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.

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.

The Smi-miR858a-SmMYB module regulates tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza.

Tanshinones and phenolic acids are two major classes of bioactive compounds in Salvia miltiorrhiza. Revealing the regulatory mechanism of their biosynthesis is crucial for quality improvement of S. miltiorrhiza medicinal materials. Here we demonstrated that Smi-miR858a-Smi-miR858c, a miRNA family previously known to regulate flavonoid biosynthesis, also played critical regulatory roles in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Overexpression of Smi-miR858a in S. miltiorrhiza plants caused significant growth retardation and tanshinone and phenolic acid reduction. Computational prediction and degradome and RNA-seq analyses revealed that Smi-miR858a could directly cleave the transcripts of SmMYB6, SmMYB97, SmMYB111, and SmMYB112. Yeast one-hybrid and transient transcriptional activity assays showed that Smi-miR858a-regulated SmMYBs, such as SmMYB6 and SmMYB112, could activate the expression of SmPAL1 and SmTAT1 involved in phenolic acid biosynthesis and SmCPS1 and SmKSL1 associated with tanshinone biosynthesis. In addition to directly activating the genes involved in bioactive compound biosynthesis pathways, SmMYB6, SmMYB97, and SmMYB112 could also activate SmAOC2, SmAOS4, and SmJMT2 involved in the biosynthesis of methyl jasmonate, a significant elicitor of plant secondary metabolism. The results suggest the existence of dual signaling pathways for the regulation of Smi-miR858a in bioactive compound biosynthesis in S. miltiorrhiza.

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.

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.

Characteristics of Salvia miltiorrhiza methylome and the regulatory mechanism of DNA methylation in tanshinone biosynthesis.

Salvia miltiorrhiza is a model medicinal plant with significant economic and medicinal value. Its roots produce a group of diterpenoid lipophilic bioactive components, termed tanshinones. Biosynthesis and regulation of tanshinones has attracted widespread interest. However, the methylome of S. miltiorrhiza has not been analysed and the regulatory mechanism of DNA methylation in tanshinone production is largely unknown. Here we report single-base resolution DNA methylomes from roots and leaves. Comparative analysis revealed differential methylation patterns for CG, CHG, and CHH contexts and the association between DNA methylation and the expression of genes and small RNAs. Lowly methylated genes always had higher expression levels and 24-nucleotide sRNAs could be key players in the RdDM pathway in S. miltiorrhiza. DNA methylation variation analysis showed that CHH methylation contributed mostly to the difference. Go enrichment analysis showed that diterpenoid biosynthetic process was significantly enriched for genes with downstream overlapping with hypoCHHDMR in July_root when comparing with those in March_root. Tanshinone biosynthesis-related enzyme genes, such as DXS2, CMK, IDI1, HMGR2, DXR, MDS, CYP76AH1, 2OGD25, and CYP71D373, were less CHH methylated in gene promoters or downstream regions in roots collected in July than those collected in March. Consistently, gene expression was up-regulated in S. miltiorrhiza roots collected in July compared with March and the treatment of DNA methylation inhibitor 5-azacytidine significantly promoted tanshinone production. It suggests that DNA methylation plays a significant regulatory role in tanshinone biosynthesis in S. miltiorrhiza through changing the levels of CHH methylation in promoters or downstreams of key enzyme genes.

Chromosome-level genome assembly of Salvia miltiorrhiza with orange roots uncovers the role of Sm2OGD3 in catalyzing 15,16-dehydrogenation of tanshinones.

