DEVELOPMENTAL & REPRODUCTIVE BIOLOGY Vol. 9 No. 2 pp: 61-68 Dec.

2000

ISSN 1004-6453/CN11-3569/Q 2000 Chin. Dev& Reprod. Biol. Socs.

An ELISA Assay for Detection of Apoptosis in Plant Cells1

NING Shun-bin, WANG Ling and SONG Yun-chun2
Key Laboratory of MOE for Plant Development, Wuhan University, Wuhan 490072, China

ABSTRACT
This paper presents the results of the ELISA and TUNEL assays for the detection of cell death in maize root tips. The results indicates that the active apoptosis process can be in-

duced in the root-tip cells of maize by isopropyl carbanilate (IPC) at moderate concentrations, and characterized by morphological and biochemical characteristics, including DNA fragmentation, nuclear condensation and chromatin marginalization (Fig.1). The ELISA tech-

nique was used for the first time for detection of apoptosis in plant system, and the results indicate it is available in plant cells. OD values of cytoplasm obtained from ELISA rose The results of dos-

gradually during early apoptotic period and then declined (Fig.2).

age-dependent tests indicate that 0.2 mg/mL IPC is most moderate for inducing apoptosis in maize root cells. Keywords: Apoptosis, Chromosome preparation, TUNEL, ELISA, Plants

1.

INTRODUCTION

Great advances on programmed cell death (PCD) in plants have achieved in recent two years. In addition to the discoveries of the activities of caspase-like proteinases [1~5] and the effect of Ca2+[5~7], it has been found that a variety of plant hormones such as GA, ABA and ethylene etc [8~13] and cytochrome C (CytC)[14~17], rice GTP-binding protein OsRac [18] and lipoid [19] play important role in signal transduction of PCD in plants. In the cause of apoptosis, the DNA of nucleosomes, produced from the dsDNA (double-stranded DNA) of chromosome, is tightly bound to nuclear histones, H2A, H2B, H3 and H4, as a compound to protect the dsDNA from degradation by nucleases. In the early period of apoptosis, the fragmentation of DNA only occurs in a few cells and the ladder pattern of DNA fragments can hardly be resolved by the biochemical methods, such as gel electrophoresis. But, by using Sandwich ELISA (enzyme-linked immu-

1 This project is granted by State Natural Science foundation (Grant No.39870423) and State Commission of Education, doctorate Spot Fund (Grant No.207980112). 2 Author for correspondence. Tel: (027)87684505. E-mail: ycsong@whu.edu..cn. Vol. 9 No. 2 (2000) 61

NING Shun-bin, , et al.

nosorbent assay) with the antibodies against histone and nuclear DNA, the detection sensitivity can be greatly improved, and is enough for detection of apoptosis at very early stage [20]. The basic principle is as follows: the characteristic oligomeric nucleosomes are released into cytoplasm as the cells undergo programmed death, and the cytoplasm is then collected by centrifugation. The anti-histone antibody is firstly conjugated to histones of the oligomeric nucleosomes and then the POD (peroxidase)-conjugated anti-DNA antibody is bound to DNA of the nucleosomes (so is the sandwich). Then the color reaction may be developed through the substrate of POD, DAB (3,3'-diaminobenzidine), and be measured by using enzyme-linked immunosorbent assay device and shown as OD (optical density) values [20]. This method has been applied extensively in animals, and the specified kit was already developed. But no application of it has been reported in plants. Because the constitutive units of nucleosomes (nucleotides and histones), especially H3 and H4, are extraordinary conserved in eucaryotes, we think hence that the antibodies against histones and DNA raised from animals can be used for detection of apoptosis in plants and can achieve the goal like that in animals. Isopropyl carbanilate (IPC) is a kind of selective herbicide for killing monocotyledon weeds but has little effect on dicotyledonous plants [21]. The meristematic cells of plant roots show the most sensitivity responded to many adverse environmental factors and are easy to be taken as experimental material for the study on the stresses from adverse environments [22]. The meristematic tissue was used in present research for two purposes as follows: 1) To research whether the IPC-induced cell death is active apoptosis in plant cells and thereby to investigate the mechanism of IPC function on cellular level; 2) to make sure whether ELISA can be available for detecting the apoptosis in plants as in animals.

2. MATERIALS AND METHODS

2.1. Experimental materials

The seeds of 'Yi Dan 6' were used as experimental material, which was kindly provided by Professor Gu Mingguang from the Institute of Genetics, Chinese Academy of Sciences.

