Plant Physiol. (1998) 117: 1515-1523
cis-Isomers of Cytokinins Predominate
in Chickpea Seeds throughout Their
Development1
Robert Joseph Neil Emery*,
Laurent Leport,
Joanne Edith Barton,
Neil Clifford Turner, and
Craig Anthony Atkins
Centre for Legumes in Mediterranean Agriculture (R.J.N.E., L.L.,
C.A.A., J.E.B., N.C.T.), and Botany Department (R.J.N.E., C.A.A.),
University of Western Australia, Nedlands, Western Australia, 6907, Australia; and University of Western Australia, Nedlands, Western Australia, 6907, AustraliaCommonwealth Scientific and Industrial Research
Organization, Division of Plant Industry, Centre for Mediterranean
Agricultural Research, Private Bag, P.O. Wembley, Western Australia,
6014, Australia (N.C.T.)
 |
ABSTRACT |
Trans-isomers of
cytokinins (CK) are thought to predominate and have greater biological
activity than corresponding cis-isomers in higher
plants. However, this study demonstrates a system within which the
predominant CK are cis-isomers. CK were measured at four
developmental stages in developing chickpea (Cicer
arietinum L. cultivar Kaniva) seeds by gas chromatography-mass
spectrometry. Concentrations were highest at an early endospermic fluid
stage and fell considerably when the cotyledons expanded. The
cis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin
riboside ([9R]Z), and zeatin (Z) were present in greater
concentrations than those of corresponding
trans-isomers: (trans)[9R-MP]Z,
(trans)[9R]Z, (trans)Z, or
dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and
the isopentenyl-type CK concentrations were either low or not
detectable. Root xylem exudates also contained predominantly cis-isomers of [9R-MP]Z and [9R]Z. Identities of
(cis)[9R]Z and (cis)Z were confirmed by
comparison of ion ratios and retention indices, and a full spectrum was
obtained for (cis)[9R]Z. Tissues were extracted under
conditions that minimized the possibility of RNase hydrolysis of tRNA
following tissue disruption, being a significant source of the
cis-CK. Since no isomerization of (trans)[2H]CK internal standards occurred,
it is unlikely that the cis-CK resulted from enzymic or
nonenzymic isomerization during extraction. Although quantities of
total CK varied, similar CK profiles were found among three different
chickpea cultivars and between adequately watered and water-stressed
plants. Developing chickpea seeds will be a useful system for
investigating the activity of cis-CK or determining the
origin and metabolism of free CK.
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INTRODUCTION |
Seed tissues were the source for isolation of the first naturally
occurring CK, trans-Z (Miller, 1961
; Letham, 1963). Seeds have turned out to be a rich source of CK, and in the past 30 years
investigators have described a range of different CK from seed tissues
(van Staden et al., 1982). This may reflect their relatively high
levels in seeds (van Staden et al., 1982), a status that is believed to
indicate a role for CK in establishing developing seeds as strong
assimilate sinks (Brenner and Cheikh, 1995). Despite a vast literature
concerning the occurrence, form, and significance of CK in plant
development, the nature and site(s) of their synthesis is yet to be
established. In fact, Holland (1997) recently proposed that CK are not
formed by plants at all but rather by bacterial symbionts that colonize
plant tissues. Although there is good evidence for the transfer of CK
synthesized by Rhizobium in legume nodules (Upadhyaya et
al., 1991), a role for bacteria in providing CK to roots or shoot
organs needs to be investigated more thoroughly. Because unequivocal
evidence for a plant isopentenyl transferase is lacking, a persistent
hypothesis, which has recently been reviewed (Prinsen et al., 1997), is
that the free CK in plants are not synthesized de novo but are released
during tRNA turnover.
Z, [9R-MP]Z, and [9R]Z have an unsaturated isopentenyl side chain
that can exist in the cis or trans conformation.
The cis-isomer occurs when the hydroxyl group of the
isopentenyl side chain is oriented toward the N-1 position
of the purine ring, whereas in the trans-isomer the hydroxyl
group is oriented away from the purine ring (Korszun et al., 1989; Fig.
1). The trans-isomers of
[9R]Z and Z are by far the more commonly reported forms and are considered the predominant isomers in higher plants (McGaw and
Burch, 1995; Prinsen et al., 1997). Systems in which the existence of
cis-CK can be demonstrated unequivocally would be
significant for two reasons. First, because cis-CK
show much lower activity than trans-CK in
bioassays (Kaminek, 1982) and their interconversion may constitute a
mechanism for reducing CK bioactivity in vivo. Second,
cis-CK provide evidence for the hypothesis that the free CK
pool in higher plants may be at least partially derived from the
breakdown of tRNA. The major criticism of this hypothesis has been the
structural distinctness between tRNA-bound CK and free-pool CK (Letham
and Palni, 1983; McGaw and Burch, 1995; Prinsen et al., 1997).
tRNA-bound CK are predominantly cis-isomers, whereas the
large majority of studies to date have reported that free CK are
predominantly or exclusively trans-isomers. It has even been
suggested by Tay et al. (1986) that where cis-isomers have been detected (Mauk and Langille, 1978; Watanabe et al., 1982; Takagi
et al., 1985) they are artifacts formed in extractions that did not
rigorously exclude the possibility of tRNA breakdown following cell
disruption.
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| Figure 1.
The stereochemical conformation of
(trans)Z and (cis)Z (adapted from Korszun
et al., 1989). Open oval, C; gray oval, N; checkered oval, O; black
oval, H.
