Genome-wide analysis of the MADS-box gene family in Lonicera japonica and a proposed floral organ identity model.

BACKGROUND: Lonicera japonica Thunb. is widely used in traditional Chinese medicine. Medicinal L. japonica mainly consists of dried flower buds and partially opened flowers, thus flowers are an important quality indicator. MADS-box genes encode transcription factors that regulate flower development. However, little is known about these genes in L. japonica. RESULTS: In this study, 48 MADS-box genes were identified in L. japonica, including 20 Type-I genes (8 Mα, 2 Mß, and 10 Mγ) and 28 Type-II genes (26 MIKCc and 2 MIKC*). The Type-I and Type-II genes differed significantly in gene structure, conserved domains, protein structure, chromosomal distribution, phylogenesis, and expression pattern. Type-I genes had a simpler gene structure, lacked the K domain, had low protein structure conservation, were tandemly distributed on the chromosomes, had more frequent lineage-specific duplications, and were expressed at low levels. In contrast, Type-II genes had a more complex gene structure; contained conserved M, I, K, and C domains; had highly conserved protein structure; and were expressed at high levels throughout the flowering period. Eleven floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in L. japonica. By integrating expression pattern and protein interaction data for these genes, we developed a possible model for floral organ identity determination. CONCLUSION: This study genome-widely identified and characterized the MADS-box gene family in L. japonica. Eleven floral homeotic MADS-box genes were identified and a possible model for floral organ identity determination was also developed. This study contributes to our understanding of the MADS-box gene family and its possible involvement in floral organ development in L. japonica.

Overexpression of a WRKY transcription factor McWRKY57-like from Mentha canadensis L. enhances drought tolerance in transgenic Arabidopsis.

BACKGROUND: Drought has become a major environmental problem affecting crop production. Members of the WRKY family play important roles in plant development and stress responses. However, their roles in mint have been barely explored. RESULTS: In this study, we isolated a drought-inducible gene McWRKY57-like from mint and investigated its function. The gene encodes a group IIc WRKY transcription factor, McWRKY57-like, which is a nuclear protein with a highly conserved WRKY domain and a C2H2 zinc-finger structure, and has transcription factor activity. Its expression levels were examined in different tissues of mint and under the treatment of mannitol, NaCl, abscisic acid, and methyl jasmonate. We found that McWRKY57-like overexpression in Arabidopsis significantly increased drought tolerance. Further studies showed that under drought stress, McWRKY57-like-overexpressing plants had higher chlorophyll, soluble sugar, soluble protein, and proline contents but lower water loss rate and malondialdehyde content than wild-type plants. Moreover, the activities of antioxidant enzymes catalase, superoxide dismutase, and peroxidase were enhanced in McWRKY57-like transgenic plants. Furthermore, qRT-PCR analysis revealed that the drought-related genes AtRD29A, AtRD29B, AtRD20, AtRAB18, AtCOR15A, AtCOR15B, AtKIN2, and AtDREB1A were upregulated in McWRKY57-like transgenic plants than in wild-type Arabidopsis under simulated drought conditions. CONCLUSION: These data demonstrated that McWRKY57-like conferred drought tolerance in transgenic Arabidopsis by regulating plant growth, osmolyte accumulation and antioxidant enzyme activities, and the expression of stress-related genes. The study indicates that McWRKY57-like plays a positive role in drought response in plants.

Application of Miscanthus substrates in the cultivation of Ganoderma lingzhi.

Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.

Characterisation of the Mentha canadensis R2R3-MYB transcription factor gene McMIXTA and its involvement in peltate glandular trichome development.

BACKGROUND: Mentha canadensis L. has important economic value for the production of essential oils, which are synthesised, secreted and stored in peltate glandular trichomes. As a typical multicellular secretory trichome, glandular trichomes are important biological factories for the synthesis of some specialised metabolites. However, little is known about the molecular mechanism of glandular trichome development in M. canadensis. RESULTS: In this study, the R2R3-MYB transcription factor gene McMIXTA was isolated to investigate its function in glandular trichome development. Bioinformatics analysis indicated that McMIXTA belonged to the subgroup 9 R2R3-MYB, with a R2R3 DNA-binding domain and conserved subgroup 9 motifs. A subcellular localisation assay indicated that McMIXTA was localised in the nucleus. Transactivation analysis indicated that McMIXTA was a positive regulator, with transactivation regions located between positions N253 and N307. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that McMIXTA formed a complex with McHD-Zip3, a trichome development-related HD-ZIP IV transcription factor. Overexpression of McMIXTA in Mentha × piperita L. caused an increase in peltate glandular trichomes density of approximately 25% on the leaf abaxial surface. CONCLUSIONS: Our results demonstrated that the subgroup 9 R2R3-MYB transcription factor McMIXTA has a positive effect on regulating peltate glandular trichome development and the MIXTA/HD-ZIP IV complexes might be conserved regulators for glandular trichome initiation. These results provide useful information for revealing the regulatory mechanism of multicellular glandular trichome development.