Salvia miltiorrhiza is well known for its clinical practice in treating heart and cardiovascular diseases. Its roots, used for traditional Chinese medicine materials, are usually brick-red due to accumulation of red pigments, such as tanshinone IIA and tanshinone I. Here we report a S. miltiorrhiza line (shh) with orange roots. Compared with the red roots of normal S. miltiorrhiza plants, the contents of tanshinones with a single bond at C-15,16 were increased, whereas those with a double bond at C-15,16 were significantly decreased in shh. We assembled a high-quality chromosome-level genome of shh. Phylogenomic analysis showed that the relationship between two S. miltiorrhiza lines with red roots was closer than the relationship with shh. It indicates that shh could not be the mutant of an extant S. miltiorrhiza line with red roots. Comparative genomic and transcriptomic analyses showed that a 1.0 kb DNA fragment was deleted in shh Sm2OGD3m. Complementation assay showed that overexpression of intact Sm2OGD3 in shh hairy roots recovered furan D-ring tanshinone accumulation. Consistently, in vitro protein assay showed that Sm2OGD3 catalyzed the conversion of cyptotanshinone, 15,16-dihydrotanshinone I and 1,2,15,16-tetrahydrotanshinone I into tanshinone IIA, tanshinone I and 1,2-dihydrotanshinone I, respectively. Thus, Sm2OGD3 functions as tanshinone 15,16-dehydrogenase and is a key enzyme in tanshinone biosynthesis. The results provide novel insights into the metabolic network of medicinally important tanshinone compounds.

Chromosome-level genome assembly of Aristolochia contorta provides insights into the biosynthesis of benzylisoquinoline alkaloids and aristolochic acids.

Aristolochic acids (AAs) and their derivatives exist in multiple Aristolochiaceae species which had been or are being used as medicinal materials. During the past decades, AAs have received increasing attention due to their nephrotoxicity and carcinogenecity. Elimination of AAs in medicinal materials using biotechnological approaches is important to improve medication safety. However, it has not been achieved because of the limited information of AA biosynthesis available. Here, we report a high-quality reference-grade genome assembly of the AA-containing vine, Aristolochia contorta. Total size of the assembly is 209.27 Mb, which is assembled into 7 pseudochromosomes. Synteny analysis, Ks distribution and 4DTv suggest absences of whole-genome duplication events in A. contorta after the angiosperm-wide WGD. Based on genomic, transcriptomic and metabolic data, pathways and candidate genes of benzylisoquinoline alkaloid (BIA) and AA biosynthesis in A. contorta were proposed. Five O-methyltransferase genes, including AcOMT1-3, AcOMT5 and AcOMT7, were cloned and functionally characterized. The results provide a high-quality reference genome for AA-containing species of Aristolochiaceae. It lays a solid foundation for further elucidation of AA biosynthesis and regulation and molecular breeding of Aristolochiaceae medicinal materials.

Genome-wide identification and functional characterization of natural antisense transcripts in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most widely used traditional medicines. Natural antisense transcripts (NATs) are a class of long noncoding RNAs that can regulate gene expression. Here, we identified 812 NATs, including 168 cis-NATs and 644 trans-NATs from twelve root, flower, and leaf samples of S. miltiorrhiza using RNA-seq. The expression profiles for 41 of 50 NATs and their sense transcripts (STs) obtained from RNA-Seq were validated using qRT-PCR. The expression profiles of 17 NATs positively correlated with their STs. GO and KEGG pathway analyses mapped the STs for cis-NATs to pathways for biosynthesis of secondary metabolites. We characterized four NATs in detail, including NAT0001, NAT0002, NAT0004, and NAT00023. Their STs are kaurene synthase-like 1 and the homologs of UDP-glucose flavonoid 3-O-glucosyltransferase 6, UDP-glycosyltransferase 90A1, and beta-glucosidase 40, respectively. The first gene is involved in the biosynthesis of bioactive tanshinones, the next two are involved in anthocyanin biosynthesis, whereas the last is involved in phenylpropanoid biosynthesis. Besides, we found seven STs that are potential targets of miRNAs. And we found two miRNAs including miR156a and miR7208, might originate from NATs, NAT0112 and NAT0086. The results suggest that S. miltiorrhiza NATs might interact with STs, produce miRNAs, and be regulated by miRNAs. They potentially play significant regulatory roles in the biosynthesis of bioactive compounds.

Noncoding RNAs in Medicinal Plants and their Regulatory Roles in Bioactive Compound Production.