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Recent evidence indicates that the occurrence and significance of
cis-CK need to be reexamined. Using an analytical procedure requiring no extraction step, Parker et al. (1989) showed in wheat and
oat that cis-CK are minor components of xylem. An enzyme
that converts cis- to trans-isomers, Z
cis-trans-isomerase, has been partially purified
and assayed in extracts of developing bean seeds (Bassil et al., 1993).
Furthermore, in potato tuber sprouts, Nicander et al. (1995) identified
cis-Z-9-glucoside, a compound that could not have been
derived directly from tRNA breakdown. Clearly, there is a need to
identify the source of cis-isomers and to establish their
significance, both as precursors for "active" CK and in general for
regulatory roles proposed for this group of compounds. Preliminary
studies of the CK composition of pulses indicated that
cis-isomers of Z and [9R]Z were minor components of
species of lupin but major components of chickpea (Cicer
arietinum L.; Emery et al., 1997). In the present study we used
GC-MS to identify and quantify CK profiles in developing chickpea
seeds, and we determined a system within which the predominant
CK are unambiguously identified as cis-isomers.
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MATERIALS AND METHODS |
Plant Material
Plants of chickpea (Cicer arietinum L.) were grown in
7.5-L free-draining polyvinylchloride pots (42.5 cm long; 15 cm in
diameter) sealed at the base, in a greenhouse at day/night temperatures of 25.5°C ± 2.7°C/16.5°C ± 1.7°C and maximum/minimum RH of
78.7% ± 20.9%/70.3% ± 12.9% at Floreat Park (Perth, Western
Australia). The pots were filled with ground, dry soil from the surface
of a native red-brown earth (U.S. Department of Agriculture, Calcic Haploxeralf) with neutral to alkaline pH, collected at Merredin, Western Australia (31o30
S,
118o12 E). The soil was mixed with 10% (w/w)
yellow sand to avoid compaction and treated with fertilizer
corresponding to 2.1 g of N, 0.8 g of P, and 1.2 g of K
per pot (1 g of commercial microelement preparation [Richgro, Canning
Vale WA, Australia], 7.51 g of KNO3, 7.13 g of NH4NO3,
10.67 g of Ca(NO3)2, and
7.61 g of triple superphosphate per 50 kg of soil). All seeds were
inoculated with commercial rhizobia inocula immediately before sowing
using group N Bradyrhizobium.
Seeds of large-seeded kabuli-type (cv Kaniva) or small-seeded desi-type
(cv Tyson or ICCV88201) chickpeas were sown to a depth of 5 cm. All
pots were irrigated every 2nd d to maintain the soil near field
capacity (17%, w/w) until the end of the flowering period. Plants of
selected pots were water stressed. Water deficit was imposed at 90 DAS
by watering once every 9 d with 200 mL per pot until 115 DAS and
then by stopping watering until terminal harvest at 157 DAS. Pods were
tagged between 69 and 87 DAS when they were 3 mm long (pod set), and
were later collected at several stages of development (1, 14, and 40 DAPS) in irrigated plants of cv Kaniva, at 30 DAPS in irrigated
and water-stressed plants of cvs Tyson and Kaniva, and at 50 DAPS in
irrigated plants of cv ICCV88201. Pods harvested at 14 DAPS were
partitioned into pod wall, seed coat, embryo with cotyledons, and
endospermic fluid. Pods harvested at 30, 40, and 50 DAPS were
partitioned into pod wall, seed coat, cotyledons, and embryonic axis.
CK were extracted from whole pods (fertilized ovaries, 1 DAPS, 30-50
mg dry weight), embryo with cotyledons, endospermic fluid and seed
coat (14 DAPS; 4-84 mg dry weight), and cotyledons (30, 40, and 50 DAPS; 16-85 mg dry weight for GC-MS-SIM and 500-900 mg dry weight for
full-scan GC-MS). All samples were freeze-dried before extraction of
CK. Harvests were chosen to correspond to previously determined
critical phases of seed development in cvs Kaniva and Tyson: start of
cell division (1 DAPS), end of cell division and commencement of seed filling (14 DAPS), maximum rate of seed filling (30 DAPS), and end of
seed filling (40 DAPS).
Collection of Xylem Exudate
Selected plants at 105 DAS were decapitated, and the root system
was enclosed in a pressure apparatus to assist in the collection of
root-bleeding sap from the cut surface of the stem. The pressure used
did not exceed 15 p.s.i. and collections were stopped after 10 min. This resulted in collection volumes of 1 to 2 mL from each plant.
Samples were frozen and stored at 
80°C before their analysis for CK
content.
Tissue Extraction
Samples were kept as cold as possible during the initial
extraction. Solvents were kept at 20°C and grinding was done on ice. Freeze-dried samples were powdered in liquid nitrogen and ground
into a slurry with cold, modified Bieleski extraction buffer 1 (Bieleski, 1964;
CH3OH:CHCl3:HCOOH:H2O
[60:15:5:20, v/v]) together with 20 ng each of
[2H6]iP,
[2H6][9R]iP,
(trans)[2H5] Z,
[2H3]DHZ,
(trans)[2H5][9R]Z, and
[2H3][9R]DHZ, and 50 ng each of
[2H6][9R-MP]iP,
(trans)[2H5][9R-MP]Z, and
[2H3][9R-MP]DHZ (Apex Organics,
Devon, UK) added as internal standards. Additional extraction buffer
was added to bring the buffer volume:sample weight ratio to 10:1; the
sample was vortexed, sonicated for 1 min, and centrifuged for 5 min to
sediment debris; and the supernatant was removed and filtered (0.45 µm). The residue was re-extracted twice more, each time in cold,
modified Bieleski extraction buffer 2 (CH3OH:HCOOH:H2O [60:5:35,
v/v]), vortexed, sonicated, and centrifuged. The supernatants were
pooled and freeze-dried. No extraction step was necessary for samples
of xylem exudate, which were directly freeze-dried and purified in the
same manner as tissue extracts.