Comparative transcriptome analysis revealed candidate genes involved in fruiting body development and sporulation in Ganoderma lucidum.

Ganoderma lucidum is an edible mushroom highly regarded in the traditional Chinese medicine. To better understand the molecular mechanisms underlying fruiting body development in G. lucidum, transcriptome analysis based on RNA sequencing was carried out on different developmental stages: mycelium (G1); primordium (G2); young fruiting body (G3); mature fruiting body (G4); fruiting body in post-sporulation stage (G5). In total, 26,137 unigenes with an average length of 1078 bp were de novo assembled. Functional annotation of transcriptomes matched 72.49% of the unigenes to known proteins available in at least one database. Differentially expressed genes (DEGs) were identified between the evaluated stages: 3135 DEGs in G1 versus G2; 120 in G2 versus G3; 3919 in G3 versus G4; and 1012 in G4 versus G5. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs identified in G1 versus G2 revealed that, in addition to global and overview maps, enriched pathways were related to amino acid metabolism and carbohydrate metabolism. In contrast, DEGs identified in G2 versus G3 were mainly assigned to the category of metabolism of amino acids and their derivatives, comprising mostly upregulated unigenes. In addition, highly expressed unigenes associated with the transition between different developmental stages were identified, including those encoding hydrophobins, cytochrome P450s, extracellular proteases, and several transcription factors. Meanwhile, highly expressed unigenes related to meiosis such as DMC1, MSH4, HOP1, and Mek1 were also analyzed. Our study provides important insights into the molecular mechanisms underlying fruiting body development and sporulation in G. lucidum.

A GPAT1 Mutation in Arabidopsis Enhances Plant Height but Impairs Seed Oil Biosynthesis.

Glycerol-3-phosphate acyltransferases (GPATs) play an important role in glycerolipid biosynthesis, and are mainly involved in oil production, flower development, and stress response. However, their roles in regulating plant height remain unreported. Here, we report that Arabidopsis GPAT1 is involved in the regulation of plant height. GUS assay and qRT-PCR analysis in Arabidopsis showed that GPAT1 is highly expressed in flowers, siliques, and seeds. A loss of function mutation in GPAT1 was shown to decrease seed yield but increase plant height through enhanced cell length. Transcriptomic and qRT-PCR data revealed that the expression levels of genes related to gibberellin (GA) biosynthesis and signaling, as well as those of cell wall organization and biogenesis, were significantly upregulated. These led to cell length elongation, and thus, an increase in plant height. Together, our data suggest that knockout of GPAT1 impairs glycerolipid metabolism in Arabidopsis, leading to reduced seed yield, but promotes the biosynthesis of GA, which ultimately enhances plant height. This study provides new evidence on the interplay between lipid and hormone metabolism in the regulation of plant height.

Transcriptome-Wide Identification and Characterization of the JAZ Gene Family in Mentha canadensis L.

Jasmonate ZIM-domain (JAZ) proteins are the crucial transcriptional repressors in the jasmonic acid (JA) signaling process, and they play pervasive roles in plant development, defense, and plant specialized metabolism. Although numerous JAZ gene families have been discovered across several plants, our knowledge about the JAZ gene family remains limited in the economically and medicinally important Chinese herb Mentha canadensis L. Here, seven non-redundant JAZ genes named McJAZ1-McJAZ7 were identified from our reported M. canadensis transcriptome data. Structural, amino acid composition, and phylogenetic analysis showed that seven McJAZ proteins contained the typical zinc-finger inflorescence meristem (ZIM) domain and JA-associated (Jas) domain as conserved as those in other plants, and they were clustered into four groups (A-D) and distributed into five subgroups (A1, A2, B1, B2, and D). Quantitative real-time PCR (qRT-PCR) analysis showed that seven McJAZ genes displayed differential expression patterns in M. canadensis tissues, and preferentially expressed in flowers. Furthermore, the McJAZ genes expression was differentially induced after Methyl jasmonate (MeJA) treatment, and their transcripts were variable and up- or down-regulated under abscisic acid (ABA), drought, and salt treatments. Subcellular localization analysis revealed that McJAZ proteins are localized in the nucleus or cytoplasm. Yeast two-hybrid (Y2H) assays demonstrated that McJAZ1-5 interacted with McCOI1a, a homolog of Arabidopsis JA receptor AtCOI1, in a coronatine-dependent manner, and most of McJAZ proteins could also form homo- or heterodimers. This present study provides valuable basis for functional analysis and exploitation of the potential candidate McJAZ genes for developing efficient strategies for genetic improvement of M. canadensis.

