BACKGROUND
panax notoginseng  f.h.
chen, which is popularly known as sanqi or tienchi ginseng, is a species of the panax genus in the araliaceae family  <cit> .
p. notoginseng has been cultivated for about 400 years in china.
it was previously considered a variety of panax pseudo-ginseng, but in  <dig> it was defined as an independent species of the panax genus by chen feng-huai and is now officially named panax notoginseng  f.h.
chen.
nowadays, more than 85% of the p. notoginseng in the worldwide market is produced in the city of wenshan, yunnan province, china.
p. notoginseng is present in several famous traditional chinese medicinal products, such as yunnan bai yao  and pian zai huang .
it is also famous for its haemostatic properties  <cit> .
the classification of p. notoginseng from the american herbal products association is class 2b and it is indicated in pregnancy because of possible haemostatic effects.
it was reported that p. notoginseng extract administered to rats after cerebral ischaemia reduced infarct volume and inhibited inflammatory inhibitors such as inducible nitric oxide synthase and cyclooxygenase  <dig> via blocking of the nf-κb pathway  <cit> , suggesting a neuroprotective effect.
moreover, saponins of p. notoginseng extract were able to modulate the expression of caspases and attenuate apoptosis in rats following focal cerebral ischaemia-reperfusion  <cit> .
in kk-ay diabetic mice injected with p. notoginseng extract, significantly lowered fasting blood glucose levels, improved glucose tolerance and lighter body weights were observed  <cit> .
besides the roots of p. notoginseng, total saponins extracted from caudexes and leaves have been commonly used for improving mental function, treating insomnia, and alleviating anxiety  <cit> .
the flower buds of p. notoginseng are also used in clinics for treating hypertension, vertigo, tinnitus and acute faucitis in china  <cit> .
chemically, the main bioactive compounds found in p. notoginseng are saponins, which have diverse biological activities such as membrane-permeabilising, immunostimulating, hypocholesterolemic, anti-carcinogenic, and anti-microbial activities  <cit> .
the p. notoginseng-derived triterpene saponins include 20-protopanaxadiol and 20-protopanaxatriol, which exhibit opposing wound healing and anti-tumour actions on the vascular system  <cit> .
notably, p. notoginseng also shares many similar chemical constituents with asian ginseng  and american ginseng   <cit> .
these panax species have species-specific saponin constituents, e.g.
pseudo-ginsenoside f <dig> is unique to american ginseng whereas ginsenoside rg <dig> is only present in asian ginseng  <cit> .
more than  <dig> chemotypes of p. notoginseng classified according to the accumulation of different ginsenosides in roots, leaves and flowers have been reported  <cit> .
p. notoginseng contains significantly higher amounts of ginsenosides rg <dig> and rb <dig> compared with other ginseng species, and the ratios of rg1/re and rg1/rb <dig> in p. notoginseng are the highest among ginseng species.
in particular, notoginsenoside r <dig> has been identified in p. notoginseng but is absent in other ginseng species.
several previous studies have suggested that the precursor molecules for triterpene saponin biosynthesis are isoprenoids, which are synthesized via the mevalonic acid  pathway, leading to the biosynthesis of  <dig> -oxidosqualene  <cit> .
this central molecule is then modified through various biochemical reactions of its triterpene skeleton, resulting in the production of various ginsenosides.
notably, ginsenosides can be isolated from different parts of p. notoginseng, e.g.
the underground parts of p. notoginseng are rich in protopanaxatriol- and protopanaxadiol-type saponins, while leaves and flowers contain protopanaxadiol-type saponins only.
on the other hand, the ginsenosides rc, rb <dig> and rb <dig> are relatively abundant in aerial parts, compared with the underground parts of p. notoginseng  <cit> .
although considerable research has been done on the pharmacological activities of ginsenosides, to date very little is known about the ginsenoside biosynthetic pathway.
some candidate genes likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis are cytochrome p450s  and glycosyltransferases , but no candidate has been identified for the cyclization step  <cit> .
in addition, p. notoginseng is a shade plant and is commonly cultivated in mountain areas of wenshan at altitudes of 1200–2000 m around  <dig> °n, 104°e  <cit> .
because of the humid and warm environment, p. notoginseng is easily infected by pests and diseases, especially in the roots.
alkaloids, which have been identified in more than  <dig>  plant species, play a role in protecting plants from pathogen and pest damage  <cit> .
however, alkaloid-related genes in p. notoginseng have not been reported previously.
transcriptomic and genomic data for p. notoginseng are very limited despite the pharmacological importance of this plant.
considering there are thousands of genes in its genome, only  <dig> mrna sequences originating from p. notoginseng could be retrieved from the nucleotide databases of the national centre for biotechnology information .
over the last decade, next-generation dna sequencing technology has provided a rapid and economical way to study the gene expression profiles of plant species.
in this study, we established transcript databases for leaves, roots and flowers from 3-year-old p. notoginseng.
moreover, we identified genes encoding enzymes involved in triterpene saponin and alkaloid biosynthesis.
differentially expressed cyp450s and gts in the three tissues are also reported.