For analyses involving the assessment of RNase activity on CK recovery
and tRNA degradation using Bieleski reagents, all glassware was baked
at 180°C for 6 h before use and gloves were worn at all times.
Extraction solvents and cation-exchange column buffers were prepared
using diethyl pyrocarbonate-treated, autoclaved water. Extractions
(cotyledons 40 DAPS, 1-4 g dry weight) were carried out as described
above, except that the modified Bieleski extraction buffers contained
10 mM RVC (Sigma-Aldrich). Degradation of tRNA was assessed
using yeast tRNA (20 µg; Sigma-Aldrich) incubated for 30 min at
37°C with 0.001 unit RNase A (Amresco, Solon, OH) in 50 mM Tris-HCl (pH 8.0) or Bieleski extraction buffer
2, with and without RVC. Reactions were snap frozen in liquid
nitrogen and freeze-dried. tRNA breakdown was assessed
quantitatively by measuring tRNA recovery following electrophoresis on
a 3% agarose gel containing 3 µg mL
1
ethidium bromide in TBE buffer (8.9 mM Tris-borate. 8.9 mM boric acid, and 0.2 mM EDTA). Gels were
photographed with a digital camera (model DC40, Kodak) and tRNA was
quantified with 1D image analysis software (Kodak).
Purification and Assay of CK
The residue from the extraction was dissolved in 3 mL of cold,
acidified water (0.1 N acetic acid) and transferred to a
10-mL of polypropylene tube, adjusted to less than pH 3.0 (with acetic acid) and passed through a sterene divinylbenzene (500 mg) SCX column
(Alltech Associates, Baulkham Hills NSW, Australia) that had been
preconditioned with 10 mL of 0.1 N acetic acid. The sample was loaded and the column washed with 10 mL of 0.1 N acetic
acid. The eluates from the load and wash steps were retained for
CK-nucleotide analysis.
Nucleoside and free-base CK were eluted from the SCX column in 20 mL of
2 N NH4OH. The eluate was dried in
vacuo (38°C), redissolved in neutral, deionized water (pH
5.0-6.0), and further purified using a syringe-tip 300-mg
C18 solid-phase extraction cartridge (Alltech
Associates). The cartridge was conditioned with 20 mL of methanol and
20 mL of neutral, deionized water before the sample was loaded. The
cartridge containing the sample was washed with 20 mL of neutral,
deionized water and the CK was eluted with 20 mL of methanol:water
(80:20, v/v). The sample was dried in vacuo (38°C) and, except for
xylem samples, further purified by HPLC on a C18
Alphabond column (Alltech Associates; length = 300 mm, i.d. = 3.9 mm, 10-µm particle size) at a flow rate of 2 mL
min1. CK eluted from a solvent program of
acetonitrile in water adjusted to pH 7.0 with triethylammonium
bicarbonate. The gradient was linear from 5% to 30% acetonitrile over
40 min. Two fractions were collected. The first, from about 15 to 20 min, contained (cis)Z, (trans)Z, DHZ,
(cis)[9R]Z, (trans)[9R]Z, and [9R]DHZ. The
second, from 24 to 26 min, contained iP and [9R]iP. Both were freeze-dried and the residues were transferred in methanol to 1.0-mL
tapered-bottom glass vials for derivatization.
CK nucleotides recovered in the acetic acid wash of the SCX column were
converted to nucleosides by incubation with alkaline phosphatase for
12 h at 37°C (type III, Sigma, 3.4 units in 1 mL of 0.1 M ethanolamine-HCl, pH 10.4). Resultant CK-nucleosides were
purified as described above.
The CK were permethylated as described previously (Emery et al., 1998),
and an aliquot in ethyl acetate was analyzed by GC-MS. The
Hewlett-Packard 5890 gas chromatograph was equipped with a split/splitless injector operating at 250°C in splitless mode and was
linked to a Hewlett-Packard 5970 series Mass Selective Detector. The GC
was fitted with a BP5 capillary column (25 m, 0.22-mm i.d.; 0.25-µm
film, 5% phenyl-95% dimethyl siloxane; SGE, Ringwood,
Victoria, Australia). The helium flow was 60 cm
s1 and the column head pressure was 1.5 p.s.i. The GC temperature program ramped from 60°C to 200°C at
20°C min1 and then at 5°C
min1 to 300°C, which was held for 5 min. Ions
for SIM mode are listed in Table I for
permethylated-iP, [9R]iP, (cis)Z, (trans)Z,
DHZ, (cis)[9R]Z,(trans)[9R]Z, and
[9R]DHZ. Individual endogenous CK levels were calculated using the
ratio of unlabeled to labeled ion pairs. Where necessary, corrections
were made for the contribution of 2H ions to
1H ions (and vice versa). Ion pairs used for
quantification of each CK are indicated in Table I. Full-scan mass
spectra were obtained for a range of m/z (40 to 300) at a rate of
0.9 scans s1. KRI values were determined using
the method of Gaskin and Macmillan (1991). The use of KRI values is
essential to distinguish between isomeric compounds that have almost
identical MS patterns but different GC characteristics. A
solution of C21- to
C36-n-alkanes was coinjected into the
gas chromatograph-mass spectrometer with authentic CK standards and
samples, and an m/z 85 mass chromatogram was added to SIM run
monitoring. KRI values were calculated according to the method of
Gaskin and Macmillan (1991).
