BackgroundPicrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs) in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput de novo transcriptome sequencing and analyses were carried out at 15°C and 25°C, the temperatures known to modulate picrosides content.ResultsUsing paired-end (PE) Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15°C and 25°C, respectively. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC) content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63%) markers.Large scale expression profiling through "read per exon kilobase per million (RPKM)", showed changes in several biological processes and metabolic pathways including cytochrome P450s (CYPs), UDP-glycosyltransferases (UGTs) and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods.ConclusionsStudy generated transcriptome of P. kurrooa at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers. Analysis also identified putative CYPs and UGTs that could help in discovering the hitherto unknown genes associated with picrosides biosynthesis.

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Light upregulates the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in Picrorhiza kurrooa, an endangered medicinal herb. Upstream sequences of HMGR of P. kurrooa (PropkHMGR) were analyzed in relation to its role in light-mediated regulation of gene expression. GATA motif in PropkHMGR exhibited stronger DNA-protein interaction with the nuclear extract of dark-exposed plants in contrast to SORLIP that exhibited stronger binding with the nuclear extract of light-exposed plants. Analysis of PropkHMGR (PropkHMGR-D1, -1,059/-1) and its deletion fragments PropkHMGR-D2 (-825/-1), PropkHMGR-D3 (-651/-1), PropkHMGR-D4 (-452/-1), and PropkHMGR-D5 (-101/-1) in Arabidopsis thaliana showed PropkHMGR to regulate gene expression [β-glucuronidase (GUS) was used as a reporter gene] at all the developmental stages but only in actively dividing tissues, excluding anthers. Whereas, PropkHMGR-D2 regulated GUS expression in relatively older seedlings but the expression was observed only in shoot apical meristem, root tips, and anthers. PropkHMGR-mediated gene expression was higher in dark as compared to that in the light in Arabidopsis across four temperatures studied. As opposed to the results in P. kurrooa, GATA motifs exhibited DNA-protein interaction with nuclear extract of light-exposed plants of Arabidopsis. SORLIP motifs in Arabidopsis also exhibited DNA-protein interaction with nuclear extract of light-exposed plants as in P. kurrooa. Data showed that (1) PropkHMGR regulated light-mediated gene expression and (2) GATA motif exhibited an inverse relationship between strength of DNA-protein interaction and the gene expression whereas the relationship was species specific for SORLIP.