Chromosome-level genome assembly of Prunella vulgaris L. provides insights into pentacyclic triterpenoid biosynthesis.

Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome-level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi-C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome-wide analysis identified 35 932 protein-coding genes (PCGs), of which 59 encode enzymes involved in 2,3-oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and ß-amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris.

Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

Genome-Wide Identification and Characterization of miRNAs and Natural Antisense Transcripts Show the Complexity of Gene Regulatory Networks for Secondary Metabolism in Aristolochia contorta.

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene-enzyme genes, NAT-STs, and NAT-miRNA-target gene-enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds.

Genome-Wide Identification and Functional Analysis of Salvia miltiorrhiza MicroRNAs Reveal the Negative Regulatory Role of Smi-miR159a in Phenolic Acid Biosynthesis.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs in plants. They play critical functions in various biological processes during plant growth and development. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant with significant medicinal, economic, and academic values. In order to elucidate the role of miRNAs in S. miltiorrhiza, six small RNA libraries from mature roots, young roots, stems, mature leaves, young leaves and flowers of S. miltiorrhiza and one degradome library from mixed tissues were constructed. A total of 184 miRNA precursors, generating 137 known and 49 novel miRNAs, were genome-widely identified. The identified miRNAs were predicted to play diversified regulatory roles in plants through regulating 891 genes. qRT-PCR and 5' RLM-RACE assays validated the negative regulatory role of smi-miR159a in SmMYB62, SmMYB78, and SmMYB80. To elucidate the function of smi-miR159a in bioactive compound biosynthesis, smi-miR159a transgenic hairy roots were generated and analyzed. The results showed that overexpression of smi-miR159a caused a significant decrease in rosmarinic acid and salvianolic acid B contents. qRT-PCR analysis showed that the targets of smi-miR159a, including SmMYB62, SmMYB78, and SmMYB80, were significantly down-regulated, accompanied by the down-regulation of SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmTAT3, SmHPPR1, SmRAS, and SmCYP98A14 genes involved in phenolic acid biosynthesis. It suggests that smi-miR159a is a significant negative regulator of phenolic acid biosynthesis in S. miltiorrhiza.

Identification of milRNAs and their target genes in Ganoderma lucidum by high-throughput sequencing and degradome analysis.

MicroRNAs (miRNAs in animals and plants or milRNAs in fungi) are endogenous noncoding RNAs that can regulate gene expression. However, little information is known about milRNAs and their target genes in Ganoderma lucidum. Here, we systematically predicted and characterised the milRNAs and their target genes across the three developmental stages of G. lucidum. A total of 168 unique milRNAs were predicted using a small RNA sequencing method. For them, 1612 target sequences corresponding to 1311 unique genes were predicted by degradome sequencing. We selected 42 predicted milRNAs and performed RT-PCR amplification and Sanger sequencing of the products. Five products were found to have sequences similar to those predicted, confirming the presence of milRNAs in G. lucidum, and demonstrating the difficulty in their validation. Among the 168 milRNAs, 111 were found to be significantly differentially expressed across the three developmental stages (q ≤ 0.05). The expression levels of 12 milRNAs were measured by stem-loop quantitative real-time polymerase chain reaction. Eight of them were in line with the sequencing results (r ≥ 0.9, p ≤ 0.05). These 12 milRNAs and their target genes form 16 milRNA-target gene pairs. The expression profiles of 8 of these 16 miRNA-target pairs were negatively correlated, according to real-time quantitative analysis, whereas the other eight pairs were positively correlated. Furthermore, the results of functional enrichment analysis showed that the target genes of milRNAs mapped to the Gene Ontology terms 'GTP binding' and 'FAD binding' were enriched in specific developmental stages. These target genes were related to the biosynthesis of triterpenes and polysaccharides and lignin degradation pathway in G. lucidum. In summary, this study indicates that milRNAs may play crucial regulatory roles in various biological processes of G. lucidum and open up new avenues for research on milRNAs' biosyntheses and functions in basidiomycetes.