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Table I.
KRIs and relative intensities of characteristic
diagnostic ions as determined by GC-MS-SIM for permethylated authentic
CK standards and putative CK purified from tissues of developing
chickpea seeds
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RESULTS |
Identification of cis-CK
The GC-MS-SIM spectra for authentic standards of
(cis)[9R]Z, (trans)[9R]Z, and
[2H5][9R]Z and a sample
from developing chickpea cotyledons are shown in Figure
2. In the cotyledon sample, a compound
was detected that had ion ratios similar to (trans)[9R]Z
in GC-MS-SIM runs but eluted from the gas chromatograph with a
retention time of approximately 0.3 min before
(trans)[9R]Z and had the same retention time as authentic
(cis)[9R]Z. Integration of SIM peaks confirmed that the
ratios of the major ions for the unknown compound,
(cis)[9R]Z and (trans)[9R]Z, were very
similar (Table I). A full spectrum of the putative
(cis)[9R]Z from chickpea cotyledons was obtained (Fig.
3), which matched closely either
(cis)[9R]Z (Fig. 3) or (trans)[9R]Z (not
shown). Since permethylated cis- and
trans-isomers of [9R]Z had almost identical fragmentation
patterns (Table I), the two isomers could not be distinguished by mass
spectra alone. KRI values were calculated for each of the compounds in
several different samples. All of the [9R]Z standards and the
putative chickpea (cis)[9R]Z eluted on GC between the
C30- and C31-alkanes (Fig.
2). KRI consistently characterized GC retention time differentials between authentic standards of isomers and confirmed that KRI values
were identical for the chickpea putative (cis)[9R]Z and authentic (cis)[9R]Z (Table I). No isomerization of
internal standard from
(trans)[2H5][9R]Z
to
(cis)[2H5][9R]Z
was observed in any of the samples.
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| Figure 2.
GC-MS-SIM spectra for putative
(cis)[9R]Z purified from cotyledons of immature
chickpea seeds (cv Kaniva, 30 DAPS) as compared with authentic
standards of (cis)[9R]Z (cis-ZR),
(trans)[9R]Z (trans-ZR), and
[2H5](trans)[9R]Z
([2H5]ZR).
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| Figure 3.
Full-scan mass spectrum of putative
(cis)[9R]Z purified from cotyledons of immature
chickpea seeds (cv Kaniva, 30 DAPS) as compared with that of an
authentic standard of (cis)[9R]Z.
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A second unknown compound with KRI values corresponding to authentic
(cis)Z was detected in extracts from developing chickpea seeds and showed a ratio of major ions from GC-MS-SIM runs similar to
those of authentic (cis)Z (Table I). A clear full spectrum for the putative (cis)Z was not obtained, since its
concentration in tissue extracts was relatively low, and a direct
comparison of spectra could not be done without increasing the scale of
extraction considerably.
Treatment of the CK-nucleotide fraction isolated from chickpea seeds by
SCX with phosphatase yielded a compound with GC-MS-SIM ion ratios
similar to [9R]Z; a GC retention time and KRI value were identical to
that of (cis)[9R]Z (Table I). This is consistent with a
cis-isomer of [9R-MP]Z being present in the original
extraction from chickpea seeds.
CK Profiles during Seed Development
CK profiles were relatively consistent across seed tissues and
stages of development (Table II). With
the exception of [9R-MP]iP, which was never detected, the CK
nucleotides were present in the greatest concentrations, followed by
the ribosides and free-base CK, respectively. iP and [9R]iP were
present in detectable quantities in only older cotyledon extracts from
seeds in which cell division had ceased.
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Table II.
Concentration of CK identified from components of
developing chickpea seeds as quantified by isotope dilution assay using
GC-MS-SIM
Data are means ± SE (n = 3-6).
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In all tissues tested from developing seeds of cv Kaniva from 1 to 40 DAPS, levels of (cis)[9R-MP]Z, (cis)[9R]Z,
and (cis)Z predominated over their corresponding
trans-isomers (Table II). Isomer differences were greatest
for [9R-MP]Z, for which the concentration of the
cis-isomer was 6- to 26-fold that of the
trans-isomer. In two cases (14-DAPS embryos and 40-DAPS
cotyledons) no (trans)[9R-MP]Z was detected.
The concentration of total CK was highest as cell division ended at 14 DAPS, ranging from 2.7 to 18.2 nmol g1 dry
weight, depending on the tissue. The highest concentrations of CK were
measured in endospermic fluid and embryos. These two tissues were from
2 to 67 times more concentrated in CK than any other tissue at all
sampling times. During rapid seed filling at 30 DAPS, the concentration
of total CK had decreased considerably and continued to decline to a
low of 0.3 nmol g1 dry weight by the end of
seed filling at 40 DAPS.
