robust phylogeny proves challenging due to the intricate evolutionary history of species, where processes such as hybridization and incomplete lineage sorting can introduce conflicting signals, thereby complicating phylogenetic inference. In this study, we conducted comprehensive sampling of Elsholtzieae, with a particular focus on its largest genus, Elsholtzia. We utilized 503 nuclear loci and complete plastome sequences obtained from 99 whole-genome sequencing datasets to elucidate the interspecific relationships within the Elsholtzieae. Additionally, we explored various sources of conflicts between gene trees and species trees. Fully supported backbone phylogenies were recovered, and the monophyly of Elsholtzia and Keiskea was not supported. Significant gene tree heterogeneity was observed at numerous nodes, particularly regarding the placement of Vuhuangia and the E. densa clade. Further investigations into potential causes of this discordance revealed that incomplete lineage sorting (ILS), coupled with hybridization events, has given rise to substantial gene tree discordance. Several species, represented by multiple samples, exhibited a closer association with geographical distribution rather than following a strictly monophyletic pattern in plastid trees, suggesting chloroplast capture within Elsholtzieae and providing evidence of hybridization. In conclusion, this study provides phylogenomic insights to untangle taxonomic problems in the tribe Elsholtzieae, especially the genus Elsholtzia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Scrub Mint clade(Lamiaceae) provides a unique system for investigating the evolutionary processes driving diversification in the North American Coastal Plain from both a systematic and biogeographic context. The clade comprisesDicerandra, Conradina, Piloblephis, Stachydeoma, and four species of the broadly defined genus Clinopodium(Mentheae; Lamiaceae), almost all of which are endemic to the North American Eastern Coastal Plain. Most species of this clade are threatened or endangered and restricted to sandhill or a mosaic of scrub habitats. We analyzed relationships in this clade to understand the evolution of the group and identify evolutionary mechanisms acting on the clade, with important implications for conservation. We used a target-capture method to sequence and analyze 238 nuclear loci across all species of scrub mints, reconstructed the phylogeny, and calculated gene tree concordance, gene tree estimation error, and reticulation indices for every node in the tree using ML methods. Phylogenetic networks were used to determine reticulation events. Our nuclear phylogenetic estimates were consistent with previous results, while greatly increasing the robustness of taxon sampling. The phylogeny resolved the full relationship between Dicerandra and Conradina and the less-studied members of the clade (Piloblephis, Stachydeoma, Clinopodium spp.). We found hotspots of gene tree discordance and reticulation throughout the tree, especially in perennial Dicerandra. Several instances of reticulation events were uncovered between annual and perennial Dicerandra, and within the Conradina + allies clade. Incomplete lineage sorting also likely contributed to phylogenetic discordance. These results clarify phylogenetic relationships in the clade and provide insight on important evolutionary drivers in the clade, such as hybridization. General relationships in the group were confirmed, while the large amount of gene tree discordance is likely due to reticulation across the phylogeny., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Terpenoids are important secondary metabolites of plants that possess both pharmacological activity and economic value. Terpene synthases(TPSs) are key enzymes in the synthesis process of terpenoids. In order to investigate the TPS gene family members and their potential functions in Schizonepeta tenuifolia, this study conducted a systematic analysis of the TPS gene family of S. tenuifolia based on the whole genome data of S. tenuifolia using bioinformatics methods. The results revealed 57 StTPS members identified from the genome database of S. tenuifolia. The StTPS family members encoded 285-819 amino acids, with protein molecular weights ranging from 32.75 to 94.11 kDa, all of which were hydrophilic proteins. The StTPS family members were mainly distributed in the cytoplasm and chloroplasts, exhibiting a random and uneven physical localization pattern. Phylogenetic analysis showed that the StTPS genes family were divided into six subgroups, mainly belonging to the TPS-a and TPS-b subfamilies. Promoter analysis predicted that the TPS gene family members could respond to various stressors such as light, abscisic acid, and methyl jasmonate(MeJA). Transcriptome data analysis revealed that most of the TPS genes were expressed in the roots of S. tenuifolia, and qRT-PCR analysis was conducted on genes with high expression in leaves and low expression in roots. Through the analysis of the TPS gene family of S. tenuifolia, this study identified StTPS5, StTPS18, StTPS32, and StTPS45 as potential genes involved in sesquiterpene synthesis of S. tenuifolia. StTPS45 was cloned for the construction of an prokaryotic expression vector, providing a reference for further investigation of the function and role of the TPS gene family in sesquiterpene synthesis.

Glechoma longituba has been frequently used in treating urolithiasis and cholelithiasis due to the presence of flavonoids, which are its major bioactive constituents. However, research on the molecular background of flavonoid biosynthesis in G. longituba is limited. In this study, we used single-molecule real-time combined with next-generation sequencing technologies to construct the complete transcriptome of G. longituba. We identified 404,648 non-redundant transcripts, including 249,697 coding sequences, 197,811 simple sequence repeats, 174,846 long noncoding RNA, and 176,554 coding RNA. Moreover, we functionally annotated 346,218 isoforms (85.56%) and identified 86,528 differentially expressed genes. We also identified 55 non-redundant full-length isoforms related to the flavonoid biosynthetic pathway. Pearson correlation analysis revealed that the expression levels of some key genes of the flavonoid biosynthesis pathway were significantly positively correlated with the flavonoid metabolites. Furthermore, we performed bioinformatics analysis (sequence and structural) of isoform_47029 (encoding flavanone 3-hydroxylase) and isoform_53692 (encoding flavonol synthase) to evaluate their potential biological functions. Finally, we validated gene expression levels of 12 flavonoid-related key enzyme genes using quantitative real-time PCR. Overall, this study provides full-length transcriptome information on G. longituba for the first time and valuable molecular resources for further research on the medicinal properties of this plant., (© 2023 Scandinavian Plant Physiology Society.)

Teak (Tectona grandis Linn. f.) is considered one of the most expensive hardwoods in the world. The dispersion of the species over the years has taken the teak beyond its first sources of diversity and little is known about the genetic origin and genetic variability. Thus, this study aimed to investigate the genetic diversity and genetic population structure existing in a representative teak germplasm bank collection. DNA was extracted from young leaves and each sample were genotyped by whole genome sequencing at 3 giga bases per sample, the sequences are aligned using the genome, and SNPcalls and quality control were made. To study the population structure of the genotypes, Bayesian variational inference was used via fastStructure, the phylogenetic tree was based on the modified Euclidean distance and the clustering by the UPGMA hierarchical method. Genetic diversity was analyzed based on the pairwise genetic divergence (Fst) of Weir and Cockerham. Genotyping by sequencing resulted in a database of approximately 1.4 million of variations SNPs were used for analysis. It was possible to identify four populations with considerable genetic variability between and within them. While the genetic variability in teak is generally known to be narrow, this study confirmed the presence of genetic variability scale in teak, which is contrary to what was initially expected., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The Elsholtzieae, comprising ca. 7 genera and 70 species, is a small tribe of Lamiaceae (mint family). Members of Elsholtzieae are of high medicinal, aromatic, culinary, and ornamentals value. Despite the rich diversity and value of Elsholtzieae, few molecular markers or plastomes are available for phylogenetics. In the present study, we employed high-throughput sequencing to assemble two Mosla plastomes, M. dianthera and M. scabra , for the first time, and compared with other plastomes of Elsholtzieae. The plastomes of Elsholtzieae exhibited a quadripartite structure, ranging in size from 148,288 bp to 152,602 bp. Excepting the absence of the pseudogene rps19 in Elsholtzia densa , the exhaustive tally revealed the presence of 132 genes (113 unique genes). Among these, 85 protein-coding genes (CDS), 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes ( rps19 and ycf1 ) were annotated. Comparative analyses showed that the plastomes of these species have minor variations at the gene level. Notably, the E. eriostchya plastid genome exhibited increased GC content regions in the LSC and SSC, resulting in an increased overall GC content of the entire plastid genome. The E. densa plastid genome displayed modified boundaries due to inverted repeat (IR) contraction. The sequences of CDS and intergenic regions (IGS) with elevated variability were identified as potential molecular markers for taxonomic inquiries within Elsholtzieae. Phylogenetic analysis indicated that four genera formed monophyletic entities, with Mosla and Perilla forming a sister clade. This clade was, in turn, sister to Collinsonia , collectively forming a sister group to Elsholtzia . Both CDS, and CDS + IGS could construct a phylogenetic tree with stronger support. These findings facilitate species identification and DNA barcoding investigations in Elsholtzieae and provide a foundation for further exploration and resource utilization within this tribe.