RESULTS
sequencing and de novo assembly
to study the transcriptomes of p. notoginseng, leaves, roots and flowers were collected from 3-year-old plants.
total rna was extracted from each part and then mrna was isolated.
each sample was sequenced using the illumina hiseq™  <dig> platform.
sequencing yielded approximately  <dig> million 90-base pair  paired end raw reads, or approximately  <dig> gbp in total.
we filtered out adapter sequences and reads that were shorter than 50 bp, and ultimately generated  <dig> ,  <dig>  and  <dig>  gbp of high-quality  reads for leaves, roots and flowers, respectively.
all of the hq sequencing reads from the three organs were deposited in ncbi and can be accessed in the sequence read archive  under the bioproject accession number prjna <dig>  the hq reads of each sample library were assembled using the trinity software  <cit>  and the tgi clustering tool   <cit> , followed by the phrap assembler  <cit>  to remove redundant trinity-generated contigs.
finally,  <dig> ,  <dig>  and  <dig>  unigenes were obtained for leaves, roots and flowers, respectively.
we also pooled the reads from all three organs together and repeated the above steps, resulting in  <dig>  contigs and  <dig>  unigenes with a mean length of 781 bp and  <dig>  bp, respectively.
the length distribution of contigs and unigenes is shown in additional file  <dig>  a summary of sequencing and assembly results is shown in table  <dig> table  <dig>
summary of illumina sequencing and assembly of
p. notoginseng
leaves
roots
flowers
three tissues together
*total number of clusters and singletons.
compared with a separate study of 4-year-old p. notoginseng  <cit> , which generated  <dig>  unigenes , we present three times more and much longer unigenes from p. notoginseng roots .
this is probably because of a 48-fold increase in sequencing throughput in this study.
the large number of novel unigenes should cover the majority of genes in the p. notoginseng genome and provide a useful resource for future studies on this pharmacologically important plant.
annotation and differential expression of transcripts in different tissues
all unigenes from leaves, roots and flowers were annotated separately using blast searches against the ncbi non-redundant protein , uniprot protein, the arabidopsis information resource , tomato genome  and plantcyc public databases.
detailed counts of the annotated unigenes are presented in additional file  <dig>  in total, there were  <dig> ,  <dig>  and  <dig>  annotated unigenes for leaves, roots and flowers, respectively, with at least one significant match in the aforementioned public databases.
figure  <dig> shows the number of unigenes annotated by all databases.
the annotation percentage for p. notoginseng unigenes  was much higher than that for p. ginseng unigenes   <cit> .
when the unigenes from different p. notoginseng tissues were compared, we found that  <dig>  unigenes were shared by all three tissues .
on the other hand,  <dig> ,  <dig>  and  <dig>  unigenes were specifically found in leaves, roots and flowers, respectively, with flowers having the highest number of unique unigenes.figure  <dig>
venn diagram of unigenes from leaves, roots and flowers annotated using various databases.
the venn diagram shows the overlapping unigenes annotated in the nr, uniprot, tair, itag and plantcyc databases in leaves , roots  and flowers .
venn diagram of the unigenes in the leaves, roots and flowers of
p. notoginseng
.
the diagram shows the overlapping unigenes in the leaves, roots and flowers.
a total  <dig>  unigenes were found in all three tissues, while some unigenes only were found in specific tissues.
we next investigated the transcriptomic similarities and differences between p. notoginseng and p. ginseng using blast searches of p. notoginseng unigenes against p. ginseng unigenes, with an e-value threshold of 1e- <dig>  <cit> .
p. ginseng transcriptome contigs were obtained under accession number gaag <dig> from the ncbi.
the results showed that  <dig>  unigenes of p. notoginseng had at least one match to the contigs of p. ginseng, suggesting that many of our unigenes are p. notoginseng-specific.
next, proteins encoded by our unigenes were predicted by the trinity software and orthologous groups were identified using orthomcl  <cit> , which groups orthologous proteins based on sequence similarity.
a total of  <dig>  protein sequences from  <dig>  unigenes were clustered into  <dig>  orthologous groups for p. notoginseng.
for p. ginseng, a total of  <dig>  protein sequences from  <dig>  contigs were clustered into  <dig>  orthologous groups.
we found that  <dig>  groups were common to both species, with  <dig>  orthologous groups specific to p. notoginseng, and  <dig> groups specific to p. ginseng.
to evaluate the abundance of transcripts in specific organs, the high quality reads were mapped back to the transcriptome generated from all three plant tissues.
among the top  <dig> most expressed unigenes in the root transcriptome , one of the most abundant genes was dammarenediol-ii synthase , which is an important player in triterpene saponin biosynthesis in p. notoginseng  <cit> .
in addition, unigenes encoding cytochrome p <dig> cyp716a <dig>  which is involved in the conversion of protopanaxadiol from dammarenediol-ii, were also present at a very high level.
this is consistent with a previous report about the co-expression of ds and cyp716a <dig> in p. ginseng  <cit> .
last but not least, reticuline oxidase-like protein-like isoform  <dig> is a predicted protein with sequence similarity to reticuline oxidase, which is involved in forming benzophenanthridine alkaloids as a pathogenic attack response.