Comparison of CK among Cultivars
Very similar profiles of CK were measured among cotyledons of
three different cultivars, with cis-isomers being
predominant in cv Kaniva at 30 DAPS (cis-CK = 89% of
total CK, trans-CK = 5%), cv Tyson at 30 DAPS
(cis-CK = 81%, trans-CK = 1%), and cv ICCV88201 at 50 DAPS (cis-CK = 92%,
trans-CK = 5%). The effect of water deficit on CK
content was tested in seeds of cvs Kaniva and Tyson at 30 DAPS and the
cultivars showed a similar response. The total CK content was markedly
reduced by water stress, with water-stressed cotyledons of cvs Kaniva
and Tyson containing 23% and 28%, respectively, of the levels
observed in well-watered controls. Despite this reduction, the CK
composition did not change greatly; the cis-CK concentration
decreased slightly (cv Kaniva cis-CK = 63%; cv Tyson
cis-CK = 75%), but the trans-CK
concentration remained relatively constant (cv Kaniva
trans-CK = 5%; cv Tyson trans-CK = 1%).
CK Profiles in Xylem Exudate
The total CK concentration was 114 pmol
mL1. The CK present were
(cis)[9R-MP]Z (81%), (cis)[9R]Z
(7%), (trans)[9R]Z(6%), and [9R]DHZ (6%). Proportions
of CK present were thus similar to the profiles determined from seed
tissue extracts.
RNase-Free Extractions
Levels of (cis)[9R]Z or (cis)Z and the
ratios of cis-CK to trans-CK were not different
between cotyledons extracted in Bieleski solvents and those extracted
in Bieleski solvents with RNase inhibitors (20 mM RVC;
Table III). Tested in vitro in Tris and
Bieleski solvents RVC were effective for preventing the breakdown of
tRNA in the presence of RNases (Fig. 4).
Bieleski solvents without RVC were also moderately effective for tRNA
protection.
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Table III.
Concentration of CK in cotyledons of developing
chickpea seeds (40 DAPS) extracted in Bieleski solvents alone or with
RNase inhibitors
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| Figure 4.
In vitro tRNA degradation assays determined in two
buffer systems (Tris, Tris-HCl, pH 8.0; BS, Bieleski solvents). tRNA
was incubated at 37°C for 30 min alone (Control) or in the presence
of RNases with (+RNase) or without (+RVC) RNase inhibitors.
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DISCUSSION |
The results establish the presence of cis-CK
{(cis)[9R-MP]Z, (cis)[9R]Z, and
(cis)Z} in developing chickpea seeds and show that the
significant levels of the cis-isomers found were not artifacts of extraction. (cis)[9R-MP]Z,
(cis)[9R]Z, and, in some cases, (cis)Z,
predominated, whereas the corresponding trans-isomers were
detected as minor constituents. Identities of (cis)[9R]Z and (cis)Z were clearly established by comparison of ion
levels and retention indices using GC-MS-SIM. In the case of
(cis)[9R]Z, a full mass spectrum identical to an authentic
standard was obtained. Likewise, (cis)[9R-MP]Z was
identified by GC-MS-SIM following its hydrolysis to
(cis)[9R]Z.
In previous studies reporting significant levels of cis-CK
in potato seedlings (Mauk and Langille, 1978), unfertilized hop cones
(Watanabe et al., 1982), rice roots and ears (Takagi et al., 1985), and
etiolated squash seedlings (Kuraishi et al., 1987, 1991), the
possibility that the cis-CK had resulted from hydrolysis of
tRNA during extraction was not rigorously excluded (Tay et al., 1986).
Although Kuraishi et al. (1987) used Bieleski solvents to reduce the
potential for enzyme hydrolysis, they used prolonged extraction times
lasting several days. In the present study cold Bieleski solvents were
used and extraction times were minimized by replacing long soaking
periods with short applications of sonication, as recommended by
Hammerton et al. (1996).
In vitro assays of tRNA integrity in the presence of RNase showed that
Bieleski solvents are capable of providing some protection for tRNA
over longer periods, and at higher temperatures, than those routinely
used in our tissue extractions. In one tissue extraction the tRNA was
further protected with the addition of the RNase inhibitor RVC to the
extraction buffers. However, the levels of cis-CK did not
decrease, as would be expected if there was significant tRNA breakdown
during extraction. The in vitro tRNA integrity assays indicated that,
even when RNase was added, negligible tRNA breakdown occurred in the
presence of RVC in Bieleski solvents. The Bieleski solvents alone
provided substantial protection for tRNA in the presence of RNase, and
it is unlikely that tRNA hydrolysis during extraction contributes to
the pool of free CK detected. These considerations are not likely to
apply to the recovery of cis-CK as the major forms in xylem
exudate. Parker et al. (1989) suggested that the minor levels of
cis-isomers in xylem exudate from cereals precluded
extraction artifacts, and this is reinforced by the data for chickpea
xylem.
Extraction artifacts other than tRNA breakdown are possible. For
example, a cis-trans-isomerase similar to the one
isolated from immature bean seeds (Bassil et al., 1993) could have
changed the CK isomer ratio following disruption of tissue or cell
compartmentation at extraction. In addition, nonenzymatic isomerization
of Z or [9R]Z is known to occur in vitro in the presence of light
(Bassil et al., 1993). These extraction artifacts are unlikely to have influenced our results. First, all extractions were carried out in cold
Bieleski solvents to minimize enzymatic activity. Second, no
isomerization of the internal standards
(trans)[2H5]Z,
(trans)[2H5][9R]Z,
or
(trans)[2H5][9R-MP]Z
was observed in any of the chickpea extracts, even though the standards
had been added before the plant tissues were disrupted with buffer.
