Plant Physiol. (1 995) 108: 1647-1 656
End-Product Control of Carbon Metabolism in
Culture-Grown Sugar Beet Plants'
Molecular and Physiological Evidence on Accelerated Leaf Development
and Enhanced Gene Expression
Yelena Kovtun and jaleh Daie*
329 Birge Hall, 430 Lincoln Drive, University of Wisconsin, Madison, Wisconsin 53706-1 381
Excessive sugar accumulation in leaves (e.g. when sink
demand is lagging behind carbon supply) results in alterations in photosynthesis and carbohydrate partitioning in
leaves (Stitt and Quick, 1989; Stitt, 1990; Krapp et al., 1991;
Goldschmidt and Huber, 1992; Daie, 1993). This end-product control is similar to the role sugars (and other end
products for that matter) play in a wide range of microorganisms and mammalian systems in altering, both at the
biochemical and molecular levels, major metabolic pathways (Entain, 1980; Lenz and Holzer, 1980; Lee, 1987; ElMaghrabi et al., 1988). Although a relatively large body of
literature provides evidence concerning the physiological
and biochemical bases of the phenomenon in plants, only
recently has molecular evidence become available about
sugar-mediated expression of carbon fixation and/or metabolism genes (Sheen, 1990, 1994).
Depending on the experimental system, the tissue, and
the gene of interest, sugars have been shown to repress
(down-regulation) or induce (up-regulation) the expression
of different genes that encode key enzymes of carbon fixation and metabolism. For example, the Suc synthase gene
in the maize endosperm and the ADP-Glc pyrophosphorylase gene in potato are up-regulated by Suc (Chourey et al.,
1986; Salanoubat and Belliard, 1989; Takeda et al., 1994),
and severa1 photosynthetic genes as well as carbon metabolism genes encoding leaf enzymes are repressed in the
presence of Suc or Glc (Sheen, 1990; Krapp et al., 1991,1993;
Kossmann et al., 1992; J.S. Lee and J. Daie, unpublished
results). Presumably, sugar-responsive regulatory sequences (cis elements) are present at the 5' region of these
genes, as has been shown in the case of a number of
photosynthetic and carbon metabolism genes (Maas et al.,
1990; Sheen, 1990; Huang et al., 1993), as well as for genes
that are not involved in carbon metabolism (patatin gene,
Wenzler et al., 1989; chalcone-A gene, Tsukaya et al., 1991;
nitrate reductase gene, Cheng et al., 1992).
Accumulation of photosynthetic products in leaves was
suggested by Wang et al. (1993) to influence the pattern of
photosynthetic gene expression in C, amaranth plants. It is
therefore conceivable that the developmental and/or physiological stage of the leaf could play a role in how leaf
carbon balance might influence patterns of gene expression
in other plants. Several lines of evidence, in fact, support
this proposition. For example, although Glc repressed expression of the rbcS in mature leaves of transgenic tobacco,
it did not repress rbcS in immature leaves of those plants
that accumulated large quantities of Glc in the leaf resulting from the overexpression of a yeast-derived invertase
(Von Schaewen et al., 1990; Dickinson et al., 1991; Krapp et
al., 1993). Likewise, Brusslan and Tobin (1992) reported
' This work was supported partially by funds from a U.S. Department of Agriculture-National Research Initiative Competitive
Grants Program grant (92-37306-8331) to J.D.
* Corresponding author; fax 1-608-265-5482.
Abbreviations: chl-FBPase, chloroplastic Fru-1,6-bisphosphatase; cyt-FBPase, cytosolic Fru-1,6-bisphosphatase; vbcS and
rbcL, small and large subunits of Rubisco, respectively; SPS, Suc
phosphate synthase.