De novo transcriptomic analysis of light-induced flavonoid pathway, transcription factors in the flower buds of Lonicera japonica.

KEY MESSAGE: Transcriptomic analysis of the relationship between gene expression patterns and flavonoid contents in the flower buds of Lonicera japonica under light-induced conditions, especially the flavonoid pathway genes and transcription factors. ABSTRACT: Flos Lonicerae Japonicae (FLJ), the flower buds of Lonicera japonica Thunb., has been used to treat some human diseases including severe respiratory syndromes and hand-foot-and-mouth diseases owing to its putative antibacterial, and antiviral effects. Luteoloside is a flavonoid that is used by the Chinese Pharmacopoeia to evaluate the quality of FLJ. Light is an important environmental factor that affects flavonoid biosynthesis in the flower buds of L. japonica. However, how light triggers increases in flavonoid production remains unclear. To enhance our understanding of the mechanism involved in light-regulated flavonoid biosynthesis, we sequenced the transcriptomes of L. japonica exposed to three different light conditions: 100% light intensity (CK), 50% light intensity (LI50), and 25% light intensity (LI25) using an Illumina HiSeq 4000 System. A total of 77,297 unigenes with an average length of 809 bp were obtained. Among them, 43,334 unigenes (56.06%) could be matched to at least one biomolecular database. Additionally, 4188, 1545 and 1023 differentially expressed genes (DEGs) were identified by comparative transcriptomics LI25-vs-CK, LI50-vs-CK, and LI25-vs-LI50, respectively. Of note, genes known to be involved in flavonoid biosynthesis, such as 4-coumarate coenzyme A ligase (4CL), and chalcone synthase (CHS) were up-regulated. In addition, a total of 1649 transcription factors (TFs) were identified and divided into 58 TF families; 98 TFs exhibited highly dynamic changes in response to light intensity. Quantitative real-time PCR (qRT-PCR) was used to test the expression profiles of the RNA sequencing (RNA-Seq) data. This study offers insight into how transcriptional expression pattern is influenced by light in the flower buds of L. japonica, and will enhance the understanding of molecular mechanisms of flavonoid biosynthesis in response to light in L. japonica.

Ectopic Expression of a R2R3-MYB Transcription Factor Gene LjaMYB12 from Lonicera japonica Increases Flavonoid Accumulation in Arabidopsis thaliana.

Lonicera japonica Thunb. is a widely used medicinal plant and is rich in a variety of active ingredients. Flavonoids are one of the important components in L. japonica and their content is an important indicator for evaluating the quality of this herb. To study the regulation of flavonoid biosynthesis in L. japonica, an R2R3-MYB transcription factor gene LjaMYB12 was isolated and characterized. Bioinformatics analysis indicated that LjaMYB12 belonged to the subgroup 7, with a typical R2R3 DNA-binding domain and conserved subgroup 7 motifs. The transcriptional level of LjaMYB12 was proportional to the total flavonoid content during the development of L. japonica flowers. Subcellular localization analysis revealed that LjaMYB12 localized to the nucleus. Transactivation activity assay indicated that LjaMYB12 was a transcriptional activator. Then, ectopic expression of LjaMYB12 in Arabidopsis could increase PAL activity and flavonoid content and promote transcription of a range of flavonoid biosynthetic genes. Interestingly, the fold changes of downstream genes in the flavonoid biosynthetic pathway were significantly higher than that of the upstream genes, which suggested that LjaMYB12 may have different regulatory patterns for the upstream and downstream pathways of flavonoid biosynthesis. The results provided here will effectively facilitate the study of subgroup 7 MYBs and transcriptional regulation of flavonoid biosynthesis in L. japonica.

