Quantitative Analysis of Cytokinin

1. Quantitative analysis for cytokinin

using cotyledons of cucumber

1-1. Introduction

We performed quantitative analysis for cytokinin using cotyledons (seed sprouts) of cucumber (Cucumis sativus L. cv.). We proposed to make E. coli produce cytokinin and needed to establish experimental system for quantitative analysis for cytokinin. Cucumber cotyledons are frequently used as samples for 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 learn methods for bioassay of cucumber cotyledons.

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

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

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

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

1-4. References

1. R. A. Fletcher and D. McCullagh. Cytokinin-Induced Chlorophyll Formation in Cucumber Cotyledons. Planta 1971;101:88-90
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 aimed to make E. coli produce iP and tZ by introducing AtIPT4 or AtIPT7 into E. coli. Before attempting the cytokinin biosynthesis in E. coli, we determined the retention times of authentic samples of iP and tZ. Then we confirmed that iP and tZ were able to be detected from the mixture of E. coli culture medium and the cytokinin solution.

2-2. Methods

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 the mixture of the E. coli culture medium and the cytokinin solution

Cultures were made 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 reached 0.5. The cells were harvested by centrifugation. The mixture of the supernatant and the 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. 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 retension times of iP and tZ standards determined by UPLC

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

The black line stands for the chromatogram of the diluted supernatant. 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. 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 the mixture of E. coli culture medium and the cytokinin solution. We believe that the method is able to be applied to confirming the biosynthesis of iP and tZ in E. coli.

2-4. References

1. O. Novák, E. Hauserová, P. Amakorová, K. Doležal, M. Strnad. (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Phytochemistry, 69, 2214–2224
2. K. Takei, H. Sakakibara, and T. Sugiyama. (2001) Identification of Genes Encoding Adenylate Isopentenyltransferase, a Cytokinin Biosynthesis Enzyme, in Arabidopsis thaliana. The Journal of Biological Chemistry, 276, 26405-26410