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Plant Physiol. (1999) 121: 245-252 Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes Due to Light/Dark Treatment1
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic (E.B., B.B.); Department of Biology, University of Antwerpen, Universiteitsplein 1, B2610 Wilrijk-Antwerpen, Belgium (E.W., W.V.D., H.A.V.O.); and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, 16502 Praha 6, Czech Republic (J.K., V.M., I.M.)
Although cytokinins (CKs)
affect a number of processes connected with chloroplasts, it has never
been rigorously proven that chloroplasts contain CKs. We isolated
intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves
and determined their CKs by liquid chromatography/tandem mass
spectroscopy. Chloroplasts from both species contained a whole spectrum
of CKs, including free bases (zeatin and isopentenyladenine), ribosides
(zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5
Cytokinins (CKs) regulate a number of growth and developmental
processes in plants, including activation of cell division, stimulation
of growth of axillary buds (suppression of apical dominance),
inhibition of root growth, and suppression of senescence (for reviews,
see Binns, 1994 CKs affect the abundance of transcripts and proteins encoded both by
nuclear and plastid genomes, the most notable being genes coding for
the small subunit of Rubisco and chlorophyll a/b binding protein (for reviews, see Link, 1988 Despite this considerable knowledge of CK effects on chloroplast
development and function, it has not yet been proven that chloroplasts
contain CKs. Davey and Van Staden (1981) The occurrence of CK-binding sites in chloroplasts has been
demonstrated previously. Romanko et al. (1986) Plant Material
Chloroplast Preparation Tobacco leaves were deribbed, cut into thin strips, and kept on ice. About 40 g was mixed with 200 mL of homogenization medium (0.33 M sorbitol, 50 mM Tris, pH 7.5, 0.4 mM KCl, 0.04 mM Na2EDTA, 0.1% [w/v] BSA, 1% [w/v] PVP, and 5 mM isoascorbic acid), which was used in a semifrozen state. Homogenization was performed with a polytron for 10 to 20 s. The brei was squeezed through two layers of muslin to remove coarse debris, and filtered through cotton wool between eight layers of muslin. The chloroplast fraction was recovered from the homogenization medium by centrifugation at 1,000g for 1 min at 4°C, washed twice with 100 mL of resuspension medium (0.33 M sorbitol, 2 mM Na2EDTA, 1 mM MgCl2,1 mM MnCl2, and 50 mM HEPES, pH 7.6), and resuspended in 4 mL of resuspension medium. This suspension was underlayed with 20 mL of 40% (w/v) and 10 mL of 80% (w/v) Percoll solution in the same medium and centrifuged at 1,000g for tobacco and 2,500 g for wheat for 15 min at 4°C. Intact chloroplasts moved to the interface between the different density Percoll solutions. Chloroplasts were washed with 10 mL of resuspension medium, and after centrifugation were resuspended in 1 to 1.5 mL of the medium (Jensen and Bassham, 1966 ) and kept frozen until analysis.
Chlorophyll Determination Chlorophyll was extracted into 80% (w/v) acetone and then centrifuged at 500g for 5 min. The absorption was measured at 652 nm and the chlorophyll concentration (in micrograms per milliliter) was calculated as A652 × 1,000/34.5 (Arnon, 1949).
Glyceraldehyde-3-P Dehydrogenase Assay The glyceraldehyde-3-P dehydrogenase assay was chosen for checking the intactness of chloroplasts (Latzko and Gibbs, 1968). The assay
solution contained the chloroplast fraction (20 µL), 0.033 M Tris/HCl buffer, pH 8.5, 17 mM sodium
arsenate, 4 mM Cys, 20 mM sodium fluoride, and
4× 10 5 M
NADP+. The reaction was initiated with 50 µL of
0.1 M glyceraldehyde-3-P. Reduction of
NADP+ was followed at 340 nm. The same assay was
run with chloroplasts disrupted with 0.01 M
MgCl2.
