Plant Physiol. (1993)103: 1001-1008
Effect of Different Carbon Sources on Relative Growth Rate,
Interna1 Carbohydrates, and Mannitol 1-0xidoreductase
Activity in Celery Suspension Cultures'
Johan Marcel Henri Stoop and David Mason Pharr*
Department of Horticultural Science and Plant Physiology Program, North Carolina State University,
Raleigh, North Carolina 27695-7609
In species such as celery (Apium graveolens L. var dulce
[Mill.] Pers.), white-ash (Fraxinus americana L.), common lilac
(Syringa vulgaris L.), and European privet (Ligustrum vulgare
L.), mannitol is a major photosynthetic product that is translocated in the phloem and can accumulate to high concentrations in various tissues (Trip et al., 1963; Keller and Matile,
1989; Davis and Loescher, 1990; Loescher et al., 1992; Stoop
and Pharr, 1992). Although the physiological roles of mannitol in higher plants are still unclear, it has been proposed
that mannitol may confer beneficia1 traits. Celery has high
photosynthetic rates, as high as 38 fim01 C 0 2 m-' leaf s-' at
light saturation, and this has been attributed to the extra
chloroplastic reductant turnover in the synthesis of mannitol
(Rumpho et al., 1983; Fox et al., 1986). Celery is also highly
tolerant of salt in the root environment, which has been
attributed to the function of mannitol as a compatible solute
(Everard et al., 1992; Stoop and Pharr, 1993, 1994). Recent
experiments with tobacco that has been genetically engineered to synthesize mannitol through the introduction of
the enzyme NAD-dependent mannitol-1 -P dehydrogenase
from Esckerickia coli (Tarczynski et al., 1992) demonstrated
increased salt tolerance as a result of the transformation
(Tarczynski et al., 1993).
The pathway of mannitol biosynthesis in photosynthetic
celery leaves has been established (Rumpho et al., 1983;
Loescher et al., 1992). Mannitol is synthesized in the cytoplasm of leaf mesophyll cells from dihydroxyacetone-P that
is exported from the chloroplasts by the Pi translocator. The
unique enzyme in this biosynthetic pathway is M6PR, which
catalyzes the NADPH-dependent reduction of Man-6-P to
mannitol-1-P. The M6PR functions primarily in a synthetic
manner, with only very low rates of mannitol-1-P oxidation
observed (Loescher et al., 1992). M6PR is distinctly different
from mannitol-metabolizing enzymes in lower organisms, a11
of which can be characterized as 2-oxidoreductases, catalyzing the interconversion of Fru or Fru-6-P to mannitol or
mannitol-1-P (Martinez et al., 1963; Ueng et al., 1976; Niehaus and Dilts, 1982; Morton et al., 1985; Teschner et al.,
1990). Many of the mannitol dehydrogenases in lower organisms can function in both directions, e.g. mannitol oxidation
or Fru reduction, depending on the organism and growth
conditions (Lewis and Smith, 1967; Lewis, 1984).
Little information exists concerning mannitol catabolism in
higher plants. Mannitol utilization in vascular plants has been
shown to occur at low rates in suspension cultures of Monterey pine and carrot (Thompson et al., 1986), as well as at
higher rates in leaves of white ash, lilac, and celery (Trip et
al., 1964; Fellman and Loescher, 1987). However, it is not
known to what extent or by what enzymic route mannitol is
assimilated in sink tissues of higher plants. Recently, an
This work was supported in part by the North Carolina Agricultural Research Service, Raleigh, NC 27695.
* Corresponding author; fax 1-919-515-7747.
Abbreviations: MDH, mannitol 1-oxidoreductase; M6PR, mannose-6-P reductase; PCV, packed cell volume; PMI, phosphomannose isomerase; RGR, relative arowth rate.
Little information exists concerning the biochemical route of
mannitol catabolism i n higher plant cells. In this study, the role of
a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery
(Apium graveolens L. var dulce [Mill.] Pers.) were successfully
grown on nutrient media with either mannitol, mannose, or sucrose
as the sole carbon source. Cell cultures grown on any of the three
carbon sources did not differ in relative growth rate, as measured
by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had
high concentrations of sucrose early in the growth cycle and
contained a substantial hexose pool. Mannose-grown cells had a
high mannose concentration early in the cycle, which decreased
during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle.
