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Reactivation of electron flow in chloroplasts of in vitro shootlets of apple through elimination of carbon source and evaluation of its activity by inhibitors of electron transport chain and chlorophyll fluorescence quenching

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Abstract

Electron transport chains (ETC) are drastically inactivated in chloroplasts of in vitro shootlets due to presence of carbon source in the growth media. This inactivation consequently creates limitations for in vitro studies on the role of chloroplasts in biotic and abiotic stresses. This research was carried out to evaluate reactivation of electron flow in chloroplasts ETC by elimination of carbon source and subsequent comparison of the effects of ETC inhibitors in the presence and absence of carbon source (i.e., sucrose) on the in vitro shootlets of apple rootstock, MM-111. All the tested ETC inhibitors, including uracil; glutaraldehyde, methyl viologen (MV) and DCMU triggered necrosis appearance on the in vitro apple shootlets exclusively on the carbon source-free media. Moreover, exposure of the in vitro shootlets to the various concentrations of ETC inhibitors demonstrated different severity of ETC inhibition by these inhibitors and their concentration-dependent effects on chloroplasts ETC. Uracil, the inhibitor of photosystem II, triggered the weakest shootlets necrosis, while DCMU exhibited no necrogenic effects at 50 mg/L and lower concentrations. MV, contrary to glutaraldehyde, did not have necrogenesis effects at 1 mg/L, but both of them caused sharp necrosis trigger on the shootlets as soon as 48–96 h after exposure to their effective concentrations. Evaluation of the shootlets H+-ATPase activity revealed that proton extrusion and pH decline in the growth media were mainly associated with the sucrose uptake by the shootlets, while no considerable pH decline observed in the presence of carbon source in the media. Finally, chloroplasts ETC activity of the shootlets was confirmed by higher percentage of O2 and less CO2 quantity in the atmospheres of the jars containing sucrose-free media, as well as by higher initial fluorescence (Fo) and maximum fluorescence (Fm) quenching parameters in the shootlets, in comparison with the sucrose-enriched condition.

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Abbreviations

ETC:

Electron transport chain

ROS:

Reactive oxygen species

MV:

Methyl viologen

DCMU:

3-(3,4-Dichlorophenyl)-1,1-dimethylurea

MM-111:

Malling-Merton 111

QL:

Quoirin and Lepoivre medium

Fo :

Initial fluorescence

Fm :

Maximum fluorescence

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Acknowledgements

This research was a collaboration project between Temperate Fruits Research Center of Horticultural Sciences Research Institute (HSRI) and Azad University of Tehran, Science and Research Unit. The authors sincerely thank all the technical stuff of fruit tissue culture and biotechnology laboratory of Temperate Fruits Research Center of Horticultural Sciences Research Institute (HSRI) for their kind supports. The funding was provided by Iranian Ministry of Agriculture, Agricultural Research Education and Extension Organization (AREEO).

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Authors and Affiliations

  1. Temperate Fruits Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

    Hamid Abdollahi & Zeynab Salehi

  2. Science and Research Unit, Faculty of Agriculture and Natural Resources, Azad University of Tehran, P.O. Box. 4933, Tehran, 14155, Iran

    Kobra Erfaninia & Zahra Ghahremani

  3. Division of Plant Molecular Genetics, Department of Agronomy and Plant Breeding, Agricultural & Natural Resources College, University of Tehran, Karaj, Iran

    Jaber Nasiri

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  1. Hamid Abdollahi
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  2. Kobra Erfaninia
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  3. Zahra Ghahremani
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  4. Jaber Nasiri
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  5. Zeynab Salehi
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Contributions

HA designed and supervised the study, wrote the manuscript and provided helpful discussions. KE performed experimental works of glutaraldehyde, methyl viologen (MV) and DCMU sections of the study. ZG carried out some parts of chloroplasts ETC activity by inhibitors alongside H+-ATPase activity section of the work. JN revised whole body of the paper, contributed in data analysis and provided micropropagation apples data alongside scientific information/helps about justification of activation of electron flow in chloroplast. SS participated in plant culture, chlorophyll fluorescence section, assay of O2/CO2 production and H2O2 generation. All the authors read and approved final version of the manuscript.

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Correspondence to Hamid Abdollahi.

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Communicated by Henryk Flachowsky.

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Abdollahi, H., Erfaninia, K., Ghahremani, Z. et al. Reactivation of electron flow in chloroplasts of in vitro shootlets of apple through elimination of carbon source and evaluation of its activity by inhibitors of electron transport chain and chlorophyll fluorescence quenching. Plant Cell Tiss Organ Cult 131, 377–389 (2017). https://doi.org/10.1007/s11240-017-1291-7

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