Cyt c Oxidase Assay To determine possible contamination of the chloroplast preparation by mitochondria, Cyt c oxidase, a mitochondrial marker enzyme, was assayed according to the method of Tolbert (1974). The
chloroplast sample (20 µL) was mixed with 10 µL of 3% (w/v) digitonin and incubated 60 s before the addition of 920 µL of 40 mM potassium phosphate buffer, pH 7.4. The
reaction was initiated with 50 µL of reduced Cyt c (4 mg/mL). Cyt c was reduced with 1 M DTT
until the
A550/A565
ratio was 9 to 10, and excess reducing agent was removed by bubbling
air through the solution. Oxidation of Cyt c was followed at
550 nm.
Cyt c Reductase Assay To determine possible contamination with membranes of the ER, Cyt c reductase, an ER marker, was assayed using oxidized Cyt c (Tolbert, 1974). The reaction mixture contained
chloroplasts (20 µL) and 50 mM Tris/MES buffer,
pH 7.5, 1 mM KCN, 0.748 mM NADH, 0.0747 mM Cyt c, and 14.8 µg/mL antimycin A. Reduction of Cyt c was followed at 550 nm.
Glc-6-P Dehydrogenase Assay To determine the presence of trace cytoplasm in the chloroplast preparation, Glc-6-P dehydrogenase, a cytosolic marker enzyme, was assayed. The reaction mixture contained 10 mM MgCl2, 0.1% (w/v) Triton X-100, 0.17 mM NaDP+, 0.33 mM Glc-6-P, and 20 mM TES NaOH buffer, pH 7.5, in a final volume of 1 mL. The reduction of NADP+ was measured by monitoring A340 (Simcox et al., 1977).
Catalase Assay H2O2 was dissolved in 50 mM sodium phosphate buffer, pH 7.4, to an A240 of 0.6 to 0.8. The reaction was initiated with a 20-µL chloroplast fraction, and the reduction of H2O2 was followed at 240 nm (Gregory and Fridovich, 1974).
Extraction and Purification of CKs Extraction and purification was performed according to the method of Redig et al. (1996). Samples were extracted with Bieleski solvent
(Bieleski, 1964) containing a mixture of deuterated standards (Z, ZR,
DZ, DZR, ZMP, DZMP, ZN7G, ZN9G, ZOG, ZROG, DZOG, DZROG, 2iP, 2iPA,
2iPMP, and 2iPN9G, Apex International, Honiton, UK) and 80%
methanol. Extracts were then purified using DEAE-Sephadex (to separate
nucleotides), C18 cartridges, and immunoaffinity columns. The combination of these methods separates CKs into three fractions: 1, free bases, ribosides, and
N9-glucosides; 2, ribotides, determined as
ribosides after cleavage by alkaline phosphatase; and 3, N7- and O-glucosides. At
the time of analyses, the standard of 2iPN7G was not available and,
thus, the separation of N7 and N9 glucosides of 2iP was not tested.
Therefore, the only 2iP glucoside that was quantified was that which
occurred in the fraction 1 together with free bases and ribosides. On
the basis of analogy with ZN7G and ZN9G, we may assume that it was
iPN9G. N3-glucosides were not determined because
standards were not available.
Quantitative Analyses of CKs Analyses were performed according to methods described by Prinsen et al. (1995) and Witters et al. (1999) using HPLC linked to a mass
spectrometer (Quatro II, Fisons, Beverly, MA) equipped with an
electro-spray interface ([+] electro-spray LC-tandem
MS). Samples were injected onto a C8 column (5 µm, 125 × 4 mm;
LiChrosphere 60 RP Select B, Merck, Rahway, NJ) and eluted with
methanol:0.01 M ammonium acetate (70:30, v/v) at 800 µL
min1. The effluent was introduced into the
electro-spray source (80°C source, +3.5 V capillary, 20 V
cone) using a post-column split 1/20. Quantitation was performed by
multiple ion monitoring (Prinsen et al., 1995). The recovery of
deuterated standards was about 25% to 50% for free bases and
ribosides, respectively, and about 10% to 20% for glucosides. All
results were calculated using the recovery value of each of the
standards in the given sample.