Celery suspension cultures on all three carbon substrates contained
an NAD-dependent MDH. Throughout the growth cycle, M D H
activity was 2- to 4-fold higher in mannitol-grown cells compared
with sucrose- or mannose-grown cells, which did not contain
detectable levels of mannitol, indicating that M D H functions predominantly in an oxidative capacity in situ. The M D H activity
observed in celery cells was 3-fold higher than the minimum
amount required to account for the observed rate of mannitol
utilization from the media. Cultures transferred from mannitol t o
mannose underwent a decrease in M D H activity over a period of
days, and transfer from mannose to mannitol resulted in an increase i n M D H activity. These data provide strong evidence that
M D H plays an important role i n mannitol utilization in celery
suspension cultures.
1001
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1002
Stoop and Pharr
NAD-dependent MDH was discovered and partially purified
from celeriac root tips (Stoop and Pharr, 1992). The enzyme
was also presenf in celery sink tissue, where it catalyzes the
oxidation of mannitol to Man, which in turn can be phosphorylated to h4an-6-P, converted to Fru-6-P, and enter
central metabolism (Stoop and Pharr, 1994). Thus, higher
plants differ in both mannitol biosynthesis and mannitol
catabolism from lower organisms.
To further assess the potential physiological importance of
the recently discovered MDH in higher plants, the growth of
celery cells on different carbon sources was characterized.
Heterotrophic plant tissue cultures of apple (Chong and
Taper, 1974; Negm and Loescher, 1979)and cucumber (Gross
et al., 1981) have been useful in defining the routes of sorbitol
and raffinose saccharide catabolism, respectively. The objectives of this study were (a) to determine if and to what extent
mannitol, the substrate of the MDH-catalyzed reaction, and
Man, the mannitol oxidation product, could support growth
of celery cells in culture; and (b) to determine if mannitol
utilization was related to the MDH activity.
MATERIALS A N D METHODS
Seed and Plant Material
Celery seed (Apium graveolens L. var dulce [Mill.] Pers.,
breeding line CE201) obtained from DNA-Plant Technology
Corp. (Cinnaminson, NJ) was surface sterilized in an 8%
(v/v) Clorox (5.25% sodium hypochlorite) solution for 20
min, followed by three rinses in sterile, deionized water.
Seeds were aseptically transferred in sterile 7.5 X 7.5 X 10
cm polypropylene vessels (Magenta, Chicago) containing
half-strength Hoagland nutrient solution (Hoagland and Arnon, 1950) at pH 6.7, solidified with 2% (w/v) Bitek agar
(Difco Laboratories, Detroit, MI). The polypropylene vessels
were placed in an environmental chamber with 8/16 h day/
night at 25OC.
lnitiation and Maintenance of Celery Suspension Cultures
Celery suspension cultures were initiated from hypocotyls
of aseptically grown celery plants. Hypocotyls were removed
from plantlets 25 d after germination and were surface sterilized in 8% (v/v) Clorox with slight agitation for 20 min.
Hypocotyls were turned over while in Clorox solution after
10 min. This was followed by three rinses in sterile, deionized
water (3-5 min per rinse). Hypocotyls were transferred onto
sterile Petri dishes (100 X 15 mm) where bleached ends were
removed. Individual hypocotyls were cut into 5-mm segments and transferred into 250-mL wide-necked Erlenmeyer
flasks containing the induction medium of 50 mL of Murashige and Skoog basal medium (inorganic salts and vitamins,
Murashige and Skoog, 1962) supplemented with 2.25 PM 2,4
D, 1.8 mg/L of kinetin, and 90 mM SUC,and adjusted to pH
5.7 before autoclaving. Cultures were held at room temperature on a gyratory shaker at 100 rpm under constant 10 to
60 pmol m-2 s-' PAR. After 25 d, cells were transferred into
500-mL Erlenmeyer flasks containing the same induction
medium. Cells were subsequently transferred to induction
medium containing mannitol (180 mM), Man (180 mM), or
Suc (90 mM) as the exclusive carbon source; the cultures were
Plant Physiol. Vol. 103, 1993
subcultured every 20 d. Because cells can hydrolyze extemal
Suc to Fru and Glc rather quickly, a 90 mM Suc concentration
was chosen that represented a theoretical equivalent of 180
mM hexoses. Each treatment was replicated four times.