identification of genes involved in triterpene saponin biosynthesis
the kyoto encyclopaedia of genes and genomes  is a tool for functional classification and pathway assignment based on gene-associated biochemical pathways.
in total,  <dig>  unigenes from all three tissues having enzyme commission numbers were assigned to  <dig> kegg pathways .
the cluster for metabolism represented the largest group, with most unigenes involved in carbohydrate metabolism, amino acid metabolism, nucleotide metabolism and metabolism of cofactors and vitamins.
among them,  <dig> unigenes were involved in the biosynthesis of various secondary metabolites , with unigenes involved in phenylpropanoid biosynthesis forming the largest group, followed by terpenoid backbone biosynthesis.table  <dig>
number of unigenes related to secondary metabolites in
p. notoginseng
secondary metabolites biosynthesis pathways
pathway id
leaves
roots
flowers
triterpene saponins are synthesized by terpenoid backbone biosynthesis, followed by sesquiterpenoid and triterpenoid biosynthesis.
according to the putative pathway, specific cyp450s and gts are involved in the formation of various ginsenosides .
from our annotation results,  <dig> unigenes were identified to encode all of the known enzymes involved in triterpene saponin biosynthesis.
there were multiple unigenes annotated to the same enzyme, which may represent different members of the same gene family.
table  <dig> shows the reads per kilobase of transcript per million reads mapped  values of genes encoding enzymes involved in triterpene saponin biosynthesis in leaves, roots and flowers.
the rpkms of all annotated isoforms for the same gene were summed as the rpkm of that gene.
we identified  <dig> out of  <dig> full-length cdna encoding enzymes involved in triterpene saponin biosynthesis and their corresponding accession numbers in ncbi nucleotide databases are listed in table  <dig>  a heat map of differentially expressed genes involved in triterpene saponin biosynthesis is shown in figure  <dig>  significant differential expression of these genes could be recognized in different p. notoginseng tissues, and most of the genes involved in triterpene saponin biosynthesis showed higher expression in roots compared with leaves or flowers.
the expression levels of selected genes  in each part were validated by real-time pcr .figure  <dig>
putative ginsenoside biosynthesis in
p. notoginseng
.
enzymes found in this study are shown between the reactions.
the expression of genes encoding these enzymes in the leaves , roots  and flowers  is shown by heatmap.
the genes were mapped using rpkm values, colour coded by increasing relative expression.
the broken arrow represents a putative ginsenoside biosynthesis step involving cyp450s and gts.
abbreviations: aact, acetyl-coa acetyltransferase; hmgs, hmg-coa synthase; hmgr, hmg-coa reductase; mk, mevalonate kinase; pmk, phosphomevalonate kinase; mdd, mevalonate diphosphate decarboxylase; ipi, isopentenylpyrophosphate isomerase; ggps, geranylgeranyl pyrophosphate synthase; ggr, geranylgeranyl diphosphate synthase; fps, farnesyl diphosphate synthase; ss, squalene synthase; se, squalene epoxidase; as, β-amyrin synthase; ds, dammarenediol-ii synthase; cyp <dig>  cytochrome p450; gt, glycosyltransferase.
discovery of unigenes involved in triterpene saponin biosynthesis in
p. notoginseng
enzymes name
ec number
abbreviation
leaves
roots
flowers
the values in different organs indicate the reads per kilobase of transcript per million reads mapped .
genes involved in triterpene saponin biosynthesis in
p. notoginseng
gene name
abbreviation
length of mrna
location of cds
accession number
*partial cds at the 5′ end.
real-time pcr analysis of selected genes involved in triterpene saponin biosynthesis.
real-time pcr was used to validate the expression levels of selected genes revealed by rna-seq.
abbreviations: aact, acetyl-coa acetyltransferase; hmgs, hmg-coa synthase; mk, mevalonate kinase; mdd, mevalonate diphosphate decarboxylase; ipi, isopentenylpyrophosphate isomerase; fps, farnesyl diphosphate synthase; ss, squalene synthase; se, squalene epoxidase.
luo et al.
<cit>  previously analysed the root transcriptome of 4-year-old p. notoginseng and discovered many partial cdnas encoding enzymes involved in triterpene saponin biosynthesis.
however, hydroxymethyl glutaryl coa synthase , a key enzyme of triterpene saponin biosynthesis, was absent in their transcriptome.
in contrast, the hmgs gene was abundant in our transcriptomes, probably because of the much greater sequencing depth achieved in our study.
therefore, compared with other published transcriptomes, the transcriptomes reported in this study will be more useful for cloning important genes involved in secondary metabolite biosynthetic pathways of p. notoginseng.
moreover, β-amyrin synthase , a key enzyme for oleanane-type ginsenoside biosynthesis, was also found in this study.
notably, as was discovered in all three tissues of 3-year-old p. notoginseng .
identification of genes related to biosynthesis of different ginsenosides
there are published reports showing that different parts of p. notoginseng differ in the synthesis of triterpene saponins.
roots are rich in protopanaxatriol- and protopanaxadiol-type saponins whereas leaves and flowers contain protopanaxadiol-type saponins only  <cit> .
