Plant Cell Physiol. 41(3): 268-273 (2000)
JSPP © 2000
Purification from Conditioned Medium and Chemical Identification of a
Factor That Inhibits Somatic Embryogenesis in Carrot
Toshihiro Kobayashi1'4' s , Katsumi Higashi 1 , Kazuo Sasaki2, Tadao Asami 3 , Shigeo Yoshida3 and
Hiroshi Kamada'
1
2
3
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572 Japan
Department of Bioscience and Biotechnology, Faculty of Engineering, Aomori University, Aomori, 030-0943 Japan
Plant Functions Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, 351-0106 Japan
Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells when the cell density is high. The inhibition is caused by factors that are
released by cells into the medium of such cultures. In this
study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of
carrot cells. The inhibitory factor with the strongest apparent activity was purified by fractionation with ethylacetate, chromatography on an octadecylsilyl (ODS) silica
gel-column and HPLC. The inhibitory factor had a single
peak of absorbance at 280 nm and was identified as 4hydroxybenzyl alcohol by mass spectrometry and 1H- and
13
C-NMR spectroscopy. Authentic 4-hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos at
a concentration equal to that in high-cell-density cultures.
These results suggest that 4-hydroxybenzyl alcohol is a
major factor that accumulates in high-cell-density cultures
of carrot cells and inhibits somatic embryogenesis.
Key words: Daucus carota — Inhibitory conditioning factor — 4-hydroxybenzyl alcohol — Somatic embryogenesis.
Many environmental and chemical factors influence
the induction and development of carrot somatic embryos
(Halperin 1967, Ammirato and Steward 1971, Kamada and
Harada 1979, Hari 1980, LoSchiavo et al. 1986, de Vries
et al. 1988a, b). Auxins are extremely important in this
context, in particular, 2,4-D. When carrot explants are
cultured on 2,4-D-containing medium for several weeks,
embryogenic cells are generated and the transfer of these
embryogenic cells to auxin-free medium results in the formation of somatic embryos (Steward et al. 1958, Reinert
1959, Kamada and Harada 1979).
Abbreviations: DW, distilled water; HCM, high-cell-density
conditioned medium; MS medium, Murashige and Skoog's medium; ODS, octadecylsilyl silica gel; PCV, packed cell volume
after centrifugation at 100 x g; TLC, thin-layer chromatography.
4
Present address: Plant Biotechnology Institute, Ibaraki Agricultural Center, Ago 3165-1, Iwama, Nishi-Ibaraki, Ibaraki, 3190292 Japan.
5
Corresponding author: e-mail, [email protected];
fax, +81 299 45 8351.
268
Cell density is also an important factor in the formation of carrot somatic embryos (Fridborg et al. 1978, Sung
and Okimoto 1981, 1983, Osuga et al. 1993). When embryogenic cells are cultured in auxin-free medium at a high
cell density, the formation of carrot somatic embryos is
strongly inhibited. Such inhibition appears to be due to
some factor(s) that is released by cells into the culture medium (Higashi et al. 1998). Conditioning factors that are
released into the culture medium stimulate the proliferation of cells in tissue culture systems derived from various
plants, including carrot (Halperin 1967, Bellincampi and
Morpurgo 1987, 1989, Huang et al. 1990, Vesseire et al.
1994). Somatic embryogenesis in carrot is known to be
promoted and stimulated by a variety of conditioning factors, such as arabinogalactan proteins (Kreuger and van
Horst 1993, Toonen et al. 1997), extracellular glycoproteins (de Vries et al. 1988a, b, Cordewener et al. 1991, de
Jong et al. 1992) and phytosulfokine-a (Kobayashi et al.
1999a). However, to our knowledge, inhibitory factors in
the culture medium of high-cell-density cultures of carrot
cells have not been isolated and characterized.
We demonstrated previously that the inhibitory factors in the medium of high-cell-density cultures of carrot
cells have molecular weights below 3,500 (Higashi et al.
1998). These factors inhibit somatic embryogenesis specifically by suppressing only the rapid division of cells that is
characteristic of the early globular stage (Kobayashi et al.
1999b). Such inhibitory factors have not previously been
described, even though several natural and synthetic compounds have been shown to inhibit somatic embryogenesis
(LoSchiavo et al. 1986, Baldan et al. 1995, Capitano et al.
1997, Toonen et al. 1997). In this study, we isolated an
inhibitory factor from conditioned medium and determined its chemical structure.