De novo origination of MIRNAs through generation of short inverted repeats in target genes.

MIRNA (MIR) gene origin and early evolutionary processes, such as hairpin precursor sequence origination, promoter activity acquirement and the sequence of these two processes, are fundamental and fascinating subjects. Three models, including inverted gene duplication, spontaneous evolution and transposon transposition, have been proposed for de novo origination of hairpin precursor sequence. However, these models still open to discussion. In addition, de novo origination of MIR gene promoters has not been well investigated. Here, I systematically investigated the origin of evolutionarily young polyphenol oxidase gene (PPO)-targeting MIRs, including MIR1444, MIR058 and MIR12112, and a genomic region termed AasPPO-as-hp, which contained a hairpin-forming sequence. I found that MIR058 precursors and the hairpin-forming sequence of AasPPO-as-hp originated in an ancient PPO gene through forming short inverted repeats. Palindromic-like sequences and imperfect inverted repeats in the ancient PPO gene contributed to initiate the generation of short inverted repeats probably by causing errors during DNA duplication. Analysis of MIR058 and AasPPO-as-hp promoters showed that they originated in the 3'-flanking region of the ancient PPO gene. Promoter activities were gained by insertion of a CAAT-box and multiple-copper-response element (CuRE)-containing miniature inverted-repeat transposable element (MITE) in the upstream of AT-rich TATA-box-like sequence. Gain of promoter activities occurred before hairpin-forming sequence origination. Sequence comparison of MIR1444, MIR058 and MIR12112 promoters showed frequent birth and death of CuREs, indicating copper could be vital for the origination and evolution of PPO-targeting MIRs. Based on the evidence obtained, a novel model for plant MIR origination and evolution is proposed.

SmPPT, a 4-hydroxybenzoate polyprenyl diphosphate transferase gene involved in ubiquinone biosynthesis, confers salt tolerance in Salvia miltiorrhiza.

KEY MESSAGE: SmPPT, which encodes 4-hydroxybenzoate polyprenyl diphosphate transferase involved in ubiquinone biosynthesis, confers salt tolerance to S. miltiorrhiza through enhancing the activities of POD and CAT to scavenge ROS. Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a key electron transporter in the mitochondrial respiratory system. UQ is composed of a benzene quinone ring and a polyisoprenoid side chain. Attachment of polyisoprenoid side chain to the benzene quinone ring is a rate-limiting step catalyzed by 4-hydroxybenzoate polyprenyl diphosphate transferase (PPT). So far, only a few plant PPT-encoding genes have been functionally analyzed. Through genome-wide analysis and subsequent molecular cloning, a PPT-encoding gene, termed SmPPT, was identified from an economically and academically important medicinal model plant, Salvia miltiorrhiza. SmPPT contained many putative cis-elements associated with abiotic stresses in the promoter region and were responsive to PEG-6000 and methyl jasmonate treatments. The deduced SmPPT protein contains the PT_UbiA conserved domain of polyprenyl diphosphate transferase and an N-terminal mitochondria transit peptide. Transient expression assay of SmPPT-GFP fusion protein showed that SmPPT was mainly localized in the mitochondria. SmPPT could functionally complement coq2 mutation and catalyzed UQ6 production in yeast cells. Overexpression of SmPPT increased UQ production and enhanced salt tolerance in S. miltiorrhiza. Under salinity stress conditions, transgenic plants accumulated less H2O2 and malondialdehyde and exhibited higher peroxidase (POD) and catalase (CAT) activities compared with wild-type plants. It indicates that SmPPT confers salt tolerance to S. miltiorrhiza at least partially through enhancing the activities of POD and CAT to scavenge ROS.

