Download Nutrient uptake and accumulation by sugarcane cell cultures in

Nutrient uptake and accumulation by sugarcane cell cultures in
relation to the growth cycle*
M. THOM,** A. MARETZKI,** E. KOMOR,*** and W.S. SAKAI****
** Hawaiian Sugar Planters' Association Experiment Station, PO Box 1057, Aiea,
Hawaii 96701, USA; ***University of Bayreuth, Universitatsstr. 30, D-8580 Bayreuth,
FRG; and **** University of Hawaii at Hilo, Hilo, Hawaii, USA
(Received 19 December 1980; in revised form 26 March 1981 )
Key words: suspension cultures, growth pattern, nutrient changes, cytoplasmic volume,
transport properties
Abstract. Growth characteristics and nutrient changes in medium and cells of batchgrown sugarcane cultures were investigated over a period of 14 days. Amino acids,
PO~- and K÷ were substantially removed from the medium during the first seven days
of culture; a strong preference for uptake of organic nitrogen over inorganic nitrogen
was observed. Sodium uptake increased during the time when K+ was becoming deficient
in the medium. The main anions taken up were SOl- and PO]-. Strong acidification
and a virtually total extraceilular hydrolysis of sucrose in the medium during the first
seven days of culture were also observed.
Tapering off of the rapid growth phase was accompanied by an increase of intracellular sucrose and a decrease of intracellular protein. As cells went from rapid growth
into stationary phase, cytoplasmic space of the cells decreased slightly in favor of vacuolar space. Overall cell volume stayed constant throughout the growth cycle, except
during a short period before onset of rapid growth.
Transport of the glucose analog 3-O-methyl glucose remained constant in terms of
Km value but the Vmax was slightly higher in rapidly growing ceils.
Introduction
Growth of plant cell suspensions in batch culture has been examined in
numerous species [4, 5, 7, 8, 10, 1 l, 13, 14, 18, 20, 21, 24, 26, 29], frequently to compare growth changes in relation to one or more metabolites.
In some instances, cells grown in batch cultures have been analyzed in terms
of weight, volume, or number, and both cells and the medium have been
analyzed for nutrient changes that accompany cell growth and expansion
[8, 20, 27]. Nutrient availability during the culture cycle may modify the
physiology and biochemistry of the cells. Furthermore, in batch culture,
growth may cease because an essential nutrient becomes exhausted from the
medium, although other environmental factors, such as pH, cell density,
accumulation of toxic factors, or rate of oxygenation also contribute to the
finite growth that typifies cells in batch culture.
Nutrient-related changes were of particular interest to us because for
some years this laboratory has been concerned with nutrient transport of
*Published with tile approval of the Director as paper no. 495 in the Journal Series of
the Experiment Station, Hawaiian Sugar Planters' Association.
3
Plant Cell Tissue Organ Culture 1.'3-14 (1981) 0167-6857/81/0011-0003 $02.25.~
© Martinus Ni]hoff/Dr IV. Junk Publishers, The Hague. Printed in the Netherlands.
organic molecules [17]. Properties of nutrient exchange between cells and
the culture medium or among cell compartments are altered during the
culture cycle, so that transport measured at any one time depends on the
composition of the medium and on the growth stage at which cells are
sampled [3, 9, 12].
In order to develop a guide for further physiological investigations, a
series of analyses of nutrient changes and measurements of physical cell
characteristics were undertaken in this investigation, using a sugarcane cell
line we have maintained in suspension culture for many years.
Materials and Methods
Suspension cultures. Sugarcane cell cultures were isolated from immature
stalk parenchyma tissue of cultivar H50-7209 in 1962 and have been growing
continuously in suspension cultures. Stock cultures have been maintained
by transferring 6 ml of cell suspension at 14-day intervals into 100ml fresh
Wlfite's basal salt medium [23] containing 0.1% yeast extract, 55 mMsucrose,
330/.tM arginine, and 9#M 2,4 dichlorophenoxyacetic acid. Cultures were
incubated at 25°C on a rotary shaker at 200 rpm in the dark.
