Interaction between isoamyl adenine and rice cytokinin oxidase

Article abstract of DOI:10.1080/00387010.2016.1168451

Cytokinin (CTK) dehydrogenase is responsible for regulating the endogenous CTK content by oxidative removal of the side chain. Herein, we have applied fluorescence method to study the interaction between CTK dehydrogenase and CTK in vitro and obtain some parameters of their interaction. We found that addition of isopentenyl adenine can quench the fluorescence of CTK dehydrogenase, and the quenching mechanism was to be a static quenching procedure. We have measured the number of binding sites n and the apparent binding constant K and have calculated the thermodynamics parameter ΔH, ΔG, and ΔS by fluorescence quenching method. Based on thermodynamics parameter’s results, we concluded that their binding reaction was both entropy driven and the enthalpy driven, and the Van der Waals force and hydrogen bond force played a major role in the interaction. Based on the synchronous fluorescence spectrometry results, we demonstrated that the binding site between isopentenyl adenine and CTK dehydrogenase is in the microenvironment of both tryptophan and tyrosine. The fluorescence signal of coenzyme, flavin adenine dinucleotide, decreases gradually with the addition of isopentenyl adenine. And this method can be used for isopentenyl adenine routine assay. Under optimized experimental parameters, the linear segment increases from 0.6?μM to 100?μM with a regression equation of ΔF?=?0.04?+?0.15c_ip (r?=?0.999, c_ip in μM) with the detection limit of 0.15?μM iP.

Full text of DOI:10.1080/00387010.2016.1168451

Spectroscopy Letters
An International Journal for Rapid Communication
ISSN: 0038-7010 (Print) 1532-2289 (Online) Journal homepage: http://www.tandfonline.com/loi/lstl20
Interaction between Isoamyl Adenine and Rice
Cytokinin Oxidase
Yujiao Fang, Yong Li, Xiaosheng Liang, Qian Xiao & Yunhua Wu
To cite this article: Yujiao Fang, Yong Li, Xiaosheng Liang, Qian Xiao & Yunhua Wu (2016):
Interaction between Isoamyl Adenine and Rice Cytokinin Oxidase, Spectroscopy Letters, DOI:
10.1080/00387010.2016.1168451
To link to this article: http://dx.doi.org/10.1080/00387010.2016.1168451
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Date: 29 March 2016, At: 14:42

Interaction between Isoamyl Adenine and Rice Cytokinin Oxidase
Yujiao Fang1, Yong Li1, Xiaosheng Liang1, Qian Xiao1, Yunhua Wu1
1Hubei Provincial Key Laboratory for Protection and Application of Special Plants in
Wuling Area of China, College of Life Science, The South Central University for
Nationalities, Wuhan , PR China
Corresponding Author: Yunhua Wu. E-mail: wuyunhua@mail.scuec.edu.cn
Abstract
Cytokinin dehydrogenase is responsible for regulating the endogenous cytokinin content
by oxidative removal of the side chain. Herein, we have applied fluorescence method to
study the interaction between cytokinin dehydrogenase and cytokinin in vitro, and obtain
some parameters of their interaction. We found that addition of isopentenyl adenine can
quench the fluorescence of cytokinin dehydrogenase and the quenching mechanism was
to be a static quenching procedure. We have measured the number of binding sites n and
apparent binding constant K, and have calculated the thermodynamics parameter ΔH, ΔG
and ΔS by fluorescence quenching method. Based on thermodynamics parameter’s results
we concluded that their binding reaction was both entropy-driven and the enthalpy-driven,
and the Van der Waals force and hydrogen bond force played major role in the interaction.
Based on the synchronous fluorescence spectrometry results we demonstrated that the
binding site between isopentenyl adenine and cytokinin dehydrogenase is in the
microenvironment of both tryptophan and tyrosine. The fluorescence signal of coenzyme,
flavin adenine dinucleotide, decreases gradually with the addition of isopentenyl adenine.
And this method can be used for isopentenyl adenine routine assay. Under optimized
experimental parameters, the linear segment increases from 0.6 μM to 100 μM with a
regression equation of ΔF = 0.04 + 0.15cip (r = 0.999, cip in μM) with the detection limit
of 0.15 μM iP.
KEYWORDS: Isopentenyl adenine; Cytokinin oxidase; Fluorescence method; Static
quenching
1

