microRNAs (miRNAs) play vital regulatory roles in many organisms through direct cleavage of transcripts, translational repression, or chromatin modification. Identification of miRNAs has been carried out in various plant species. However, no information is available for miRNAs from Panax ginseng, an economically significant medicinal plant species. Using the next generation high-throughput sequencing technology, we obtained 13,326,328 small RNA reads from the roots, stems, leaves and flowers of P. ginseng. Analysis of these small RNAs revealed the existence of a large, diverse and highly complicated small RNA population in P. ginseng. We identified 73 conserved miRNAs, which could be grouped into 33 families, and 28 non-conserved ones belonging to 9 families. Characterization of P. ginseng miRNA precursors revealed many features, such as production of two miRNAs from distinct regions of a precursor, clusters of two precursors in a transcript, and generation of miRNAs from both sense and antisense transcripts. It suggests the complexity of miRNA production in P. gingseng. Using a computational approach, we predicted for the conserved and non-conserved miRNA families 99 and 31 target genes, respectively, of which eight were experimentally validated. Among all predicted targets, only about 20% are conserved among various plant species, whereas the others appear to be non-conserved, indicating the diversity of miRNA functions. Consistently, many miRNAs exhibited tissue-specific expression patterns. Moreover, we identified five dehydration- and ten heat-responsive miRNAs and found the existence of a crosstalk among some of the stress-responsive miRNAs. Our results provide the first clue to the elucidation of miRNA functions in P. ginseng.

Background

Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date.

Results

To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated.

Conclusions

We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.

Background

MicroRNAs (miRNAs) play key roles in diverse developmental processes, nutrient homeostasis and responses to biotic and abiotic stresses. The biogenesis and regulatory functions of miRNAs have been intensively studied in model angiosperms, such as Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. However, global identification of Pinus densata miRNAs has not been reported in previous research.

Results

Here, we report the identification of 34 conserved miRNAs belonging to 25 miRNA families from a P. densata mRNA transcriptome database using local BLAST and MIREAP programs. The primary and/or precursor sequences of 29 miRNAs were further confirmed by RT-PCR amplification and subsequent sequencing. The average value of the minimal folding free energy indexes of the 34 miRNA precursors was 0.92. Nineteen (58%) mature miRNAs began with a 5' terminal uridine residue. Analysis of miRNA precursors showed that 19 mature miRNAs were novel members of 14 conserved miRNA families, of which 17 miRNAs were further validated by subcloning and sequencing. Using real-time quantitative RT-PCR, we found that the expression levels of 7 miRNAs were more than 2-fold higher in needles than in stems. In addition, 72 P. densata mRNAs were predicted to be targets of 25 miRNA families. Four target genes, including a nodal modulator 1-like protein gene, two GRAS family transcription factor protein genes and one histone deacetylase gene, were experimentally verified to be the targets of 3 P. densata miRNAs, pde-miR162a, pde-miR171a and pde-miR482a, respectively.

Conclusions

This study led to the discovery of 34 conserved miRNAs comprising 25 miRNA families from Pinus densata. These results lay a solid foundation for further studying the regulative roles of miRNAs in the development, growth and responses to environmental stresses in P. densata.

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.

Abstract

Background

Digitalis purpurea is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome.

Results

Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in D. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of D. purpurea mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other.

Conclusions

Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in D. purpurea.

The modification or degradation of RNAs including miRNAs may play vital roles in regulating RNA functions. The polyadenylation- and exosome-mediated RNA decay is involved in the degradation of plant RNAs including the primary miRNA processing intermediates. However, plant miRNA levels are not affected by exosome depletion. Here, we report the cloning of a large number of 5′ and/or 3′ truncated versions of the known miRNAs from various tissues of Populus trichocarpa (black cottonwood). It suggests that plant miRNAs may be degraded through either 5′ to 3′ or 3′ to 5′ exonucleolytic digestion. We also show that a significant portion of the isolated miRNAs contains, at the 3′-end, one or a few post-transcriptionally added adenylic acid residues, which are distinct in length from the polyadenylate tail added to other plant RNAs for exosome-mediated degradation. Using an in vitro miRNA degradation system, where synthesized miRNA oligos were degraded in extracts of P. trichocarpa cells, we revealed that the adenylated miRNAs were degraded slower than others without adenylation. It indicates that addition of adenylic acid residues on the 3′-end plays a negative role in miRNA degradation. Our results provide new information for understanding the mechanism of miRNA degradation.

In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants. Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown. Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters. Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics. The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts. Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants. Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.