Plant Physiol. (1999) 120: 1043-1048
The Endogenous Sulfated Pentapeptide Phytosulfokine-
Stimulates Tracheary Element Differentiation of
Isolated Mesophyll
Cells of Zinnia1
Yoshikatsu Matsubayashi*,
Leiko Takagi,
Naomi Omura,
Akiko Morita, and
Youji Sakagami
Laboratory of Bioactive Natural Products Chemistry, Graduate School
of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya
464-8601, Japan
 |
ABSTRACT |
Dispersed zinnia (Zinnia
elegans) mesophyll cells cannot differentiate into tracheary
elements (TEs) at low cell density conditions even if auxin and
cytokinin are present in the medium, indicating the involvement of
intercellular interactions during the initiation and/or subsequent
progresses in TE differentiation. When zinnia cells were incubated at a
low density (2.5 × 104 cells mL
1) in
TE-inductive medium in the presence of various concentrations of
phytosulfokine (PSK)-
, which was originally identified as an
intercellular signal peptide involved in cell proliferation, TE
differentiation was strongly stimulated in a dose-dependent fashion;
more than 35% of the living cells differentiated into TEs by 5 d
of culture in the presence of 10 nM PSK-. Enzyme-linked immunosorbent assay and mass spectroscopy confirmed that cultured zinnia cells produce nanomolar levels of PSKs under inductive conditions. These results suggest that PSK- is a factor responsible for TE differentiation of zinnia mesophyll cells.
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INTRODUCTION |
TE formation in culture has been used as a model system for the
study of cell differentiation of higher plants (for review, see Fukuda,
1992
). In mechanically isolated mesophyll cells of zinnia (Zinnia
elegans) in liquid culture, differentiation into TEs can be
readily induced by phytohormones and clearly distinguished on the basis
of morphological features (Fukuda and Komamine, 1980a). Because of the
nature of this system, individual cells respond homogeneously to
physiological and chemical stimuli, and synchronously differentiate at
relatively high frequency. Many researchers have investigated the
determining factor(s) of TE differentiation at the cellular level and
found that differentiation necessitates at least two exogenous factors,
an auxin and a cytokinin (for review, see Fukuda and Komamine, 1985).
In particular, cytokinins have been considered an absolute requirement
in zinnia mesophyll cells (Fukuda and Komamine, 1980a).
The initial cell density is also a determining factor in TE
differentiation in zinnia mesophyll cell cultures (Fukuda and Komamine,
1980a). Differentiation occurs synchronously at high frequency above an
initial cell density of 4.2 × 104 cells
mL1, but is significantly suppressed below this
threshold, suggesting that intercellular interactions are involved in
the initiation and/or subsequent progresses in TE differentiation. An
oligosaccharide-like factor in zinnia conditioned medium appear to play
an important role in cell expansion and metaxylem-like TE
differentiation (Roberts et al., 1997), but the active principle has
not been chemically identified.
The relative growth rate of the plant cells in culture also strictly
depends on the initial cell density, even if sufficient amounts of
auxins and cytokinins are present in the medium, so additional factors
must play a role in cell proliferation (Bellincampi and Morpurgo, 1987;
Birnberg et al., 1988). In 1996, we first isolated one of these factors
from conditioned medium derived from mesophyll culture of asparagus,
and determined its structure to be a sulfated
pentapeptide,H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH (Matsubayashi and Sakagami, 1996). This peptide, named PSK-, compensates for cell growth suppression in low-density cultures at a
concentration as low as 1.0 nM. PSK- has also been
identified in conditioned medium derived from rice and maize cell
cultures, apparently promoting cell growth by interacting with specific binding sites distributed upon plasma membranes (Matsubayashi et al.,
1997; Matsubayashi and Sakagami, 1999).
Although the generality of PSK- in plant kingdom has not yet been
well clarified, its influence on cell differentiation and proliferation
are clearly of interest. In the present study, we investigated the
physiological effects of PSK- on TE differentiation and
proliferation of zinnia mesophyll cells, and also determined, using
ELISA and MS, whether zinnia cells themselves produce PSK-.
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MATERIALS AND METHODS |
Materials
Sep-Pak cartridges were purchased from Millipore and Develosil
ODS-5 and Develosil ODS-10 reverse-phase columns were purchased from
Nomura Chemicals (Seto, Japan). Twenty four-well microplates were
obtained from Iwaki (Chiba, Japan). Polystyrene, 96-well plates for
ELISA were from Nunc (Naperville, IL). All of the other inorganic and
organic chemicals were obtained from Wako Pure Chemicals (Osaka).