Test for CK Absorption A mixture of 3H-labeled zeatin, zeatin riboside, and isopentenyladenosine (each, 50 kBq mL1) was prepared by Dr. J. Hanu of the
Isotope Laboratory, Institute of Experimental Botany (Prague) by
alkylation of [3H]adenosine with
4-tert-butoxy-3-methyl-trans-but-2-enyl bromide, followed by
Dimroth rearrangement and hydrolysis of the tert-butyl group. This
mixture was incubated with 1 mL of the chloroplast preparation at
20°C for 1 h. Chloroplasts were then sedimented at
1,000g and washed three times with resuspension solution.
The radioacivity of the chloroplast fraction was measured by
scintillation counting.
CK Oxidase Activity The method described by Motyka and Kamínek (1994) was used
to measure CK oxidase activity. The chloroplast suspension was mixed
with 0.1 M Tris-HCl, pH 7.5, in the presence of insoluble PVP. After filtration, centrifugation, and removal of nucleic acids by Polymin P (Serva Feinbiochemico, Heidelberg), proteins were
precipitated with 80% (w/v) saturated ammonium sulfate. Protein content was determined according to the method of Bradford (1976) using
BSA as a standard. The assay of CK oxidase activity was based on
the conversion of [2,8-3H]isopentenyladenine to
adenine. Tritiated isopentenyladenine was prepared by Dr. J. Hanu as described above. Separation of substrate from the
product was achieved by TLC on microcrystalline cellulose plates
developed with the upper phase of the 4:1:2 (v/v) mixture of
ethylacetate:n-propanol:water (Motyka and Kamínek, 1994).
Presentation of the Results Each experiment was repeated two to three times. Results of one or two representative studies of each type are given. The other experiments showed similar tendencies, including changes in individual CKs between leaves and chloroplasts or between light and dark, but the absolute values resulting from each study were different, so no statistical analyses of the data are given.
Chloroplast Preparation All of the chloroplast preparations used for CK analysis were determined to be intact using the Glc-6-P-dehydrogenase test, with only approximately 10% of the chloroplasts disrupted. The measurements of the other marker enzymes showed the following impurities in the chloroplast fraction: less than 5% ER, less than 4% mitochondria, and less than 2% cytosol and microbodies. These results show that highly purified intact chloroplasts were obtained.Content of CKs in Chloroplasts from Tobacco Upper and Lower Leaves Chloroplasts isolated from tobacco leaves contain a whole spectrum of CKs, including free bases (2iP), ribosides (2iPA and ZR), ribotides (2iPMP, ZMP, and DZMP), and N-glucosides (ZN9G, DZN9G, ZN7G, and 2iPNG). No O-glucosides were detected (Table I). The bases and ribosides represent 36.2% of total CKs in chloroplasts from both young and mature leaves, ribotides 38.4% and 31.8%, respectively, and N-glucosides 25.5% and 32.12%, respectively (Table II). Younger leaves contained 70% more CKs than mature leaves, while chloroplasts isolated from younger leaves contained 81% more CKs than those from mature leaves. These results show that although the age of the leaves affected the total level of CKs in chloroplasts, the proportions of individual CKs were not affected by leaf age.
Content of CKs in Chloroplasts from Tobacco and Wheat Leaves in Relation to Light/Dark Treatment In tobacco leaves the level of all CKs determined was about three times higher at the end of the dark period than at the end of the light period, which shows that the changes observed in C. rubrum (Machá ková et al., 1993) are not general and may be
correlated with the photoperiodic type of the plant. The relative abundance of individual CKs remained almost unchanged (Tables III and
IV). However, in chloroplasts isolated
from leaves at the end of either a light or dark period the differences
were more pronounced: chloroplasts from dark-treated leaves accumulated more CKs than those from light-treated leaves. The differences were
moderate for free bases and ribosides but dramatic for glucosides, with
a 25.6- and 25.3-fold increase in the content of ZN9G and DZN9G in the
dark (Table IV). Some ZROG and DZROG were also detected in these
chloroplasts. This was the only case where O-glucosides were
found. The recovery for glucoside determinations was relatively low, so
some lower levels might have escaped detection. This is a significant
result because CK O-glucosides have been previously reported
to occur in vacuoles (Fusseder and Ziegler, 1988).