Transcriptome Analysis of JA Signal Transduction, Transcription Factors, and Monoterpene Biosynthesis Pathway in Response to Methyl Jasmonate Elicitation in Mentha canadensis L.

Mentha canadensis L. has important economic value for its abundance in essential oils. Menthol is the main component of M. canadensis essential oils, which is certainly the best-known monoterpene for its simple structure and wide applications. However, the regulation of menthol biosynthesis remains elusive in M. canadensis. In this study, transcriptome sequencing of M. canadensis with MeJA treatment was applied to illustrate the transcriptional regulation of plant secondary metabolites, especially menthol biosynthesis. Six sequencing libraries were constructed including three replicates for both control check (CK) and methyl jasmonate (MeJA) treatment and at least 8 Gb clean bases was produced for each library. After assembly, a total of 81,843 unigenes were obtained with an average length of 724 bp. Functional annotation indicated that 64.55% of unigenes could be annotated in at least one database. Additionally, 4430 differentially expressed genes (DEGs) with 2383 up-regulated and 2047 down-regulated transcripts were identified under MeJA treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that "Monoterpenoid biosynthesis" was one of the most significantly enriched pathways in metabolism. Subsequently, DEGs involved in JA signal transduction, transcription factors, and monoterpene biosynthesis were analyzed. 9 orthologous genes involved in menthol biosynthesis were also identified. This is the first report of a transcriptome study of M. canadensis and will facilitate the studies of monoterpene biosynthesis in the genus Mentha.

Identification of the mulberry genes involved in ethylene biosynthesis and signaling pathways and the expression of MaERF-B2-1 and MaERF-B2-2 in the response to flooding stress.

The phytohormone ethylene is essential to plant growth and development. It plays crucial roles in responses to biotic and abiotic stress. The mulberry tree is an important crop plant in countries in which people rear silkworms for silk production. The availability of the mulberry genome has made it possible to identify mulberry genes involved in ethylene biosynthesis and signal pathways. A total of 145 mulberry genes were identified by both homology-based and hidden Markov model (HMM) search, including 29 genes associated with ethylene biosynthesis and 116 genes in the AP2/ERF family. Studies on gene structure have provided a genetic basis for understanding the functions of these genes. The differences in gene expression were also observed in different tissues. The expression of two mulberry genes in the AP2/ERF family, MaERF-B2-1 and MaERF-B2-2, was found to be associated with the response to flooding stress.

Cloning and expression analyses of the anthocyanin biosynthetic genes in mulberry plants.

Anthocyanins are natural food colorants produced by plants that play important roles in their growth and development. Mulberry fruits are rich in anthocyanins, which are the most important active components of mulberry and have many potentially beneficial effects on human health. The study of anthocyanin biosynthesis will bring benefits for quality improvement and industrial exploration of mulberry fruits. In the present study, nine putative genes involved in anthocyanin biosynthesis in mulberry plants were identified and cloned. Sequence analysis revealed that the mulberry anthocyanin biosynthetic genes were conserved and had counterparts in other plants. Spatial transcriptional analysis showed detectable expression of eight of these genes in different tissues. The results of expression and UPLC analyses in two mulberry cultivars with differently colored fruit indicated that anthocyanin concentrations correlated with the expression levels of genes associated with anthocyanin biosynthesis including CHS1, CHI, F3H1, F3'H1, and ANS during the fruit ripening process. The present studies provide insight into anthocyanin biosynthesis in mulberry plants and may facilitate genetic engineering for improvement of the anthocyanin content in mulberry fruit.

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry (Morus notabilis).

Jasmonate (JA) is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry (Morus notabilis) in conjunction with genome sequencing of silkworm (Bombyx mori) provides an opportunity to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophytodienoic acid reductase (OPRs) and jasmonate ZIM-domain (JAZs) was performed and the results indicated that the mulberry genome contains a relatively small number of JA biosynthetic and signaling pathway genes. A gene encoding an important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information will facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.

Genome-wide identification and expression analyses of cytochrome P450 genes in mulberry (Morus notabilis).

Cytochrome P450s play critical roles in the biosynthesis of physiologically important compounds in plants. These compounds often act as defense toxins to prevent herbivory. In the present study, a total of 174 P450 genes of mulberry (Morus notabilis C.K.Schn) were identified based on bioinformatics analyses. These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner. These genes were compared to the P450 genes in Arabidopsis thaliana. Families CYP80, CYP92, CYP728, CYP733, CYP736, and CYP749 were found to exist in mulberry, and they may play important roles in the biosynthesis of mulberry secondary metabolites. Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids, other secondary metabolites, xenobiotics, amino acids, cofactors, vitamins, terpenoids, and polyketides. These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes.