1. INTRODUCTION
Cytokinins (CTKs) are derivatives of adenine and are classified on the basis of the
variable substituent at the adenine C6 position [1]. They belong to the one of five types of
plant hormones (i.e., auxin, gibberellin, cytokinin, abscisic acid and ethylene) [2]. Some
derivatives of adenine are present in various tissues as free low-molecular-weight
components, whereas some are an integral part of specific tRNA molecules. CTKs
participate in regulating plant growth and development, especially play an important role
in cell division and bud differentiation, lateral bud development, fruit development [3]
.
The mechanism of CTKs in regulating is still not clear entirely. CTKs, which produced
by the change of purine in tRNA, exist in tRNAs close to anticodons. CTKs involve in
tRNA and ribosome mRNA complex formation in the process of protein synthesis.
However, exogenous CTKs are not involved in tRNA, but promote the production of
nitrate reductase, protein and nucleic acid [4]
.
The metabolism of CTKs regulates their own dynamic balance in plant cells. The CTKs
synthetic way has been studied extensively [4]. The degradation of CTKs is largely
dependent on cytokinin oxidase (CKO). The breakdown of the active isopentenyl adenine
yields adenine and an unsaturated aldehyde, 3-methyl-2-butenal (Figure 1). The CKO is
the only known enzyme that catalyzes natural isoamyl alkene CTKs and nucleoside
irreversible degradation including different pentenyl adenine (isopentenyl adenine, iP),
zeatin (zeatin, Z) and nucleotide. CKO genes from corn [5], arabidopsis thaliana [6]
,
2

dendrobium[7], barley[8]a nd wheat[9] have been cloned. Transgenic experiments show that
the change of the plant CKO gene expression regulates the content of endogenous
hormone CTK, results in affecting the growth of plants. Ashikari M [10] reported that the
decreased expression of CKO gene OsCKX2 in the rice inflorescence meristem caused
increased level of cytokinin and yield improvement. However, the CKO’s role in the
regulation of plant growth and development process has less been reported. CKO
degrades extracellular CTKs, then makes the CTKs sensory system restore to the original
level. In the process of cell cycle the CKO activity changes with the concentration of
endogenous CTK during mitosis. And it indicated that the CTKs degraded rapidly and
regulated the cell cycle [11]
.
To our knowledge, however, to date the interaction parameters between CKO and iP in
vitro hasn’t been investigated. Fluorescence is powerful tool for the study of the reactions
of chemical and biological systems since it allows non-intrusive measurements of
substances in low concentration under physiological conditions. The aim of our work was
to determine the affinity of isoamyl adenine to the protein of rice cytokinin oxidase in
vitro, and to obtain some parameters of their interactions by fluorescence technique. We
found that iP itself has no fluorescence, but it affected the CKO fluorescence signal. In
the experiments we have studied the interaction between the CKO and different
concentration of iP under different temperatures because temperature will influence the
collision and electron transfer process between molecules, resulting in different
3

fluorescence signal change of CKO with binding iP. Based on the results, we have
inferred the binding mode and binding constant between them. Furthermore, we have
investigated the fluorescence signal change of coenzyme, FAD, with addition of iP. We
have described how CKO can be used to assay the concentration of iP in solution. Till
now, the method for the determination of endogenous cytokinins (CKs) is mainly based
on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS)
[12]. In this paper we have developed the fluorescence method for determination iP based
on CKO.
EXPERIMENTAL
2.1 Apparatus
All fluorescence measurements were made with a Hitachi F-2500 spectrofluorimeter
(Tokyo, Japan) equipped with a 1.0 cm quartz cell and a thermostat bath. All pH
measurements were made with a pHs-3 digital pH-meter (Shanghai Lei Ci Device Works,
Shanghai, China) with a combined glass electrode.
2.2 Reagents
All starting materials were analytical reagent grade and double distilled water was used
for all the measurements. 0.2 mol L-1 phosphate buffer solution of pH 7.0 was prepared.
N6-isoamyl adenine (Sigma) was directly dissolved in 50% ethanol to prepare stock
4