DetermhTation of cell weight, volume, and number. Dry weights were taken
on lyophilized cells. Total cell volume was determined as the water-accessible
but dextran-excluding space [28]. Cell number was determined by a method
developed by G.M. Richards (unpublished results). An aliquot of cell suspension (0.1-0.5 ml packed cell volume) was washed with water and centrifuged for 5 rain at 800g. The supernatant was removed by aspiration and
cells were fixed in 4% formaldehyde-10% ethanol for 30min, followed
by an additional 30-min fixation period in 4% formaldehyde-20% ethanol.
The fixative was removed by centrifugation and aspiration and cells were
washed with water. Cupriethylenediamine was prepared by adjusting a
0.5M CuSO4 '5H20 solution to pH 7 with NaOH. The Cu(OH)2 precipitate
was washed with water and dissolved in 6.8ml of 99% ethylenediamine.
The deep blue cupriethylenediamine solution was diluted to 0.5M solution
with water. KI (5 mg/ml) was added to stabilize the solution, which can be
stored for three months. Cell aggregates were separated by incubating the
fixed cells in 5 ml of 0.5M cupriethylenediamine for 18 h (timing is critical).
The cell suspension was diluted and counted on a Sedgewick-Rafter counting
chamber. At least ten fields of 1 5 - 3 0 cells were counted with a 10x diameter
objective. Diameter of the microscope field was determined with a stage
micrometer and the volume was calculated using the formula, 2nr z x depth.
Determination of relative cytoplasmic and vacuolar size. Cells from 7- to
12-day-old cultures were fixed in 2.5% glutaraldehyde in 0.1M phosphate
buffer, pH 6.0, and postfixed in 1% osmium tetroxide in the same buffer.
Samples were dehydrated in an acetone series and embedded in Epon 812.
Sections approximately I/~m thick were mounted on glass slides, stained with
toluidine blue-O, and photographed. The photographs were printed on
8 x 10 inch paper. Micrographs of sectioned cells were divided into cytoplasm, nucleus, and vacuole and the relative size of the compartments was
determined from the weight of the cut-out paper area of each portion.
Approximately 200 cells of each age were measured.
Analysis of cell and medium composition. Total N was determined by a
Kjeldahl procedure [1]. Nitrate was determined by a Kjeldahl procedure
after reduction of NO3 to NH~ with Devarda's alloy. Ammonium nitrogen
was determined with Nessler's reagent. Potassium and Ca z÷ were determined
by flame photometry (Technicon Instruments Corp.), Mgz÷ and Na÷ by
atomic absorption (Perk.in-Elmer Corp.), PO 3- by reaction with molybdate, SO]- as BaSO4 [2], and C1- was measured with a chloridometer
(Buchler).
Amino acid composition of the medium was determined using a Technicon amino acid analyzer. Peptides were determined as an increase in amino
acids (analyzed by using a Technicon amino acid analyzer) after hydrolysis
of medium for 24h in 3NHC1. Total amino acids were determined with
a ninhydrin procedure [19]. Reducing sugars were determined by reduction
of alkaline copper sulfate [22]. Sucrose was determined by the anthrone
reaction after destruction of reducing sugars by strong alkali [30]. Protein
was determined according to the method of Lowry [15] after precipitation
with tfichloroacetic acid. Crystalline bovine serum albumin (Sigma Chemical
Co.) was used as a standard for protein.
Uptake measurements. Cells were transferred to White's basal salt medium
without sugars and incubated for 18 h. For uptake measurements, cells were
harvested, washed with water, and resuspended in fresh White's basal medium
without sugars. Uptake measurements were started by addition of 14C 3-Omethyl glucose. Samples were withdrawn at l-rain intervals for 4 rain, rapidly
filtered on paper discs, and washed with water. Cells were transferred to a
vial containing scintillation fluid consisting of PPO, POPOP, and 10% Biosolve BBS-3 (Beckman instrument Corp.) in toluene. Radioactivity was
determined by scintillation spectrometry with a counting efficiency of 65%.