Growth Determination
Crowth curves were established by plotting PCV over days
after transfer. PCV measures the volume of cells in a given
culture volume by collecting the cells by centrifugation. Cell
cultures were grown for 19 to 20 d before being subcultured.
The volume of cells to be subcultured was determined sci that
the PCV at d O was close to 0.05 mL cell/mL culture. PCV
was measured at severa1 time intervals during the growth
cycle by aseptically sampling 5 or 10 mL of culture and
centrifuging it for 2 min at 1000 rpm in a table-top centrifuge
(IEC HN-SII, Needham, MA). PCV was measured and the
supernatant was decanted and kept for analysis of media
carbohydrates. Cells were rinsed by adding 1O:l (v/v) ddonized water, stirring the mixture, and centrifuging it at 1000
rpm for 2 min. The resulting supernatant was discardedl and
the cells were used for carbohydrate and enzyme analysis.
The RGR was determined by plotting the PCV over time,
resulting in an exponential growth curve of which the E'XPOnent represents the RGR ([mL cell d-'1 X mL-' cell).
Carbohydrate Analysis
Ethanolic extractions of cells from each of the samples
were used for measurements of SUC,hexose sugars, and
mannitol. Elapsed time between aseptic cell harvest and
ethanolic extraction varied between 30 min and 1 h. The
procedures for ethanolic extraction were followed as described by Hubbard et al. (1990). Carbohydrate conteiit of
the cells was determined on an HPLC system using a BC100 Ca2+column (Benson, Reno, NV) operated at 75OC with
deionized, degassed water as the Cls eluent. The column was
preceded by a Waters Bondapak Cls/Corasil guard and a set
of anion and cation de-ashing cartridges (Bio-Rad). Media
carbohydrates were analyzed on the same HPLC system.
When the concentration of media carbohydrates ( y axis) was
regressed against the volume of cells in the culture ( X axi~s),a
linear relationship was obtained, of which the slope (dy/dx)
represents the amount of carbohydrates used (mg) to produce
1 mL of cells. Slopes were statistically compared using the
estimates of slopes and their SE values obtained from the
analysis of variance model (SAS, Cary, NC).
Enzyme Extraction and Assays
Celery suspension cells were harvested as described above
and ground in a chilled mortar using a 1:4 (v/v) tissue:buffer
ratio. The extraction buffer contained 50 mM Mops (pH '7.5),
5 mM MgC12, 1 mM EDTA, 5 mM DTT, and 1%Triton X-100.
Homogenates were transferred to microcentrifuge tubes and
centrifuged at 10,OOOg for 1 min and desalted by centrifugal
filtration on a Sephadex G-25-50 column equilibrated with
50 m~ Mops-NaOH (pH 7.5), 5 mM MgC12, and 5 mM DTT
prior to assay for MDH activity. Samples were stored on ice.
MDH activity was assayed by monitoring the reduction of
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Copyright © 1993 American Society of Plant Biologists. All rights reserved.
Mannitol 1-0xidoreductase in Celery Suspension Cultures
NAD spectrophotometrically at 340 nm. Assays were conducted at room temperature (25OC) in a total volume of 1
mL. The reaction mixture contained 100 mM Bis-Tris propane
(pH 9.0), 2 mM NAD+, enzyme extract, and 150 mM Dmannitol. The reactions were initiated with mannitol. One
unit of activity was defined as the amount of enzyme that
catalyzes the reduction of 1 pmol NAD+/h. Initial rates of
oxidation were used to calculate MDH activity. Protein concentrations were determined by the method of Bradford
(1976) using BSA as a standard.
The product of the oxidation of mannitol catalyzed by
MDH from celery suspension cultures was purified and identified as described for celeriac root tips by Stoop and Pharr
(1992).
RESULTS
RGR
Celery cell-suspension cultures were successfully established and they grew on mannitol, Man, or SUCas the sole
carbon source. Regardless of the carbon source, cells grew
exponentially during the entire experimental period (Fig. 1).
Growth curves similar to that shown in Figure 1 were obtained during the 12-month period of this study. The RGR
of cells grown on mannitol or Man did not statistically differ
from the RGR of cells grown on SUC(Table I).