The Pentatricopeptide Repeat Gene Family in Salvia miltiorrhiza: Genome-Wide Characterization and Expression Analysis.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants and plays important roles in posttranscriptional regulation. In this study, we combined whole genome sequencing and transcriptomes to systematically investigate PPRs in Salvia miltiorrhiza, which is a well-known material of traditional Chinese medicine and an emerging model system for medicinal plant studies. Among 562 identified SmPPRs, 299 belong to the P subfamily while the others belong to the PLS subfamily. The majority of SmPPRs have only one exon and are localized in the mitochondrion or chloroplast. As many as 546 SmPPRs were expressed in at least one tissue and exhibited differential expression patterns, which indicates they likely play a variety of functions in S. miltiorrhiza. Up to 349 SmPPRs were salicylic acid-responsive and 183 SmPPRs were yeast extract and Ag⁺-responsive, which indicates these genes might be involved in S. miltiorrhiza defense stresses and secondary metabolism. Furthermore, 23 salicylic acid-responsive SmPPRs were co-expressed with phenolic acid biosynthetic enzyme genes only while 16 yeast extract and Ag⁺-responsive SmPPRs were co-expressed with tanshinone biosynthetic enzyme genes only. Two SmPPRs were co-expressed with both phenolic acid and tanshinone biosynthetic enzyme genes. The results provide a useful platform for further investigating the roles of PPRs in S. miltiorrhiza.

Identification and Characterization of Flavonoid Biosynthetic Enzyme Genes in Salvia miltiorrhiza (Lamiaceae).

Flavonoids are a class of important secondary metabolites with a broad spectrum of pharmacological functions. Salviamiltiorrhiza Bunge (Danshen) is a well-known traditional Chinese medicinal herb with a broad diversity of flavonoids. However, flavonoid biosynthetic enzyme genes have not been systematically and comprehensively analyzed in S.miltiorrhiza. Through genome-wide prediction and molecular cloning, twenty six flavonoid biosynthesis-related gene candidates were identified, of which twenty are novel. They belong to nine families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavone synthase (FNS), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), respectively. Analysis of intron/exon structures, features of deduced proteins and phylogenetic relationships revealed the conservation and divergence of S.miltiorrhiza flavonoid biosynthesis-related proteins and their homologs from other plant species. These genes showed tissue-specific expression patterns and differentially responded to MeJA treatment. Through comprehensive and systematic analysis, fourteen genes most likely to encode flavonoid biosynthetic enzymes were identified. The results provide valuable information for understanding the biosynthetic pathway of flavonoids in medicinal plants.

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Genome-wide analysis, molecular cloning and expression profiling reveal tissue-specifically expressed, feedback-regulated, stress-responsive and alternatively spliced novel genes involved in gibberellin metabolism in Salvia miltiorrhiza.