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ELISA Assays for Detection of Apoptosis in Plant Cells

2.2. Induction of apoptosis in meristematic cells of roots-tip in maize

Following the method of Katsuhara et al. [22], the seeds of maize were soaked in double-distilled water for 1 day, and transferred to the culture solution (4 mM KNO3, 1 mM MgSO4, 1 mM CaCl2, 1 mg/L ferrous citrate, pH 5.5 ) after germination and cultured in dark condition until the young root-tip developed into around 2 cm in length and then again transferred into 0.01-20 mg/mL IPC solution for treatments for 1-7 days, respectively.
2.3. Chromosome preparation and TUNEL in individual cells

The chromosome preparation and the in situ labeling followed the methods of Ning Shunbin et al. (1999) with modifications [23]. The root tips were cut at 2 mm in length in control and IPC-treated roots and immediately fixed in methanol : acetic acid (3:1) solution for around 3 h, then the root caps were cut off and discarded. After fully washing with dd-H2O, the tips were enzymolysis with the mixture of 2% pectinase and 2% cellulase for around 3 h., the slide was then dried over flame. For in situ labeling, the Fluorescein-dUTP was substituted for Biotin-dUTP, and TdT enzyme (terminal deoxynucleotidyl transferase) for Klenow enzyme (TUNEL Detection Kit, purchased from Boehringer Mannheim Co., In situ Cell Death Detection Kit, Fluorescein). The reaction was carried out under 37 for 1 h. After labeling, the slides were washed in PBS for 35 min, and then counterstained with PI (propidium iodide), and observed under fluorescence microscope (BX60, Japan).
2.4. Detection of apoptosis in cell population by using ELISA technique

The ELISA detection kit was purchased from Boehringer Mannheim Co, containing anti-histone antibody (clone H 11-4), and POD-linked anti-DNA antibody (Anti-DNA-POD, clone MCA-33). This kit was designed for in vitro cultured animal cells. Hence, in our experiment, the material, root-tip tissue of maize plant, was pretreated before ELISA as follows: the root tips without caps (around 2 mm in length) used in control and IPC-treated sets were immediately ground into fine powder in liquid nitrogen, and then, 600 l lysis solution was added. This mixture was taken for ice-bath for 30 min and then centrifuged at 15,000 rpm for 10 min. 400 l of supernatant (cytoplasm) was carefully pipetted and used in antibody sandwich ELISA test, as described in the protocol provided by the manufacture. The reaction performed under room temperature at dark for 15 min. The non-stressed cell sample was used as negaVol. 9 No. 2 (2000) 63

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tive control, and all the reactants, including that of negative control and substrate buffer solution (400 l of each), were diluted 5 times, the final volume of test samples was 2 ml, respectively. Then, using a spectrometer (PERKIN-ELMER LAMBDA BIO20/1.0 nm UV/VIS spectrometer (1.20)) the OD values for each sample were tested at the 405 nm of the spectrum band. The OD value of diluted substrate buffer was used as the background. The difference between OD values of each tested sample and that of background were defined as the net OD values for each apoptotic cell sample and negative control.

3.

RESULTS

3.1. Apoptosis on individual cell level

Fig.1 Nuclear morphological and biochemical change during IPC-induced apoptosis in individual meristematic cells of maize roots detected by TUNEL assay (3300). (A) Control, round, red with PI, no FITC fluorescence. (B) Treated for 2d, DNA breaks are marked with FITC (yellowish green in color). Obvious nucleus condensation is not observed yet and nucleoli dont disappear. (C) Treated for 3d, nuclear substance is condensed, and serious DNA breakage occurred. Marginalization of chromatin and DNA-free region are seen, and nucleolus also disappears. (D) Treated for 4d. Obvious fragmentation of condensed nuclear substances is showed. (E & F) Treated for 5d. Nuclear substances degrades, very concentrated FITC fluorescence granules appear.
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ELISA Assays for Detection of Apoptosis in Plant Cells

The in situ TUNEL labeling which is mediated by terminal deoxynucleotidyl transferase (TdT) is one of the traditional methods of quantifying DNA breakage in situ. It has been commonly used in both animal and plant systems. Due to that the application of TUNEL labeling on chromosome preparation slides can overcome the shortcomings of low penetration, high background and time-consuming procedure in paraffin section and make the accurate quantification on individual cell instead of at tissue level (Fig.1).
1.2

OD values

Apoptotic
1 0.8 0.6 0.4 0.2 0 1 2 3 4 Time (d) 5 6 7 Control

Fig.2 ELISA assay for apoptosis in maize root-tip cells, induced by 0.2 mg/mL IPC. Each data point represents the mean SD.