Results
Cell growth
Growth of sugarcane cells in suspension undergoes a typical sigrnoidal growth
pattern, both in terms of cell weight and cell number (Fig. 1A). Cell volume
per cell more than doubled by the time cells had been in the fresh medium
L
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D A Y S IN CULTURE
Figure 1A and B. Growth patterns of sugarcane ceils in suspension cultures. (A) Cell
number and weight of sugarcane cell suspension during successive stages of the cell growth
cycle in terms of cells/ml (o) and in terms of dry weight/ml (o). See Materials and
Methods for details of procedure. (B) Cell volume of sugarcane cell suspensions in terms
of cen number (o) and weight (e). See Materials and Methods for details of procedure.
7
for 48 h. Over the same period,.a less dramatic increase (25%) in dry weight
per cell was noted (data not shown). Over the subsequent 48 h, cell volume
and weight per cell decreased to the original value and remained constant
thereafter (Fig. 1B).
Though overall cell size remained constant after the initial dramatic
changes, there were changes in the relative sizes of intracellular compartments
after the rapid growth phase (Table 1). Cytoplasmic space in sugarcane
cells was calculated using photographs of sectioned cells (Table 1). The
assumption was made that by using a large number of cells (approx. 200)
from which micrograph sections were randomly selected, sectioning would
be representative of relative compartment size. This method has been previously employed using a smaller number of cell sections [25]. Cytoplasm
occupied less of the cellular volume in 14-day-old tl~an in 7-day-old cultures
(24% vs 32%), a result to be expected if there is maturation of a large percentage of the older cell population during the culture cycle. However, even
in older cells, cytoplasm still occupied almost one-quarter of the ~~.al cellular
space. Since nuclear and cell sizes remained constant, vacuolar si:" increased
as a percentage of the total cell space in the aging cell population.
Nutrient changes in the medium and in the cells
The initial pH of the YE medium was 6.12. During the first four days of
culture, the pH dropped more than 1 pH unit to 4.94; there was, therefore,
an approximately 27-fold increase in I-I+ concentration of the medium during
this period. After day 4 of culture, the pH of the medium decreased at a
much slower rate.
Nitrogen concentrations in the nutrient medium and in the cells were
determined at two different stages during the growth cycle (Table 2). Of the
total N available, 23% was in the form of NO~-N, a small amount in the form
of NH~-N, and the remainder was organic N. Free amino acid-N constituted
approximately 34% of the total N supplied, whereas peptide-N accounted for
about 13%, thus representing a significant fraction of the total N in the yeast
extract preparation. No trichloroacetic acid precipitable protein was detected
in the medium.
Disappearance of most free amino acids from the medium was rapid
(Table 3) and the disappearance of arginine was particularly rapid. Arginine,
Table 1. Cytoplasmic, vacuolar, and nuclear sizesa in terms of percentage of total cell
volume in sugarcane cell suspensions
Day
Percent distribution of cell volume in cell suspensions
Age of culture
Vacuole
Nucleus
Cytoplasm
7
12
32
24
62
71
asizes were determined from photomicrographs.
6
5
8
Table 2. Concentration (mM)a of N components in the medium used for growth of
sugarcane cells and corresponding changes in the cells
Medium
Cells
Days of incubation
0
7
14
7
14
Total N
NH~-N
NO~-N
Free amino-N
'Peptide' anaino-N
12.86
0.24
2.93
4.34
1.65
4.29
0.27
2.50
0
NDb
2.14
0.30
0.36
0
ND
304.30
3.57
4.64
46.00
ND
162.90
3.21
1.79
18.00
ND
a Intracellular concentrations were based on total cell volume determined as the wateraccessible but dextran-excluding space.
bNot determined.
