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Light and temperature regulated terpene biosynthesis: hepatoprotective monoterpene picroside accumulation in Picrorhiza kurrooa

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Abstract

Picrorhiza (Picrorhiza kurrooa) is an endangered medicinal plant with well-known hepatoprotective activity attributed to monoterpenoid picrosides. The present article details on regulatory genes of terpenoid metabolism, 3-hydroxy-3-methylglutaryl coenzyme A reductase (pkhmgr) and 1-deoxy-D-xylulose-5-phosphate synthase (pkdxs) from picrorhiza. Since no molecular information was available, these genes were cloned to full-length by degenerate primers and rapid amplification of cDNA ends, followed by cloning of the upstream sequences that showed the presence of core sequences for light and temperature responsiveness. Electrophoretic mobility shift assay confirmed binding of protein to these motifs. Expression of pkhmgr and pkdxs was up-regulated at 15°C as compared to at 25°C as well as under light as compared to dark conditions. Picrosides content exhibited the trend similar to gene expression. To rule out the possible limitation of carbon pool under dark condition, plantlets of picrorhiza were raised in vitro in Murashige and Skoog medium supplemented with 3% sucrose. Results showed similar up-regulation of both the genes and the higher picrosides content in in vitro raised plantlets in the presence of light. Data suggested the important roles played by light and temperature in regulating pkhmgr and pkdxs, and the picrosides level in picrorhiza.

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Abbreviations

aa:

amino acid

DXS:

1-deoxy-D-xylulose-5-phosphate synthase

EMSA:

electrophoretic mobility shift assay

GDP:

geranyl diphosphate

HMGR:

3-hydroxy-3-methylglutaryl coenzyme A reductase

MEP:

methylerythritol phosphate

MVA:

mevalonate

PLACE:

plant cis-acting regulatory elements

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Acknowledgments

We acknowledge financial support from Council of Scientific and Industrial Research (CSIR) for work on gene cloning through Mission Mode program entitled “Development of medicinal plant chemotypes for enhanced marker and value added compounds (COR002)”. Financial support from the Department of Biotechnology (DBT), Govt. of India is duly acknowledged for funding the project entitled “Molecular cloning and characterization of regulatory genes involved in picrosides metabolism in Picrorhiza kurrooa” sanctioned vide sanction order number BT/PR6614/PBD/17/425/2005 to work on promoters. TK thanks CSIR, India, for awarding CSIR Diamond Jubilee Research Intern Award; HS thanks CSIR for awarding junior and senior research fellowships. AK, KD, and SKS gratefully acknowledge DBT for providing assistantship. Technical help provided by Mr. Digvijay Singh in gene sequencing is duly acknowledged. The manuscript represents IHBT communication number 0812.

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Author notes

  1. Harsharan Singh

    Present address: Biotechnology Division, Lyallpur Khalsa College, Jalandhar, Punjab, India

  2. Amit Kumar

    Present address: Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

  3. Sunil Kumar Sharma

    Present address: Biotechnology Division, Jaypee University of Information Technology, Waknaghat, Distt., Solan, Himachal Pradesh, India

Authors and Affiliations

  1. Biotechnology Division, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur, 176061, Himachal Pradesh, India

    Tabasum Kawoosa, Harsharan Singh, Amit Kumar, Sunil Kumar Sharma, Kiran Devi, Som Dutt, Madhu Sharma, Paramvir Singh Ahuja & Sanjay Kumar

  2. Biodiversity Division, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur, 176061, Himachal Pradesh, India

    Surender Kumar Vats

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  1. Tabasum Kawoosa
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Correspondence to Sanjay Kumar.

Electronic supplementary material

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Electronic Supplementary Table S1

Comparison of predicted secondary structures of HMGR and DXS of picrorhiza with those from other plants. Protein sequences were retrieved from the NCBI databank and secondary structural prediction was carried out using ExPASy server tool SOPMA (http://www.npsa-pbil.ibcp.fr/) by keeping similarity threshold 8 and conformational states 4. (DOC 74 kb)

Electronic Supplementary Fig. S1

Multiple sequence alignment of deduced amino acids encoded by pkhmgr (3-hydroxy-3-methylglutaryl coenzyme A reductase; S1 a) and pkdxs (1-deoxy-D-xylulose-5-phosphate synthase; S1 b) of P. kurrooa. Alignment was performed by Accelrys software tool (Accelrys Gene software, version 2.5 of Accelrys software Inc., USA). Sequences denoted are: P. kurrooa -PK (ABC74565), Nicotiana tabacum -NT (AAB87727), Eucommia ulmoides -EU (AAV54051), Andrographis paniculata -AP (AAP14352), Litchi chinensis -LC (ABF56181), Mentha x piperita -MP (AAC33513), Antirrhinum majus -AM (AAW28999), Tagetes erecta -TE (AAG10432), Catharanthus roseus -CR (CAA09804). Accession number of the deduced amino acid sequences is shown in the respective parenthesis. (PPT 394 kb)

Electronic Supplementary Fig. S2

Conserved domains of deduced proteins pkHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase) and pkDXS (1-deoxy-D-xylulose-5-phosphate synthase) of picrorhiza. Deduced amino acid sequences were analyzed for the location of conserved domains using Conserved Domain Database available at NCBI (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi). (PPT 39 kb)

Electronic Supplementary Fig. S3

Predicted secondary structure of deduced proteins, pkHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase) and pkDXS (1-deoxy-D-xylulose-5-phosphate synthase) using ExPASy server tool, SOPMA (http://www.npsa-pbil.ibcp.fr/) by keeping similarity threshold 8 and conformational states 4. Helices, sheets, turns, and coils are indicated with the longest, the second longest, the second shortest and the shortest vertical lines, respectively. (PPT 104 kb)

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Kawoosa, T., Singh, H., Kumar, A. et al. Light and temperature regulated terpene biosynthesis: hepatoprotective monoterpene picroside accumulation in Picrorhiza kurrooa . Funct Integr Genomics 10, 393–404 (2010). https://doi.org/10.1007/s10142-009-0152-9

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