CK Oxidase Activity in Tobacco Chloroplasts
Chloroplasts contain a wide spectrum of CKs and a relatively high activity of CK oxidase, and they react to darkness with specific changes in the level of various CK metabolites. Our results suggest that the metabolism of CKs in chloroplasts may be of importance for their function in these organelles.
* Corresponding author; e-mail machackova{at}ueb.cas.cz; fax 420-2-20390456. Received May 27, 1999;
accepted June 7, 1999.
The authors thank Prof. Dennis Baker (Wye College, London) and Dr. Laura Zonia (Institute of Experimental Botany, Prague) for language correction of the manuscript and Prof. Miroslav Kamínek (Institute of Experimental Botany, Prague) for critical reading of the manuscript.
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  G. Lochmanova, Z. Zdrahal, H. Konecna, S. Koukalova, J. Malbeck, P. Soucek, M. Valkova, N. S. Kiran, and B. Brzobohaty Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis J. Exp. Bot., October 1, 2008; 59(13): 3705 - 3719. [Abstract] [Full Text] [PDF]
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 Y. O. Zubo, M. V. Yamburenko, S. Y. Selivankina, F. M. Shakirova, A. M. Avalbaev, N. V. Kudryakova, N. K. Zubkova, K. Liere, O. N. Kulaeva, V. V. Kusnetsov, et al. Cytokinin Stimulates Chloroplast Transcription in Detached Barley Leaves Plant Physiology, October 1, 2008; 148(2): 1082 - 1093. [Abstract] [Full Text] [PDF]
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F. A. Brovko, V. S. Vasil'eva, A. O. Shepelyakovskaya, S. Yu. Selivankina, G. R. Kudoyarova, A. V. Nosov, D. A. Moshkov, A. G. Laman, K. M. Boziev, V. V. Kusnetsov, et al. Cytokinin-binding protein (70 kDa): localization in tissues and cells of etiolated maize seedlings and its putative function J. Exp. Bot., July 1, 2007; 58(10): 2479 - 2490. [Abstract] [Full Text] [PDF]
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L. Polanska, A. Vicankova, M. Novakova, J. Malbeck, P. I. Dobrev, B. Brzobohaty, R. Vankova, and I. Machackova Altered cytokinin metabolism affects cytokinin, auxin, and abscisic acid contents in leaves and chloroplasts, and chloroplast ultrastructure in transgenic tobacco J. Exp. Bot., February 1, 2007; 58(3): 637 - 649. [Abstract] [Full Text] [PDF]
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 Y. Kodama and H. Sano Evolution of a Basic Helix-Loop-Helix Protein from a Transcriptional Repressor to a Plastid-resident Regulatory Factor: INVOLVEMENT IN HYPERSENSITIVE CELL DEATH IN TOBACCO PLANTS J. Biol. Chem., November 17, 2006; 281(46): 35369 - 35380. [Abstract] [Full Text] [PDF]
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N. S. Kiran, L. Polanska, R. Fohlerova, P. Mazura, M. Valkova, M. Smeral, J. Zouhar, J. Malbeck, P. I. Dobrev, I. Machackova, et al. Ectopic over-expression of the maize {beta}-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco J. Exp. Bot., March 1, 2006; 57(4): 985 - 996. [Abstract] [Full Text] [PDF]
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 T. Werner, V. Motyka, V. Laucou, R. Smets, H. Van Onckelen, and T. Schmulling Cytokinin-Deficient Transgenic Arabidopsis Plants Show Multiple Developmental Alterations Indicating Opposite Functions of Cytokinins in the Regulation of Shoot and Root Meristem Activity PLANT CELL, November 1, 2003; 15(11): 2532 - 2550. [Abstract] [Full Text] [PDF]
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 M. LEXA, T. GENKOV, J. MALBECK, I. MACHACKOVA, and B. BRZOBOHATY Dynamics of Endogenous Cytokinin Pools in Tobacco Seedlings: a Modelling Approach Ann. Bot., April 1, 2003; 91(5): 585 - 597. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-monophosphate, zeatin
riboside-5
70°C.
ímalová,
eds, Physiology and Biochemistry of Cytokinins in Plants.
SPB Academic Publishing, The Hague, The Netherlands, pp 255-260