Quantitative Analysis of Cytokinin

1. Quantitative analysis of cytokinins

using cotyledons of cucumber

1-1. Introduction

We performed quantitative analysis of cytokinin using cotyledons (seed sprouts) of cucumber (Cucumis sativus L. cv.). We have proposed to make E. coli produce cytokinin. We need to establish experimental system for quantitative analysis of cytokinin. Cucumber cotyledons bioassay is frequently used as a simple and rapid bioassay for cytokinin (Fletcher et al., 1971; 1982). Previous works indicated that cytokinin enhanced chlorophyll levels in plant cells. By using cytokinin samples, we attempted to acquire the technique of cucumber cotyledons bioassay.

1-2. Materials and Methods

Fig. 3-6-1. Samples of cytokinin

1. Cucumber seeds were planted on absorbent cotton dampened with water and germinated in the dark at 27°C for 5 days.
2. The cotyledons were excised in dim red light. Two cotyledons were from one seed. One cotyledon was placed in 3.5 cm plastic dish containing 0.4 mL of 100 microM cytokinin and 0.1% (v/v) of dimethylsulfoxide (DMSO) solution. The other cotyledon was placed in 3.5 cm plastic dish containing only 0.1% (v/v) of DMSO solution as a negative control. 6 cotyledons were placed together in one dish.
3. The dishes were returned to the dark at 27°C for 24 h and then moved under fluorescent light with an intensity of about 40 micromol /m²S¹ (photosynthetic photon flux density).
4. After 24 h, the weight of cotyledons was measured per dish.
5. The 6 cotyledons in the dish were homogenized together and the chlorophyll was extracted in 3 mL 80% (v/v) of cold acetone. The volume was brought up to 5 mL with the acetone. The extract was centrifuged (2,000 rpm, 5 min, 4°C). The absorbances of the supernatant were read at 663.6 and 646.6 nm. Calculation of chlorophyll concentration was carried out following the formula shown below (Porra et al., 1989).

   Chlorophyll concentration (microg/mL) = 17.76 X A (646.6 nm) + 7.34 X A (663.6 nm)

Fig. 3-6-2. Extraction of chlorophyll

1-3. Results

Two pictures of cytokinin-treated cotyledons are shown in Fig. 3-6-3. Cytokinin had effects of hypertrophy and greening on cotyledons. The weight ratio and the chlorophyll concentration ratio are shown in Fig. 3-6-4. Both weight and chlorophyll concentration of cytokinin-treated cotyledons were higher than those of non-treated cotyledons.

Fig. 3-6-3. Pictures of cotyledons of 24 h after the treatment of cytokinin samples.

Fig. 3-6-4. Weight ratio of cotyledons.

Fig. 3-6-5. Chlorophyll concentration ratio of cotyledons.

1-4. References

1. R. A. Fletcher and Dlanne McCullagh. Cytokinin-Induced Chlorophyll Formation in Cucumber Cotyledons. Planta 1971;101:88-90
2. 2. R. A. Fletcher, V. Kallidumbil and P. Steele. An Improved Bioassay for Cytokinin Using Cucumber Cotyledons. Plant Physiol 1982;69:675-677
3. R.J. Porra, W.A. Thompson and P.E. Kridemann. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents : verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta 1989;975:384-394

2. Identification of cytokinin

by ultra-performance liquid chromatography (UPLC)

Fig. 3-6-6. iP : 6-(γ, γ-Dimethylallylamino) purine and tZ : trans-zeatin

2-1. Introduction

In order to confirm that our E. coli synthesizes cytokinin (especially iP : 6-(γ, γ-Dimethylallylamino) purine, tZ : trans-zeatin), we planned to use ultra-performance liquid chromatography (UPLC). We aim to make E. coli produce iP and tZ by introducing AtIPT4 or AtIPT7 into E. coli. In order to confirm that E. coli synthesize iP and tZ, we will use ultra-performance liquid chromatography (UPLC). Before attempting the cytokinin biosynthesis in E. coli, we determined the retention times of iP and tZ which were from authentic samples. Then we confirmed that iP and tZ were able to be detected from the mixture of E. coli culture medium and cytokinin solution.

2-2. Method

2-2-1. Determination of retention times of iP and tZ

UPLC was carried out as described by Novák et al. (2008) [1]. Samples (5 microM) were prepared by diluting each cytokinin DMSO solution with a mobile phase (initial conditions). 5 microL of each sample was injected onto a reversed phase column (BEH C18, 2.1 X 100 mm, 1.7 microm; Waters). The samples were eluted with an 8 min. linear gradient of 90:10 = A:B to 50:50 = A:B (v/v) where A was 15 mM ammonium formate and B was methanol at a flow rate of 0.25 mL / min. The column temperature was set to 40°C. At the end of the gradient, the column was washed with 100% B for 1 min. and equilibrated to initial conditions for 3 min. Under these conditions, the retention times for the monitored compounds ranged from 2.5 to 7.5 min. The effluent was passed through an ultraviolet detector at 268 nm.

2-2-2. Detection of iP and tZ from mixture of E. coli culture medium and cytokinin solution

Culture was carried out as described by Takei et al. (2001) [2]. E. coli (JM109) transformed with pSB6A1-Promoterless-atipt7 were grown in M9 minimal medium, which is supplemented with 20 mg/mL ampicillin, 1 M sorbitol, 1% (w/v) casamino acid, 2% (w/v) sucrose, 2.5 mM betaine, 5 mg/mLthiamine, 1 mM MgSO₄, and 0.1 mM CaCl₂. The cultures were incubated at 25°C with shaking until those OD600 were 0.5. The cells were harvested by centrifugation. Mixture of the supernatant and cytokinin solution were prepared and used for UPLC.

2-3. Results

2-3-1. Determination of retention times of iP and tZ

The peak of tZ standard was detected at 5.0 min. And the peak of iP standard was detected at 9.1 min. These results are shown in Fig. 3-6-7.

Fig. 3-6-7. The UPLC chromatogram of tZ and iP standards.

2-3-2. Detection of iP and tZ from mixture of E. coli culture medium and cytokinin solution

The black line stands for the chromatogram of the diluted supernatant. And the red line stands for the chromatogram of the diluted supernatant containing 1 microM cytokinin standards. Results are shown in Fig. 3-6-8.

Fig. 3-6-8. The UPLC chromatograms. The black line stands for the chromatogram of the diluted supernatant. And the red line stands for the chromatogram of the diluted supernatant containing 1 microM cytokinin standards.

This result suggests that using UPLC, iP and tZ were detected from mixture of E. coli culture medium and cytokinin solution. We believe that the method is able to apply to confirming E. coli synthesis iP and tZ.

2-4. References

1. Ondrˇej Novák, Eva Hauserová, Petra Amakorová, Karel Dolezˇal, Miroslav Strnad (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Phytochemistry, 69, 2214–2224
2. Kentaro Takei, Hitoshi Sakakibara, and Tatsuo Sugiyama. (2001) Identification of Genes Encoding Adenylate Isopentenyltransferase, a Cytokinin Biosynthesis Enzyme, in Arabidopsis thaliana. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 26405-26410