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Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

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Abstract

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.

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Abbreviations

ABA:

Abscisic acid

APX:

Ascorbate peroxidase

AtCKX2:

Transgenic tobacco overexpressing the Arabidopsis gene for CKX2

CAT:

Catalase

cisZ:

cis-zeatin

cisZ7G:

cis-zeatin-7-glucoside

CKX:

Cytokinin oxidase/dehydrogenase

DCPIP:

2,6-dichlorophenolindophenol

DTT:

Dithiotreitol

EDTA:

Ethylendiaminetetraacetic acid

GR:

Glutathione reductase

GSSG:

Oxidised glutathione

iP:

N6-(Δ2-isopentenyl)adenine

iP7G:

N6-(Δ2-isopentenyl)adenine-7-glucoside

MTT:

3(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide

NBT:

3,3′-(3,3′-dimethoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride]

PAR:

Photosynthetically active radiation

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

TEMED:

N,N,N′,N′-tetramethylethylenediamine

XTT:

2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, sodium salt

Z:

trans-zeatin

Z7G:

trans-zeatin-7-glucoside

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Acknowledgments

This work was supported by the Grant Agency of the Czech Republic (522/03/0312), the Grant Agency of the Academy of Sciences of the Czech Republic (IAA600380701), the Ministry of Education, Youth and Sports CR (LC 06034) and AVOZ50380511. The authors thank Dr. Miroslav Kamínek for critical reading of manuscript and Ms. Marie Korecká and Ms. Lenka Kolčabová for excellent technical assistance.

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Authors and Affiliations

  1. Faculty of Science, Charles University, Albertov 6, 128 43, Praha 2, Czech Republic

    Zuzana Lubovská

  2. Academy of Sciences of the Czech Republic, v.v.i., Institute of Experimental Botany, Na Karlovce 1a, 160 00, Praha 6, Czech Republic

    Zuzana Mýtinová, Daniel Haisel, Zuzana Lubovská & Naďa Wilhelmová

  3. Academy of Sciences of the Czech Republic, Institute of Experimental Botany, Rozvojová 263, 165 02, Praha 6, Czech Republic

    Václav Motyka, Alena Trávníčková & Petre Dobrev

  4. Academy of Sciences of the Czech Republic, Institute of Experimental Botany, Vídeňská 1083, 142 20, Praha 4, Czech Republic

    Josef Holík

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  1. Zuzana Mýtinová
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  2. Václav Motyka
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  3. Daniel Haisel
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  4. Zuzana Lubovská
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  5. Alena Trávníčková
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  7. Josef Holík
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  8. Naďa Wilhelmová
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Correspondence to Naďa Wilhelmová.

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Z. Mýtinová and V. Motyka contributed equally to this work.

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Mýtinová, Z., Motyka, V., Haisel, D. et al. Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion. Plant Growth Regul 65, 23–34 (2011). https://doi.org/10.1007/s10725-011-9571-4

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