Biogeographic disjunctions, including intercontinental disjunctions, are frequent across plant lineages and have been of considerable interest to biologists for centuries. Their study has been reinvigorated by molecular dating and associated comparative methods. One of the "classic" disjunction patterns is that between Eastern Asia and North America. It has been speculated that this pattern is the result of vicariance following the sundering of a widespread Acrto-Teritary flora. Subtribe Nepetinae in the mint family (Lamiaceae) is noteworthy because it contains three genera with this disjunction pattern: Agastache, Dracocephalum, and Meehania. These disjunctions are ostensibly the result of three separate events, allowing for concurrent testing of the tempo, origin, and type of each biogeographic event. Using four plastid and four nuclear markers, we estimated divergence times and analyzed the historical biogeography of Nepetinae, including comprehensive sampling of all major clades for the first time. We recover a well-supported and largely congruent phylogeny of Nepetinae between genomic compartments, although several cases of cyto-nuclear discordance are evident. We demonstrate that the three disjunctions are pseudo-congruent, with unidirectional movement from East Asia at slightly staggered times during the late Miocene and early Pliocene. With the possible exception of Meehania, we find that vicariance is likely the underlying driver of these disjunctions. The biogeographic history of Meehania in North America may be best explained by long-distance dispersal, but a more complete picture awaits deeper sampling of the nuclear genome and more advanced biogeographical models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Powdery mildew (PM) is a widespread plant disease that causes significant economic losses in thousands crops of temperate climates, including Lamiaceae species. Multiple scientific studies describe a peculiar form of PM-resistance associated at the inactivation of specific members of the Mildew Locus O (MLO) gene family, referred to as mlo-resistance. The characterization of Lamiaceae MLO genes, at the genomic level, would be a first step toward their potential use in breeding programs. We carried out a genome-wide characterization of the MLO gene family in 11 Lamiaceae species, providing a manual curated catalog of 324 MLO proteins. Evolutionary history and phylogenetic relationships were studied through maximum likelihood analysis and motif patter reconstruction. Our approach highlighted seven different clades diversified starting from an ancestral MLO domain pattern organized in 18 highly conserved motifs. In addition, 74 Lamiaceae putative PM susceptibility genes, clustering in clade V, were identified. Finally, we performed a codon-based evolutionary analysis, revealing a general high level of purifying selection in the eleven Lamiaceae MLO gene families, and the occurrence of few regions under diversifying selection in candidate susceptibility factors. The results of this work may help to address further biological questions concerning MLOs involved in PM susceptibility. In follow-up studies, it could be investigated whether the silencing or loss-of-function mutations in one or more of these candidate genes may lead to PM resistance.

Background: The chloroplast (cp) genome has unique and highly conserved characteristics and is therefore widely used in species identification and classification, as well as to improve the in-depth understanding of plant evolution., Methods: In this study, the cp genomes of 13 Lamiaceae plants in the Tibet Autonomous Region of China were sequenced, assembled and annotated using bioinformatics methods. Phylogenetic trees were constructed to reveal the phylogenetic relationship of related species in the Lamiaceae., Results: The results showed that all 13 cp genomes had a typical four-segment structure, including one large single-copy (LSC) region, one pair of inverted repeat (IR) regions and one small single-copy (SSC) region. The sequence lengths of the 13 cp genomes were between 149,081 bp and 152,312 bp, and the average GC content was 37.6%. These genomes contained 131-133 annotated genes, including 86-88 protein-coding genes, 37-38 tRNA genes, and 8 rRNA genes. A total of 542 SSR loci were detected using MISA software. The repeat types were mostly single-nucleotide repeats, accounting for 61% of simple repeats. A total of 26,328-26,887 codons were detected in 13 cp genomes. According to the RSCU value analysis, the codons mostly ended with A/T. Analysis of IR boundaries showed that the other species were relatively conserved, except for Nepeta laevigata (D. Don) Hand.-Mazz., which differed in gene type and location on both sides of the boundary. By analysing nucleotide diversity, two highly mutated regions located in the LSC and SSC regions were identified in the 13 cp genomes., Conclusions: Using the cp genome of Lycium ruthenicum Murray as the outgroup, 97 cp genomes of the Lamiaceae were used to construct an Maximum Likehood (ML) phylogenetic tree, in which these species were divided into eight major clades, corresponding to eight subfamilies based on morphological classification. The phylogenetic results based on monophyletic relationships were consistent with the morphological classification status at the tribe level., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Our previous study was the first to confirm that the predominant conformation of mitochondrial genome (mitogenome) sequence of Salvia species contains two circular chromosomes. To further understand the organization, variation, and evolution of Salvia mitogenomes, we characterized the mitogenome of Salvia officinalis . The mitogenome of S. officinalis was sequenced using Illumina short reads and Nanopore long reads and assembled using a hybrid assembly strategy. We found that the predominant conformation of the S. officinalis mitogenome also had two circular chromosomes that were 268,341 bp (MC1) and 39,827 bp (MC2) in length. The S. officinalis mitogenome encoded an angiosperm-typical set of 24 core genes, 9 variable genes, 3 rRNA genes, and 16 tRNA genes. We found many rearrangements of the Salvia mitogenome through inter- and intra-specific comparisons. A phylogenetic analysis of the coding sequences (CDs) of 26 common protein-coding genes (PCGs) of 11 Lamiales species and 2 outgroup taxa strongly indicated that the S. officinalis was a sister taxon to S. miltiorrhiza , consistent with the results obtained using concatenated CDs of common plastid genes. The mapping of RNA-seq data to the CDs of PCGs led to the identification of 451 C-to-U RNA editing sites from 31 PCGs of the S. officinalis mitogenome. Using PCR amplification and Sanger sequencing methods, we successfully validated 113 of the 126 RNA editing sites from 11 PCGs. The results of this study suggest that the predominant conformation of the S. officinalis mitogenome are two circular chromosomes, and the stop gain of rpl 5 was found through RNA editing events of the Salvia mitogenome.