Media Depletion and Interna1 Carbohydrate
Concentrations
Cells grown on mannitol differed from Suc-grown cells in
that they depleted the external carbon source without converting it to hexose prior to uptake (Fig. 2A). Suc-grown cells
depleted SUCbut also hydrolyzed SUCto Fru and Glc, which
accumulated in the media through d 13 (Fig. 28). Thereafter,
external hexoses and SUCwere further depleted to support
cell growth. Man-grown cells responded similarly to mannitol-grown cells by depleting the external Man without converting it to other hexoses prior to uptake (Fig. 2C).
Cells grown on mannitol, Man, or SUCwere able to take
up external carbohydrate rather quickly, since intemal car-
LI o
C
2-
&I
mannitol
E 0.40 -O0 mannose
o
i V
V sucrose
v
-5
0.3W
>
=
0.20
O
al
o
0.10
Y
o
2
0.00
O
4
8
12
16
20
day after transfer
Figure 1. PCV (mL cell/mL culture) of a typical culture cycle of
celery suspension cells grown on mannitol, SUC, or Man as their
sole carbon source. Data represent mean SE of four replications.
[pcv = 0.05 x e(0.106xDAT). , ? = 0.98.1 (DAT, days after transfer.)
*
1003
Table 1. RCR [(mL cell d-').mL-' cell] of celery suspension cells
grown on three different carbon sources
Data represent mean -+ S E of five different growth cycles (four
replications per cycle). Means with the same letter are not significantly different at (Y = 0.05.
Carbon
Source
RCR
(ml cell d-').ml-' cell
Mannitol
O.1 1 O
Man
0.107 & 0.003a
0.102 f 0.006a
SUC
k
0.004a
bohydrate pools, determined 30 min to 1 h after transfer to
the new media, were high on d O compared with their levels
prior to cell transfer (Fig. 2, D-F). Cells from the end of the
previous cycle had carbohydrate levels similar to those at d
19 (Fig. 2, D-F). Celery suspension cells grown on different
carbon sources differed drastically in intemal soluble carbohydrate concentrations. The internal soluble carbohydrate
pool of mannitol-grown cells consisted almost entirely of
mannitol and was extremely low in hexose (Glc and Fru) and
in SUC(Fig. 2D). Man was present at about 5 - to 10-fold
lower concentration than Glc or Fru in mannitol-grown cells
(data not shown). Early in the growth cycle the internal
soluble carbohydrate pool of Suc-grown cells consisted
mainly of SUC(Fig. 2E). Toward the end of the growth cycle
SUCconcentrations dropped substantially, but a minimum
SUCpool remained (+ 2 mg/mL). Hexose levels peaked at d
5 but remained at least 2-fold lower than SUClevels, and Fru
was always higher in concentration than Glc (Fig. 2E). Mangrown cells had a high initial Man pool, which decreased
during the growth cycle, whereas their internal Suc leve1
remained relatively constant over the growth cycle (Fig. 2F).
The hexose concentration of Man-grown cells was higher
than that of mannitol-grown cells and lower than that of
Suc-grown cells. Neither SUC-nor Man-grown cells contained
detectable mannitol. In a11 cell cultures, internal carbohydrate
concentrations seemed to be related to media carbohydrate
concentrations as internal carbohydrate pools decreased with
decreasing external pools (Fig. 2).
When cells grown for severa1 months on Man, SUC,or
mannitol were transferred to a solely mannitol-containing
medium, the internal carbohydrate pool of cells from a11
treatments was similar to that of the mannitol-grown culture
depicted in Figure 2D. When cells adapted to growth on any
one of the three carbon sources were transferred to either
SUCor Man medium, internal carbohydrate pools were similar
to that of SUCor Man-grown cells depicted in Figure 2, E or
F, respectively. Therefore, the intemal carbohydrate pool of
cells grown in suspension culture is affected by the current
carbon source in the media much more strongly than by the
external carbon source used in previous growth cycles (data
not shown).
