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Overexpression of Artemisia annua sterol C-4 methyl oxidase gene, AaSMO1, enhances total sterols and improves tolerance to dehydration stress in tobacco

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Abstract

Biosynthesis of sterols is a multistep process in higher plants where the precursor cycloartenol gets converted into functional phytosterols after removal of two methyl groups at C-4 by an enzyme complex involving a sterol C-4 methyl oxidase (SMO). We identified and cloned a cDNA from Artemisia annua designated as AaSMO1 showing similarity to SMO. The cDNA predicted to encode a polytopic protein with characteristic histidine-rich motifs and an ER retrieval signal. GFP-AaSMO1 fusion protein was localized in endoplasmic reticulum of transformed protoplast and onion epidermal cells. AaSMO1 expression was drastically induced upon osmotic/dehydration stress and its promoter showed the presence of abscisic acid responsive element. Transgenic tobacco plants ectopically overexpressing AaSMO1 were raised, and various biochemical and physiological analyses of transgenics revealed increased total sterol, better germination and growth in subsequent generations. They also exhibited reduced sensitivity towards osmotic/dehydration stress which may be attributed to enhanced SMO1 activity. Our studies demonstrated that apart from acting as phytohormones, plant sterols also participate in providing capability to plants for improved growth and adaptation during stress conditions. AaSMO1 can be used as an excellent candidate for generating dehydration/drought tolerant plants.

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Abbreviations

4-MU:

4-Methylumbelliferone

ABA:

Abscisic acid

ABRE:

Abscisic acid responsive element

DRE/CRT:

Dehydration-responsive element/C-RepeaT

ER:

Endoplasmic reticulum

GA:

Gibberellic acid

GFP:

Green fluorescent protein

GUS:

β-Glucuronidase

MeJa:

Methyl jasmonate

MS:

Murashige and Skoog medium

MUG:

4-Methylumbelliferyl glucuronide

RT-qPCR:

Reverse transcription quantitative real-time PCR

SMO:

Sterol-4α-methyl-oxidase/sterol C-4 methyl oxidase

TCA:

Trans-cinnamic acid

TLC:

Thin layer chromatography

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Acknowledgments

The initial work was supported by CSIR, Govt. of India, under NWP08 project, and further funding was provided by SERB, DST, Govt. of India under GAP265 project. Research fellowships from CSIR and UGC, Govt. of India, are acknowledged. The authors wish to express their sincere thanks to the Director, CSIR-CIMAP for providing necessary facilities. Help from Drs. R. K. Shukla, A. K. Shasany, A. S. Negi and Ms. Pooja Sharma is duly acknowledged. Dr. Samir Sawant from CSIR-NBRI, Lucknow and Dr. Jitendra Thakur from NIPGR, New Delhi, are acknowledged for extending their help in confocal microscopy.

Conflict of interest

The authors declare no competing interests.

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Authors and Affiliations

  1. Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India

    Alka Singh, Sunita Jindal, Bendangchuchang Longchar & Vikrant Gupta

  2. Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India

    Feroz Khan

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  1. Alka Singh
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Correspondence to Vikrant Gupta.

Electronic supplementary material

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11240_2014_692_MOESM1_ESM.ppt

Supplemental Fig. S1 Comparative and phylogenetic analyses of AaSMO1. a Predicted amino acid sequence was aligned with selected sterol C-4 methyl oxidases reported in NCBI database by using ClustalW. Identical and similar amino acid residues are shaded in black and grey, respectively. Dashes denote gaps inserted to maximize the alignment. Signature characteristic motifs (domain I, II and III) of sterol C-4 methyl oxidases are boxed and ER retrieval signal is underlined. b Phylogenetic relationship (unrooted neighbor-joining tree) of AaSMO1 with related homologs from other plant species. The sequences used for alignment or phylogenetic analysis in ‘a’ and ‘b’ include: A. thaliana (CAB78254; NP_567670; NP_001077975; AAM65428; AAM64961; NP_567669; NP_850133; CAA62079; AAQ13424), A. tripolium (BAC57961), Brachypodium distachyon (XP_003574284), Glycine max (NP_001276171), Gossypium arboreum (AAO13795), Hordeum vulgare (BAK02768; BAJ99813), Komagataella pastoris/Pichia pastoris (XP_002492261), Medicago truncatula (XP_003608797; XP_003613314), N. benthamiana (AAQ83691), N. tabacum (AAD04034; AAD20458), Oryza sativa (AAN62786), P. trichocarpa (XP_002304440), Ricinus communis (EEF51010; XP_002509623), S. cerevisiae (EDN61653; P32353; AAA16608), Sorghum bicolor (XP_002455692), V. vinifera (XP_002282305; XP_002282653; XP_002270978), Z. mays (NP_001105744; NP_001148435; NP_001148698) and A. annua (AaSMO1; GQ847864). (PPT 565 kb)

11240_2014_692_MOESM2_ESM.ppt

Supplemental Fig. S2 Kyte-Doolittle hydrophobicity plot of AaSMO1. Amino acid regions with values above zero are hydrophobic in nature. The horizontal scale indicates the position of amino acids. Hydrophobic regions identified in the plot are encircled. (PPT 152 kb)

11240_2014_692_MOESM3_ESM.doc

Supplemental Fig. S3 In silico docking of AaSMO1 with 24-methylene cycloartanol and 24-ethylidene lophenol (i.e. substrates of SMO1 and SMO2, respectively) by homology modelling, and its comparison with that of the binding affinity of AtSMO1-1 isoform. (DOC 701 kb)

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Singh, A., Jindal, S., Longchar, B. et al. Overexpression of Artemisia annua sterol C-4 methyl oxidase gene, AaSMO1, enhances total sterols and improves tolerance to dehydration stress in tobacco. Plant Cell Tiss Organ Cult 121, 167–181 (2015). https://doi.org/10.1007/s11240-014-0692-0

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