BACKGROUND: Gibberellin (GA), a classical phytohormone, plays significant roles in plant growth and development. It shares the important intermediate diphosphate precursor, GGPP, with the main lipophilic bioactive components, diterpenoid tanshinones in Salvia miltiorrhiza Bunge, one of the most important Traditional Chinese Medicine materials and an emerging model medicinal plant. Analysis of GA metabolism and regulation may help to demonstrate the biological functions of GAs and the crosstalk between GA metabolism and tanshinone biosynthesis in S. miltiorrhiza. However, genes involved in the conversion of ent-kaurene to GAs have not been systematically studied. RESULTS: Through genome-wide prediction and molecular cloning, twenty two candidate gibberellin metabolism pathway genes were systematically identified for the first time. It includes a SmKO, two SmKAOs, six SmGA20oxs, two SmGA3oxs and eleven SmGA2oxs, of which twenty genes are novel. The deduced proteins showed sequence conservation and divergence. Gibberellin metabolism pathway genes exhibited tissue-specific expression patterns and responded differentially to exogenous GA3 treatment, indicating differential regulation of gibberellin metabolism in different tissue types in S. miltiorrhiza. SmKAO1, SmKAO2, SmGA2ox2, and SmGA2ox4-SmGA2ox7 were significantly up-regulated; SmGA20ox2, SmGA3ox1, SmGA2ox1, SmGA2ox8, SmGA2ox10 and SmGA2ox11 were significantly down-regulated; while the responses of many other genes varied among different tissue-types and time-points of GA3 treatment, suggesting the complexity of feedback regulation. Tanshinone biosynthesis-related genes, such as SmCPS1 and SmKSL1, were up-regulated in response to GA3 treatment. Among the 22 identified genes, nine responded to yeast extract and Ag(+)-treatment in S. miltiorrhiza hairy roots. Moreover, tissue-specifically expressed splice variants were identified for SmKO, SmGA20ox3, SmGA2ox3 and SmGA2ox11, of which SmKOv1, SmGA20ox3v and SmGA2ox11v1 were GA3-responsive, suggesting the importance of alternative splicing in regulating GA metabolism. CONCLUSIONS: The results show tissue-specifically expressed, feedback-regulated, stress-responsive and alternatively spliced novel genes and reveal multiple layer regulation of GA metabolism and crosstalk between gibberellin metabolism and tanshinone biosynthesis in S. miltiorrhiza.

Molecular cloning and expression analysis of WRKY transcription factor genes in Salvia miltiorrhiza.

BACKGROUND: WRKY proteins comprise a large family of transcription factors and play important regulatory roles in plant development and defense response. The WRKY gene family in Salvia miltiorrhiza has not been characterized. RESULTS: A total of 61 SmWRKYs were cloned from S. miltiorrhiza. Multiple sequence alignment showed that SmWRKYs could be classified into 3 groups and 8 subgroups. Sequence features, the WRKY domain and other motifs of SmWRKYs are largely conserved with Arabidopsis AtWRKYs. Each group of WRKY domains contains characteristic conserved sequences, and group-specific motifs might attribute to functional divergence of WRKYs. A total of 17 pairs of orthologous SmWRKY and AtWRKY genes and 21 pairs of paralogous SmWRKY genes were identified. Maximum likelihood analysis showed that SmWRKYs had undergone strong selective pressure for adaptive evolution. Functional divergence analysis suggested that the SmWRKY subgroup genes and many paralogous SmWRKY gene pairs were divergent in functions. Various critical amino acids contributed to functional divergence among subgroups were detected. Of the 61 SmWRKYs, 22, 13, 4 and 1 were predominantly expressed in roots, stems, leaves, and flowers, respectively. The other 21 were mainly expressed in at least two tissues analyzed. In S. miltiorrhiza roots treated with MeJA, significant changes of gene expression were observed for 49 SmWRKYs, of which 26 were up-regulated, 18 were down-regulated, while the other 5 were either up-regulated or down-regulated at different time-points of treatment. Analysis of published RNA-seq data showed that 42 of the 61 identified SmWRKYs were yeast extract and Ag(+)-responsive. Through a systematic analysis, SmWRKYs potentially involved in tanshinone biosynthesis were predicted. CONCLUSION: These results provide insights into functional conservation and diversification of SmWRKYs and are useful information for further elucidating SmWRKY functions.

Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that are important regulators of gene expression, and play major roles in plant development and their response to the environment. Root extracts from Panax notoginseng contain triterpene saponins as their principal bioactive constituent, and demonstrate medicinal properties. To investigate the novel and conserved miRNAs in P. notoginseng, three small RNA libraries constructed from 1-, 2-, and 3-year-old roots in which root saponin levels vary underwent high-throughput sequencing. METHODS: P. notoginseng roots, purified from 1-, 2-, and 3-year-old roots, were extracted for RNA, respectively. Three small libraries were constructed and subjected to next generation sequencing. RESULTS: Sequencing of the three libraries generated 67,217,124 clean reads from P. notoginseng roots. A total of 316 conserved miRNAs (belonging to 67 miRNA families and one unclassified family) and 52 novel miRNAs were identified. MIR156 and MIR166 were the largest miRNA families, while miR156i and miR156g showed the highest abundance of miRNA species. Potential miRNA target genes were predicted and annotated using Cluster of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Comparing these miRNAs between root samples revealed 33 that were differentially expressed between 2- and 1-year-old roots (8 increased, 25 decreased), 27 differentially expressed between 3- and 1-year-old roots (7 increased, 20 decreased), and 29 differentially expressed between 3- and 2-year-old roots (8 increased, 21 decreased). Two significantly differentially expressed miRNAs and four miRNAs predicted to target genes involved in the terpenoid backbone biosynthesis pathway were selected and validated by quantitative reverse transcription PCR. Furthermore, the expression patterns of these six miRNAs were analyzed in P. notoginseng roots, stems, and leaves at different developmental stages. CONCLUSIONS: This study identified a large number of P. notoginseng miRNAs and their target genes, functional annotations, and gene expression patterns. It provides the first known miRNA profiles of the P. notoginseng root development cycle.

Identification and characterization of mRNA-like noncoding RNAs in Salvia miltiorrhiza.

MAIN CONCLUSION: Identification and characterization of 5,446 mlncRNAs from Salvia miltiorrhiza showed that the majority of identified mlncRNAs were stress responsive, providing a framework for elucidating mlncRNA functions in S. miltiorrhiza. mRNA-like noncoding RNAs (mlncRNAs) are transcribed by RNA polymerase II and are polyadenylated, capped and spliced. They play important roles in plant development and defense responses. However, there is no information available for mlncRNAs in Salvia miltiorrhiza Bunge, the first Chinese medicinal material entering the international market. To perform a transcriptome-wide identification of S. miltiorrhiza mlncRNAs, we assembled over 8 million RNA-seq reads from GenBank database and 5,624 ESTs from PlantGDB into 44422 unigenes. Using a computational identification pipeline, we identified 5446 S. miltiorrhiza mlncRNA candidates from the assembled unigenes. Of the 5446 mlncRNAs, 2 are primary transcripts of conserved miRNAs, and 2030 can be grouped into 470 families with at least two members in a family. Quantitative real-time PCR analysis of mlncRNAs with at least 900 nt showed that the majority were differentially expressed in roots, stems, leaves and flowers and responsive to methyl jasmonate (MeJA) treatment in S. miltiorrhiza. Analysis of published RNA-seq data showed that a total of 3,044 mlncRNAs were expressed in hairy roots of S. miltiorrhiza and the expression of 1,904 of the 3,044 mlncRNAs was altered by yeast extract and Ag(+) treatment. The results indicate that the majority of mlncRNAs are involved in plant response to stress. This study provides a framework for understanding the roles of mlncRNAs in S. miltiorrhiza.

Genome-wide analysis reveals dynamic changes in expression of microRNAs during vascular cambium development in Chinese fir, Cunninghamia lanceolata.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in the process of plant development. To date, extensive studies of miRNAs have been performed in a few model plants, but few efforts have focused on small RNAs (sRNAs) in conifers because of the lack of reference sequences for their enormous genomes. In this study, Solexa sequencing of three sRNA libraries obtained from dormant, reactivating, and active vascular cambium in Chinese fir (Cunninghamia lanceolata) using tangential cryosectioning identified 20 known miRNA families and 18 novel potential miRNAs, of which nine novel miRNA precursors were validated by RT-PCR and sequencing. More than half of these novel miRNAs displayed stage-specific expression patterns in the vascular cambium. Furthermore, analysing the 103 miRNAs and their predicted targets indicated that about 70% appeared to negatively regulate their targets, of which two target genes involved in the regulation of cambial cell division were validated via RNA ligase-mediated rapid amplification of 5'-cDNA ends (RLM 5'-RACE) and transient co-expression in Nicotiana benthamiana leaves. Interestingly, miRNA156 and miRNA172 may regulate the phase transition in vascular cambium from dormancy to active growth. These results provide new insights into the important regulatory functions of miRNAs in vascular cambium development and wood formation in conifers.