Materials and Methods
Plant materials and culture conditions—Details of the methods used for the culture of embryogenic cells of Daucus carota L.
cv. US-Harumakigosun have been described previously (Kamada
and Harada 1979, 1984). Embryogenic cells obtained from hypocotyls were subcultured at two-week intervals in liquid Murashige
and Skoog's (MS) medium (Murashige and Skoog 1962) that
contained 1 mg liter ! of 2,4-D. For induction of somatic embryogenesis, small clusters of cells (37-63 //m in diameter) were
Inhibitor of somatic embryogenesis in carrot
collected by passage of two-week-old suspension cultures through
stainless-steel sieves (pore size, 37 and 63 /urn). The clusters were
washed with an excess of phytohormone-free MS medium and
then suspended in phytohormone-free liquid MS medium at a low
cell density (0.2 ml PCV liter '). Cell density was denned in terms
of the packed cell volume (PCV) in ml after centrifugation at
lOOxg of one liter of culture medium (ml PCV liter ~l).
Details of the preparation of conditioned cell-free medium
have been described previously (Higashi et al. 1998). Conditioned
medium was prepared by passing two-week-old high-cell-density
cultures (5.0 ml PCV liter l) through a filter (GF/F; Whatman,
Maidstone, England). The resultant conditioned cell-free medium
was designated high-cell-density conditioned medium (HCM).
All cultures were incubated on a gyratory shaker (70 rpm) at
25°C in darkness.
Fractionation of HCM with ethylacetate—HCM (100 ml) was
subjected to fractionation with an equal volume of ethylacetate
and this procedure was repeated three times. pH of HCM was
approximately 4.5. In a control experiment, MS medium was used
instead of HCM. The aqueous and ethylacetate fractions were
evaporated to dryness in vacuo and residues were dissolved in 100
ml of distilled water. Each solution was added to an equal volume
of 2,4-D-free double-strength MS medium. The pH of the resultant medium was adjusted to 5.7 and the medium was sterilized by
passage through a cellulose acetate membrane with 0.45 /urn pores
(DISMIC-25cs; Advantec, Tokyo). Small clusters (37-63 /urn in
diameter) of embryogenic cells, collected as described above, were
cultured in the medium at an initial cell density of 0.2 ml PCV
liter"1. The number of somatic embryos in each culture was determined on the 14th d of culture.
Purification of inhibitory factors by column chromatography—The ethylacetate fraction prepared from 100 ml of HCM
was evaporated to dryness in vacuo. The residue was dissolved in
1 ml of 10% (v/v) ethanol and subjected to chromatography on a
column (10 mm inner diameter, x 15 cm) of octadecylsilyl (ODS)
silica gel (Silica Gel ODS-Q3; Wako, Osaka, Japan). The inhibitory factors were eluted with 50 ml of 10%, 20% and 40% (v/v)
ethanol and then with 150 ml of 100% methanol. Successive 5-ml
fractions were collected and lyophilized. Each residue was dissolved in 500 [il of distilled water. To determine how to combine
the fractions for bioassays, an aliquot of each fraction was spotted on a silica gel plate (60 F254, Art. 5715; Merck & Co., Inc.,
Germany) with a mixture of isopropyl alcohol and ethylacetate
(4 : 6, v/v) as the mobile phase. The pattern of spots upon irradiation with UV light was examined. According to the pattern
of spots in each fraction, fractions were combined to give nine
pooled fractions as follows; 0-50 ml of 10% ethanol, 0-15 ml and
15-50 ml of 20% ethanol, 0-20 ml and 20-50 ml of 40% ethanol,
0-25 ml, 25-50 ml, 50-100 ml and 100-150 ml of 100% methanol.
Then, each pooled fraction was added to 100 ml of MS medium.
Small clusters of embryogenic cells were then added to medium
containing each sample at 0.2 ml PCV liter"1. Somatic embryos
were counted 14 d later.