Table 3. Concentration (u.M) of individual amino acids in the growth medium during the
first six days after cell transfer of sugarcane cell suspensions
Aspartic acid
Threonine
Serine
Glutamic acid
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Amino acids in medium
Day 0
Day 1
Day 2
Day 3
Day 4
129.8
84.5
200.1
398.1
157.3
376.8
208.3
34.3
168.8
247.4
44.1
125.3
146.9
54.8
429.5
1.03.3
74.1
98.5
340.2
125.4
228.3
156.2
10.4
85.1
111.6
17.9
55.0
98.5
30.1
97.2
98.8
67.5
89.1
230.4
101.2
204.9
137.7
5.0
83.9
100.9
16.7
40.4
0
28.2
0
95.0
15.1
13.6
146.0
14.9
23.2
0
0
15.8
9.5
0
0
0
0
0
116.3
82.7
129.6
404.6
153.6
254.3
204.6
20.7
132.9
196.4
26.2
95.8
139.8
43.3
171.1
No amino acids detected on days 5 and 6.
initially present at a higher concentration than any of the other amino acids
(430/zM), was completely removed from the medium by day 3 after cell
transfer; and, in fact, approximately 60% of the available arginine was removed by the cells from the medium within one day. Most other amino acids
were not appreciably decreased by day 2, but thereafter, with the exception
of aspartic acid, disappeared from the medium at a rapid rate over the following two days, the period during which rapid cell division commenced. By
day 6, no amino acids could be detected in the medium. These changes in
the medium were reflected in the status of the endogenous amino acid
pool at various times during the 14-day culture period (Table 4). A tenfold
increase in free amino acids occurred during the first three days of culture,
followed by a rate of amino acid pool depletion that was almost as rapid over
the subsequent four days.
9
Table 4. Concentrations of extractable protein and free amino acids in sugarcane cells
during their growth in suspension culture
Days of
incubation
Protein
~g'(mg dry weight)- 1
Amino acids
mM a
1
3
7
10
14
17
26.58
73.15
43.02
35.13
17.34
15.22
134
198
47
27
18
NDb
a Intracellular concentrations were based on total cell volume determined as the wateraccessible but dextran-excluding space.
bNot determined.
Table 5. Concentration (mM)a of reducing sugars and sucrose in sugarcane cells and
medium during growth of suspension cultures
Days of
incubation
1
3
7
10
14
17
Reducing sugars
Sucrose
Cells
Medium
Cells
Medium
10.0
5.4
14.9
14.6
10.4
9.2
6.0
12.0
86.0
82.0
64.0
NDb
26.5
7.3
24.9
48.0
57.5
55.6
54.0
52.0
7.0
1.0
1.0
ND
a Intracellular concentrations were based on total cell volume determined as the wateraccessible but dextran-excluding space.
bNot determined.
The cellular protein concentration reached a maximum after cells had
been in the fresh culture medium for three days (Table 4), and therefore
correlated with the rapid uptake of amino acids immediately following
transfer to the new medium. Protein content dropped rapidly in cells cultured
for more than three days.
Removal of sugars from the medium by the cells was initially much less
spectacular than that of amino acids, in part at least because of the high
concentration of sucrose ( 5 5 m M ) present in the medium at time of inoculation. Sucrose disappeared rapidly from the medium after a four- to
five-day lag; its disappearance was accompanied by a corresponding conversion of sucrose to reducing sugars (Table 5). Cell wall invertase activity
is typically high in sugarcane cells and presumably accounted for the present
observations. While sucrose concentration in the medium dropped from
55rnM to 7.5 mM between days 2 and 8, reducing sugar concentration
increased from 7 mM at day 2 to a maximum value of 80 mM at day 8.
Since the initial equivalent hexose concentration in the medium was about
110mM, the concentration of hexoses actually removed from the medium
amounted to about 3 0 m M by day 8. Therefore, approximately 3 . 0 m m o l
10
exogenous hexoses per 100 ml suspension were accumulated or metabolized
by the ceils within the first eight days of culture. By day 10, no sucrose
remained in the medium and hexoses began to decrease after day 8 as sugar
utiliz::~tion by the cells continued.