Identification of mRNA-like non-coding RNAs and validation of a mighty one named MAR in Panax ginseng.

Increasing evidence suggests that long non-coding RNAs (lncRNAs) play significant roles in plants. However, little is known about lncRNAs in Panax ginseng C. A. Meyer, an economically significant medicinal plant species. A total of 3,688 mRNA-like non-coding RNAs (mlncRNAs), a class of lncRNAs, were identified in P. ginseng. Approximately 40% of the identified mlncRNAs were processed into small RNAs, implying their regulatory roles via small RNA-mediated mechanisms. Eleven miRNA-generating mlncRNAs also produced siRNAs, suggesting the coordinated production of miRNAs and siRNAs in P. ginseng. The mlncRNA-derived small RNAs might be 21-, 22-, or 24-nt phased and could be generated from both or only one strand of mlncRNAs, or from super long hairpin structures. A full-length mlncRNA, termed MAR (multiple-function-associated mlncRNA), was cloned. It generated the most abundant siRNAs. The MAR siRNAs were predominantly 24-nt and some of them were distributed in a phased pattern. A total of 228 targets were predicted for 71 MAR siRNAs. Degradome sequencing validated 68 predicted targets involved in diverse metabolic pathways, suggesting the significance of MAR in P. ginseng. Consistently, MAR was detected in all tissues analyzed and responded to methyl jasmonate (MeJA) treatment. It sheds light on the function of mlncRNAs in plants.

Genome-wide characterization and comparative analysis of R2R3-MYB transcription factors shows the complexity of MYB-associated regulatory networks in Salvia miltiorrhiza.

BACKGROUND: MYB is the largest plant transcription factor gene family playing vital roles in plant growth and development. However, it has not been systematically studied in Salvia miltiorrhiza, an economically important medicinal plant. RESULTS: Here we report the genome-wide identification and characterization of 110 R2R3-MYBs, the largest subfamily of MYBs in S. miltiorrhiza. The MYB domain and other motifs of SmMYBs are largely conserved with Arabidopsis AtMYBs, whereas the divergence of SmMYBs and AtMYBs also exists, suggesting the conservation and diversity of plant MYBs. SmMYBs and AtMYBs may be classified into 37 subgroups, of which 31 include proteins from S. miltiorrhiza and Arabidopsis, whereas 6 are specific to a species, indicating that the majority of MYBs play conserved roles, while others may exhibit species-specialized functions. SmMYBs are differentially expressed in various tissues of S. miltiorrhiza. The expression profiles are largely consistent with known functions of their Arabidopsis counterparts. The expression of a subset of SmMYBs is regulated by microRNAs, such as miR159, miR319, miR828 and miR858. Based on functional conservation of MYBs in a subgroup, SmMYBs potentially involved in the biosynthesis of bioactive compounds were identified. CONCLUSIONS: A total of 110 R2R3-MYBs were identified and analyzed. The results suggest the complexity of MYB-mediated regulatory networks in S. miltiorrhiza and provide a foundation for understanding the regulatory mechanism of SmMYBs.

Molecular characterization of the SPL gene family in Populus trichocarpa.