When cells adapted to growth on SUCor mannitol were
grown in a mixture of SUCand mannitol (50:50, w/w), no
significant difference in RGR or in the amount of carbohydrate used (mg) per volume of cells produced (mL) could be
observed when compared with cells grown solely on mannitol
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Stoop and Pharr
1004
Figure 2. Change:, in concentration of the me-
Plant Physiol. Vol. 103, 1993
Media Carbohydrates
lnternal Carbohydrates
dia carbohydrates of celery suspensioncultures
grown o n mannitol (A), SUC(B), or Man (C) as
the sole carbon source. Suc-grown cells hydrolyzed external Suc (V) to Fru (O) and Glc (A),
with (+) representing the sum of these external
sugars (B). lnternal mannitol (O),SUC(V), Man
(O),Clc (A),and Fru (O) concentrations of celery suspensioncultures grown on mannitol (D),
SUC(E), or Man ( F ) as the sole carbon source.
Mannitol was not detected in SUC- or Mangrown cells (E, F). IData represent mean 4 S E of
four replications.
6
$Y 4
m
o
a
22
2
E 4
O
m
O
E
O
32
28
24
20
m
16
20
I
T
Y
o
m
a
z2
a
4
O
32
12
& 4
“ 8
E
8
4
6
-3
E l6
g 12
m
4
-
E
O
O
4
16
20
O
4
O
4
8
12
16
day after transfer
20
O
4
8
12
8
12
16
20
8
12
16
day after transfer
20
I
5 28
E 24
.
2
2 20
O
g
16
12
E
8
E O4
or Suc (Table 11). However, early in the growth cycle (up to
d 8) mannitol- and Suc-adapted cells differed in the relative
amount of mannitol and sugar (sum of external SUC,Fru, and
Glc) depletion (Fig. 3). Mannitol-adapted cells depleted mannitol and SUCin about equal amounts starting immediately
after cell transfer (Fig. 3A). In contrast, the Suc-adapted cells
preferentially used Suc above mannitol, especially during the
first 4 d (Fig. 3B). When the amount of mannitol used (mg)
to produce 1 mL of cells and the amount of sugar used to
produce 1 mL of cells was determined by regression analysis
of mannitol or sugar concentration in the media versus cell
volume, using data up to d 8, a significant difference could
be observed between mannitol- and Suc-adapted cells (Table
11). Suc-adapted cells used more sugar and less mannitol
compared with the mannitol-adapted cells. After 8 d the two
cell lines did not differ significantly (data not shown), which
suggested that early in the growth cycle mannitol-adapted
cells had the capacity to utilize mannitol together with SUC,
Table II. RCR and carbohydrate used of celery suspension cells grown on mixed carbon sources
Data represent mean -t SE of six replications. Means with the same letters within the same column
,are not significantly different at cy = 0.07.
Carbon
Source
Prior to
Transfer
First 8 d
Carbon Source
during Crowth
SUC
SUC
Mannitol
Mannitol
Sugar Usedb
Mannitol
Used
d- ’
mg mL-’ cell
mg mL-’ cell
mg mL-’ cell
502
445
436
504
395 f 39a
243 k 27b
152 f 39b
243 k 29a
0,101 f 0.010a
0.105 f 0.004a
0.103 f 0.003a
0.106 f 0.003a
Mannitol + SUC
Mannitol Mannitol + Sue
a Carbohydrate = SUC+ Glc + Fru
SUC
RCR
Carbohydrate
Used”
+ mannitol.
k 32a
f 25a
f 23a
k 23a
Sugar = SUC+ Glc + Fru.
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Mannitol 1-0xidoreductase in Celery Suspension Cultures
1005
b= 0.32
I= 0.75
O
20
2
4
6
8
10 12 14 16
to
O
B sucrose adapted cells
20 40 60 80 100120140160180200
MDH (pmolMlask)
Figure 5. Relationship between the rate of mannitol consumption
and MDH activity of mannitol-grown celery suspension cultures.
Data represent values obtained from three different growth cycles.
The slope of the regression curve is represented by b; r represents
the correlation coefficient.
O
2
4 6 8 10 12 14 16
day after transfer
Figure 3. Depletion of mannitol and sugars (SUC+ Fru + Glc) from
the medium of mannitol-adapted cells (A) and Suc-adapted cells
(B) transferred to a medium containing both SUC and mannitol
(5050, w/w) at d O. Data represent mean f SE of four replications.
whereas Suc-adapted cells were more restricted to SUCutilization. Later in the growth cycle, Suc-adapted cells utilized
mannitol as well as SUC,indicating the possibility for a
requirement of an induction period to provide the mechanism
that enables those cells to utilize mannitol.