Third, extractions of immature lupin seed tissue under conditions
identical to those of the present study yielded predominantly
trans-CK, whereas the cis-CK were relatively low
or, in some cases, undetectable (R.J.N. Emery, J.E. Barton, and C.A.
Atkins, unpublished data).
Four studies have quantified CK in chickpea without reporting the
occurrence of cis-isomers. Martin et al. (1987a, 1987b) separated CK from germinating seeds with TLC and used a bioassay that
measured chlorophyll synthesis in cucumber cotyledons to identify CK.
They would have been unlikely to detect cis-CK given the
reported reduction or lack of biological activity of
cis-isomers. Saha and Sircar (1996) quantified [9R]Z, DHZ,
Z, [9R]iP, and iP in germinating seeds using HPLC and UV absorbance.
Although [9R]Z was listed as the major CK, no distinction between the
isomers was made. It was unclear whether their HPLC protocol could have resolved the isomers. Turnbull et al. (1997) reported
(trans)[9R]Z and (trans)Z as among the major CK
in lateral branch buds. However, the antibodies used in their
immunoassay would have been unlikely to detect even quite substantial
levels of cis-isomers. The polyclonal antibodies used show
negligible cross-reactivity with cis-CK, except for those
against [9R]DHZ, which cross-react with (cis)[9R]Z at
about 5% (C.G.N. Turnbull, personal communication; Turnbull and Hanke,
1985; Parker et al., 1989). Other CK detected included iP, [9R]iP,
[9R-MP]iP, [9R-MP]Z, and [9G]Z. In common with the present study,
samples had low levels of dihydro-CK (DHZ, [9R-MP]DHZ, and
[9R]DHZ).
The present study has shown that levels of total CK may vary markedly
over seed development and with water supply, but the profile of
individual CK remains relatively constant, with the cis-isomers, especially (cis)[9R]Z and
(cis)[9R-MP]Z, predominating. It is interesting to note
that, although cis-isomers could be detected in developing
lupin seeds, they were minor rather than major constituents of the CK
spectrum (R.J.N. Emery, J.E. Barton, and C.A. Atkins, unpublished
data). Clearly, a more complete understanding of the role of CK in pod
set and seed development in legumes will require an appreciation of the
relative levels and bioactivity of both cis- and
trans-isomers. It also appears that not all species will
show the same relationship between the isomers, and it seems reasonable
to suspect that they will also vary in their mechanistic significance.
The unique profile of CK in chickpea seeds raises two issues relating
to CK biosynthesis and metabolism. The first is the source of these
cis-isomers. The second is the level of bioactivity cis-isomers have in developing chickpea seeds.
With respect to the source of the cis-isomers, there appear
to be a few possibilities. Xylem exudates collected from the root system contained predominantly cis-isoforms; therefore,
deposition in pods as a result of transpiration could be one source.
However, Zhang and Letham (1990) used estimates for xylem delivery
alone to developing lupin seeds and calculated that xylem accounts for only minor delivery (about 1%) of the total CK. Furthermore,
developing legume fruits are mainly phloem fed and it is possible that
the assimilate stream also carries CK to the seeds. There is no
information regarding CK content of phloem in chickpea; therefore, it
is difficult to assess the overall significance of translocation.
cis-CK could arise from a de novo synthetic pathway or as a
result of tRNA turnover in situ (Prinsen et al., 1997), and either
could be due to plant metabolism or to the activity of symbiotic
bacteria colonizing the shoots (Holland, 1997). Current models of de
novo CK biosynthesis (Binns, 1994; Jameson, 1994, figure 9-3) proceed
from [9R-MP]iP to either [9R]iP or (trans)[9R-MP]Z,
which are either not found or are present only as minor constituents of
chickpea seeds. No metabolic pathway has been described that accounts
for (cis)[9R]Z or (cis)[9R-MP]Z synthesis de
novo. In plant systems that show major levels of cis-isomers
(i.e. rice, squash, or chickpea) there may be a CK pathway involving
initial production and subsequent modification of cis-CK
nucleotides. Otherwise, considering Holland's (1997) hypothesis for
bacterial CK synthesis, it is perhaps significant that in bacteria,
which are symbionts of, or are otherwise associated with, plants
(Morris et al., 1991; Upadhyaya et al., 1991; Holland and Polacco,
1994), cis-CK can be a significant component of the free CK
pool. Should transfer to the tissues of the seed take place this might
lead to an accumulation of cis-isomers.
The contribution of tRNA to the CK complement of plant tissues also
remains to be resolved. Binns (1994) and Hall (1973) argued that tRNA
turnover was not sufficiently rapid to account for the observed CK
levels. Furthermore, unlike the vast majority of reported plant systems
that contain trans-CK, the cis-isomers should
predominate following CK release from tRNA (McGaw and Burch, 1995;
Prinsen et al., 1997). Clearly, tissues such as those of developing
chickpea seeds, which accumulate cis-CK, challenge this
idea. The demonstration of an isomerase that would interconvert the
isomers (Bassil et al., 1993) also indicates that the
cis-configuration may not be an impediment to a tRNA source.
In the case of chickpea seeds it might be argued that the level of
isomerase is very low.