BACKGROUND: SPLs, a family of transcription factors specific to plants, play vital roles in plant growth and development through regulation of various physiological and biochemical processes. Although Populus trichocarpa is a model forest tree, the PtSPL gene family has not been systematically studied. RESULTS: Here we report the identification of 28 full-length PtSPLs, which distribute on 14 P. trichocarpa chromosomes. Based on the phylogenetic relationships of SPLs in P. trichocarpa and Arabidopsis, plant SPLs can be classified into 6 groups. Each group contains at least a PtSPL and an AtSPL. The N-terminal zinc finger 1 (Zn1) of SBP domain in group 6 SPLs has four cysteine residues (CCCC-type), while Zn1 of SPLs in the other groups mainly contains three cysteine and one histidine residues (C2HC-type). Comparative analyses of gene structures, conserved motifs and expression patterns of PtSPLs and AtSPLs revealed the conservation of plant SPLs within a group, whereas among groups, the P. trichocarpa and Arabidopsis SPLs were significantly different. Various conserved motifs were identified in PtSPLs but not found in AtSPLs, suggesting the diversity of plant SPLs. A total of 11 pairs of intrachromosome-duplicated PtSPLs were identified, suggesting the importance of gene duplication in SPL gene expansion in P. trichocarpa. In addition, 18 of the 28 PtSPLs, belonging to G1, G2 and G5, were found to be targets of miR156. Consistently, all of the AtSPLs in these groups are regulated by miR156. It suggests the conservation of miR156-mediated posttranscriptional regulation in plants. CONCLUSIONS: A total of 28 full-length SPLs were identified from the whole genome sequence of P. trichocarpa. Through comprehensive analyses of gene structures, phylogenetic relationships, chromosomal locations, conserved motifs, expression patterns and miR156-mediated posttranscriptional regulation, the PtSPL gene family was characterized. Our results provide useful information for evolution and biological function of plant SPLs.

Genome-wide analysis and molecular dissection of the SPL gene family in Salvia miltiorrhiza.

SQUAMOSA promoter binding protein-likes (SPLs) are plant-specific transcription factors playing vital regulatory roles in plant growth and development. There is no information about SPLs in Salvia miltiorrhiza (Danshen), a significant medicinal plant widely used in Traditional Chinese medicine (TCM) for >1,700 years and an emerging model plant for TCM studies. Through genome-wide identification and subsequent molecular cloning, we identified a total 15 SmSPLs with divergent sequence features, gene structures, and motifs. Comparative analysis showed sequence conservation between SmSPLs and their Arabidopsis counterparts. A phylogenetic tree clusters SmSPLs into six groups. Many of the motifs identified commonly exist in a group/subgroup, implying their functional redundancy. Eight SmSPLs were predicted and experimentally validated to be targets of miR156/157. SmSPLs were differentially expressed in various tissues of S. milltiorrhiza. The expression of miR156/157-targeted SmSPLs was increased with the maturation of S. miltiorrhiza, whereas the expression of miR156/157 was decreased, confirming the regulatory roles of miR156/157 in SmSPLs and suggesting the functions of SmSPLs in S. miltiorrhiza development. The expression of miR156/157 was negatively correlated with miR172 during the maturation of S. miltiorrhiza. The results indicate the significance and complexity of SmSPL-, miR156-, and miR172-mediated regulation of developmental timing in S. miltiorrhiza.

Systematic characterization of gene families and functional analysis of PvRAS3 and PvRAS4 involved in rosmarinic acid biosynthesis in Prunella vulgaris.

Prunella vulgaris is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses showed that 17 of them could be involved in RA biosynthesis. In vitro enzymatic assay showed that PvRAS3 catalyzed the condensation of p-coumaroyl-CoA and caffeoyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was higher than caffeoyl-CoA. PvRAS4 catalyzed the condensation of p-coumaroyl-CoA with pHPL and DHPL. Its affinity toward p-coumaroyl-CoA was lower than PvRAS3. UPLC and LC-MS/MS analyses showed the existence of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid in P. vulgaris. Generation and analysis of pvras3 homozygous mutants showed significant decrease of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid and significant increase of DHPL and pHPL. It suggests that PvRAS3 is the main enzyme catalyzing the condensation of acyl donors and acceptors during RA biosynthesis. The role of PvRAS4 appears minor. The results provide significant information for quality control of P. vulgaris medicinal materials.