Purification of an inhibitory factor by HPLC—The fraction
eluted in 20% ethanol from the ODS column (starting volume of
HCM, 100 ml) was lyophilized. The residue was dissolved in 100
jul of 10% (v/v) methanol and the resultant solution was subjected
to HPLC on a column (TSK gel-ODS80TM CTR; 4.6 mm innerdiameter x 10 cm; Tosoh Co. Ltd., Tokyo) with isocratic elution
in 10% (v/v) methanol at a flow rate of O.Smlmin" 1 . The absorbance of the eluate was monitored with a photodiode-array
detector (991J; Waters Associated, Milford, MS, U.S.A.). Successive 0.8-ml fractions were collected and lyophilized. Each resi-
269
due was then added to 100 ml of MS medium. Small clusters of
embryogenic cells were added to each sample of medium at 0.2 ml
PCV liter x and somatic embryos were counted 14 d later.
Mass spectrometry and NMR spectroscopy—The inhibitory
fraction after ODS-HPLC was collected and 1.5 mg of a purified
compound was obtained after lyophilization. The electron impact
mass spectrum of the purified compound was obtained at 70
eV with a JEOL DX303 mass spectrometer in a direct mode.
'H-NMR (300 MHz) and 13C-NMR (75 MHz) spectra were recorded on a Brucker AC-300 Plus spectrometer in a methanold4 solution. Chemical shifts were recorded as 5 ppm relative to
tetramethylsilane as an internal standard.
Effect of 4-hydroxybenzyl alcohol on somatic embryogenesis—4-Hydroxybenzyl alcohol (extra pure grade) was obtained
from Nacalai tesque, Inc. (Kyoto, Japan). It was dissolved in distilled water and the solution was sterilized by filtration (DISMIC25cs; Advantec) and added to MS medium at various concentrations. Small clusters of embryogenic cells were added to the
medium at 0.2 ml PCV liter"1 and somatic embryos were counted
14 d later.
In all experiments, cultures were incubated in 50-ml flasks
that contained 15 ml of test medium, with four flasks for each
experiment. Somatic embryos were counted in 500-//1 aliquots of
culture in a counting chamber two times and averages of results
are shown. Standard deviation value in each experiment was calculated against the total number of somatic embryos.
Results and Discussion
Inhibitory factors were purified from the conditioned
medium prepared from high-cell-density cultures, which
was designated HCM. HCM contained nutrients and sucrose that were components of the original MS medium.
High concentrations of such nutrients and sucrose inhibit
somatic embryogenesis and, therefore, HCM was subjected
to fractionation with ethylacetate.The ethylacetate fraction
and the aqueous fraction were added separately to the induction medium for somatic embryogenesis, and formation of somatic embryos was strongly inhibited by addition
of the former, but not of the latter fraction (Fig. lc). In a
control experiment, the aqueous or the ethylacetate fraction of MS medium had a weak inhibitory effect (Fig. lb).
This weak inhibition might be due to residual ethylacetate.
However, the inhibitory effect of ethylacetate fraction of
HCM was much stronger than that in other fractions.
The ethylacetate fraction of HCM was fractionated by
chromatography on an ODS-column. Inhibitory factors
were eluted stepwise with 10%, 20% and 40% ethanol and
finally with 100% methanol. According to the spot pattern
of these fractions by TLC analysis (data not shown), successive 5-ml fractions were collected and pooled as nine
fractions; 0-50 ml of 10% ethanol, 0-15 ml and 15-50 ml
of 20% ethanol, 0-20 ml and 20-50 ml of 40% ethanol,
0-25 ml, 25-50 ml, 50-100 ml and 100-150 ml of 100%
methanol. Formation of somatic embryos was inhibited by
the fractions eluted in 20% ethanol and in 100% methanol
(Fig. 2). This suggested that at least two compounds were
acting as inhibitors. The inhibitory effect of the fraction
Inhibitor of somatic embryogenesis in carrot
270
120
3* 100
O JO
I!
_T
80
60
CD ®
.> o
«
ss
CD E
DC O
to
40
20
0
DWMS
A
E
A
E
C
1 0 % 2 0 % 40%
Ethanol
100%
Methanol
Fig. 1 Effect of fractionated HCM on the formation of somatic
embryos. HCM was fractionated with ethylacetate. The aqueous
and ethylacetate fractions evaporated to dryness in vacuo. Each
residue was dissolved in distilled water and added to an equal
volume of double-strength MS medium. These media were tested
for the formation of somatic embryos, a: distilled water (DW) and
MS medium (MS) supplemented with double-strength MS medium, b and c: Aqueous (A) and ethylacetate (E) fractions of MS
medium (b) and of HCM (c). Closed columns, globular embryos;
striped columns, heart-shaped embryos; open columns, torpedoshaped embryos. The number of somatic embryos is given relative
to the number in DW supplemented with double-strength MS
medium, which was 100%. Results represent means with standard
deviations (n=4).