it has been suggested that putative candidate genes involved in triterpene saponins biosynthesis are mainly cyp450s and gts, which may account for the synthesis and accumulation of triterpene saponins in specific organs  <cit> .
in this study,  <dig> and  <dig> members of the cyp <dig> and gt gene families, respectively, were identified .
it has been reported that the cytochrome p <dig> cyp716a53v <dig> participates in the formation of protopanaxatriol from protopanaxadiol in p. ginseng  <cit> .
in our transcriptome, one unigene annotated as cyp716a53v <dig> was found and subsequent sequence analysis showed that it was the full-length homologue in p. notoginseng.
figure  <dig> shows the alignment of the predicted protein sequences of cyp716a53v <dig> from p. notoginseng, p. ginseng and p. quinquefolius.
the accession numbers of the sequences are jx <dig> for p. ginseng and kc <dig> for p. quinquefolius in the genbank database.
all three cyp <dig> genes had  <dig> amino acid residues and showed 98% identity between p. notoginseng and p. ginseng, 96% identity between p. notoginseng and p. quinquefolius, and 97% identity between p. ginseng and p. quinquefolius.
it was also very encouraging to find that the expression level of the putative cyp716a53v <dig> in the root  was much higher than that in the leaf  or flower  of p. notoginseng .
to confirm the differential expression of the putative cyp716a53v <dig> gene, we analysed its expression level in different tissues by real-time pcr.
the result obtained was consistent with the rpkm values, showing that the expression of the putative cyp716a53v <dig> in roots was significantly higher than in leaves or flowers .
this may explain, at least partially, why the root has a much higher concentration of protopanaxatriol-type saponins.
moreover, this result also indicates that the abundance of target genes in our transcriptomes closely reflects the actual gene expression level.figure  <dig>
alignment of cyp716a53v <dig> amino acid sequences from
p. notoginseng
,
p. ginseng
and
p. quinquefolium
.
alignment results showed 98% identity between p. notoginseng and p. ginseng, 96% identity between p. notoginseng and p. quinquefolius, and 97% identity between p. ginseng and p. quinquefolius.
expression levels of target cyp450s in different
p. notoginseng
tissues.
comparison of relative abundance of target cyp <dig> unigenes in the leaf, root and flower according to real time pcr  and rpkm values .
identification of candidate genes involved in alkaloid biosynthesis
alkaloids, which are found in about 20% of plant species, are a diverse group of low-molecular-weight compounds acting as poisonous agents in the defence of plants against herbivores and pathogens  <cit> .
p. notoginseng grows sub-optimally in direct sunlight and so is often planted under tree canopies, but the shady and humid growing conditions favour infection by numerous phytopathogens, which can cause root rot, black spot or round spot diseases  <cit> .
in this study, three pathways involved in alkaloid biosynthesis were found in our transcriptomes , including isoquinoline alkaloid biosynthesis , indole alkaloid biosynthesis  and tropane, piperidine and pyridine alkaloid biosynthesis .
the annotation results from the three transcriptomes were used to identify genes encoding enzymes involved in various alkaloid biosynthetic pathways.
in total,  <dig> unigenes were assigned to six enzymes involved in alkaloid biosynthesis .
according to the rpkm values of the genes encoding enzymes involved in alkaloid biosynthesis in leaves, roots and flowers shown in table  <dig>  most of the enzymes were expressed at the lowest level in roots, in particular polyphenol oxidase , which has a role in plant resistance to stress and pathogens.
it is notable that wounding and herbivore attacks have also been shown to induce ppo activity  <cit> .
ppo activation is thought to involve proteolytic processing, but many mature ppos appear to remain in a latent form  <cit> .
root rot disease is the most damaging disease that can plague p. notoginseng over its long growth period, leading to production loss and quality reduction.
it is mainly caused by fungal pathogens such as cylindrocarpon destructans, cylindrocarpon didymum and fusarium solani  <cit> .
in this study, the expression levels of aspartate transaminase, strictosidine synthase and histidinol-phosphate transaminase in different tissues were validated by real-time pcr .
further investigation of the relationship between alkaloid biosynthesis genes and plant defence should provide novel insights into the complex disease resistance mechanisms of p. notoginseng.table  <dig>
discovery of unigenes involved in alkaloid biosynthesis in
p. notoginseng
enzymes
ec number
leaves
roots
flowers
the values in different organs indicate the reads per kilobase of transcript per million reads mapped .
real-time pcr analysis of genes involved in the alkaloid pathway.
the expression levels of selected genes in the alkaloid pathway were validated by real-time pcr.
CONCLUSIONS
p. notoginseng is a widely used medicinal herb.
in this study, leaf, root and flower transcriptomes from p. notoginseng are presented.
the resulting unigene dataset will provide a large number of transcripts for gene discovery and genetic analyses in this medicinal plant.
notably, many genes involved in triterpene saponin biosynthesis were identified in this study.
more importantly, a large number of unigenes were annotated as cyp450s and gts.
in summary, this study provides comprehensive information on the transcriptional regulation of functionally important genes in p. notoginseng.