PSK- was prepared by solid-phase synthesis as previously described
(Matsubayashi et al., 1996).
Plants
Seeds of zinnia (Zinnia elegans cv Canary bird; Takii
Shubyo, Kyoto) were grown on moist sterile soil at 25 ± 2°C
with a 16-h light period (approximately 20,000 lux at the plant level).
Isolation of Single Cells from the Mesophyll
Zinnia leaves were sterilized for 10 min in a solution of 0.05%
(w/v) NaOCl containing 0.05% (w/v) Tween 20, then rinsed three times
with sterile water. Single cells were liberated by homogenization with
a glass homogenizer in 0.2 M mannitol solution. The
homogenate was filtered through a 37-µm stainless-steel mesh, and the
filtrate was centrifuged at 100g for 3 min. The separated
single cells were washed with 0.2 M mannitol
three times and used in the following experiments.
Cell Culture and Bioassay Methods
The basal medium used for TE differentiation experiments was
prepared according to the method of Fukuda and Komamine (1980a), except
that NH4Cl was eliminated to improve the cell
viability under low-density conditions. The medium was adjusted to pH
5.5 with 1.0 N KOH. Isolated single mesophyll cells were
suspended in 0.2 M mannitol, adjusted to twice the final
cell density using a hemocytometer, and dispensed into 24-well
microplates at a volume of 250 µL per well. Culture medium (125 µL)
prepared at a 4-fold concentration and various sample solutions (125 µL) were sterilized by filtration and then added to the cell
suspension in each well. The plates were sealed with laboratory film to
avoid evaporation of the medium, and were then incubated in the dark at
25°C with continuous rotary shaking at 120 rpm. The TE
differentiation frequency was determined by counting the numbers of
undifferentiated and differentiated cells under an inverted microscope.
The TE differentiation frequency for each well was calculated by
dividing the number of TE-differentiated cells by the total number of
undifferentiated and differentiated cells. The cell viability for each
well was calculated by dividing the number of living cells (determined by bromphenol blue; Suzuki et al., 1992) by the number of total cells.
For the preparation of conditioned medium, single zinnia mesophyll
cells were suspended in 200 mL of culture medium at a density of
2.0 × 105 cells
mL1. This suspension was cultured in 500-mL
Erlenmeyer flasks in the dark at 25°C with rotary shaking at 120 rpm.
After 6 d of culture, conditioned medium was collected by
filtration and stored at 
20°C until use.
Competition ELISA Procedure
Preparation of anti-PSK- polyclonal antibodies and the
procedure for competitive ELISA were described previously
(Matsubayashi et al., 1999). Two different PSK- conjugated proteins,
Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-(Gly)3-Cys-linker-KLH (antigen A) and
Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-(Ala)3-Lys-linker-BSA (antigen B) were prepared by coupling the peptides with the
corresponding proteins. Rabbits were immunized with antigen A, and the
obtained antibodies were purified with an immunoaffinity column
containing Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-Cys-linker-resin.
For quantification of PSK-, polystyrene 96-well plates were coated
with antigen B and blocked with 0.1% (w/v) BSA in PBS (10 mM phosphate buffer [pH 7.0] containing 8.0 g
L1 NaCl). Purified antibody and
samples were then added and the plates were incubated at 37°C for
1.5 h. During this period, competition occurred for the antibody
between antigen B bound to the plate and free PSK- in solution.
After washing with PBS containing 0.1% (w/v) Tween 20, plates were
incubated for 1.5 h with a solution of horseradish
peroxidase-coupled anti-rabbit immunoglobulin. After three washings,
orthophenylenediamine solution containing 0.01% hydrogen peroxide was
added, and the plates were incubated for 20 min at 37°C. Color
development was terminated with sulfuric acid, and optical density was
recorded at a wavelength of 490 nm with a plate reader.