The non-specific lipid transfer protein McLTPII.9 of Mentha canadensis is involved in peltate glandular trichome density and volatile compound metabolism.

Mentha canadensis L. is an important spice crop and medicinal herb with high economic value. The plant is covered with peltate glandular trichomes, which are responsible for the biosynthesis and secretion of volatile oils. Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family involved in various plant physiological processes. Here, we cloned and identified a non-specific lipid transfer protein gene (McLTPII.9) from M. canadensis, which may positively regulate peltate glandular trichome density and monoterpene metabolism. McLTPII.9 was expressed in most M. canadensis tissues. The GUS signal driven by the McLTPII.9 promoter in transgenic Nicotiana tabacum was observed in stems, leaves, and roots; it was also expressed in trichomes. McLTPII.9 was associated with the plasma membrane. Overexpression of McLTPII.9 in peppermint (Mentha piperita. L) significantly increased the peltate glandular trichome density and total volatile compound content compared with wild-type peppermint; it also altered the volatile oil composition. In McLTPII.9-overexpressing (OE) peppermint, the expression levels of several monoterpenoid synthase genes and glandular trichome development-related transcription factors-such as limonene synthase (LS), limonene-3-hydroxylase (L3OH), geranyl diphosphate synthase (GPPS), HD-ZIP3, and MIXTA-exhibited varying degrees of alteration. McLTPII.9 overexpression resulted in both a change in expression of genes for terpenoid biosynthetic pathways which corresponded with an altered terpenoid profile in OE plants. In addition, peltate glandular trichome density was altered in the OE plants as well as the expression of genes for transcription factors that were shown to be involved in trichome development in plants.

Transcriptome Analysis of Light-Regulated Monoterpenes Biosynthesis in Leaves of Mentha canadensis L.

Light is a key environmental aspect that regulates secondary metabolic synthesis. The essential oil produced in mint (Mentha canadensis L.) leaves is used widely in the aromatics industry and in medicine. Under low-light treatment, significant reductions in peltate glandular trichome densities were observed. GC-MS analysis showed dramatically reduced essential oil and menthol contents. Light affected the peltate glandular trichomes' development and essential oil yield production. However, the underlying mechanisms of this regulation were elusive. To identify the critical genes during light-regulated changes in oil content, following a 24 h darkness treatment and a 24 h recovery light treatment, leaves were collected for transcriptome analysis. A total of 95,579 unigenes were obtained, with an average length of 754 bp. About 56.58% of the unigenes were annotated using four public protein databases: 10,977 differentially expressed genes (DEGs) were found to be involved in the light signaling pathway and monoterpene synthesis pathway. Most of the TPs showed a similar expression pattern: downregulation after darkness treatment and upregulation after the return of light. In addition, the genes involved in the light signal transduction pathway were analyzed. A series of responsive transcription factors (TFs) were identified and could be used in metabolic engineering as an effective strategy for increasing essential oil yields.

Characterization of the Glehnia littoralis Non-specific Phospholipase C Gene GlNPC3 and Its Involvement in the Salt Stress Response.

Glehnia littoralis is a medicinal halophyte that inhabits sandy beaches and has high ecological and commercial value. However, the molecular mechanism of salt adaptation in G. littoralis remains largely unknown. Here, we cloned and identified a non-specific phospholipase C gene (GlNPC3) from G. littoralis, which conferred lipid-mediated signaling during the salt stress response. The expression of GlNPC3 was induced continuously by salt treatment. Overexpression of GlNPC3 in Arabidopsis thaliana increased salt tolerance compared to wild-type (WT) plants. GlNPC3-overexpressing plants had longer roots and higher fresh and dry masses under the salt treatment. The GlNPC3 expression pattern revealed that the gene was expressed in most G. littoralis tissues, particularly in roots. The subcellular localization of GlNPC3 was mainly at the plasma membrane, and partially at the tonoplast. GlNPC3 hydrolyzed common membrane phospholipids, such as phosphotidylserine (PS), phosphoethanolamine (PE), and phosphocholine (PC). In vitro enzymatic assay showed salt-induced total non-specific phospholipase C (NPC) activation in A. thaliana GlNPC3-overexpressing plants. Plant lipid profiling showed a significant change in the membrane-lipid composition of A. thaliana GlNPC3-overexpressing plants compared to WT after the salt treatment. Furthermore, downregulation of GlNPC3 expression by virus-induced gene silencing in G. littoralis reduced the expression levels of some stress-related genes, such as SnRK2, P5SC5, TPC1, and SOS1. Together, these results indicated that GlNPC3 and GlNPC3-mediated membrane lipid change played a positive role in the response of G. littoralis to a saline environment.