E~,dogenous concentrations of sugars changed dramatically during the
first several days after the cells had been transferred into a fresh medium
(Table 5). Initial sucrose content was decreased by 50% within the first day
when compared with 14- or 17-day-old cells. Sugarcane cells cannot take
up sucrose (Komor et al., in preparation) and the level of reducing sugars
in the medium appeared to be insufficient to maintain a constant level of
endogenous reducing sugars in the metabolic pool until after the third day
in culture. Stored sucrose is likely to be broken down to supply the high
sugar demands which are reflected in the high respiration rates typical of
young cells [e.g., 20]. As cells underwent.further division and t h e population of older cells increased, a relatively constant level of internal reducing
sugars was attained. Cellular sucrose, on the other hand, tripled between
days 3 and 7, reaching a final concentration of 0.21 mg/mg dry weight of
cells, i.e., more than 20% of the cell dry weight.
Inorganic nutrients in the medium were determined 7 and 14 days after
transfer to a fresh medium (Table 6). Phosphate was completely exhausted
from the medium within seven days. The cells may have been depleted of
PO43-. The effects of PO4a- depletion on cell metabolism is not known. Low
PO,3- concentration in the medium of older cultures has been noted in other
cell cultures [e.g., 20]. All other inorganic nutrients appeared to be in excess.
The concentration of Na* had remained constant in the medium during the
first seven days. However, uptake of Na* between 7 and 14 days may indicate
a substitution of Na÷ for K ÷. The net effect of this substitution would be
charge equilibration when K ÷ concentration becomes low in the medium
even though the internal K ÷ concentration did not suggest depletion. The
decreased concentration of Mg 2÷ in the cells, in spite of its apparently adequate supply in the medium, may be a sign of depletion, or it may reflect
dilution of an excess. In spite of an apparent adequate supply of C1- in the
Table 6. Concentration (raM) a of ions in sugarcane cells and medium during growth
of suspension cultures
Medium
Cells
Days of incubation
0
7
14
7
14
PO~K÷
Mg~÷
Na÷
CISO~-
0.42
3.12
0.95
5.26
1.63
2.97
0
1.28
0.46
5.18
1.60
2.43
0
0.01
0.14
1.57
1.37
1.00
22.25
51.28
2.18
46.65
4.62
11.33
10.06
40.51
1.67
112.00
4.97
21.88
alntracellular concentrations were based on total cell volume determined as the wateraccessible but dextran-excluding space.
11
Table 7. Kinetics of 3-O-methyl glucose uptake during growth of sugarcane cell suspensions
No. of days
in culture
Krn
gM
Vmax
nmol'min'(mg dry wt)-1
5
7
9
12
14
17
22
17
22
10
0.286
0.500
0.286
0.182
0.154
medium, C1- content of the cells was low. Compared to the C1- concentration,
endogenous SO~- content was high and this anion doubled during the latter
part of the growth cycle when PO~- concentration had decreased by more
than 50%.
Sugar transport during growth cycle
A comparison was made of the rates at which 3-O-methyl glucose was transported by cultures of different ages (Table 7). Maximum velocity decreased
by 69% between days 7 and 14, whereas Krn values showed no such trend
with culture age. A change of uptake rate depending on cell age has also been
noted by Blackman and McDaniels [3].
Discussion
Cells grown in batch culture will sequentially deplete the medium of nutrients. In the medium used for the present investigation, a striking preferential
uptake of organic N over inorganic N was observed early during the cell
growth cycle. This is in agreement with a distinct preference for reduced N
in the form of NNg over NO3 by cultures of Ipomoea [26]. With the exception of aspartic and glutamic acids, amino acids were virtually depleted
from the medium within four days, a period during which the cells underwent
only one or two divisions. The particularly rapid disappearance of arginine
from the medium within two days agreed with prior findings that arginine
is rapidly taken up by sugarcane cells [16]. In addition, selectively rapid
uptake of several other amino acids was noted. Presumably the amino acids
were being used as precursors for N-containing polymers during this phase
of cell growth [7, 20]. Rose et al. [26] have shown that accumulation of N
during the early growth cycle stages of Ipomoea cells accounted for a much
higher percentage of the cell dry weight increments than it did during the
later stages.