MDH
Celery suspension cultures grown on mannitol, Man, and
SUCcontained an NAD-dependent MDH. The product of the
oxidation of mannitol, catalyzed by the MDH, was shown to
be exclusively Man by protocols similar to those published
for the celeriac root enzyme (Stoop and Pharr, 1992). At any
time during the growth cycle, MDH activity was 2- to 3-fold
higher in mannitol-grown cells compared with Suc-grown
cells, whereas Man-grown cells had very low to undetectable
MDH activities (Fig. 4). Similar relationships were observed
for specific activities (rmol h-‘ mg-’ protein) of MDH (data
not shown). The MDH activity peaked 4 to 7 d after transfer
and decreased during the rest of the culture cycle (Fig. 4).
The question of whether the cells contained adequate MDH
activity to account for the observed rate of depletion of
mannitol from the culture medium was addressed, and data
relevant are shown in Figure 5. The rate of depletion of
mannitol from the media of individual flasks for discrete 4-d
time intervals over the culture cycle was plotted as a function
of the mean MDH activity during each interval (Fig. 5). The
regression analysis indicated that the activity of MDH was
positively correlated with the rate of mannitol depletion.
Furthermore, the slope of the regression indicated that cells
contained about a 3-fold excess of the minimum amount of
enzyme activity necessary to account for the observed rate of
mannitol depletion.
When mannitol-adapted cells were transferred to media
containing SUCor Man as the sole carbon source, MDH
activity of the developing culture was suppressed compared
with the MDH activity of cells grown continuously on mannitol (Fig. 6A). This suggests that the current carbon source
strongly influences the MDH activity of the cells. Man- or
Suc-grown cells transferred to mannitol media underwent an
increase in MDH activity over a period of severa1 days (Fig.
6, B and C). No major differences were observed when Mangrown cells were transferred to SUCmedia and vice versa.
DISCUSSION
O
4
8
12
16
day after transfer
20
Figure 4. MDH activity of celery suspension cultures grown on
mannitol (O),SUC(V),or Man (O) as the sole carbon source.
The results demonstrate that celery suspension cells are
capable of utilizing SUC,mannitol, or Man (the oxidation
product of mannitol) as the sole carbon source for growth.
Cells grown on mannitol or Man had growth rates similar to
cells grown on the more commonly used carbon source, SUC.
Celery cells are different from those of many species in that
mannitol and Man are rapidly taken up by the cells and
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1006
Stoop and Pharr
O
2
4
6
8 1 0 1 2
O
2
4
6
8 1 0 1 2
O
2
4
6
8 1 0 1 2
day after transfer
8,
-
o
Figure 6 . Effect of changing carbon sources on MDH activity.
Celery suspension cultures maintained in media with mannitol (A),
Man (B), or Suc (C) as the sole carbon source were transferred to
media containing mannitol (Mt), Man (Ms),or Suc (S). Data represent
mean ? SE of four replications.
utilized. Mannitol is widely used as an experimental osmoticum and is believed to be nearly metabolically inert because
it is very slowly taken up by a wide range of plant cells
(Thimann et al., 1960; Yuri, 1988; Nade1 et al., 1989). Although use of mannitol as an osmoticum may be valid in
short-term studies (hours), it is of questionable use for longterm studies (days:)(Thompson et al., 1986; Yuri, 1988).
Wolter and Skoog (1966) reported that yields of F. americana callus were similar when mannitol was substituted for
Suc in the growth medium. Sorbitol, a polyol that occurs
abundantly in species of the Rosaceae, has been shown to be
a good carbon substrate for cultures of Malus species and
Prunus persica (Chong and Taper, 1972) and was able to
support growth of 11 Rosaceae species (Coffin et al., 1976).
Therefore, it is probable that the ability of cultured cells to
grow on mannitol might be restricted to species that form
and translocate this polyol to sinks. In many plant species,
Man is readily phosphorylated but not further metabolized
Plant Physiol. Vol. 103, .I 993
due to the low activity of or the absence of PMI, a key
enzymc’ that allows Man to enter the pool of glycolytic
intermediates (Herold and Lewis, 1977). In these species,
Man feeding results in lowered endogenous Pi and decreased
ATP availability, which induces inhibitory effects on various
metabolic processes (Herold and Lewis, 1977). Celery differs
from these species in that it contains very high PMI activities
in source and sink tissues (Stoop and Pharr, 1994). A ‘high
PMI activity (162 pmol h-’ mL-’ cell) was also observed in
celery suspension cultures.
