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

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 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 μ mol * m-2 * S-1 (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% of cold acetone. The volume was brought up to 5 ml with the acetone. The extract was centrifuged (2000 rpm, 5 min, 4°C). The absorbance of the supernatant was read at 663.6 and 646.6 nm. Calculation of chlorophyll concentration was carried out following the formula shown below (Porra. 1989). Chlorophyll concentration (microg/ml) = 17.76 * A(646.6 nm) + 7.34 * A(663.6 nm)

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

(1) R. A. Fletcher and Dlanne 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 Cytokinins 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

We aim to make E. coli to produce cytokinins (especially iP : 6-(γ, γ-Dimethylallylamino) purine, tZ : trans-zeatin) 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, we determined the retention times of iP and tZ by using authentic samples.　

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). 10 microL of each sample was injected onto a reversed phase column (BEH C18, 2.1 * 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 (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.

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 shown in Fig. 2.

(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