The second issue relates to the bioactivity of the
cis-isomers in tissues such as those of chickpea seeds in
which CK have always been regarded as establishing a strong "sink"
to attract assimilates. It has been proposed that CK controls seed size
by influencing cell number in very young, developing seeds. Increased cell number would enhance storage capacity (Morris et al., 1993; Brenner and Cheikh, 1995). Studies of corn, wheat, and rice (Morris et
al., 1993) show that CK content is highest during developmental stages
that encompass periods of the most rapid nuclear and cell division of
the endosperm. CK levels are greatest at the early endospermic fluid
stages of seed growth in lupin (Davey and van Staden, 1978). Our data
demonstrate that CK levels in chickpea are also greatest over phases of
rapid cell division. However, current thinking presumes that the CK
activity is still low, since the increase is predominantly in
cis-isomers. Nonetheless, whereas bioassays used in earlier
studies found that the cis-isomers were relatively inactive
compared with the trans forms of CK, none of them was based
on activity measurement with chickpea tissues (Kaminek, 1982). The
possibility that cis forms are active in species such as
chickpea and trans-isomers are active in other species such
as lupins and soybeans, cannot be ignored. Furthermore, manipulation of
endogenous CK-isomer ratios in chickpea and other pulses may offer a
means to examine the stereochemical-activity relationships of CK and
determine what potential may exist to improve the sink strength of
growing seeds. Genetic manipulation of enzymes of CK metabolism,
especially of the cis-trans-isomerase, offers a
means to explore both questions.
| |
FOOTNOTES |
1
This research was funded by the Australian
Cooperative Research Centre for Legumes in Mediterranean Agriculture
and the Grains Research and Development Corporation of Australia.
*
Corresponding author; e-mail rjnemery{at}cyllene.uwa.edu.au; fax
61-8-9380-1001.
Received February 27, 1998;
accepted May 18, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CK, cytokinin(s).
DHZ, dihydrozeatin.
[9R]DHZ, dihydrozeatin riboside.
[9R-MP]DHZ, dihydrozeatin nucleotide.
DAPS, days after pod set.
DAS, days after sowing.
iP, isopentenyl-adenine.
[9R]iP, isopentenyl-adenosine.
[9R-MP]iP, isopentenyl-adenine
nucleotide.
KRI, Kovat's retention index.
m/z, mass to charge
ratio.
RVC, RNase vanadyl complexes.
SCX, strong cation-exchange
solid-phase extraction column.
SIM, selected ion monitoring.
Z, zeatin.
[9R]Z, zeatin riboside.
[9R-MP]Z, zeatin nucleotide.
| |
LITERATURE CITED |
Bassil NV,
Mok DWS,
Mok MC
(1993)
Partial purification of a cis-trans-isomerase of zeatin from immature seed of Phaseolus vulgaris L.
Plant Physiol
102:
867-872
[Abstract]
Bieleski RL
(1964)
The problem of halting enzyme action when extracting plant tissues.
Anal Biochem
9:
431-442
[CrossRef][ISI][Medline]
Binns AN
(1994)
Cytokinin accumulation and action: biochemical, genetic, and molecular approaches.
Annu Rev Plant Physiol Plant Mol Biol
45:
173-196
[CrossRef][ISI]
Brenner ML,
Cheikh N
(1995)
The role of hormones in photosynthate partitioning and seed filling.
In
PJ Davies,
eds, Plant Hormones, Physiology, Biochemistry, and Molecular Biology, Ed 2.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 649-670
Davey J,
van Staden J
(1978)
Cytokinin activity in Lupinus albus. III. Distribution in fruits.
Physiol Plant
43:
87-93
Emery RJN,
Leport L,
Atkins CA,
Turner NC
(1997)
cis-Isomers of cytokinins predominate in developing seeds of Cicer arietinum (abstract no. 179).
Plant Physiol
114:
S-53
Emery RJN,
Longnecker NE,
Atkins CA
(1998)
Branch development in Lupinus angustifolius L. II. Relationship with endogenous ABA, IAA and cytokinins in axillary and main stem buds.
J Exp Bot
49:
555-562
[Abstract/Free Full Text]
Gaskin P, Macmillan J (1991) GC-MS of the Gibberellins and Related
Compounds: Methodology and a Library of Spectra. University of Bristol
(Cantocks Enterprises), Bristol, UK
Hall RH
(1973)
Cytokinin as a probe of developmental processes.
Annu Rev Plant Physiol
24:
425-444
Holland MA
(1997)
Occam's razor applied to hormonology. Are cytokinins produced by plants?
Plant Physiol
115:
865-868
[ISI][Medline]
Holland MA,
Polacco JC
(1994)
PPFMs and other covert contaminants: is there more to plant physiology than just plant?
Annu Rev Plant Physiol Plant Mol Biol
45:
197-209
[CrossRef][ISI]
Hammerton RD,
Nicander B,
Tillberg E
(1996)
Identification of some major cytokinins in Phaseolus vulgaris and their distribution.
Physiol Plant
96:
77-94
[CrossRef]
Jameson PE
(1994)
Cytokinin metabolism and compartmentation.
In
DS Mok,
MC Mok,
eds, Cytokinins: Chemistry, Activity and Function.
CRC Press, Boca Raton, FL, pp 113-128
Kaminek M (1982) Mechanisms preventing the interference of tRNA
cytokinins in hormonal regulation. In PF Wareing, ed, Plant
Growth Substances 1982. Academic Press, New York, pp 215-224
Korszun ZR,
Knight C,
Chen C-M
(1989)
A stereochemical model for cytokinin activity.