Purification of PSK- from Conditioned Medium
Conditioned medium derived from zinnia suspension cultures (400 mL) was concentrated to one-third volume, adjusted to pH 8.0 with 6.0 N KOH, and then applied to a DEAE Sephadex A-25 column (3.2 × 12 cm, flow rate 100 mL h1)
equilibrated with 20 mM Tris-HCl buffer, pH 8.0. The column was washed with 200 mL of the buffer, and fractions were eluted successively with 200 mL of this buffer containing 400, 800, or 1,200 mM KCl. Fractions of 800 and 1,200 mM were
concentrated to one-half volume and TFA was added to this combined
fraction at a final concentration of 0.1% (v/v). This solution was
applied to C18 cartridges (Sep-Pak Vac, 12 mL × 2, flow rate 150 mL h1) that had
been equilibrated with 0.1% TFA. After washing with 80 mL of 0.1%
(v/v) TFA in water, fractions were eluted with 30% (v/v) acetonitrile
containing 0.1% (v/v) TFA. After lyophilization, materials were
dissolved in 1.0 mL of 20 mM
KH2PO4-KOH buffer, pH 5.8, and then applied to a Bio-Gel P-2 extra-fine column (1.7 × 42 cm), previously equilibrated with the same buffer. Five-milliliter fractions were collected and assayed by ELISA. Positive fractions recovered from the Bio-Gel column were lyophilized, dissolved in 200 µL of 10% (v/v) acetonitrile in 0.1% TFA, and chromatographed on a
Develosil ODS-5 column (4.6 × 250 mm, Nomura Chemicals) by isocratic elution with 10% (v/v) acetonitrile in 0.1% TFA at a flow
rate of 1.0 mL/min with monitoring of the UV
A220. Fractions were collected every
1.0 min and assayed by ELISA after lyophilization.
MS
Positive fractions determined by ELISA were dissolved in 20 µL
of water and directly analyzed with a vacuum generator platform quadrupole mass spectrometer (Fisons, Cheshire, UK) equipped for electrospray ionization. The source temperature was maintained at
70°C, and the range m/z 50 to 1,000 was scanned
over 1.9 s.
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RESULTS AND DISCUSSION |
Effects of Initial Cell Density on TE Differentiation
Although Fukuda and Komamine's medium (Fukuda and Komamine,
1980a) has been widely used in TE differentiation experiments, zinnia
cells cultured at low cell density often show relatively low viability
in this medium (approximately 35% at 2.5 × 104 cells mL1). This
phe-nomenon is likely attributed to ammonium ions, because cell
growth of mesophyll primary cultures under low cell density conditions
is strongly inhibited by ammonium ions even if sufficient amounts of
PSK- are added to the medium (Matsubayashi and Sakagami, 1998).
Therefore, we modified Fukuda and Komamine's medium to eliminate
NH4Cl.
The TE differentiation frequency of single zinnia cells in suspension
culture was markedly dependent on the initial cell density (Fig.
1). The TEs formed at relatively high
frequency at the initial cell density of 1.0 × 105 and 5.0 × 104
cells mL1, reaching approximately 30% after
5 d of culture. In contrast, TE differentiation was markedly
inhibited at densities below 2.5 × 104
cells mL1, and the frequencies remained
approximately 5% or less of these values even after 5 d of
culture. A similar observation was made in a previous report (Fukuda
and Komamine, 1980a), suggesting the presence of intercellular
communication mediated by chemical, if not physical, signals.
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| Figure 1.
Effects of the initial cell density on TE
differentiation. Single zinnia cells were suspended in liquid medium,
dispensed into 24-well culture plates at a final volume of 500 µL per
well, and incubated at 25°C with shaking at 120 rpm. The TE
differentiation frequency for each well was calculated by dividing the
number of TEs by the total number of living cells. Data are mean values
of three replicates ± SD.  , 1 × 105
cells/mL;  , 5 × 104 cells/mL;  , 2.5 × 104 cells/mL;  , 1.3 × 104 cells/mL;  ,
6.2 × 103 cells/mL;  , 3.1 × 103
cells/mL.
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Effects of PSK- on TE Differentiation
On the assumption that a chemical factor(s) other than auxin or
cytokinin is involved in TE differentiation, we investigated the
effects of PSK-, an endogenous peptide growth factor shown to
compensate for growth suppression observed at low cell density, on TE
differentiation of zinnia cells.