 panax notoginseng f.h. chen, which is popularly known as sanqi or tienchi ginseng, is a species of the panax genus in the araliaceae family. p. notoginseng has been cultivated for about 400 years in china. it was previously considered a variety of panax pseudo-ginseng, but in it was defined as an independent species of the panax genus by chen feng-huai and is now officially named panax notoginseng f.h. chen. nowadays, more than 85% of the p. notoginseng in the worldwide market is produced in the city of wenshan, yunnan province, china. p. notoginseng is present in several famous traditional chinese medicinal products, such as yunnan bai yao and pian zai huang. it is also famous for its haemostatic properties. the classification of p. notoginseng from the american herbal products association is class 2b and it is indicated in pregnancy because of possible haemostatic effects. it was reported that p. notoginseng extract administered to rats after cerebral ischaemia reduced infarct volume and inhibited inflammatory inhibitors such as inducible nitric oxide synthase and cyclooxygenase via blocking of the nf-κb pathway, suggesting a neuroprotective effect. moreover, saponins of p. notoginseng extract were able to modulate the expression of caspases and attenuate apoptosis in rats following focal cerebral ischaemia-reperfusion. in kk-ay diabetic mice injected with p. notoginseng extract, significantly lowered fasting blood glucose levels, improved glucose tolerance and lighter body weights were observed. besides the roots of p. notoginseng, total saponins extracted from caudexes and leaves have been commonly used for improving mental function, treating insomnia, and alleviating anxiety. the flower buds of p. notoginseng are also used in clinics for treating hypertension, vertigo, tinnitus and acute faucitis in china. chemically, the main bioactive compounds found in p. notoginseng are saponins, which have diverse biological activities such as membrane-permeabilising, immunostimulating, hypocholesterolemic, anti-carcinogenic, and anti-microbial activities. the p. notoginseng-derived triterpene saponins include 20-protopanaxadiol and 20-protopanaxatriol, which exhibit opposing wound healing and anti-tumour actions on the vascular system. notably, p. notoginseng also shares many similar chemical constituents with asian ginseng and american ginseng. these panax species have species-specific saponin constituents, e.g. pseudo-ginsenoside f is unique to american ginseng whereas ginsenoside rg is only present in asian ginseng. more than chemotypes of p. notoginseng classified according to the accumulation of different ginsenosides in roots, leaves and flowers have been reported. p. notoginseng contains significantly higher amounts of ginsenosides rg and rb compared with other ginseng species, and the ratios of rg1/re and rg1/rb in p. notoginseng are the highest among ginseng species. in particular, notoginsenoside r has been identified in p. notoginseng but is absent in other ginseng species. several previous studies have suggested that the precursor molecules for triterpene saponin biosynthesis are isoprenoids, which are synthesized via the mevalonic acid pathway, leading to the biosynthesis of -oxidosqualene. this central molecule is then modified through various biochemical reactions of its triterpene skeleton, resulting in the production of various ginsenosides. notably, ginsenosides can be isolated from different parts of p. notoginseng, e.g. the underground parts of p. notoginseng are rich in protopanaxatriol- and protopanaxadiol-type saponins, while leaves and flowers contain protopanaxadiol-type saponins only. on the other hand, the ginsenosides rc, rb and rb are relatively abundant in aerial parts, compared with the underground parts of p. notoginseng. although considerable research has been done on the pharmacological activities of ginsenosides, to date very little is known about the ginsenoside biosynthetic pathway. some candidate genes likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis are cytochrome p450s and glycosyltransferases, but no candidate has been identified for the cyclization step. in addition, p. notoginseng is a shade plant and is commonly cultivated in mountain areas of wenshan at altitudes of 1200–2000 m around °n, 104°e. because of the humid and warm environment, p. notoginseng is easily infected by pests and diseases, especially in the roots. alkaloids, which have been identified in more than plant species, play a role in protecting plants from pathogen and pest damage. however, alkaloid-related genes in p. notoginseng have not been reported previously. transcriptomic and genomic data for p. notoginseng are very limited despite the pharmacological importance of this plant. considering there are thousands of genes in its genome, only mrna sequences originating from p. notoginseng could be retrieved from the nucleotide databases of the national centre for biotechnology information. over the last decade, next-generation dna sequencing technology has provided a rapid and economical way to study the gene expression profiles of plant species. in this study, we established transcript databases for leaves, roots and flowers from 3-year-old p. notoginseng. moreover, we identified genes encoding enzymes involved in triterpene saponin and alkaloid biosynthesis. differentially expressed cyp450s and gts in the three tissues are also reported.  