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Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants

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Abstract

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.

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Abbreviations

CMS:

Cytoplasmic male sterility

GMS:

Genetic male sterility

CGMS:

Cytoplasmic genetic male sterility

PET1:

Helianthus petiolaris cytoplasm based male sterile line in sunflower

CaMV35S:

Cauliflower mosaic virus 35S promoter

TA29:

Tapetum-specific promoter from tobacco

orfH522:

Open reading frame associated with PET1-CMS of sunflower

PCD:

Programmed cell death

Rf :

Restorer of fertility

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Acknowledgments

The financial help by Indian Council of Agricultural Research, New Delhi through a research grant (Code no. 030558005) to V.D.K. for carrying out this work is acknowledged. The help given by Dr. Nagamalleswari, Associate Professor, Ruska lab, ANGRAU, Hyderabad, India in microtomy work is appreciated. Authors are thankful to Dr. A.R.G. Ranganatha, Principal Scientist, Directorate of Oilseeds Research, Hyderabad for providing the seed material of sunflower CMS and fertility restorer lines. Suggestions given by Dr. S.R. Bhat, Principal Scientist, NRCPB, IARI, New Delhi during the course of this investigation are acknowledged. We are highly thankful to the two anonymous reviewers whose probing questions, suggestions and comments helped us in improving the clarity and quality of the manuscript significantly.

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Authors and Affiliations

  1. Directorate of Oilseeds Research, Rajendranagar, Hyderabad, 500 030, India

    Narasimha Rao Nizampatnam, Sujatha Mulpuri & Dinesh Kumar Viswanathaswamy

  2. Tata Institute of Fundamental Research, Mumbai, India

    Harinath Doodhi

  3. Regional Agricultural Research Station, ANGRAU, Warangal, Andhra Pradesh, India

    Yamini Kalinati Narasimhan

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  1. Narasimha Rao Nizampatnam
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  2. Harinath Doodhi
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  3. Yamini Kalinati Narasimhan
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  4. Sujatha Mulpuri
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  5. Dinesh Kumar Viswanathaswamy
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Correspondence to Dinesh Kumar Viswanathaswamy.

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425_2009_888_MOESM1_ESM.tif

Supplementary Fig. 1 Primers used for different PCR/RT-PCR for confirmation of the presence or expression of transgene are indicated. Details of the primers are in Table 1. The figure is not to scale.

The mitochondrial transit sequence indicated as dotted boxes was the first 25 amino acid sequence of COXIV from yeast, orfH522 is indicated by open box, grey colored boxes are nos terminator and open arrow indicates the TA29 promoter from tobacco

P1: SphI TA29 For, P2: SalI CoxIV For, P3: KpnI orfH522 For, P4: orf316 For, P5: SacI orfH522 Rev, P6: orf316 Rev

PCR confirmation of the presence of transgene cassette (for which the data has been provided) was carried out with P1 and P5 in T0 generation and with P2 and P5 in the T1 generation. Apart from these sets, other sets of primers were used for the analyses, but the data are not provided. For RT-PCR analysis of T0 plants of TCON and TON constructs, P3 and P5 were used while with T1 plants of TCON construct, P3 and P6 were used. For probe preparation, primers P4 and P6 were used. For confirmation of the cloning of gene cassettes during vector construction, all the primer sets were used. (TIFF 1169 kb)

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Nizampatnam, N.R., Doodhi, H., Kalinati Narasimhan, Y. et al. Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants. Planta 229, 987–1001 (2009). https://doi.org/10.1007/s00425-009-0888-4

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