Fig. 2 Effect of fractions of ODS-column chromatography on
the formation of somatic embryos. The ethylacetate fraction prepared from HCM was subjected to chromatography on an ODScolumn. Inhibitory factors were eluted with 50 ml of 10%, 20%
and 40% ethanol and with 150 ml of 100% methanol. Successive
5-ml fractions were collected and pooled to give nine fractions as
follows; 0-50 ml of 10% ethanol, 0-15 ml and 15-50 ml of 20%
ethanol, 0-20 ml and 20-50 ml of 40% ethanol, 0-25 ml, 25-50
ml, 50-100 ml and 100-150 ml of 100% methanol. These fractions
were added to MS medium and tested for the formation of somatic embryos. Concentrations were adjusted to be equal to those
in the initial HCM. C, MS medium. Closed columns, globular
embryos; striped columns, heart-shaped embryos; open columns,
torpedo-shaped embryos. The number of somatic embryos is
given relative to the number in MS medium, which was 100%.
Results represent means with standard deviations (n=4).
eluted in 20% ethanol was stronger than that of the fraction eluted in 100% methanol.
The material eluted in 20% ethanol fraction was subjected to HPLC on an ODS column. The profile of UV
absorbance of the eluate, as detected by the photodiodearray detector, is shown in Figure 3a. Successive 0.8-ml
fractions were collected as indicated. The inhibitory effect
was associated with fractions 8 and 9 (Fig. 3b). The formation of somatic embryos was not affected by addition of
fractions after 12min (data not shown). These fractions
contained a major component with a single peak of absorbance at 280 nm (Fig. 3a, arrow).
We used about 30 liters of HCM to purify the inhibitory factor by HPLC on an ODS column. The yield was 1.5
mg and the factor was analyzed. The factor had an m/z of
124 (Fig. 4), a result that is consistent with our previous
observation that the inhibitory factors had molecular
weights below 3,500 (Higashi et al. 1998). The high-resolution mass spectrum (m/z 124.0504) corresponded to
C7H8O2 (124.0524). 1H- and 13C-NMR spectra of the factor
are shown in Figures 5a and 5b, respectively. The 'H-NMR
spectral data were as follows: <57.20 (2H, d, 7=8.5 Hz, 2H), 6.78 (2H, d, 7=8.5 Hz, 3-H) and 4.51 (2H, s, 7-H).
The 13C-NMR spectral data were as follows: <5157.98 (4-C),
133.56 (1-C), 129.86 (2-C), 116.12 (3-C) and 65.16 (7-C).
These results suggested that the purified factor was 4-hydroxybenzyl alcohol. The mass spectrum and ! H- and
13
C-NMR spectra of authentic 4-hydroxybenzyl alcohol
were identical to those of the purified factor (data not
shown).
4-Hydroxybenzyl alcohol has been found in seedlings
of muskmelon (Cucurbita moschata), bulbs of soldier orchid {Orchis militaris) and roots of another orchid, Galeola
faberi Rolfe (Hardegger et al. 1963, Li et al. 1993a, b).
However, little is known about the physiological functions
of 4-hydroxybenzyl alcohol. Therefore, we examined the
effects of 4-hydroxybenzyl alcohol on somatic embryogenesis in carrot. When authentic 4-hydroxybenzyl alcohol
was added to the induction medium for somatic embryogenesis at 10~5 M, the formation of somatic embryos was
strongly inhibited (Fig. 6). Slight inhibition was observed at
10"8 M and the number of somatic embryos decreased with
Inhibitor of somatic embryogenesis in carrot
5
Retention time (min)
10
15
20
271
25
,124
ioo95
123,
77
50-
,51
0-
i.l..
65
.ll,
ll
l
80
.Mil
1
100
m/z
Fig. 4 Mass spectrum of the purified inhibitory factor. See text
for details.