Fig.1 indicates that the death of cell of maize roots, induced by IPC is characterized by typical apoptotic characteristics in animals. These characteristics include the specified breakage of DNA breakage (Fig.1, B~F), the marginalization of chromatin (Fig.1, B&C), the formation of DNA-free region in center (Fig.1, C); the nuclear substance condensation (Fig.1, C~F), the breakage (Fig.1, D), formation of fragments (Fig.1, D) and sequent degradation of nuclear substance (Fig.1, E&F); and the loss of nucleoli (Fig.1, C~F). Sometimes, the condensed granule could be found (Fig.1, F). While in control, the cell, still showing the nucleus in round shape, is not labeled by TUNEL, only displayed PI red (Fig.1). Before the disappearance of nucleoli, the form of nucleus is called deformation and, is called degradation after the former disappeared. Such morphological and biochemical characteristics indicate that IPC at certain concentration can induce apoptosis in root-tip cells of maize. These events that the morphological changes and the breakage of DNA occurred gradually and orderly indicate that
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the above-mentioned apoptosis is a kind of controlled programmed death.

3.2. Apoptosis in cell population

The results of ELISA assay indicates that, like that in animal, along with the induction time prolonged, the OD value of cytoplasm sample rises gradually (Fig. 2&3). It indicates that the chromosomes are degraded specifically into (oligomeric) nucleosomes, which were released gradually into cytoplasm.
1.0

OD values

Apoptotic Control
0.8

0.6

0.4

0.2

0.0 0.01 0.1 1 10

IPC concentration (mg/ml)

Fig.3 ELISA assay for apoptosis in maize root-tip cells, which have been treated for 4d by IPC at gradient concentration (0.01~20 mg/mL). the mean SD. Each data point represents

4.

DISCUSSION

The results indicate that in maize, a monocotyledon plant, apoptosis can be induced by IPC at a certain concentration, and is characterized by typical morphological and biochemical change. It provides the possible explanation for the mechanism of weed control by IPC at cytological level and certain instructive guide for agricultural practice. In recent years, the research on apoptosis in plants is one of the new emerging topics in the field of cell biology in plants. But it still needs to advance in depth and the method which was been originally available for animals should be improved or modified when they are adopted for plant system. Providing another feasible quantitative method for detecting apoptosis in plants, our results proved firstly that ELISA is available for plant
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system. It indicates in our results that in the OD values of cytoplasm increases gradually before 4 d treatments with 0.2 mg/mL IPC and begins to decline after a certain period of time. It illustrates that the amount of oligomeric nucleosomes in the cytoplasm decrease gradually after it reached the maximum. From the previous report [20], it is presumed that, that is caused by the following two reasons: (1) The gradual degradation of chromosomes at earlier period of apoptosis caused the gradual release of (oligomeric) nucleosomes into cytoplasm, and the amount of nucleosomes can keep stable as the chromosomes were fully degraded. Then, along with the time of IPC induction prolonged, the (oligomeric) nucleosomes were further degraded into lower molecular weight substances, nucleotides and amino acids, and the separation of histone and DNA caused the failure of detection by using ELISA. (2) Along with the induction time prolonged, a small fraction of cells undergo necrosis, rather than apoptosis. Because the chromosomal degradation of necrotic cells is non-specific, it was hard to form (oligomeric) nucleosome, and such non-specific degradation could not be detected by using ELISA technique. Thus, it demonstrates that the ELISA method is highly specific to (oligomeric) nucleosomes produced during apoptosis, and can be used to quantity the (oligomeric) nucleosomes through OD values. Various reports on apoptosis in animals and plants have shown that modest abiotic stress treatments can cause apoptosis, but the same abiotic stresses at a high level cause rapid necrosis with cell swelling and lysis [16,17,22,24]. In our experiment, the treatments with gradient concentration of IPC indicated that the sample were treated 4 d on IPC at 0.2 mg/mL level gave the maximum OD values, and at the concentration over 0.2 mg/mL, the OD values of the sample decreased gradually. It demonstrates that the induction of apoptosis is dose-dependent too.

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ELISA TUNEL

430072

TUNEL DNA ( 1) ELISA ELISA OD ( 2)0.2mg/ml ( 3)

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