Correction: Chen et al. Genome-Wide Analysis of Terpene Synthase Gene Family in Mentha longifolia and Catalytic Activity Analysis of a Single Terpene Synthase. Genes 2021, 12, 518.

The authors have requested that the following changes be made to their paper [...].

Genome-Wide Analysis of Terpene Synthase Gene Family in Mentha longifolia and Catalytic Activity Analysis of a Single Terpene Synthase.

Terpenoids are a wide variety of natural products and terpene synthase (TPS) plays a key role in the biosynthesis of terpenoids. Mentha plants are rich in essential oils, whose main components are terpenoids, and their biosynthetic pathways have been basically elucidated. However, there is a lack of systematic identification and study of TPS in Mentha plants. In this work, we genome-widely identified and analyzed the TPS gene family in Mentha longifolia, a model plant for functional genomic research in the genus Mentha. A total of 63 TPS genes were identified in the M. longifolia genome sequence assembly, which could be divided into six subfamilies. The TPS-b subfamily had the largest number of genes, which might be related to the abundant monoterpenoids in Mentha plants. The TPS-e subfamily had 18 members and showed a significant species-specific expansion compared with other sequenced Lamiaceae plant species. The 63 TPS genes could be mapped to nine scaffolds of the M. longifolia genome sequence assembly and the distribution of these genes is uneven. Tandem duplicates and fragment duplicates contributed greatly to the increase in the number of TPS genes in M. longifolia. The conserved motifs (RR(X)8W, NSE/DTE, RXR, and DDXXD) were analyzed in M. longifolia TPSs, and significant differentiation was found between different subfamilies. Adaptive evolution analysis showed that M. longifolia TPSs were subjected to purifying selection after the species-specific expansion, and some amino acid residues under positive selection were identified. Furthermore, we also cloned and analyzed the catalytic activity of a single terpene synthase, MlongTPS29, which belongs to the TPS-b subfamily. MlongTPS29 could encode a limonene synthase and catalyze the biosynthesis of limonene, an important precursor of essential oils from the genus Mentha. This study provides useful information for the biosynthesis of terpenoids in the genus Mentha.

Selection and Validation of Reference Genes for Normalisation of Gene Expression in Glehnia littoralis.

Glehnia littoralis is an important medicinal halophyte-the dried root of which is used as Chinese herbal medicine. However, the use, selection and stability of reference genes are rarely verified in studies of G. littoralis, which hampers investigation of its salt tolerance and metabolism. In this study, we selected 13 candidate reference genes from the transcriptome data of G. littoralis-serine/threonine-protein phosphatase PP2A (PP2A), polyubiquitin 10 (UBQ10), actin (ACT), elongation factor 1-α (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-tubulin (α-TUB), ß-tubulin (ß-TUB), polypyrimidine tract-binding protein 1 (PTBP1), expressed protein 1 (EXP1), expressed protein 2 (EXP2), TIP41-like (TIP41), SAND family (SAND), and cyclophilin 2 (CYP2), and used qRT-PCR to analyse their expression levels in roots of G. littoralis treated with NaCl, polyethylene glycol (PEG), abscisic acid (ABA), and methyl jasmonate (MeJA), as well as in various organs of G. littoralis. The ΔCt, geNorm, NormFinder, and BestKeeper algorithms were used to assess the expression stability of the candidate reference genes and the results were then used to generate a comprehensive rank list with the RankAggreg R package. The most stable reference genes for normalisation were EXP1 and PP2A in response to NaCl, EXP2 and PP2A in response to ABA, CYP2 and α-TUB in response to MeJA, and ACT and EXP1 in the PEG and the organ subsets. GAPDH, ß-TUB, and UBQ10 exhibited low stability and so were unsuitable for normalisation. This study is the first systematic analysis of candidate reference genes in G. littoralis and will facilitate further investigation of normalisation of gene expression in G. littoralis.