sequencing and de novo assembly to study the transcriptomes of p. notoginseng, leaves, roots and flowers were collected from 3-year-old plants. total rna was extracted from each part and then mrna was isolated. each sample was sequenced using the illumina hiseq™ platform. sequencing yielded approximately million 90-base pair paired end raw reads, or approximately gbp in total. we filtered out adapter sequences and reads that were shorter than 50 bp, and ultimately generated, and gbp of high-quality reads for leaves, roots and flowers, respectively. all of the hq sequencing reads from the three organs were deposited in ncbi and can be accessed in the sequence read archive under the bioproject accession number prjna the hq reads of each sample library were assembled using the trinity software and the tgi clustering tool, followed by the phrap assembler to remove redundant trinity-generated contigs. finally,, and unigenes were obtained for leaves, roots and flowers, respectively. we also pooled the reads from all three organs together and repeated the above steps, resulting in contigs and unigenes with a mean length of 781 bp and  bp, respectively. the length distribution of contigs and unigenes is shown in additional file a summary of sequencing and assembly results is shown in table  table summary of illumina sequencing and assembly of p. notoginseng leaves roots flowers three tissues together *total number of clusters and singletons. compared with a separate study of 4-year-old p. notoginseng, which generated unigenes, we present three times more and much longer unigenes from p. notoginseng roots. this is probably because of a 48-fold increase in sequencing throughput in this study. the large number of novel unigenes should cover the majority of genes in the p. notoginseng genome and provide a useful resource for future studies on this pharmacologically important plant. annotation and differential expression of transcripts in different tissues all unigenes from leaves, roots and flowers were annotated separately using blast searches against the ncbi non-redundant protein, uniprot protein, the arabidopsis information resource, tomato genome and plantcyc public databases. detailed counts of the annotated unigenes are presented in additional file in total, there were, and annotated unigenes for leaves, roots and flowers, respectively, with at least one significant match in the aforementioned public databases. figure  shows the number of unigenes annotated by all databases. the annotation percentage for p. notoginseng unigenes was much higher than that for p. ginseng unigenes. when the unigenes from different p. notoginseng tissues were compared, we found that unigenes were shared by all three tissues. on the other hand,, and unigenes were specifically found in leaves, roots and flowers, respectively, with flowers having the highest number of unique unigenes.figure venn diagram of unigenes from leaves, roots and flowers annotated using various databases. the venn diagram shows the overlapping unigenes annotated in the nr, uniprot, tair, itag and plantcyc databases in leaves, roots and flowers. venn diagram of the unigenes in the leaves, roots and flowers of p. notoginseng. the diagram shows the overlapping unigenes in the leaves, roots and flowers. a total unigenes were found in all three tissues, while some unigenes only were found in specific tissues. we next investigated the transcriptomic similarities and differences between p. notoginseng and p. ginseng using blast searches of p. notoginseng unigenes against p. ginseng unigenes, with an e-value threshold of 1e-. p. ginseng transcriptome contigs were obtained under accession number gaag from the ncbi. the results showed that unigenes of p. notoginseng had at least one match to the contigs of p. ginseng, suggesting that many of our unigenes are p. notoginseng-specific. next, proteins encoded by our unigenes were predicted by the trinity software and orthologous groups were identified using orthomcl, which groups orthologous proteins based on sequence similarity. a total of protein sequences from unigenes were clustered into orthologous groups for p. notoginseng. for p. ginseng, a total of protein sequences from contigs were clustered into orthologous groups. we found that groups were common to both species, with orthologous groups specific to p. notoginseng, and groups specific to p. ginseng. to evaluate the abundance of transcripts in specific organs, the high quality reads were mapped back to the transcriptome generated from all three plant tissues. among the top most expressed unigenes in the root transcriptome, one of the most abundant genes was dammarenediol-ii synthase, which is an important player in triterpene saponin biosynthesis in p. notoginseng. in addition, unigenes encoding cytochrome p cyp716a which is involved in the conversion of protopanaxadiol from dammarenediol-ii, were also present at a very high level. this is consistent with a previous report about the co-expression of ds and cyp716a in p. ginseng. last but not least, reticuline oxidase-like protein-like isoform is a predicted protein with sequence similarity to reticuline oxidase, which is involved in forming benzophenanthridine alkaloids as a pathogenic attack response. identification of genes involved in triterpene saponin biosynthesis the kyoto encyclopaedia of genes and genomes is a tool for functional classification and pathway assignment based on gene-associated biochemical pathways. in total, unigenes from all three tissues having enzyme commission numbers were assigned to kegg pathways. the cluster for metabolism represented the largest group, with most unigenes involved in carbohydrate metabolism, amino acid metabolism, nucleotide metabolism and metabolism of cofactors and vitamins. among them, unigenes were involved in the biosynthesis of various secondary metabolites, with unigenes involved in phenylpropanoid biosynthesis forming the largest group, followed by terpenoid backbone biosynthesis.table number of unigenes related to secondary metabolites in p. notoginseng secondary metabolites biosynthesis pathways pathway id leaves roots flowers triterpene saponins are synthesized by terpenoid backbone biosynthesis, followed by sesquiterpenoid and triterpenoid biosynthesis. according to the putative pathway, specific cyp450s and gts are involved in the formation of various ginsenosides. from our annotation results, unigenes were identified to encode all of the known enzymes involved in triterpene saponin biosynthesis. there were multiple unigenes annotated to the same enzyme, which may represent different members of the same gene family. table  shows the reads per kilobase of transcript per million reads mapped values of