solutions(1mg mL-1) and then stored at 4。C. We have obtained the cytokinin oxidase gene
from RNA of var nipponbare by RT-PCR and constructed the recombinant plasmid
pET-28a-CKO. Rice cytokinin oxidase has been expressed in E.coli. BL21, then stored at
-80 ˚C for following use [13]
.
2.3 Procedures For Fluorescence Spectrum Measurements
A 3.0-mL solution in 1.0-cm quartz cells, containing appropriate concentration of rice
CKO, was titrated by successive additions of 1mg mL-1 stock solution of N6-isoamyl
adenine. Titration was done manually by using microinjector. The aliquot of each
injection was 10 µL to avoid the change of the volume. Before measurements, the tubes
were shaken up and placed into a thermostat water bath for 5 min. Then the assay
solution was transferred into a quartz cell, and fluorescence measurements were
performed. Fluorescence quenching spectra of CKO were obtained at excitation
wavelengths of λex =280 nm and recorded λem = 297-330 nm range. Synchronous
fluorescence spectrometry of Tyr and Trp were obtained by setting Δλ = 20 nm and Δλ =
80 nm, respectively. The excitation and emission slit widths were set at 10.0 nm, and 0.2
mol L-1 pH 7.0 phosphate buffer were subtracted to correct background fluorescence.
5

3. RESULTS AND DISCUSSION
3.1 The Fluorescent Quenching Of CKO Caused By Ip
For macromolecules, the fluorescence measurements can give some information of the
binding of small substances to protein. Fluorescence spectrum of the interaction between
iP and rice CKO is shown in Figure 2 Under the excited wavelength of 280 nm, the
.
maximum fluorescence emission peak of rice CKO appeared at 335 nm and iP have no
fluorescence in the wavelength range from 300 nm to 500 nm (Figure 2). The
fluorescence signal of CKO is decreased with the increasing of iP addition, but the
maximum emission wavelength and peak shape remains unchanged. These results
indicated that CKO interacted with iP and the interaction resulted in fluorescence
quenching of protein.
3.1.1 Analysis Of The Fluorescence Quenching Process
Fluorescence quenching is the fluorescence signal decrease caused by the physical or
chemical process between fluorescent molecules and solvent molecules, or solute
molecules. Fluorescence quenching process is divided into two kinds: a dynamic
quenching or a static quenching. For dynamic quenching, the decrease in signal is
described by Stern-Volmer equation:
6

(1)
( 2)
F / F 1 Kqt0 Q
1 KSV Q
0
Kq KSV / t0
where F and F0 are the fluorescence intensity in the absence and in the presence of
quencher, Kq is the quenching rate constant of the biomolecule, Ksv is the Stern-Volmer
quenching constant, τ0 is the average lifetime of the biomolecule without quencher, [Q] is
the concentration of quencher. In general, dynamic quenching is caused by the interaction
between quencher Q and fluorescent molecular in the excited state. Increase in
temperature will intensify the effective collision and electron transfer process between
molecules, resulting in the increase of KSV with the temperature. And static quenching is
due to the compounds formation between quencher and fluorescent molecular in the
ground state. A rise in temperature will reduce the stability of the compounds and the
quenching constant KSV will decrease [14]. In this work the effect of different
concentration of iP on the fluorescence signal of the CKO is investigated under three
different temperatures of the 288 K, 298 K and 308 K. The Stern-Volmer curve of F0/F
versus the concentration of iP at various temperatures is plotted. The Stern-Volmer
equation and quenching constant KSV under different temperatures is listed in Table 1.
The Stern-Volmer curves of quenching of CKO with iP had linear relationships at
concentration of iP range from 0 µM to 16 µM indicated in Table 1. This linear
relationship indicated only one binding site or one kind of fluorescence quenching existed
7