1 2 3 4 5 6 7 8 9101112
Fraction number
C1
2 3 4 5 6 7 8 9
Fraction number
as a result of the absence of some other inhibitory factors
that were present in the HCM. Our results suggest that 4-
3
2
5
6
10 11 12
-OH
Fig. 3 Effect of fractions of HPLC on the formation of somatic
embryos. The active fraction recovered from the ODS column
(eluate in 20% ethanol) was subjected to HPLC as described in
the text, a: The UV absorbance of the eluate as detected by the
photodiodearray detector. Successive 0.8-ml of fractions were
collected as indicated. The arrow indicates the peak of UV absorbance that corresponded to the inhibitory activity, b: The
fractions were added to MS medium and tested for their effects on
the formation of somatic embryos. Concentrations were adjusted
to be equal to those in the original HCM. C, MS medium. Closed
columns, globular embryos; striped columns, heart-shaped embryos; open columns, torpedo-shaped embryos. The number of
somatic embryos is given relative to the number in MS medium,
which was 100%. Results represent means with standard deviations (n=4).
increases in the concentration of 4-hydroxybenzyl alcohol.
We obtained 1.5 mg of purified 4-hydroxybenzyl alcohol
from 30 liters of HCM. When authentic 4-hydroxybenzyl
alcohol was dissolved in MS medium, the total recovery of
4-hydroxybenzyl alcohol was more than 80% after three
steps of purification. Thus, the concentration of 4-hydroxybenzyl alcohol in HCM was estimated to be at least 4.0 x
10"7 M. 4-Hydroxybenzyl alcohol at a concentration equal
to that in HCM significantly inhibited the formation of
somatic embryos. The inhibitory effect at this concentration was slightly weaker than that of HCM itself, perhaps
T
PPM
200
150
100
PPM
50
Fig. 5 'H-NMR (a) and 13C-NMR (b) spectra of the purified inhibitory factor. Arrowheads indicate signals due to the purified
factor. Other signals were due to solvent.
272
Inhibitor of somatic embryogenesis in carrot
ever, inhibition appears to be stronger than stimulation
since we observed strong inhibition in the presence of
HCM. In an attempt to clarify the mechanism of inhibition
of somatic embryogenesis in high-cell-density cultures, we
are now analyzing details of the physiological effects of 4hydroxybenzyl alcohol on somatic and zygotic embryogenesis in carrot.
This work was supported in part by Grants-in-Aid for Special
Research on Priority Areas from the Ministry of Education,
Science, Culture and Sports, Japan, by the Program for Promotion of Basic Research Activities for Innovative Biosciences, and
by the Special Coordination Fund of the Science and Technology
Agency, Japan.
o id9io8id7io6io5id4
Concentration (M)
Fig. 6 Effects of authentic 4-hydroxybenzyl alcohol on the formation of somatic embryos. Small clusters of embryogenic cells
were cultured at 0.2 ml PCV liter"1 in the medium that contained
authentic 4-hydroxybenzyl alcohol at various concentrations. The
number of somatic embryos was determined on the 14th day of
culture. Closed columns, globular embryos; striped columns,
heart-shaped embryos; open columns, torpedo-shaped embryos.
The number of somatic embryos is given relative to the number
without addition of 4-hydroxybenzyl alcohol, which was 100%.
Results represent means with standard deviations (n=4).
hydroxybenzyl alcohol is a major factor that accumulates
in high-cell-density cultures of carrot cells and inhibits somatic embryogenesis.
Several phytohormones and chemicals suppress somatic embryogenesis in carrot (Fridborg et al. 1978, LoSchiavo et al. 1986, Baldan et al. 1995, Capitano et al.
1997). The existence and concentrations of inhibitors in
conditioned medium have not been clarified, except in
the case of 4-hydroxybenzoic acid. Fridborg et al. (1978)
reported that 4-hydroxybenzoic acid accumulates in the
medium during carrot somatic embryogenesis and inhibits
the formation of somatic embryos. However, we did not
detect 4-hydroxybenzoic acid in HCM or in our inhibitory
fractions; and, authentic 4-hydroxybenzoic acid had a
much smaller inhibitory effect on somatic embryogenesis
than 4-hydroxybenzyl alcohol (data not shown). Thus, 4hydroxybenzoic acid was not essential for inhibition of
somatic embryogenesis in our high-cell-density cultures.
Some factors that stimulate the formation of carrot
somatic embryos have been recognized in carrot conditioned medium (Higashi et al. 1998, Kobayashi et al.
1999a, Matsubayashi et al. personal communication).
Thus, somatic embryogenesis from carrot cells appears to
be influenced by the balance between levels of stimulatory
and inhibitory factors in the conditioned medium. How-
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(Received September 27, 1999; Accepted December 14, 1999)