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai-Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata . In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS , EF-1α , and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP , TFIIS , and CYP22 were the optimal reference genes in the roots of L. rotata . The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

Background: The plants of the genus Clerodendrum L. have great potential for development as an ornamental and important herbal resource. There is no significant morphological difference among many species of the genus Clerodendrum, which will lead to confusion among the herbs of this genus and ultimately affect the quality of the herbs. The chloroplast genome will contribute to the development of new markers used for the identification and classification of species., Methods and Results: Here, we obtained the complete chloroplast genome sequences of Clerodendrum chinense (Osbeck) Mabberley and Clerodendrum thomsoniae Balf.f. using the next generation DNA sequencing technology. The chloroplast genomes of the two species all encode a total of 112 unique genes, including 80 protein-coding, 28 tRNA, and four rRNA genes. A total of 44-42 simple sequence repeats, 19-16 tandem repeats and 44-44 scattered repetitive sequences were identified. Phylogenetic analyses showed that the nine Clerodendrum species were classified into two clades and together formed a monophyletic group. Selective pressure analyses of 77 protein-coding genes showed that there was no gene under positive selection in the Clerodendrum branch. Analyses of sequence divergence found two intergenic regions: trnH-GUG-psbA, nhdD-psaC, exhibiting a high degree of variations. Meanwhile, there was no hypervariable region identified in protein coding genes. However, the sequence identities of these two intergenic spacers (IGSs) are greater than 99% among some species, which will result in the two IGSs not being used to distinguish Clerodendrum species. Analysis of the structure at the LSC (Large single copy) /IR (Inverted repeat) and SSC (Small single copy)/IR boundary regions showed dynamic changes. The above results showed that the complete chloroplast genomes can be used as a super-barcode to identify these Clerodendrum species. The study lay the foundation for the understanding of the evolutionary process of the genus Clerodendrum., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Perilla frutescens (Lamiaceae) is an important herbal plant with hundreds of bioactive chemicals, among which perillaldehyde and rosmarinic acid are the two major bioactive compounds in the plant. The leaves of red perilla are used as traditional Kampo medicine or food ingredients. However, the medicinal and nutritional uses of this plant could be improved by enhancing the production of valuable metabolites through the manipulation of key enzymes or regulatory genes using genome editing technology. Here, we generated a high-quality genome assembly of red perilla domesticated in Japan. A near-complete chromosome-level assembly of P. frutescens was generated contigs with N50 of 41.5 Mb from PacBio HiFi reads. 99.2% of the assembly was anchored into 20 pseudochromosomes, among which seven pseudochromosomes consisted of one contig, while the rest consisted of less than six contigs. Gene annotation and prediction of the sequences successfully predicted 86,258 gene models, including 76,825 protein-coding genes. Further analysis showed that potential targets of genome editing for the engineering of anthocyanin pathways in P. frutescens are located on the late-stage pathways. Overall, our genome assembly could serve as a valuable reference for selecting target genes for genome editing of P. frutescens., (© The Author(s) 2022. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites., (© 2023. The Author(s).)

Molecular biodiversity surveys have been increasingly applied in hyperdiverse tropical regions as an efficient tool for rapid species assessment of partially undiscovered fauna and flora. This is done by overcoming shortfalls in knowledge or availability of reproductive structures during the sampling period, which often represents a bottleneck for accurate specimens' identification. DNA sequencing technology is intensifying species discovery, and in combination with morphological identification, has been filling gaps in taxonomic knowledge and facilitating species inventories of tropical ecosystems. This study aimed to apply morphological taxonomy and DNA barcoding to assess the occurrence of Lamiaceae species in converted land-use systems (old-growth forest, jungle rubber, rubber, and oil palm) in Sumatra, Indonesia. In this species inventory, we detected 89 specimens of Lamiaceae from 18 species distributed in seven subfamilies from the Lamiaceae group. One third of the species identified in this study lacked sequences in the reference database for at least one of the markers used (matK, rbcL, and ITS). The three loci species-tree recovered a total of 12 out of the 18 species as monophyletic lineages and can be employed as a suitable approach for molecular species assignment in Lamiaceae. However, for taxa with a low level of interspecific genetic distance in the barcode regions used in this study, such as Vitex gamosepala Griff. and V. vestita Wall. ex Walp., or Callicarpa pentandra Roxb. and C. candidans (Burm.f.) Hochr., the use of traditional taxonomy remains indispensable. A change in species composition and decline in abundance is associated with an increase in land-use intensification at the family level (i.e., Lamiaceae), and this tendency might be constant across other plant families. For this reason, the maintenance of forest genetic resources needs to be considered for sustainable agricultural production, especially in hyperdiverse tropical regions. Additionally, with this change in species composition, accurate species identification throughout molecular assignments will become more important for conservation planning., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Halmschlag et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We sequenced the leaf and inflorescence transcriptomes of 10 Elsholtzia species to mine genes related to the volatile terpenoid metabolic pathway. A total of 184.68 GB data and 1,231,162,678 clean reads were obtained from 20 Elsholtzia samples, and 333,848 unigenes with an average length of at least 1440 bp were obtained by Trinity assembly. KEGG pathway analysis showed that there were three pathways related to volatile terpene metabolism: terpenoid backbone biosynthesis (No. ko00900), monoterpenoid biosynthesis (No. ko00902), and sesquiterpenoid and triterpenoid biosynthesis (No. ko00909), with 437, 125, and 121 related unigenes, respectively. The essential oil content and composition in 20 Elsholtzia samples were determined by gas chromatography-mass spectrometry. The results showed that there were obvious interspecific differences among the 10 Elsholtzia species, but there were no significant differences between the different tissues among species. The expression levels of seven candidate genes involved in volatile terpenoid biosynthesis in Elsholtzia were further analyzed by quantitative real-time PCR. The results showed that HMGS had the highest expression among all genes, followed by GGPS4. In addition, there was not a significant correlation between the seven genes and the components with high essential oil contents. Combined with the essential oil components detected in this study, the possible biosynthetic pathway of the characteristic components in Elsholtzia plants was speculated to be a metabolic pathway with geraniol as the starting point and elsholtzione as the end product. Phylogenetic analysis was conducted using the nucleotide sequences of the geranyl diphosphate synthase candidate genes, and the results showed that genes related to the volatile terpenoid biosynthetic pathway may be more suitable gene fragments for resolving the Elsholtzia phylogeny., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Hd-Zip, a unique transcription factor in plant kingdom, influences the growth, development, and secondary metabolism of plants. Hd-zip Ⅳ is thought to play an important role in trichome development of Schizonepeta tenuifolia. This study aims to explore the functions of StHD1 and StHD8 in Hd-zip Ⅳ subfamily in peltate glandular trichome development. To be specific, the expression patterns of the two genes and interaction between the proteins encoded by them were analyzed based on transcriptome sequencing and two-hybrid screening. The subcellular localization was performed and functions of the genes were verified in tobacco and S. tenuifolia. The results showed that StHD1 and StHD8 had high similarity to HD-Zip Ⅳ proteins of other plants and they all had the characteristic conserved domains of HD-Zip Ⅳ subfamily. They were located in the nucleus. The two genes mainly expressed in young tissues and spikes, and StHD1 and StHD8 proteins interacted with each other. The density and length of glandular trichomes increased significantly in tobacco plants with the overexpression of StHD1 and StHD8. Inhibiting the expression of StHD1 and StHD8 by VIGS(virus-induced gene silencing) in S. tenuifolia resulted in the reduction in the density of peltate glandular trichomes, the expression of key genes related to mono-terpene synthesis, and the relative content of limonene and pulegone, the main components of monoterpene. These results suggested that StHD1 and StHD8 of S. tenuifolia formed a complex to regulate glandular trichomes and affect the biosynthesis of monoterpenes.