genes encoding enzymes involved in triterpene saponin biosynthesis in leaves, roots and flowers. the rpkms of all annotated isoforms for the same gene were summed as the rpkm of that gene. we identified out of full-length cdna encoding enzymes involved in triterpene saponin biosynthesis and their corresponding accession numbers in ncbi nucleotide databases are listed in table  a heat map of differentially expressed genes involved in triterpene saponin biosynthesis is shown in figure  significant differential expression of these genes could be recognized in different p. notoginseng tissues, and most of the genes involved in triterpene saponin biosynthesis showed higher expression in roots compared with leaves or flowers. the expression levels of selected genes in each part were validated by real-time pcr.figure putative ginsenoside biosynthesis in p. notoginseng. enzymes found in this study are shown between the reactions. the expression of genes encoding these enzymes in the leaves, roots and flowers is shown by heatmap. the genes were mapped using rpkm values, colour coded by increasing relative expression. the broken arrow represents a putative ginsenoside biosynthesis step involving cyp450s and gts. abbreviations: aact, acetyl-coa acetyltransferase; hmgs, hmg-coa synthase; hmgr, hmg-coa reductase; mk, mevalonate kinase; pmk, phosphomevalonate kinase; mdd, mevalonate diphosphate decarboxylase; ipi, isopentenylpyrophosphate isomerase; ggps, geranylgeranyl pyrophosphate synthase; ggr, geranylgeranyl diphosphate synthase; fps, farnesyl diphosphate synthase; ss, squalene synthase; se, squalene epoxidase; as, β-amyrin synthase; ds, dammarenediol-ii synthase; cyp cytochrome p450; gt, glycosyltransferase. discovery of unigenes involved in triterpene saponin biosynthesis in p. notoginseng enzymes name ec number abbreviation leaves roots flowers the values in different organs indicate the reads per kilobase of transcript per million reads mapped. genes involved in triterpene saponin biosynthesis in p. notoginseng gene name abbreviation length of mrna location of cds accession number *partial cds at the 5′ end. real-time pcr analysis of selected genes involved in triterpene saponin biosynthesis. real-time pcr was used to validate the expression levels of selected genes revealed by rna-seq. abbreviations: aact, acetyl-coa acetyltransferase; hmgs, hmg-coa synthase; mk, mevalonate kinase; mdd, mevalonate diphosphate decarboxylase; ipi, isopentenylpyrophosphate isomerase; fps, farnesyl diphosphate synthase; ss, squalene synthase; se, squalene epoxidase. luo et al. previously analysed the root transcriptome of 4-year-old p. notoginseng and discovered many partial cdnas encoding enzymes involved in triterpene saponin biosynthesis. however, hydroxymethyl glutaryl coa synthase, a key enzyme of triterpene saponin biosynthesis, was absent in their transcriptome. in contrast, the hmgs gene was abundant in our transcriptomes, probably because of the much greater sequencing depth achieved in our study. therefore, compared with other published transcriptomes, the transcriptomes reported in this study will be more useful for cloning important genes involved in secondary metabolite biosynthetic pathways of p. notoginseng. moreover, β-amyrin synthase, a key enzyme for oleanane-type ginsenoside biosynthesis, was also found in this study. notably, as was discovered in all three tissues of 3-year-old p. notoginseng. identification of genes related to biosynthesis of different ginsenosides there are published reports showing that different parts of p. notoginseng differ in the synthesis of triterpene saponins. roots are rich in protopanaxatriol- and protopanaxadiol-type saponins whereas leaves and flowers contain protopanaxadiol-type saponins only. it has been suggested that putative candidate genes involved in triterpene saponins biosynthesis are mainly cyp450s and gts, which may account for the synthesis and accumulation of triterpene saponins in specific organs. in this study, and members of the cyp and gt gene families, respectively, were identified. it has been reported that the cytochrome p cyp716a53v participates in the formation of protopanaxatriol from protopanaxadiol in p. ginseng. in our transcriptome, one unigene annotated as cyp716a53v was found and subsequent sequence analysis showed that it was the full-length homologue in p. notoginseng. figure  shows the alignment of the predicted protein sequences of cyp716a53v from p. notoginseng, p. ginseng and p. quinquefolius. the accession numbers of the sequences are jx for p. ginseng and kc for p. quinquefolius in the genbank database. all three cyp genes had amino acid residues and showed 98% identity between p. notoginseng and p. ginseng, 96% identity between p. notoginseng and p. quinquefolius, and 97% identity between p. ginseng and p. quinquefolius. it was also very encouraging to find that the expression level of the putative cyp716a53v in the root was much higher than that in the leaf or flower of p. notoginseng. to confirm the differential expression of the putative cyp716a53v gene, we analysed its expression level in different tissues by real-time pcr. the result obtained was consistent with the rpkm values, showing that the expression of the putative cyp716a53v in roots was significantly higher than in leaves or flowers. this may explain, at least partially, why the root has a much higher concentration of protopanaxatriol-type saponins. moreover, this result also indicates that the abundance of target genes in our transcriptomes closely reflects the actual gene expression level.figure alignment of cyp716a53v amino acid sequences from p. notoginseng, p. ginseng and p. quinquefolium. alignment results showed 98% identity between p. notoginseng and p. ginseng, 96% identity between p. notoginseng and p. quinquefolius, and 97% identity between p. ginseng and p. quinquefolius. expression levels of target cyp450s in different p. notoginseng tissues. comparison of relative abundance of target cyp unigenes in the leaf, root and flower according to real time pcr and rpkm