BACKGROUND: Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and long noncoding RNAs (lncRNAs), play significant regulatory roles in plant development and secondary metabolism and are involved in plant response to biotic and abiotic stresses. They have been intensively studied in model systems and crops for approximately two decades and massive amount of information have been obtained. However, for medicinal plants, ncRNAs, particularly their regulatory roles in bioactive compound biosynthesis, are just emerging as a hot research field. OBJECTIVE: This review aims to summarize current knowledge on herbal ncRNAs and their regulatory roles in bioactive compound production. RESULTS: So far, scientists have identified thousands of miRNA candidates from over 50 medicinal plant species and 11794 lncRNAs from Salvia miltiorrhiza, Panax ginseng, and Digitalis purpurea. Among them, more than 30 miRNAs and five lncRNAs have been predicted to regulate bioactive compound production. CONCLUSION: The regulation may achieve through various regulatory modules and pathways, such as the miR397-LAC module, the miR12112-PPO module, the miR156-SPL module, the miR828-MYB module, the miR858-MYB module, and other siRNA and lncRNA regulatory pathways. Further functional analysis of herbal ncRNAs will provide useful information for quality and quantity improvement of medicinal plants.

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.

Systematic Analysis of Alkaline/Neutral Invertase Genes Reveals the Involvement of Smi-miR399 in Regulation of SmNINV3 and SmNINV4 in Salvia miltiorrhiza.

Alkaline/neutral invertases (NINVs), which irreversibly catalyze the hydrolysis of sucrose into fructose and glucose, play crucial roles in carbohydrate metabolism and plant development. Comprehensive insights into NINV genes are lacking in Salvia miltiorrhiza, a well-known traditional Chinese medicinal (TCM) plant with significant medicinal and economic value. Through genome-wide prediction, nine putative SmNINV genes, termed SmNINV1-SmNINV9, were identified. Integrated analysis of gene structures, sequence features, conserved domains, conserved motifs and phylogenetic trees revealed the conservation and divergence of SmNINVs. The identified SmNINVs were differentially expressed in roots, stems, leaves, flowers, and different root tissues. They also responded to drought, salicylic acid, yeast extract, and methyl jasmonate treatments. More importantly, computational prediction and experimental validation showed that SmNINV3 and SmNINV4 were targets of Smi-miR399, a conserved miRNA previously shown to affect Pi uptake and translocation through the cleavage of PHOSPHATE2 (PHO2). Consistently, analysis of 43 NINV genes and 26 miR399 sequences from Arabidopsis thaliana, Populus trichocarpa, Manihot esculenta, and Solanum lycopersicum showed that various AtNINV, PtNINV, MeNINV, and SlNINV genes were regulated by miR399. It indicates that the miR399-NINV module exists widely in plants. Furthermore, Smi-miR399 also cleaved SmPHO2 transcripts in S. miltiorrhiza, suggesting the complexity of NINVs, PHO2, and miR399 networks.

Transcriptomic analysis reveals potential genes involved in tanshinone biosynthesis in Salvia miltiorrhiza.

Tanshinones are important bioactive components in Salvia miltiorrhiza and mainly accumulate in the periderms of mature roots. Tanshinone biosynthesis is a complicated process, and little is known about the third stage of the pathway. To investigate potential genes that are responsible for tanshinone biosynthesis, we conducted transcriptome profiling analysis of two S. miltiorrhiza cultivars. Differential expression analysis provided 2,149 differentially expressed genes (DEGs) for further analysis. GO and KEGG analysis showed that the DEGs were mainly associated with the biosynthesis of secondary metabolites. Weighted gene coexpression network analysis (WGCNA) was further performed to identify a "cyan" module associated with tanshinone biosynthesis. In this module, 25 cytochromes P450 (CYPs), three 2-oxoglutarate-dependent dioxygenases (2OGDs), one short-chain alcohol dehydrogenases (SDRs) and eight transcription factors were found to be likely involved in tanshinone biosynthesis. Among these CYPs, 14 CYPs have been reported previously, and 11 CYPs were identified in this study. Expression analysis showed that four newly identified CYPs were upregulated upon application of MeJA, suggesting their possible roles in tanshinone biosynthesis. Overall, this study not only identified candidate genes involved in tanshinone biosynthesis but also provided a basis for characterization of genes involved in important active ingredients of other traditional Chinese medicinal plants.