When the cells, cultured at a density of 2.5 × 104 cells mL1, were
treated with PSK- at a concentration range from 0.1 nM
to 1.0 µM, TE differentiation was strongly stimulated in
a dose-dependent manner (Fig. 2A). The
TE-formation-stimulating activity of PSK- was detected at
concentrations as low as 0.01 µM, where more than 35% of
the living cells differentiated into TEs by 5 d of culture in the
presence of PSK-. In contrast, the frequency of TE differentiation in the absence of PSK- remained at approximately 1% by 5 d,
indicating that PSK- compensates for the suppression of TE
differentiation observed at low cell densities.
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| Figure 2.
Effects of PSK- on TE differentiation (A) and
cell viability (B). Single zinnia cells were incubated in liquid medium
at a density of 2.5 × 104 cells mL1 in
the presence of various concentrations of PSK-. The TE
differentiation frequency for each well was calculated by dividing the
number of TEs by the total number of living cells, and cell viability
was calculated by dividing the number of living cells by the number of
total cells. Data are means of three replicates ± SD.
, 1.0 µM; , 0.1 µM; , 0.01 µM; , 1.0 nM; , 0.1 nM;
, control.
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Zinnia mesophyll cells have an absolute requirement for an auxin and a
cytokinin for TE differentiation (Fukuda and Komamine, 1980a). To
determine how these two phytohormones and PSK- are involved in the
stimulation of TE differentiation, we cultured mesophyll cells in media
that contained 1.0 µM PSK- and four combinations of
plant hormones: NAA and 6-BA, NAA only, 6-BA only, and no hormones.
Elimination of NAA and/or BA completely suppressed TE differentiation
(0%) even when the culture medium contained a sufficient amount of
PSK- for the stimulation. We conclude that PSK- requires both
auxin and cytokinin to stimulate TE differentiation of zinnia cells.
TE differentiation stimulated by PSK- is not a secondary effect
caused by inhibiting cell death, because cell viabilities were not
altered by changing the PSK- concentration (Fig. 2B). It is also
unlikely that PSK- promoted the differentiation by increasing a cell
division, because more than 80% of TEs differentiated directly from
the dispersed mesophyll cells without intervening cell division (Table
I; Fig. 3).
Direct TE differentiation without intervening cell division has been
reported in many studies of colchicine-treated (Fukuda and Komamine,
1980b) or 
-irradiated cells (Phillips, 1981; Sugiyama et al., 1986),
as well as after serial observation of single mesophyll cells (Fukuda
and Komamine, 1980b).
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Table I.
Percentages of TEs differentiated without
intervening cell division
Zinnia mesophyll cells were cultured at an initial density of 2.5 × 104 cells mL1 in media containing PSK-
at various concentrations. The TE differentiation frequency was
determined after 5 d of culture. Data are mean values of three
replicates ± SD.
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| Figure 3.
Micrographs of zinnia mesophyll cells cultured in
the presence or absence of PSK-. Single zinnia cells were incubated
for 5 d in medium containing PSK- at a concentration of 10 nM
(A) or in the absence of PSK- (B). Bar =100 µm.
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The fact that the minimum density required for zinnia cell division in
this medium was approximately 103 cells
mL1, which is far lower than that required for
TE differentiation, further supports the independence of
differentiation and proliferation. At a density of 2.5 × 104 cells mL1, more than
90% of the viable cells that had not differentiated into TEs had
divided by 5 d of culture regardless of the concentration of
PSK-. Thus, it may be concluded that PSK- stimulated TE
differentiation by recruiting more cells into the TE developmental
pathway without altering cell viability or cell division.
Identification of PSKs in Conditioned Medium Derived from
Zinnia Suspension Culture
Because PSK- stimulates TE differentiation of zinnia
mesophyll cells, we next investigated whether zinnia mesophyll cells
themselves produce PSK-. As a first step, we tested the effects of
crude conditioned medium on TE differentiation by adding aliquots to
bioassay media. As shown in Figure 4,
TE differentiation was stimulated (compared with control) by
conditioned medium; the maximum frequency was about 10% after 8 d
of the start of bioassay when the conditioned medium concentration was
12.5% (v/v), indicating that zinnia conditioned medium may contain
PSK- or PSK--like compounds. In contrast, the addition of
conditioned medium at a concentration above 12.5% resulted in
inhibition of TE differentiation (data not shown). Conditioned
medium may contain inhibitory factor(s) for TE differentiation as well
as stimulatory factor(s).
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| Figure 4.