between quencher and the protein fluorescence groups [15]. With the increase of
temperature, the CKO quenching curve's slope decreased. Dynamic quenching depended
on diffusion, since higher temperature resulted in larger diffusion coefficients, the
bimolecular quenching constants were expected to increase with increasing temperature.
By inference, the procedure maybe is not dynamic quenching. And increasing
temperature will lead to the stability of the complexes reduced, so the static quenching
constant will decrease with the increase of temperature. Suppose KSV is dynamic
quenching constants, because fluorescence lifetime τ0 of biological molecules
approximately is 10-8 s, and the maximum value of Kq for quencher to biological is
2.0×1010 L mol-1 s-1. According to Table 1 it shows that quenching rate constant of iP to
CKO is greater than the value. Thus, we can conclude the fluorescence quenching
process of iP to CKO is static quenching process.
3.1.2 Calculation Of The Binding Sites And Binding Constant
Binding constant KA and binding sites n are very important parameters to study the
interaction of a small molecule with protein. For static quenching, the apparent binding
constant
and binding sites can be obtained from the equation:
KA
n
(3)
lg F F / F lgKA nlg Q
0
where F and F0 are the fluorescence intensities in the absence and in the presence of
quencher, [Q] is the concentration of quencher. KA is the binding constant of CKO and iP,
8

n is the binding number, which can be determined by the intercept and slope of the plot
lg(F0-F) /F = lgKA + n lg[Q] as shown in Table 2.
Because the complexes’ stability reduced with the increasing temperature, static
quenching constant decreased. Table 2 indicated binding constant decreased with the
increasing temperature, and binding site of CKO and iP approximately to be 1.
3.1.3 Determination Of The Binding Mode
Considering the dependence of binding constant on temperature, a thermodynamic
process was considered to be responsible for the formation of the complex of CKO and iP.
Therefore, the thermodynamic parameters dependent on temperatures were analyzed in
order to further characterize the acting forces between them. In general, the binding force
between small molecules and biological macromolecules are mainly based on hydrogen
bond, van der Waals force, electrostatic forces or hydrophobic interaction force. The
thermodynamic parameters, enthalpy ΔH, entropy ΔS and free energy change ΔG, are the
main evidences to estimate the binding mode. If ΔS>0 and ΔH>0, the process of the
interaction is the endothermic reaction, the type of reaction between the two is mainly the
hydrophobic. If ΔS>0，ΔH<0, the process of the interaction is the exothermic reaction, the
type of reaction between the two is mainly electrostatic attraction. If ΔS<0 and ΔH<0, the
type of reaction between the two is van der Waals force or hydrogen bond [16]. A plot of
lnK versus 1/T gives a straight line according to the van’t Hoff equation:
9

H
S
ln K
(4)
RT
R
where constants K are analogous to the Stern-Volmer quenching constants KSV at the
corresponding temperature [17] (the temperatures used were 288, 298, and 308 K). The
free energy change ΔG is estimated from the following relationship:
G
H T S
(5)
A linear relationship was obtained as following: lnKSV = 286.36 / T + 0.48 by plotting the
values of lnKSV versus 1/T. The value of ΔG was further calculated based on equation 4.
The results are shown in Table 3. From Table 3, it can be seen that the negative sign for
free energy ΔG means that the interaction process is spontaneous. The negative enthalpy
ΔH and entropy ΔS values of the interaction of iP and CKO, indicate that the binding is
both entropy-driven and the enthalpy-driven, the hydrogen bond and van der Waals force
played major roles in the reaction [16]
.
3.2 The Effect Of Ip On The CKO’s Conformation
3.2.1 Synchronous Fluorescence Spectra
Synchronous fluorescence spectra of protein have been widely used to determine protein
conformation change [18]. Spectral features of protein Tyr residues and Trp residues was
obtained at Δλ = 20 nm and Δλ = 80 nm, respectively. Since maximum emission
wavelength of residues was relevant to the polarity of their environment, the change of
protein conformation could be figured by change of the emission wavelength [19]. Like
10