The northern temperate genus Dracocephalum consists of approximately 70 species mainly distributed in the steppe-desert biomes of Central and West Asia and the alpine region of the Qinghai-Tibetan Plateau (QTP). Previous work has shown that Dracocephalum is not monophyletic and might include Hyssopus and Lallemantia. This study attempts to clarify the phylogenetic relationships, diversification patterns, and the biogeographical history of the three genera (defined as Dracocephalum s.l.). Based on a sampling of 66 taxa comprising more than 80% from extant species of Dracocephalum s.l., morphological, phylogenetic (maximum parsimony, likelihood, and Bayesian inference based on nuclear ITS and ETS, plastid rpl32-trnL, trnL-trnF, ycf1, and ycf1-rps15, and two low-copy nuclear markers AT3G09060 and AT1G09680), molecular dating, diversification, and ancestral range estimation analyses were carried out. Our results demonstrate that both Hyssopus and Lallemantia are embedded within Dracocephalum and nine well-supported clades can be recognized within Dracocephalum s.l. Analyses of divergence times suggest that the genus experienced an early rapid radiation during the middle to late Miocene with major lineages diversifying within a relatively narrow timescale. Ancestral area reconstruction analyses indicate that Dracocephalum s.l. originated in Central and West Asia and southern Siberia, and dispersed from Central and West Asia into the QTP and adjacent areas twice independently during the Pliocene. The aridification of the Asian interior possibly promoted the rapid radiation of Dracocephalum within this region, and the uplift of the QTP appears to have triggered the dispersal and recent rapid diversification of the genus in the QTP and adjacent regions. Combining molecular phylogenetic and morphological evidence, a revised infrageneric classification of Dracocephalum s.l. is proposed, which recognizes nine sections within the genus., (© 2022 Willi Hennig Society.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Patchouli (Pogostemon cablin (Blanco) Benth.), a member of the Lamiaceae family, is an important aromatic plant that has been widely used in medicine and perfumery. Here, we report a 1.94 Gb chromosome-scale assembly of the patchouli genome (contig N50 = 7.97 Mb). The gene annotation reveals that tandem duplication of sesquiterpene biosynthetic genes may be a major contributor to the biosynthesis of patchouli bioactivity components. We further phase the genome into two distinct subgenomes (A and B), and identify a chromosome substitution event that have occurred between them. Further investigations show that a burst of universal LTR-RTs in the A subgenome lead to the divergence between two subgenomes. However, no significant subgenome dominance is detected. Finally, we track the evolutionary scenario of patchouli including whole genome tetraploidization, subgenome divergency, hybridization, and chromosome substitution, which are the key forces to determine the complexity of patchouli genome. Our work sheds light on the evolutionary history of patchouli and offers unprecedented genomic resources for fundamental patchouli research and elite germplasm development., (© 2022. The Author(s).)

Mint ( Mentha L., Lamiaceae) is a strongly scented herb of the family Lamiaceae that is grown mostly by clonal propagation, making it a valuable species for the study of somaclonal variation and its phenotypic consequences. The recent introduction of a few species of mint in South America, followed by a presumably rampant propagation, make this region particularly ideal for studying the extent of somaclonal genetic diversity. Hence, the objective of this work was to offer a preliminary characterization of somaclonal genetically coding diversity of the mint in the northern Andes in order to address the question of whether somaclonal variants may have emerged despite relatively recent introductions in a region where mint is not native. A total of 29 clonally propagated specimens, collected in mint export farms in the province of Antioquia, a major region for mint production in the northwest Andes of Colombia, were genotyped using RNA sequencing (RNA-Seq). SNP calling was carried out from the leaves' transcriptome profiles of each plant by combining the GATK4 and TRINITY protocols, obtaining a total of 2033 loci across 912 transcripts with a minimum read depth of 20X and 4% of missing data. Unsupervised machine learning algorithms considered the K -means, AGNES and UPGMA approaches, all of which suggested three genetic clusters for M. spicata and a unique cluster for M. × piperita . The results indicate that at least two different origins of M. spicata reached the eastern region of the Antioquia province, clonally propagated in the locality ever since for local consumption and export. One of these ancestries had more population structure, possibly due to environmental or anthropological pressures that intervened in the fragmentation of this genetic group or to a higher somaclonal mutation rate. This work offers a first step into the study of the accumulation and transmission of presumably quasi-neutral somatic mutations at coding regions in an herbaceous clonally propagated scented species such as mint, likely favored by an expected population expansion after its Andean introduction. These ad hoc hypotheses warrant further study as part of future research.

In the present study, we depicted the complete mitochondrial genome of a valuable medicinal plant, Vitex rotundifolia . The mitochondrial genome of V. rotundifolia , mapped as a circular molecule, spanned 380,980 bp in length and had a GC content of 45.54%. The complete genome contained 38 protein-coding genes, 19 transfer RNAs (tRNAs), and 3 ribosomal RNAs (rRNAs). We found that there were only 38.73% (147.54 kb), 36.28% (138.23 kb), and 52.22% (198.96 kb) of the homologous sequences in the mitochondrial genome of V. rotundifolia , as compared with the mitochondrial genomes of Scutellaria tsinyunensis , Boea hygrometrica , and Erythranthe lutea , respectively. A multipartite structure mediated by the homologous recombinations of the three direct repeats was found in the V. rotundifolia mitochondrial genome. The phylogenetic tree was built based on 10 species of Lamiales, using the maximum likelihood method. Moreover, this phylogenetic analysis is the first to present the evolutionary relationship of V. rotundifolia with the other species in Lamiales, based on the complete mitochondrial genome.

Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus, and the outgroup Paulownia tomentosa, further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi-iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Background: Clerodendranthus spicatus (Thunb.) C. Y. Wu ex H. W. Li is one of the most important medicines for the treatment of nephrology in the southeast regions of China. To understand the taxonomic classification of Clerodendranthus species and identify species discrimination markers, we sequenced and characterized its chloroplast genome in the current study., Methods and Results: Total genomic DNA were isolated from dried leaves of C. spicatus and sequenced using an Illumina sequencing platform. The data were assembled and annotated by the NOVOPlasty software and CpGAVAS2 web service. The complete chloroplast genome of C. spicatus was 152,155 bp, including a large single-copy region of 83,098 bp, a small single-copy region of 17,665 bp, and a pair of inverted repeat regions of 25,696 bp. The Isoleucine codons are the most abundant, accounting for 4.17% of all codons. The codons of AUG, UUA, and AGA demonstrated a high degree of usage bias. Twenty-eight simple sequence repeats, thirty-six tandem repeats, and forty interspersed repeats were identified. The distribution of the specific rps19, ycf1, rpl2, trnH, psbA genes were analyzed. Analysis of the genetic distance of the intergenic spacer regions shows that ndhG-ndhI, accD-psaI, rps15-ycf1, rpl20-clpP, ccsA-ndhD regions have high K2p values. Phylogenetic analysis showed that C. spicatu is closely related to two Lamiaceae species, Tectona grandis, and Glechoma longituba., Conclusions: In this study, we sequenced and characterized the chloroplast genome of C. spicatus. Phylogenomic analysis has identified species closely related to C. spicatus, which represent potential candidates for the development of drugs improving renal functions., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Hymenocrater longiflorus (surahalala) is a wild plant species with potential pharmaceutical and ornamental interest. To date, the genomics of this plant is unknown and the gene expression profiling of the genes related to its metabolite has never been studied before. In order to study the responses of in vitro-grown surahalala plants to abiotic stresses and the differential expression of the genes related to its essential oils under exogenous proline application; three levels of PEG600 (0, 10, and 20%) and five levels of proline (0, 5, 10, 15, and 20 µm) were combined in the culture media. Thus, water deficit increased oxidants levels and decreased fresh weight of surahalala tissues, whereas addition of proline up to 15 µm was able to relatively compensate the negative effect of water deficit. Contrarily, high proline level (20 µm) had a negative effect on surahalala plants probably due to the stress simulation (nutrition) under high proline concentration. In addition, the best combination for achieving highest essential oils content was 10 µm proline plus 10% PEG. The expressional profiling of the genes TPS27, L3H, TPS2, TPS1, OMT and GDH3 were successfully carried out and their involvement in 1,8-cineole, carvone, α-pinene, thymol, estragole and β-Citronellol biosynthesis, respectively, was verified. In addition, our results indicated that these genes could also be involved in the synthesis of other metabolites under water deficit condition., (© 2022. The Author(s).)

Dragon's head plant (Lallemantia iberica), is a flowering species belongs to the mint family (Lamiaceae). The species contains valuable essential oils, mucilage and oil which are used in pharmaceutical and food industries. Tissue culture is a feasible strategy to attain large-scale production of plantlets with a huge potential to produce plants with superior quality. The objective of this study was to develop a simple and efficient method for regeneration and transformation of L. iberica. To reach this goal, the regeneration ability of various explants including leaf, cotyledonary node, hypocotyl and cotyledon segments was investigated in MS medium supplemented with diverse concentrations of NAA (Naphthalene acetic acid) and BAP (6-Benzyl Amino Purine). According to the results, cotyledonary nodes showed the best regeneration response. The maximum rate of regeneration (and number of induced shoots was achieved in 1 mg l -1 BAP in combination with 0.05 mg l -1 NAA from the cotyledonary nodes. Additionally, through the optimized regeneration technique Agrobacterium-mediated transformation of L. iberica was successfully accomplished. Gene transfer was assessed on leaf samples from regenerated plantlets under a fluorescent microscope to detect the GFP signals. Moreover, transgene integration and its expression were confirmed by PCR and RT-PCR analysis, respectively. The establishment of these efficient regeneration and genetic transformation methods paved the way for further application such as plant improvement, functional analysis and gene editing., (© 2022. The Author(s).)

Background: Salvia macrosiphon is an aromatic perennial species of Lamiaceae family that grows naturally in different parts of Iran. This herb is widely used in folk and modern medicine. Although in Flora Iranica and Flora of Iran, no infraspecific taxonomic rank has been detected for S. macrosiphon, some infraspecific taxonomic ranks have been reported. In the current study, we evaluated the genetic diversity and structure of 11 populations of this species to detect inter and intrapopulation genetic diversity and to survey the possibility of infraspecific taxonomic ranks in this species., Methods and Results: We utilized the modified C-TAB protocol for DNA extraction and amplified the genomes using several SCoT molecular markers. We calculated of genetic diversity and polymorphism parameters using GenAlex 6.4, Geno-Dive ver.2, PopGene, PopART and Structure 2.3.4. The parameters of genetic polymorphism differed between the populations. Moreover, a low rate of gene flow supported a moderate level of population's genetic diversity and differentiation. According to haplotypes network (TCS) analysis, a high level of genetic mutation has occurred among the individuals of some populations leading to high intrapopulation diversity. On the basis of structure analysis and Nei's genetic distance, the examined populations were classified into four genotypes., Conclusions: The clustering pattern of the populations in each group was not related to geographical distance or phytogeography. It seems that the wide geographic distribution, a small gene flow rate and the occurrence of a high level of genetic mutation lead to infraspecific genetic differentiation in the species and we suppose some infraspecific ranks exist for it., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Natural compounds have historically had a wide application in nutrition. Recently, a fundamental role has been identified for essential oils extracted from aromatic plants for their nutritional, antimicrobial, and antioxidant properties, and as food preservatives. In the present study, essential oils (EOs) from ten aromatic plants grown in Calabria (Italy), used routinely to impart aroma and taste to food, were evaluated for their antibacterial activity. This activity was investigated against Escherichia coli strain JM109, and its derived antibiotic-resistant cells selected by growing the strain at low concentrations of ampicillin, ciprofloxacin, and gentamicin by measuring the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Although all the essential oils showed bactericidal activity, those from Clinopodium nepeta , Origanum vulgare , and Foeniculum vulgare displayed the greatest inhibitory effects on the bacterial growth of all cell lines. It is plausible that the antibacterial activity is mediated by epigenetic modifications since the tested essential oils induce methylation both at adenine and cytosine residues in the genomes of most cell lines. This study contributes to a further characterization of the properties of essential oils by shedding new light on the molecular mechanisms that mediate these properties.

Platostoma palustre (Blume) A.J.Paton is an annual herbaceous persistent plant of the Labiatae family. However, there is a lack of genomic data for this plant, which severely restricts its genetic improvement. In this study, we performed genome survey sequencing of P. palustre and developed simple sequence repeat (SSR) markers based on the resulting sequence. K-mer analysis revealed that the assembled genome size was approximately 1.21 Gb. A total of 15,498 SSR motifs were identified and characterized in this study; among them, dinucleotide, and hexanucleotide repeats had the highest and lowest, respectively. Among the dinucleotide repeat motifs, AT/TA repeat motifs were the most abundant, and GC/CG repeat motifs were rather rare, accounting for 44.28% and 0.63%, respectively. Genetic similarity coefficient analysis by the UPMGA methods clustered 12 clones, of P. palustre and related species into two subgroups. These results provide helpful information for further research on P. palustre resources and variety improvements., (© 2022. The Author(s).)