values. identification of candidate genes involved in alkaloid biosynthesis alkaloids, which are found in about 20% of plant species, are a diverse group of low-molecular-weight compounds acting as poisonous agents in the defence of plants against herbivores and pathogens. p. notoginseng grows sub-optimally in direct sunlight and so is often planted under tree canopies, but the shady and humid growing conditions favour infection by numerous phytopathogens, which can cause root rot, black spot or round spot diseases. in this study, three pathways involved in alkaloid biosynthesis were found in our transcriptomes, including isoquinoline alkaloid biosynthesis, indole alkaloid biosynthesis and tropane, piperidine and pyridine alkaloid biosynthesis. the annotation results from the three transcriptomes were used to identify genes encoding enzymes involved in various alkaloid biosynthetic pathways. in total, unigenes were assigned to six enzymes involved in alkaloid biosynthesis. according to the rpkm values of the genes encoding enzymes involved in alkaloid biosynthesis in leaves, roots and flowers shown in table  most of the enzymes were expressed at the lowest level in roots, in particular polyphenol oxidase, which has a role in plant resistance to stress and pathogens. it is notable that wounding and herbivore attacks have also been shown to induce ppo activity. ppo activation is thought to involve proteolytic processing, but many mature ppos appear to remain in a latent form. root rot disease is the most damaging disease that can plague p. notoginseng over its long growth period, leading to production loss and quality reduction. it is mainly caused by fungal pathogens such as cylindrocarpon destructans, cylindrocarpon didymum and fusarium solani. in this study, the expression levels of aspartate transaminase, strictosidine synthase and histidinol-phosphate transaminase in different tissues were validated by real-time pcr. further investigation of the relationship between alkaloid biosynthesis genes and plant defence should provide novel insights into the complex disease resistance mechanisms of p. notoginseng.table discovery of unigenes involved in alkaloid biosynthesis in p. notoginseng enzymes ec number leaves roots flowers the values in different organs indicate the reads per kilobase of transcript per million reads mapped. real-time pcr analysis of genes involved in the alkaloid pathway. the expression levels of selected genes in the alkaloid pathway were validated by real-time pcr.  p. notoginseng is a widely used medicinal herb. in this study, leaf, root and flower transcriptomes from p. notoginseng are presented. the resulting unigene dataset will provide a large number of transcripts for gene discovery and genetic analyses in this medicinal plant. notably, many genes involved in triterpene saponin biosynthesis were identified in this study. more importantly, a large number of unigenes were annotated as cyp450s and gts. in summary, this study provides comprehensive information on the transcriptional regulation of functionally important genes in p. notoginseng. 
after assembling the high quality sequencing reads into unigenes, biochemical pathways were predicted and unigenes were assigned to kegg pathways. among them, unigenes were identified to be involved in triterpene saponin biosynthesis. in addition, and unigenes were predicted to encode cytochrome p450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. candidate genes involved in ginsenoside biosynthesis, including putative cytochrome p450s and glycosyltransferases were obtained. hence, genomic and transcriptomic details about p. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now. the transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of p. notoginseng. in this study, rna sequencing of three different p. notoginseng tissues was performed using next generation dna sequencing. in particular, one unigene predominately expressed in the root was annotated as cyp716a53v which probably participates in the formation of protopanaxatriol from protopanaxadiol in p. notoginseng. we have established a global transcriptome dataset for p. notoginseng and provided additional genetic information for further genome-wide research and analyses. the major bioactive molecules are triterpene saponins, which are also known as ginsenosides. the differential expression of this gene was further confirmed by real-time pcr.
 panax notoginseng f.h. chen is one of the most highly valued medicinal plants in the world. the major bioactive molecules are triterpene saponins, which are also known as ginsenosides. however, its large genome size has hindered the assembly of a draft genome by whole genome sequencing. hence, genomic and transcriptomic details about p. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now.  in this study, rna sequencing of three different p. notoginseng tissues was performed using next generation dna sequencing. after assembling the high quality sequencing reads into unigenes, biochemical pathways were predicted and unigenes were assigned to kegg pathways. among them, unigenes were identified to be involved in triterpene saponin biosynthesis. in addition, and unigenes were predicted to encode cytochrome p450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. in particular, one unigene predominately expressed in the root was annotated as cyp716a53v which probably participates in the formation of protopanaxatriol from protopanaxadiol in p. notoginseng. the differential expression of this gene was further confirmed by real-time pcr.  we have established a global transcriptome dataset for p. notoginseng and provided additional genetic information for further genome-wide research and analyses. candidate genes involved in ginsenoside biosynthesis, including putative cytochrome p450s and glycosyltransferases were obtained. the transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of p. notoginseng. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords panax notoginsengtranscriptomeginsenosidesissue-copyright-statement© the author 2015 