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Proteolytic activities in cortex of apical parts of Vicia faba ssp. minor seedling roots during kinetin-induced programmed cell death

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Abstract

Programmed cell death (PCD) is a crucial process in plant development. In this paper, proteolytically related aspects of kinetin-induced PCD in cortex cells of Vicia faba ssp. minor seedlings were examined using morphological, fluorometric, spectrophotometric, and fluorescence microscopic analyses. Cell viability estimation after 46 μM kinetin treatment of seedling roots showed that the number of dying cortex cells increased with treatment duration, reaching maximum after 72 h. Weight of the apical root segments increased with time and was about 2.5-fold greater after 96 h, while the protein content remained unchanged, compared to the control. The total and cysteine-dependent proteolytic activities fluctuated during 1–96-h treatment, which was not accompanied by the changes in the protein amount, indicating that the absolute protein amounts decreased during kinetin-induced PCD. N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), and Z-Leu-Leu-Nva-H (MG115), the respective cysteine, serine, and proteasome inhibitors, suppressed kinetin-induced PCD. PMSF significantly decreased serine-dependent proteolytic activities without changing the amount of proteins, unlike NEM and MG115. More pronounced effect of PMSF over NEM indicated that in the root apical segments, the most important proteolytic activity during kinetin-induced PCD was that of serine proteases, while that of cysteine proteases may be important for protein degradation in the last phase of the process. Both NEM and PMSF inhibited apoptotic-like structure formation during kinetin-induced PCD. The level of caspase-3-like activity of β1 proteasome subunit increased after kinetin treatment. Addition of proteasome inhibitor MG-115 reduced the number of dying cells, suggesting that proteasomes might play an important role during kinetin-induced PCD.

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Abbreviations

Ctrl:

control

Kin:

Kinetin

MG115:

Z-Leu-Leu-Nva-H

NEM:

N-ethylmaleimide

PCD:

Programmed cell death

PMSF:

Phenylmethylsulfonyl fluoride

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Acknowledgments

We thank Ms. M. Fronczak for the help in preparing the manuscript in English. This work was supported by a grant from the University of Łódź, no. 506/1141 and no. B1551000000988.02.

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Authors and Affiliations

  1. Department of Cytophysiology, Faculty of Biology and Environmental Protection, The University of Łódź, Pomorska 141/143, 90-236, Łódź, Poland

    Andrzej Kaźmierczak, Magdalena Doniak & Anita Kunikowska

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  1. Andrzej Kaźmierczak
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  2. Magdalena Doniak
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  3. Anita Kunikowska
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Correspondence to Andrzej Kaźmierczak.

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Handling Editor: Pavel Dráber

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Fig. S1

Simple exemplification of existence signalling, killing and execution phase of Kin-induced of death of cortex cells of the root apical segments of V. faba ssp. minor seedlings in a triangular form. The scheme shows that kinetin induced death signal via cytokinin receptors, then the proteolytic machinery is activated activating the nuclei and cell wall degradation. (GIF 36 kb)

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Kaźmierczak, A., Doniak, M. & Kunikowska, A. Proteolytic activities in cortex of apical parts of Vicia faba ssp. minor seedling roots during kinetin-induced programmed cell death. Protoplasma 254, 2273–2285 (2017). https://doi.org/10.1007/s00709-017-1119-7

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