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p -cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p -cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Lucilia sericata is a medical and veterinary important insect species because its larvae feed on tissues of vertebrates including humans. Very few microsatellite makers have been reported from the species to illuminate its genetic variability and population genetic structure., Methods and Results: In this study, L. sericata samples were collected from four different localities in Korea to develop the microsatellite markers to provide basic information on the genetic variability and population genetic structure in Korea of this species. In total, ten new microsatellite markers were sequenced and analyzed. Genetic diversity was performed using these microsatellite markers. The observed heterozygosity varied from 0.205 to 0.824, with an average of 0.546. The expected heterozygosity ranged from 0.579 to 0.886, with an average of 0.804. PIC value varied from 0.553 to 0.876., Conclusions: The markers developed in the present study are expected as informative for estimating genetic diversity of L. sericata., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Phlomis lychnitis is a mostly endemic species of the Iberian Peninsula that frequently hybridizes with the narrow endemic P. crinita subsp. malacitana in southern Spain. Whenever they coexist they form homoploid hybrid zones. Unlike hybridization at the polyploid level, the process of hybridization at the homoploid level is much less well known., Methods and Results: In this study we report the development of 22 microsatellite markers through next-generation sequencing technologies for P. lychnitis. We characterize the genetic diversity for two populations of this species for the 10 markers that resulted to be polymorphic. Further, we check the transferability of these polymorphic markers to one population of P. crinita subsp. malacitana to verify the potential use of these markers for hybridization studies. The values of expected heterozygosity for P. lychnitis were higher than in P. crinita subsp. malacitana, and the three analyzed populations displayed negative values for the inbreeding coefficient which is compatible with the frequent instances of hybridization and introgression between species., Conclusions: This set of polymorphic markers are useful for further studies aiming at a deeper understanding of the homoploid hybrid process between these species. Additionally, this is the first panel of microsatellite markers developed for the genus Phlomis, a genus very rich in endemic species and with medicinal properties that could benefit from the use of these new markers., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Nepeta tenuifolia Briq. (Lamiaceae) is a medicinal plant historically used in the East Asia region to treat cold and fever, and it is currently used as a clinically effective treatment for respiratory diseases. We previously found that monoterpenoids are the dominant volatile secondary metabolites in N. tenuifolia and their biosynthesis occurs in peltate glandular trichomes. To gain an insight into the molecular mechanisms underlying monoterpenoid biosynthesis in N. tenuifolia, we conducted transcriptome sequencing and examined the expression differences in monoterpene molecular pathway-related genes in different tissues and growth stages by qRT-RCR. In total, six p-menthane monoterpene biosynthetic genes in the (+)-menthone pathway were identified and cloned successfully based on transcriptome data. Moreover, the major constituents, including (+)-limonene, (-)-pulegone and (+)-menthone showed greater accumulation in the spikes than in other organs, such as the expression levels of related key enzyme genes. Additionally, the relative expression of pulegone reductase was the highest at 84 days, showing an inverse trend from (-)-pulegone relative content and leading to (+)-menthone accumulation in peltate glandular trichomes. Finished cloning of the gene for limonene 3-hydroxylase in N. tenuifolia (NtL3OH), heterologous expression in yeast, and in vitro assays were performed for functional characterization. Our study provides an important resource for further research of secondary metabolism of monoterpenes in peltate glandular trichomes of N. tenuifolia and other homologous species., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Nowadays, with the development and advancement of next-generation sequencing technologies, a new path has been provided for transcriptomic studies. In this study, the transcriptome of Dracocephalum kotschyi Boiss., as an endemic and endangered plant which is contained a large amount of valuable secondary metabolites with antioxidant and anticancer properties, was sequenced. Then functional annotation and gene ontology analysis for 165,597 assembled transcripts were performed, most were associated with the metabolic pathways. This might be because there are various active biochemical pathways in this plant. Furthermore, after comprehensive transcript annotation, the putative genes involved in the main metabolic pathways of D. kotschyi were identified. Then, the biosynthetic pathway of its valuable methoxylated flavones was proposed. Finally, the accumulations of important methoxylated-flavone metabolites in three different tissues were quantified by HPLC. The relative expression of the genes involved in the proposed pathway was investigated by qRT-PCR, which indicated high expression levels in the bud tissue. The present results may lead to the design strategies to preserve the genetic diversity of endangered D. kotschyi plants and apply the new methods for engineering its valuable methoxylated-flavones pathway., (© 2021. The Author(s).)

Terpenoids are the largest class of natural products with complex structures and extensive bioactivities; their scaffolds are generated by diverse terpenoid synthases (TPSs) from a limited number of isoprenoid diphosphate precursors. Promiscuous TPSs play important roles in the evolution of terpenoid chemodiversity, but they remain largely unappreciated. Here, an extremely promiscuous terpenoid synthase (CcTPS1) of the TPS-b subfamily was cloned and functionally characterized from a leaf-specific transcriptome of the Lamiaceae plant Colquhounia coccinea var. mollis . CcTPS1 is the first sester-/di-/sesqui-/mono-TPS identified from the plant kingdom, accepting C 25 /C 20 /C 15 /C 10 diphosphate substrates to generate a panel of sester-/di-/sesqui-/mono-terpenoids. Engineered Escherichia coli expressing CcTPS1 produced three previously unreported terpenoids (two sesterterpenoids and a diterpenoid) with rare cyclohexane-containing skeletons, along with four sesquiterpenoids and one monoterpenoid. Their structures were elucidated by extensive nuclear magnetic resonance spectroscopy. Nicotiana benthamiana transiently expressing CcTPS1 also produced the diterpenoid and sesquiterpenoids, demonstrating the enzyme's promiscuity in planta . Its highly leaf-specific expression pattern combined with detectable terpenoid products in leaves of C . coccinea var. mollis and N. benthamiana expressing CcTPS1 suggested that CcTPS1 was mainly responsible for diterpenoid and sesquiterpenoid biosynthesis in plants. CcTPS1 expression and the terpenoid products could be induced by methyl jasmonate, suggesting their possible role in plant-environment interaction. CcTPS1 was localized to the cytosol and may differ from mono-TPSs in subcellular compartmentalization and substrate tolerance. These findings will greatly aid our understanding of plant TPS evolution and terpenoid chemodiversity; they also highlight the enormous potential of transcriptome mining and heterologous expression for the exploration of unique enzymes and natural products hidden in plants., (© 2021 The Author(s).)

According to the present knowledge, this is the first report on establishing transformed root cultures of Leonotis nepetifolia after Rhizobium rhizogenes -mediated transformation. The preliminary phytochemical analysis showed differences in the content of phenols and flavonoids in transformed and nontransformed roots. The dominant compounds in the analyzed extracts were (+)-catechin (5464 and 6808 µg/g DW), p -coumaric acid (2549 and 4907 µg/g DW), m -coumaric acid (1508 and 2048 µg/g DW) and rosmarinic acid (1844 and 2643 µg/g DW) for nontransformed (LNNR) and transformed (LNTR4) roots, respectively. Initial biological studies carried out on LNNR, and LNTR4 extracts showed a cytotoxic effect on the A549 lung, HCC1937 breast and leukemia NALM-6 cell lines, antioxidants, as well as repair and protection against DNA damage induced by H 2 O 2 in HUVEC cells. Due to the stronger effect of the LNTR4 root extract, which can be a relatively efficient and cheap source of bioactive secondary metabolites, further biological analyses are needed to discover in detail their potentially valuable biological properties.

Cell wall invertase (CWIN) activity and the expression of the corresponding gene were previously observed to be significantly elevated in a Cu-tolerant population of Elsholtzia haichowensis relative to a non-tolerant population under copper stress. To understand the differences in CWIN gene regulation between the two populations, their CWIN promoter β -glucuronidase (GUS) reporter vectors were constructed. GUS activity was measured in transgenic Arabidopsis in response to copper, sugar, and phytohormone treatments. Under the copper treatment, only the activity of the CWIN promoter from the Cu-tolerant population was slightly increased. Glucose and fructose significantly induced the activity of CWIN promoters from both populations. Among the phytohormone treatments, only salicylic acid induced significantly higher ( p < 0.05) activity of the Cu-tolerant CWIN promoter relative to the non-tolerant promoters. Analysis of 5'-deletion constructs revealed that a 270-bp promoter fragment was required for SA induction of the promoter from the Cu-tolerant population. Comparison of this region in the two CWIN promoters revealed that it had 10 mutation sites and contained CAAT-box and W-box cis -elements in the Cu-tolerant promoter only. This work provides insights into the regulatory role of SA in CWIN gene expression and offers an explanation for differences in CWIN expression between E. haichowensis populations.