other proteins, specific fluorescence absorption peaks of Tyr and Trp residues of rice
CKO were investigated. But fluorescence spectrum of these amino acid residues
overlapped each other, and it made fluorescence spectrometry difficult to distinguish.
Therefore, synchronous fluorescence spectrometry was used to study the interaction
between iP and rice CKO.
The synchronous fluorescence spectra of CKO are shown In Figure 3. The results show
that emission peaks of Tyr and Trp appeared at 290 nm and 280 nm, respectively. After
interacting with iP, fluorescence signal of Tyr and Trp residues decreased and the
maximum emission peaks obviously red shift at the same time, indicating that the polarity
of the both group's environment of CKO increased with the increase of iP concentration.
But emission peak of Trp decreased and the maximum emission peaks red shifted more
greatly than that of Tyr. The total numbers of Tyr and Trp residues in rice cytokinin
oxidase are 16 and 8, respectively. Thus, the more emission peak shift of Trp is not
caused by the more numbers of Trp residues in rice cytokinin oxidase. The experimental
results just indicated that the binding sites of iP with rice CKO approached the
microenvironment of Tyr and Trp residues but more close to Trp group.
3.2.2 Fluorescence Spectra Of CKO’s Coenzyme FAD
11

The fluorescence spectra of coenzyme FAD is shown in Figure 4. Under aerobic
conditions when excited at 440 nm, the coenzyme FAD of CKO fluoresces weakly
between 510 and 520 nm. However, qualitative additions of iP induce the fluorescence
signal
there is a blue shift (~5 nm) in the emission
peak of the fluorescence spectra and an attenuation of the overall fluorescence signal.
This indicated that the conformational alterations in the CKO are induced by iP binding
with FAD.
The fluorescence spectral response of the CK
concentration
of iP under aerobic conditions. A 2 ml sample of approximately 5 μM CKO in 100 mM
pH 7.2 phosphate buffers was titrated with a saturated solution of iP (10 uL additions) in
the same buffer causing a decrease in the fluorescence signal of CKO (Figure 4). The
FAD-iP complex remained stable and the fluorescence signal was unaltered after 10
scans under these conditions.
The fluorescence signal change of 5 μM CKO with addition of 4.8 μM iP in 0.1 mol L−1
phosphate buffer with different pH values were studied. It shows that the fluorescence
signal of CKO obviously increase when pH value gradually increases from 2.0 to 7.0.
However, the signal begins to remakably decrease as further increasing pH from 8.0 to
10.0. The fluorescence signal is highest at pH 7.0. Thus, pH 7.0 phosphate buffer was
used for the routine assay of iP to achieve higher sensitivity.
12

The effect of the concentration of CKO on the routine assay of iP was also studied. As
the concentration of CKO value gradually improving from 2.0 μM to 6.0 μM, the
fluorescence signal change of CKO with addition of 4.8 μM iP gradually increases.
Considering the cost of CKO, 5.0 μM of CKO was chosen for the routine assay of iP in
the following experiments.
Under optimized experimental parameters, the calibration curve was obtained in pH 7.0
phosphate buffer. The linear segment increases from 0.6 μM to 100 μM with a regression
equation of ΔF = 0.04 + 0.15cip (r = 0.999, cip in μM). It is found that this method can
detect 0.15 μM iP. The variation coefficient was 3.5% for 7 successive assays of 5 µM iP.
It showed good reproducibility for the determination of iP in the linear range.
4. CONCLUSIONS
We have studied the interaction of rice CKO and iP by fluorescence spectrometry. The
results show that iP has a strong ability to quench the CKO fluorescence mainly through
a static quench procedure. The binding constant K and the number of binding site n were
calculated according to the fluorescence quenching results. Based on thermodynamic
parameters’ calculation, the binding mode was determined to be the van der Waals or
hydrogen bond force. The effect of iP on the CKO conformation was investigated by
synchronous fluorescence spectra and fluorescence spectra of CKO coenzyme FAD. The
results indicated that the conformational alterations in the CKO were induced by iP
13