Organic N in the commercial yeast extract preparation used for the medium
on which cells were grown consists of peptides as well as amino acids. An
additional 29% of the organic N in the yeast extract is contributed by unknown sources such as vitamins, purines, and pyrimidines. These and the
12
peptides may be unavailable to the cells and, if so, organic N could become
a limiting factor for optimum growth.
There is a doubling of cell volume over the first two days after transfer.
Particularly in view of the fact that the volume per cell remained quite
constant thereafter, this is a remarkably large change. The fact that this
transient pronounced change in cell size during this period has not been
noted previously in cell cultures may be due to the determination of packed
cell volume rather than the water-accessible but dextran-excluding space of
cells. The latter method eliminates the problem of variable interstitial space
in the cell culture due to the density of cell aggregation when packed total
cell volume is determined by sedimentation. Solute as well as water concentration must increase in the cells during this predivision phase. The decrease
in reducing sugars and sucrose concentrations are more than offset by very
large increases in soluble amino acids during the first 1 - 3 days. However,
amino acid concentrations alone cannot explain the large cell volume increases. Since fresh weight-dry weight ratios remain relatively constant,
it is likely that water concentration in the cells also increases significantly.
The period of initial solute changes is followed by changes of similar magnitude, but in the opposite direction in amino acid and carbohydrate pools.
Although the composition of White's inorganic salts supplement in the
medium used for sugarcane cell suspensions is considerably lower in concentrations of most nutrient ions than either Murashige-Skoog or B-5 media
[6], there is no conclusive evidence that cell growth is sustained for a longer
period of time in a richer medium (data not shown). Nevertheless, it is
important for transport studies to ensure that an adequate supply of ions
such as K ÷, which can exchange for other essential nutrients across the
membranes, is available.
Carbohydrates remained in ample supply throughout the culture period.
This appears to be the case for other plant cultures as well [11, 27]. Structural cell components take on greater significance during the later stages of
cell development [26]. Sugarcane cells accumulated a significantly high
concentration of sucrose during the maturation phase, in spite of the fact
that the ratio of cytoplasmic to vacuolar space remained relatively high.
Itis tempting to correlate sucrose concentration in these maturing cells with
increased accumulation of sucrose in parenchyma cells of the plant as the
tissue matures. However, the region of the sugarcane stalk where cytoplasm
occupies 24% of the total cellular space is very close to the apical dome and
this tissue has not been sufficiently characterized for composition of nutrients.
Our findings illustrate the importance of defining carefully the growth
stage of cells in batch culture when such cultures are used for kinetic studies
of nutrient uptake. Compositions of internal pools affect the velocity of
nutrient uptake and possibly also Krn values. Furthermore, our f'mdings
show that relative compartment sizes vary with cell age. In subsequent
13
r e p o r t s (in p r e p a r a t i o n ) , we will b e e v a l u a t i n g c o n c e n t r a t i o n s o f n u t r i e n t s
in cellular c o m p a r t m e n t s in r e l a t i o n to u p t a k e m e c h a n i s m s . F o r this p u r p o s e ,
a c c u r a t e i n f o r m a t i o n o n relative c o m p a r t m e n t sizes at t h e time u p t a k e
m e a s u r e m e n t s are m a d e b e c o m e s o f p a r a m o u n t i m p o r t a n c e .
Acknowledgments. This work was supported by a research grant to E. Komor by the
Deutsche Forschungsgemeinschaft, and by a grant from the Governor's Agriculture
Coordinating Committee, State of Hawaii, to A. Maretzki.
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