Several species of the Lamiaceae family are the primary source of bioactive aromatic oils and secondary metabolites, having broader applications in the cosmetics, pharmaceuticals, food, confectionery and liquor industries. Due to the scarcity of raw materials and high costs of this family's economically vital species, its products often adulterated to cater to the market's high demand. The present study provides a DNA based approach for identifying different species of this family. Henceforth, the performance of three already proposed barcode loci (matK, trnH-psbA and trnL) was examined for their PCR amplification and species recognition efficacy on various Lamiaceae species and cultivars using three different approaches such as pairwise genetic distance method, BLASTn and phylogenetic tree based on maximum likelihood (ML) analysis. Results illustrate that among all the DNA barcoding loci, matK locus can accurately and efficiently distinguish all the studied species followed by trnH-psbA and trnL. Present investigation may help diminish the illegal trade and events of adulteration of medicinally important plants species in genus Mentha, Ocimum and Plectranthus. This investigation will also help fulfil the scarcity of sequences of barcode loci of these species in the NCBI database. Apart from providing a molecular level reference for identifying processed herbal products, this technique also offers a convenient method for species identification and germplasm conservation of the Lamiaceae family.

Mesona chinensis Benth (MCB) is one of the main economic crops in tropical and subtropical areas. To understand the codon usage bias (CUB) in M. chinensis Benth, chloroplast genome is essential to study its genetic law, molecular phylogenetic relationships, and exogenous gene expression. Results showed that the GC content of 53 CDS sequences was 37.95%, and GC1, GC2, and GC3 content were 46.02%, 38.26%, and 29.85%, respectively. The general GC content order was GC1>GC2>GC3. Moreover, the majority of genes had an effective number of codon (ENC) value greater than 40, except ndhE, rps8, and rps18. Correlation analysis results revealed that the GC content was significantly correlated with GC1, GC2, GC3, and ENC. Neutrality plot analysis, ENC-plot analysis, and PR2-plot analysis presented that the CUB of M. chinensis Benth chloroplast genome was mainly affected by mutation and selection. In addition, GGG, GCA, and TCC were found to be the optimal codons. Furthermore, results of cluster analysis and evolutionary tree showed that M. chinensis Benth was closely related to Ocimum basilicum, indicating that there was a certain correlation between the CUB of the chloroplast gene and the genetic relationship of plant species. Overall, the study on the CUB of chloroplast genome laid a basis for genetic modification and phylogenetic research of M. chinensis Benth chloroplast genome.

Background: A robust molecular phylogeny is fundamental for developing a stable classification and providing a solid framework to understand patterns of diversification, historical biogeography, and character evolution. As the sixth largest angiosperm family, Lamiaceae, or the mint family, consitutes a major source of aromatic oil, wood, ornamentals, and culinary and medicinal herbs, making it an exceptionally important group ecologically, ethnobotanically, and floristically. The lack of a reliable phylogenetic framework for this family has thus far hindered broad-scale biogeographic studies and our comprehension of diversification. Although significant progress has been made towards clarifying Lamiaceae relationships during the past three decades, the resolution of a phylogenetic backbone at the tribal level has remained one of the greatest challenges due to limited availability of genetic data., Results: We performed phylogenetic analyses of Lamiaceae to infer relationships at the tribal level using 79 protein-coding plastid genes from 175 accessions representing 170 taxa, 79 genera, and all 12 subfamilies. Both maximum likelihood and Bayesian analyses yielded a more robust phylogenetic hypothesis relative to previous studies and supported the monophyly of all 12 subfamilies, and a classification for 22 tribes, three of which are newly recognized in this study. As a consequence, we propose an updated phylogenetically informed tribal classification for Lamiaceae that is supplemented with a detailed summary of taxonomic history, generic and species diversity, morphology, synapomorphies, and distribution for each subfamily and tribe., Conclusions: Increased taxon sampling conjoined with phylogenetic analyses based on plastome sequences has provided robust support at both deep and shallow nodes and offers new insights into the phylogenetic relationships among tribes and subfamilies of Lamiaceae. This robust phylogenetic backbone of Lamiaceae will serve as a framework for future studies on mint classification, biogeography, character evolution, and diversification.

Availability of comprehensive phylogenetic tree for flowering plants which includes many of the economically important crops and trees is one of the essential requirements of plant biologists for diverse applications. It is the first study on the use of chloroplast genome of 3265 Angiosperm taxa to identify evolutionary relationships among the plant species. Sixty genes from chloroplast genome was concatenated and utilized to generate the phylogenetic tree. Overall the phylogeny was in correspondence with Angiosperm Phylogeny Group (APG) IV classification with very few taxa occupying incongruous position either due to ambiguous taxonomy or incorrect identification. Simple sequence repeats (SSRs) were identified from almost all the taxa indicating the possibility of their use in various genetic analyses. Large proportion (95.6%) of A/T mononucleotide was recorded while the di, tri, tetra, penta and hexanucleotide amounted to less than 5%. Ambiguity of the taxonomic status of Tectona grandis L.f was assessed by comparing the chloroplast genome with closely related Lamiaceae members through nucleotide diversity and contraction/expansion of inverted repeat regions. Although the gene content was highly conserved, structural changes in the genome was evident. Phylogenetic analysis suggested that Tectona could qualify for a subfamily Tectonoideae. Nucleotide diversity in intergenic and genic sequences revealed prominent hyper-variable regions such as, rps16-trnQ, atpH-atpI, psc4-psbJ, ndhF, rpl32 and ycf1 which have high potential in DNA barcoding applications.

Dracocephalum tanguticum and Dracocephalum moldavica are important herbs from Lamiaceae and have great medicinal value. We used the Illumina sequencing technology to sequence the complete chloroplast genome of D. tanguticum and D. moldavica and then conducted de novo assembly. The two chloroplast genomes have a typical quadripartite structure, with the gene's lengths of 82,221 bp and 81,450 bp, large single-copy region's (LSC) lengths of 82,221 bp and 81,450 bp, and small single-copy region's (SSC) lengths of 17,363 bp and 17,066 bp, inverted repeat region's (IR) lengths of 51,370 bp and 51,352 bp, respectively. The GC content of the two chloroplast genomes was 37.80% and 37.83%, respectively. The chloroplast genomes of the two plants encode 133 and 132 genes, respectively, among which there are 88 and 87 protein-coding genes, respectively, as well as 37 tRNA genes and 8 rRNA genes. Among them, the rps2 gene is unique to D. tanguticum , which is not found in D. moldavica . Through SSR analysis, we also found 6 mutation hotspot regions, which can be used as molecular markers for taxonomic studies. Phylogenetic analysis showed that Dracocephalum was more closely related to Mentha ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Junjun Yao et al.)