The composition of the internal carbohydrate pool of cdery
cells was strongly dependent on the carbon source supplied
in the media. Mannitol-grown cells contained high levels of
mannitol and extremely low levels of SUC,Man, and hexoses.
Suc-grown cells had high levels of Suc early in the growth
cycle and substantial hexose pools that peaked at mid-cycle.
Man-grown cells accumulated primarily Man and Suc and
contained low concentrations of Fru and Glc. This drastic
effect of exogenous carbon on internal carbohydrate pools
differs from cucumber (Gross et al., 1981) or sugarcane cell
cultures (Maretzki and Thom, 1978), where, regardless of
carbon source, cells predominantly accumulate SUC,Glc, and
Fru, except for Gal-grown cultures, which accumulate G,d in
addition to other soluble sugars (Maretzki and Thom, 1978).
Interna1 and media carbohydrate concentrations decreased in
a correlated manner throughout the culture cycle, indicating
that externa1 carbohydrate concentration might affect the
magnitude of the internal carbohydrate concentration.
Data presented here support the hypothesis that mannitol
utilization may occur by the NAD-dependent MDH activity.
The MDH activity observed in celery cells was 3-fold higher
than the minimum amount required to account for the observed rate of mannitol utilization from the media. SUC-and
Man-grown cells did not contain detectable levels of maiinitol, indicating that MDH functions predominantly in an
oxidative capacity in situ. Cells transferred from mannitol to
Man underwent a decrease in MDH activity over a period of
days, and transfer from Man to mannitol resulted in an
increase in MDH activity. This is, to some extent, analogous
to substrate induction and repression of mannitol-I-P dehydrogenase in bacteria (Martinez De Drets and Arias, 1970).
However, in bacteria the variation in enzyme activity in
response to substrates is much greater than in these celery
cells. The decrease in MDH activity of mannitol-adapted cells
grown on Man and the increase in MDH activity of Manadapted cells grown on mannitol suggest that the observed
MDH activity might be caused by a direct or indirect effect
of the current carbon source. Alternatively, these changes in
MDH activity could be from selection for specific biochemical
cell type from a heterogeneous mixture of cells differing in
their ability to metabolize mannitol.
MDH might represent the initial step of the enzymic rcute
of mannitol assimilation in sink tissues by converting mannitol to Man. Man can be readily phosphorylated and further
metabolized by PMI to Fru-6-P, which can enter central
metabolism. However, these data cannot answer the question
of whether an additional route of mannitol catabolism exists
in these cells. Such a question may be highly relevant when
considering sink metabolism, because multiple invertases, as
well as Suc synthases, exist simultaneously in sink tissuee of
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Mannitol 1-Oxidoreductase in Celery Suspension Cultures
Suc-translocating plants (Ho, 1988; Sung et al., 1988), and
a11 of these enzymes may operate simultaneously in some
sinks (Hubbard et al., 1989). Multiple a-galactosidases as
well as the Suc-degrading enzymes exist in the sinks of
raffinose saccharide-translocating plants (Pharr and Sox,
1984), whereas sorbitol dehydrogenase and sorbitol oxidase
have been reported in sinks of sorbitol-translocating plants
(Yamaki and Ishikawa, 1986; Moriguchi et al., 1990).
The ability of celery cells to grow on mannitol and its
oxidation product Man as the sole carbon source provides a
useful system to further study mannitol catabolism in vitro.
Furthermore, it may prove useful that Suc-grown cells do not
contain an internal mannitol pool whereas mannitol-grown
cells do contain an internal pool of mannitol. This characteristic is currently being used to study the role of internal
mannitol in salt tolerance at the cellular level.
ACKNOWLEDCMENTS
The authors wish to thank Mara Massel for her technical assistance
and Dr. Frank Blazich for the use of his tissue culture facilities.
Received June 22, 1993; accepted August 3, 1993.
Copyright Clearance Center: 0032-0889/93/lO3/l001/08.
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