Effects of conditioned medium on TE
differentiation. Single zinnia cells were incubated in liquid medium at
a density of 2.5 × 104 cells mL1 in the
presence of various concentrations of conditioned medium. Data are the
means of three replicates ± SD. , 12.5%; ,
3.2%; , 0.8%; , control.
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To determine if conditioned medium contains PSK-, we fractionated
zinnia conditioned medium by stepwise elution from a DEAE Sephadex
column. Fractions of 800 and 1,200 mM KCl were desalted on
a Sep-Pak column and on Bio-Gel P-2 by gel-permeation chromatography. Each eluted fraction (5.0 mL) was assayed by ELISA, and positive fractions (40-55 mL) were pooled and lyophilized. These fractions were
further purified by reverse-phase HPLC, with monitoring of UV
A220 (Fig.
5A). Fractions (1.0 mL) were collected
from 0 to 20 min and assayed by ELISA. Significant amounts of PSKs were detected in the 9.0 to 11.0 min and the 15.0 to 16.0 min fractions (Fig. 5B). A combined fraction of 9.0 to 11.0 min had a pseudomolecular ion of m/z 845 corresponding to
[M-H] and a fragment ion of
m/z 765 corresponding to
[M-H-80], as shown by MS, confirming that
this fraction contains PSK- (Fig. 5C). By comparing the retention
time of eluted peak and that of synthetic PSK- (data not shown),
a peak eluting at 10.2 min was determined to be PSK-. A similar
procedure showed that a peak eluting at 15.7 min was PSK-
, a
C-terminal truncated peptide of PSK-. A control experiment revealed
that the total recovery of PSK- by this purification procedure was
15%. Therefore, the total amount of PSKs contained in 400 mL of
conditioned medium was estimated to be 3.0 nmol equivalent to PSK-.
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| Figure 5.
Identification of PSKs in conditioned medium
derived from zinnia mesophyll cell culture. A, HPLC profile of purified
conditioned medium. Conditioned medium derived from TE-inductive
culture was separated by two steps of open-column chromatography and
reverse-phase HPLC. Fractions were collected every minute. B, Results
of competitive ELISA. The amount of PSKs contained in each fraction was
determined by competitive ELISA based on anti-PSK- antibodies. Peaks
eluting at 10.2 and 15.7 min were estimated to be PSK- and PSK-,
respectively. C, Comparison of mass spectrum of natural sample and
synthetic PSK-. A combined fraction (9.0-11.0 min) was concentrated
and analyzed by MS (1st row). A pseudomolecular ion of
m/z 845 corresponds to
[M-H] and a fragment ion of
m/z 765 corresponds to
[M-H-80] of PSK-, coinciding well with the spectrum
of synthetic PSK- (2nd row). A fraction (15.0-16.0 min) was
concentrated and analyzed by MS (3rd row). A pseudomolecular ion of
m/z 717 corresponds to
[M-H], and a fragment ion of
m/z 637 corresponds to
[M-H-80] of PSK-, coinciding well with the spectrum
of synthetic PSK- (4th row).
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We have shown that PSK- stimulates TE differentiation of dispersed
zinnia mesophyll cells without intervening cell division in the
presence of auxin and cytokinin. We also confirmed that zinnia cells in
culture produce considerable amounts of PSK-. Although this peptide
was originally isolated as a mitogenic factor from conditioned medium
derived from an asparagus mesophyll culture (Matsubayashi and Sakagami,
1996), current results indicate that PSK- stimulates cell
differentiation instead of cell division under the specified
conditions.
What is the fundamental function of PSK-? One possibility is that
PSK- makes cells receptive to signals that ultimately determine the cell destiny, i.e. cell division or cell differentiation. Research into PSK- receptors transmitting secondary messages that
activate a specific set of genes appears to be warranted.
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FOOTNOTES |
1
This research was supported by the Program for
Promotion of Basic Research Activities for Innovative Biosciences.
*
Corresponding author; e-mail matsu{at}agr.nagoya-u.ac.jp; fax
81-52-789-4118.
Received February 11, 1999;
accepted April 30, 1999.
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ABBREVIATIONS |
Abbreviations:
PSK, phytosulfokine.
TE, tracheary element.
TFA, trifluoroacetic acid.
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ACKNOWLEDGMENTS |
We thank Dr. Hiroo Fukuda and Hiroyasu Motose (Graduate
School of Sciences, University of Tokyo) for useful discussions.
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Abstract
Introduction