Background: Plants exhibit wide chemical diversity due to the production of specialized metabolites that function as pollinator attractants, defensive compounds, and signaling molecules. Lamiaceae (mints) are known for their chemodiversity and have been cultivated for use as culinary herbs, as well as sources of insect repellents, health-promoting compounds, and fragrance., Findings: We report the chromosome-scale genome assembly of Callicarpa americana L. (American beautyberry), a species within the early-diverging Callicarpoideae clade of Lamiaceae, known for its metallic purple fruits and use as an insect repellent due to its production of terpenoids. Using long-read sequencing and Hi-C scaffolding, we generated a 506.1-Mb assembly spanning 17 pseudomolecules with N50 contig and N50 scaffold sizes of 7.5 and 29.0 Mb, respectively. In all, 32,164 genes were annotated, including 53 candidate terpene synthases and 47 putative clusters of specialized metabolite biosynthetic pathways. Our analyses revealed 3 putative whole-genome duplication events, which, together with local tandem duplications, contributed to gene family expansion of terpene synthases. Kolavenyl diphosphate is a gateway to many of the bioactive terpenoids in C. americana; experimental validation confirmed that CamTPS2 encodes kolavenyl diphosphate synthase. Syntenic analyses with Tectona grandis L. f. (teak), a member of the Tectonoideae clade of Lamiaceae known for exceptionally strong wood resistant to insects, revealed 963 collinear blocks and 21,297 C. americana syntelogs., Conclusions: Access to the C. americana genome provides a road map for rapid discovery of genes encoding plant-derived agrichemicals and a key resource for understanding the evolution of chemical diversity in Lamiaceae., (© The Author(s) 2020. Published by Oxford University Press.)

Species within the mint family, Lamiaceae, are widely used for their culinary, cultural, and medicinal properties due to production of a wide variety of specialized metabolites, especially terpenoids. To further our understanding of genome diversity in the Lamiaceae and to provide a resource for mining biochemical pathways, we generated high-quality genome assemblies of four economically important culinary herbs, namely, sweet basil (Ocimum basilicum L.), sweet marjoram (Origanum majorana L.), oregano (Origanum vulgare L.), and rosemary (Rosmarinus officinalis L.), and characterized their terpenoid diversity through metabolite profiling and genomic analyses. A total 25 monoterpenes and 11 sesquiterpenes were identified in leaf tissue from the 4 species. Genes encoding enzymes responsible for the biosynthesis of precursors for mono- and sesqui-terpene synthases were identified in all four species. Across all 4 species, a total of 235 terpene synthases were identified, ranging from 27 in O. majorana to 137 in the tetraploid O. basilicum. This study provides valuable resources for further investigation of the genetic basis of chemodiversity in these important culinary herbs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Considering titanium dioxide nanoparticles (TiO 2 NPs) role in plant growth and especially in plant tolerance against abiotic stress, a greenhouse experiment was carried out to evaluate TiO 2 NPs effects (0, 50, 100 and 200 mg L -1 ) on agronomic traits of Moldavian balm (Dracocephalum moldavica L.) plants grown under different salinity levels (0, 50 and 100 mM NaCl). Results demonstrated that all agronomic traits were negatively affected under all salinity levels but application of 100 mg L -1 TiO 2 NPs mitigated these negative effects. TiO 2 NPs application on Moldavian balm grown under salt stress conditions improved all agronomic traits and increased antioxidant enzyme activity compared with plants grown under salinity without TiO 2 NP treatment. The application of TiO 2 NPs significantly lowered H 2 O 2 concentration. In addition, highest essential oil content (1.19%) was obtained in 100 mg L -1 TiO 2 NP-treated plants under control conditions. Comprehensive GC/MS analysis of essential oils showed that geranial, z-citral, geranyl acetate and geraniol were the dominant essential oil components. The highest amounts for geranial, geraniol and z-citral were obtained in 100 mg L -1 TiO 2 NP-treated plants under control conditions. In conclusion, application of 100 mg L -1 TiO 2 NPs could significantly ameliorate the salinity effects in Moldavian balm.

The families Lamiaceae and Verbenaceae comprise several closely related species that possess high morphological synapomorphic traits. Hence, there is a tendency of species misidentification using only the morphological characters. Herein, we evaluated the discriminatory power of the universal DNA barcodes (matK and rbcL) for 53 species spanning the two families. Using these markers, we inferred phylogenetic relationships and conducted species delimitation analysis using four delimitation methods: Automated Barcode Gap Discovery (ABGD), TaxonDNA, Bayesian Poisson Tree Processes (bPTP) and General Mixed Yule Coalescent (GMYC). The phylogenetic reconstruction based on the matK gene resolved the relationships between the families and further suggested the expansion of the Lamiaceae to include some core Verbanaceae genus, e.g., Gmelina . The rbcL marker using the TaxonDNA method displayed high species delimitation resolutions, while the ABGD, GMYC, and bPTP generated different number of Operational Taxonomic Units/genetic clusters. Our results underscored the efficiency of the matK and rbcL genes as reliable markers for resolving phylogenetic relationships and species delimitation of both families, respectively. The current study provides insights into the DNA barcode applications in these families, at the same time contributing to the current understanding of genetic divergence patterns in angiosperms.

Virus-induced gene silencing (VIGS) is a versatile tool for genetic studies that has been applied to a variety of plant species. With the advent of more accessible genomic and transcriptomic technology applied to an increasing range of plants, tools such as VIGS are being adapted to more non-model plants to explore genes relevant to agriculture and chemical discovery. In this protocol, we adapted VIGS technology to target genes in Nepeta cataria (catnip) and Nepeta mussinii (catmint). These plants carry biochemical and economical value for their production of nepetalactone, an iridoid which provokes a strong reaction in both house cats and aphids. We describe a method to target magnesium chelatase subunit H (CHlH), a gene often targeted as a visual marker for VIGS. Furthermore, we describe a method to simultaneously target two genes in a single plant, which aids in the study of genes found in key biochemical steps in the production of nepetalactone. This approach, which was successfully applied in two members of the Lamiaceae family (mint), could be adapted to other members of the mint family with economical and chemical value.

NAC proteins are one of the largest families of plant-specific transcription factors (TFs). They regulate diverse complex biological processes, including secondary xylem differentiation and wood formation. Recent genomic and transcriptomic studies of Tectona grandis L.f. (teak), one of the most valuable hardwood trees in the world, have allowed identification and analysis of developmental genes. In the present work, T. grandis NAC genes were identified and analyzed regarding to their evolution and expression profile during wood formation. We analyzed the recently published T. grandis genome, and identified 130 NAC proteins that are coded by 107 gene loci. These proteins were classified into 23 clades of the NAC family, together with Populus , Eucalyptus, and Arabidopsis. Data on transcript expression revealed specific temporal and spatial expression patterns for the majority of teak NAC genes. RT-PCR indicated expression of VND genes ( Tg11g04450-VND2 and Tg15g08390-VND4 ) related to secondary cell wall formation in xylem vessels of 16-year-old juvenile trees. Our findings open a way to further understanding of NAC transcription factor genes in T. grandis wood biosynthesis, while they are potentially useful for future studies aiming to improve biomass and wood quality using biotechnological approaches., Competing Interests: The authors declare no conflicts of interest.