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Execution of programmed cell death by singlet oxygen generated inside the chloroplasts of Arabidopsis thaliana

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Abstract

Absorption of excess excitation energy induces overproduction of singlet oxygen (1O2) in plants. The major sources of singlet oxygen production are chlorophyll and its intermediates located in the chloroplast. Over-accumulation of the chlorophyll biosynthetic intermediate protochlorophyllide by the exogenous application of 5-aminolevulinic acid (ALA), the precursor of tetrapyrrole, induced singlet oxygen production in the plastidic membranes. Over-expression of protochlorophyllide oxidoreductase C (PORC) in Arabidopsis thaliana resulted in efficient light-induced photo-transformation of protochlorophyllide to chlorophyllide that limited the accumulation of protochlorophyllide. Consequently, the 1O2 generation decreased in the PORC overexpressors (PORCx) and their cell death was minimal. Conversely, porC-2 over-accumulated protochlorophyllide in response to ALA treatment and generated higher amounts of 1O2 in light and had highest cell death as monitored by Evans blue staining. The protoplasts isolated from PORCx plants, when treated with ALA, generated minimal amounts of 1O2 as revealed by singlet oxygen sensor green (SOSG) fluorescence emission from chloroplasts. Conversely, the protoplasts of porC-2 mutants under identical conditions generated the maximum SOSG fluorescence in their chloroplasts and cytosol surrounding the chloroplasts most likely due to the leakage from the organelle. The membrane blebbing, a hallmark of programmed cell death, was clearly visible in WT and porC-2 protoplasts. Similarly, the nick end labelling (TUNEL) assay revealed nicks in the DNA. The TUNEL-positive nuclei after 30 min of light exposure were highest in porC-2 and lowest in PORCx protoplasts. The results demonstrate that higher amounts of singlet oxygen produced in the chloroplasts play an important role in programmed cell death.

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Abbreviations

1O2 :

Singlet oxygen

ALA:

5-Aminolevulinic acid

Chlide:

Chlorophyllide

Chls:

Chlorophylls

DAPI:

4′,6-diamidino-2-phenylindole

DIC:

Differential interference contrast

FDA:

Fluorescein diacetate

FITC:

Fluorescein isothiocyanate

Fv/Fm:

Ratio of variable fluorescence to maximal fluorescence

PCD:

Programmed cell death

Pchlide:

Protochlorophyllide

PSI:

Photosystem I

PSII:

Photosystem II

POR:

Protochlorophyllide oxidoreductase

ROS:

Reactive oxygen species

SOSG:

Singlet oxygen sensor green

TUNEL:

Terminal deoxynucleotide transferase dUTP nick end labelling

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Funding

This work was supported by a grant from the Department of Science and Technology, Government of India (EMR/2016/004976) and BSR Faculty Fellowship from the University Grants Commission to BCT. BST received financial support from DST/SERB (No EMR/2016/002732).

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  1. Vivek Ambastha and Garima Chauhan contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

    Vivek Ambastha, Garima Chauhan & Baishnab C Tripathy

  2. School of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba, Gandhinagar, Gujarat, 382007, India

    Budhi Sagar Tiwari

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  1. Vivek Ambastha
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  2. Garima Chauhan
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  4. Baishnab C Tripathy
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Correspondence to Baishnab C Tripathy.

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Ambastha, V., Chauhan, G., Tiwari, B.S. et al. Execution of programmed cell death by singlet oxygen generated inside the chloroplasts of Arabidopsis thaliana. Protoplasma 257, 841–851 (2020). https://doi.org/10.1007/s00709-019-01467-y

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