Analysis of the laccase gene family and miR397-/miR408-mediated posttranscriptional regulation in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most commonly used traditional Chinese medicine materials. It contains important bioactive phenolic compounds, such as salvianolic acids, flavonoids and anthocyanins. Elucidation of phenolic compound biosynthesis and its regulatory mechanism is of great significance for S. miltiorrhiza quality improvement. Laccases (LACs) are multicopper-containing enzymes potentially involved in the polymerization of phenolic compounds. So far, little has been known about LAC genes in S. miltiorrhiza. Through systematic investigation of the whole genome sequence and transcriptomes of S. miltiorrhiza, we identified 65 full-length SmLAC genes (SmLAC1-SmLAC65). Phylogenetic analysis showed that 62 of the identified SmLACs clustered with LACs from Arabidopsis and Populus trichocarpa in seven clades (C1-C7), whereas the other three fell into one S. miltiorrhiza-specific clade (C8). All of the deduced SmLAC proteins contain four conserved signature sequences and three typical Cu-oxidase domains, and gene structures of most LACs from S. miltiorrhiza, Arabidopsis and P. trichocarpa were highly conserved, however SmLACs encoding C8 proteins showed distinct intron-exon structures. It suggests the conservation and diversity of plant LACs in gene structures. The majority of SmLACs exhibited tissue-specific expression patterns, indicates manifold functions of SmLACs played in S. miltiorrhiza. Analysis of high-throughput small RNA sequences and degradome data and experimental validation using the 5' RACE method showed that 23 SmLACs were targets of Smi-miR397. Among them, three were also targeted by Smi-miR408. It suggests the significance of miR397 and miR408 in posttranscriptional regulation of SmLAC genes. Our results provide a foundation for further demonstrating the functions of SmLACs in the production of bioactive phenolic compounds in S. miltiorrhiza.

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.

Functional Analysis of Polyprenyl Diphosphate Synthase Genes Involved in Plastoquinone and Ubiquinone Biosynthesis in Salvia miltiorrhiza.

Polyprenyl diphosphate synthase (PPS) plays important roles in the biosynthesis of functionally important plastoquinone (PQ) and ubiquinone (UQ). However, only few plant PPS genes have been functionally characterized. Through genome-wide analysis, two PPS genes, termed SmPPS1 and SmPPS2, were identified from Salvia miltiorrhiza, an economically significant Traditional Chinese Medicine material and an emerging model medicinal plant. SmPPS1 and SmPPS2 belonged to different phylogenetic subgroups of plant trans-long-chain prenyltransferases and exhibited differential tissue expression and light-induced expression patterns. Computational prediction and transient expression assays showed that SmPPS1 was localized in the chloroplasts, whereas SmPPS2 was mainly localized in the mitochondria. SmPPS2, but not SmPPS1, could functionally complement the coq1 mutation in yeast cells and catalyzed the production of UQ-9 and UQ-10. Consistently, both UQ-9 and UQ-10 were detected in S. miltiorrhiza plants. Overexpression of SmPPS2 caused significant UQ accumulation in S. miltiorrhiza transgenics, whereas down-regulation resulted in decreased UQ content. Differently, SmPPS1 overexpression significantly elevated PQ-9 content in S. miltiorrhiza. Transgenic lines showing a down-regulation of SmPPS1 expression exhibited decreased PQ-9 level, abnormal chloroplast and trichome development, and varied leaf bleaching phenotypes. These results suggest that SmPPS1 is involved in PQ-9 biosynthesis, whereas SmPPS2 is involved in UQ-9 and UQ-10 biosynthesis.

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.