FEBS Lett
243:
53-56
[CrossRef]
Kuraishi S,
Ohara T,
Sakuri N
(1987)
Gas chromatography thermionic detector analysis of cytokinins in etiolated squash seedlings using 14C-BA as an internal standard.
Plant Cell Physiol
28:
1033-1041
[Abstract/Free Full Text]
Kuraishi S,
Tasaki K,
Sakuri N,
Sadatoku K
(1991)
Changes in levels of cytokinins in etiolated squash seedlings after illumination.
Plant Cell Physiol
32:
585-591
[Abstract/Free Full Text]
Letham DS
(1963)
Zeatin, a factor inducing cell division from Zea mays.
Life Sci
8:
569-573
Letham DS,
Palni LMS
(1983)
The biosynthesis and metabolism of cytokinins.
Annu Rev Plant Physiol
34:
163-197
[ISI]
Martin L,
Diez A,
Nicolas G,
Legaz ME,
Villalobos N
(1987a)
Cytokinins in chick-pea seeds. Identification and transformation during germination and seedling growth.
J Plant Physiol
128:
133-140
Martin L,
Diez A,
Nicolas G,
Villalobos N
(1987b)
Variation of the levels and transport of cytokinins during germination of chick-pea seeds.
J Plant Physiol
128:
141-151
Mauk CS,
Langille AR
(1978)
Physiology of tuberization in Solanum tuberosum L.
Plant Physiol
62:
438-442
[Abstract/Free Full Text]
McGaw BA,
Burch LR
(1995)
Cytokinin biosynthesis and metabolism.
In
PJ Davies,
eds, Plant Hormones, Physiology, Biochemistry, and Molecular Biology, Ed 2.
Kluwer Academic, Dordrecht, The Netherlands, pp 98-117
Miller CO
(1961)
A kinetin-like compound in maize.
Proc Natl Acad Sci USA
47:
170-174
[Free Full Text]
Morris RO,
Blevins DG,
Dietrich JT,
Durley RC,
Gelvin SB,
Gray J,
Hommes NG,
Kaminek M,
Mathews LJ,
Meilan R,
and others
(1993)
Cytokinins in plant pathogenic bacteria and developing cereal grains.
Aust J Plant Physiol
20:
621-637
Morris RO,
Jameson PE,
Laloue M,
Morris JW
(1991)
Rapid identification of cytokinins by an immunological method.
Plant Physiol
95:
1156-1161
[Abstract/Free Full Text]
Nicander B,
Bjorkman P-O,
Tillberg E
(1995)
Identification of an N-glucoside of cis-zeatin from potato tuber sprouts.
Plant Physiol
109:
513-516
[Abstract]
Parker CW,
Badenoch-Jones J,
Letham DS
(1989)
Radioimmunoassay for quantifying the cytokinins cis-zeatin and cis-zeatin riboside and its application to xylem sap samples.
J Plant Growth Regul
8:
93-105
Prinsen E,
Kaminek M,
van Onckelen HA
(1997)
Cytokinin biosynthesis: a black box?
J Plant Growth Regul
23:
3-15
[CrossRef]
Saha S,
Sircar PK
(1996)
Cytokinin profile during early phase of germination of gram seeds after application of benzylaminopurine.
Biol Plant
38:
293-296
Takagi M,
Yokota T,
Murofushi N,
Ota Y,
Takahashi N
(1985)
Fluctuation of endogenous cytokinin contents in rice during its life cycle-quantification of cytokinins by selected ion monitoring using deuterium-labeled internal standards.
Agric Biol Chem
49:
3271-3277
Tay SAB,
MacLeod J,
Palni LMS
(1986)
On the reported occurrence of cis-zeatin riboside as a free cytokinin in tobacco shoots.
Plant Sci
43:
131-134
[CrossRef]
Turnbull CGN,
Hanke DE
(1985)
The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins.
Planta
165:
366-376
Turnbull CGN,
Raymond MAA,
Dodd IC,
Morris SE
(1997)
Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance.
Planta
202:
271-276
[CrossRef][ISI]
Upadhyaya NM,
Parker CW,
Letham DS,
Scott KF,
Dart PJ
(1991)
Evidence for cytokinin involvement in Rhizobium (IC3342)-induced leaf curl syndrome of pigeonpea (Cajanus cajan Millsp.).
Plant Physiol
95:
1019-1025
[Abstract/Free Full Text]
van Staden J, Davey J, Brown NAC (1982) Endogenous cytokinins in
seed development and germination. In AA Khan, ed, The
Physiology and Biochemistry of Seed Development, Dormancy and
Germination. Elsevier Biomedical Press, Amsterdam, The Netherlands, pp
137-156
Watanabe N,
Yokota T,
Takahashi N
(1982)
Transfer RNA, a possible supplier of free cytokinins, ribosyl-cis-zeatin and ribosyl-2-methylthiozeatin: quantitative comparison between free and transfer cytokinins in various tissues of the hop plant.
Plant Cell Physiol
23:
479-488
[Abstract/Free Full Text]
Zhang R,
Letham DS
(1990)
Cytokinin translocation and metabolism in lupin species. III. Translocation of xylem cytokinin into the seeds of lateral shoots of Lupinus angustifolius.
Plant Sci
70:
65-71
[CrossRef]
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Abstract
Introduction