binding. And the binding site of iP and CKO was close to both Tyr and Trp residues. The
fluorescence signal of FAD in CKO decreases gradually with the addition of iP. A linear
response over the iP concentration range from 0.6 µM to 100 µM was obtained, with a
detection limit of 0.15 uM. The method can be applied to determine of iP in solution.
ACKNOWLEDGMENTS
We are gratefully acknowledged the financial support from the National Natural Science
Foundation of China (No. 20777094), the Hubei Provincial Natural Science Foundation
(2014CFB381); the 3551 Schema for Talents in Optics Valley and the Special Fund for
Basic Scientific Research of Central Colleges, South-central University for Nationalities
(CZW14008).
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nanoparticles-based magnetic solid-phase extraction, J. Chromatogr. A 2014, 1340:
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16

Table 1 The Stern-Volmer quenching constant of isopentenyl adenine to cytokinin
oxidase at various temperatures.
KSV ×104 (L mol-1)
T(K) Regression
equations
correlation
coefficients
0.9975
Y = 1.03+3.38×104
288
3.38
2.38
1.80
X
Y =1.01+2.38×104
298
308
0.9995
0.9987
X
Y = 1.00 +1.80×104
X
17

Table 2 The binding constant and the binding number of isopentenyl adenine and
cytokinin oxidase at different temperatures
KA ( L mol−1)
14757
T( K)
288
n
R
0.986
0.914
0.907
0.9972
0.9974
0.9978
298
13772
308
8452
18

Table 3 Thermodynamic parameters of isopentenyl adenine - cytokinin oxidase system
T（K）
ΔG(KJ mol-1) ΔH((KJ mol-)
ΔS(KJ
mol-K-1)
288
298
308
-24.969
-24.966
-25.090
-2725.388
-120.895
19

Figure 1. Scheme of the reaction catalysed by cytokinin oxidase enzyme with reference
to the isopentenyl adenine.
20

Figure 2. The fluorescence spectra of 2 µM cytokinin oxidase in 100 mM pH 7.4
phosphate buffer with the addition of 0 µM; 1.6 µM, 3.2 µM, 4.8 µM, 6.4 µM, 8.0 µM,
9.6 µM, 11.2 µM, 12.8 µM, 14.4 µM and 16 µM isopentenyl adenine, respectively (curve
1-11). The excited wavelength is 280 nm.
21

Figure 3. The synchronous fluorescent spectra of 2 µM cytokinin oxidase in 100 mM pH
7.4 phosphate buffer with the addition of 0 µM; 1.6 µM, 3.2 µM, 4.8 µM, 6.4 µM, 8.0
µM, 9.6 µM, 11.2 µM, 12.8 µM, 14.4 µM and 16 µM isopentenyl adenine, respectively
(curve 1-11). The difference of excitation wavelengths and emission wavelength, Δλ = 20
nm (a) and Δλ = 80 nm (b).
22

Figure 4. The fluorescence spectra of 5 µM cytokinin oxidase in 100 mM pH 7.4
phosphate buffer with the addition of 0 µM; 1.6 µM, 3.2 µM, 4.8 µM, 6.4 µM, 8.0 µM,
9.6 µM, 11.2 µM, 12.8 µM, 14.4 µM and 16 µM isopentenyl adenine, respectively (curve
1-11). The excited wavelength is 440 nm.
23