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Characterization of MYB35 regulated methyl jasmonate and wound responsive Geraniol 10-hydroxylase-1 gene from Bacopa monnieri

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Abstract

Main conclusion

BmG10H-1 transcript from B. monnieri was functionally active. BmG10H-1 promoter drives GUS activity in response to MeJA and wounding. BmMYB35 regulates BmG10H-1 transcript by binding to its promoter.

Geraniol 10-hydroxylase (G10H) is one of the important regulatory cytochrome P450 monooxygenase, which is involved in the biosynthesis of monoterpene alkaloids. However, G10H is not characterized at the enzymatic or at the regulatory aspect in B. monnieri. In the present study, we have identified two transcripts of BmG10H (BmG10H-1and BmG10H-2) and characterized the methyl jasmonate (MeJA) and wound responsive BmG10H-1 transcript from B. monnieri. BmG10H-1 showed induced expression after 3 h of MeJA and wounding treatment in the shoot. Yeast purified recombinant BmG10H-1 protein is enzymatically active, having Vmax of 0.16 µMsec−1 μg−1 protein and catalyzes the hydroxylation of geraniol to 10-hydroxy geraniol. The BmG10H-1 promoter was isolated by using the genome walking method. BmG10H-1 promoter can drive GUS expression in transgenic Arabidopsis thaliana. GUS activity of MeJA and wound-treated Arabidopsis seedlings were found to be increased as compared to the control untreated seedlings, whereas no GUS activity was found in deleted MeJA responsive and W-box cis-elements. This shows that the BmG10H-1 promoter contains functional MeJA (TGACG) and wound responsive (TGACCT) cis-elements. Further, shoot specific and MeJA responsive recombinant BmMYB35 protein was purified, which binds with the MYB recognition cis-element (TGGTTA) present in the BmG10H-1 promoter and transcriptionally activates the reporter gene in yeast. In conclusion, the characterization of MeJA and wound responsive BmG10H-1 provides novel information about its transcriptional regulation by binding with MYB transcription factor in B. monnieri.

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Acknowledgments

The authors acknowledge the national gene bank of the Central Institute of Medicinal and Aromatic Plants (CIMAP) Lucknow for providing B. monnieri plants. RKS acknowledge Director CSIR-CIMAP for providing necessary facilities. RKS acknowledge CSIR-FBR (MLP0005) for funding. GSJ acknowledges UGC for fellowship. We are thankful to analytic CSIR-CIMAP, Dr. Karuna Shanker, Dr. Neerja Tiwari, and Namita Gupta (Central Instrumentation facility, CSIR-CIMAP) for providing the HPLC facility.

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Authors and Affiliations

  1. Biotechnology Division (CSIR-CIMAP), Central Institute of Medicinal and Aromatic Plants, (CSIR-CIMAP), P.O. CIMAP, Near Kukrail Picnic Spot, Lucknow, 226015, India

    Gajendra Singh Jeena, Sunil Kumar & Rakesh Kumar Shukla

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  1. Gajendra Singh Jeena
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  2. Sunil Kumar
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  3. Rakesh Kumar Shukla
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Correspondence to Rakesh Kumar Shukla.

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Communicated by Anastasios Melis.

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Supplementary Information

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425_2021_3614_MOESM1_ESM.pptx

Melting curve analysis of internal reference genes actin and ubiquitin for normalization of genes in the qRT-PCR experiment (PPTX 381 KB)

425_2021_3614_MOESM2_ESM.pptx

Linearity plot of the standard using HPLC. The calibration curve of the standard product (10 hydroxy-geraniol) based upon the peak area at different concentrations was plotted (PPTX 38 KB)

425_2021_3614_MOESM3_ESM.pptx

Promoter analysis and primer designing. BmG10H-1 promoter sequence obtained after sequencing. Forward and reverse primer sequences used in the study are shown and highlighted in green (PPTX 100 KB)

425_2021_3614_MOESM4_ESM.pptx

(a) Phylogenetic analysis of BmG10H from different plant species. The phylogenetic tree was constructed by using MEGA6.06 software with a pair wise distance and neighbor-joining algorithm. The characterized G10H sequences from different plant species with maximum homology were utilized to generate the phylogenetic trees. The numbers represent the bootstrap values. (b) Multiple sequence alignment of BmG10H protein by using Clustal Omega (PPTX 610 KB)

425_2021_3614_MOESM5_ESM.pptx

Substrate saturation curve of the BmG10H enzyme for substrate geraniol. Vmax value was calculated based upon the Michaelis Menten saturation plot (PPTX 34 KB)

425_2021_3614_MOESM6_ESM.pptx

In silico analysis of BmG10H-1 promoter. (a) A 939 bp fragment of upstream promoter sequence containing the start codon is marked by red color. Some important regulatory cis-elements are marked with different colors and underlined. The potential MYB binding site is highlighted and marked with an asterisk symbol. Arrow indicates the direction of the promoter. The region downstream of ATG that matches with the BmG10H-1 gene is shown with dark red color. Plus sign indicates the nucleotide position upstream to start codon. (b) Schematic representation of the promoter elements along with their different truncations. D1 (deletion-1 represents MeJA responsive cis-element deleted), D2 (represents W-box cis-element deleted), and ID3 (represents MYB binding cis-element deleted) (PPTX 165 KB)

425_2021_3614_MOESM7_ESM.pptx

Generation of BmG10H-1 promoter transgenic lines in Arabidopsis thaliana. The transgenic lines were selected in kanamycin containing MS media and confirmed by genomic DNA PCR using nptII specific primers. An expected band of 786bp was obtained in the PCR from genomic DNA. Positive lines were further grown in selection media to obtained T3 homozygous seeds (PPTX 1421 KB)

425_2021_3614_MOESM8_ESM.pptx

Histochemical localization of GUS activity in roots of transgenic Arabidopsis after MeJA and wound treatment. Scale bar = 1mm (PPTX 265 KB)

425_2021_3614_MOESM9_ESM.pptx

Differential gene expression data of BmMYB35 obtained from B. monnieri transcriptome. BmMYB35 is uniquely expressed in shoot tissue as compared to root tissue with a significant P-value (PPTX 39 KB)

425_2021_3614_MOESM10_ESM.pptx

Protein induction in E. coli Bl21. The recombinant BmMYB35-GST protein was induced by using different concentrations of IPTG at 370C for 5 hours along with vector control. The size of (GST)-BmMYB35 is 61.6 KD, which is purified by using affinity beads and run in SDS-PAGE (PPTX 516 KB)

. List of primers (XLSX 12 KB)

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Jeena, G.S., Kumar, S. & Shukla, R.K. Characterization of MYB35 regulated methyl jasmonate and wound responsive Geraniol 10-hydroxylase-1 gene from Bacopa monnieri. Planta 253, 89 (2021). https://doi.org/10.1007/s00425-021-03614-3

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