Sugar beet (Beta vulgaris L.) seedlings were grown on media
containing 90 t o 300 mM sucrose or glucose. Compared to controls,
sugar-grown plants had higher growth rate, photosynthesis, and leaf
sugar levels. The steady-state level of transcripts increased significantly for the small subunit of ribulose-1 ,s-bisphosphate carboxylase/oxygenase (Rubisco) (rbcS) and the cytosolic fructose-l,6bisphosphatase and moderately for the Rubisco large subunit (rbcl).
l h e transcript level of sucrose phosphate synthase remained unchanged. Fructose-1,6-bisphosphatase and Rubisco activities did
not change in the presence of sugars, but that of sucrose phosphate
synthase increased (44 and 90% under selective and nonselective
assay conditions, respectively). Accelerated leaf development was
indicated by (a) autoradiograms of leaves that showed that sucrose
loading occurred earlier, (b) export capacity that also occurred
earlier but, after about 2 weeks, differences were not detectable,
and (c) sucrose synthase activity that declined significantly. Several
conclusions emerged: (a) response was nonosmotic and gene and
sugar specific, (b) sugars caused accelerated leaf development and
sink-to-source transition, ( c ) enhanced gene expression was due t o
advanced leaf development, and (d) whereas Rubisco and cytosolic
fructose-l,6-bisphosphatase genes were sugar repressed in mature
leaves of greenhouse-grown plants, they were unaffected in mature,
culture-grown leaves. To our knowledge, these data provide the first
evidence in higher plants that, depending on the physiological/
developmental context of leaves, sugars lead to differential regulation of the same gene.
1647
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Kovtun and Daie
1648
that the cab-1 gene was not repressed in etiolated Arabidopsis seedlings (grown on Suc-containing media) despite
reports of its sugar repression in mature plants. Evidence
from other (nonplant) systems, however, is more definitive
and shows that, depending on the physiological and/or
metabolic context of the cell and developmental stage of
the system, molecular signals may serve to either up- or
down-regulate the same gene (Miesfeld et al., 1987; Sakai et
al., 1988; Diamond et al., 1990).
As part of our efforts to investigate the role of sugars in
metabolic repression of transcription in sugar beet leaves,
we developed a culture-grown sugar beet seedling system
that would allow better control of the variables. Surprisingly, but consistent with the conclusions of Diamond et al.
(1990), rather than the expected repression by sugars,
which we had observed in mature leaves of greenhousegrown sugar beets, enhanced expression of the same genes
occurred (in a highly gene-specific manner) in the culturegrown seedlings. In this paper we report molecular and
physiological evidence for sugar-induced accelerated leaf
development, sink-to-source transition, and enhanced expression of some carbon metabolism genes in the leaves of
culture-grown sugar beet plants. Moreover, we provide the
first evidence, to our knowledge, that genes that are sugar
repressed in mature leaves of greenhouse/soil-grown
plants are either enhanced (in developing leaves) or unaffected (in mature leaves) in culture-grown plants.
MATERIALS A N D METHODS
Plant Physiol. Vol. 108, 1995
Vein Accumulation of SUC and Development of
Loading Capacity
At least three plants were used for each treatment. Uniformly labeled ['4ClSuc was delivered through the transpiration stream under continuous light conditions (same as
growing conditions). A plant stem (with at least two leaf
pairs on it) was placed in a solution containing 10 mM
unlabeled SUC,5 mM Mes (pH 5.5), and 60 kBq mL-' of
['4C]Suc (specific activity, 671 mCi/mM). Uptake via the
transpiration stream was allowed to continue for 6 h. After
exposure to labeled SUC,leaves were cut and washed three
times in chilled water (removing most of the label in the
apoplastic space). For dark experiments, plants were
placed in a chamber in a dark room for 3 h before they were
placed in labeled Suc solution for another 6 h of uptake in
the dark. Autoradiograms were obtained by placing leaves
against x-ray films (Kodak Industrex M) for 10 to 14 d at
-80°C. To demonstrate the leaf ability to export SUC,a
solution containing 10 mM Suc spiked with 20 kBq ['4C]Suc
was applied to the leaf surface, which had been abraded
with Carborundum. Translocation of label out of the leaf
was allowed to occur in the light for 4 to 10 h, after which
excess radioactivity on the leaf surface was removed (by
washing the leaf three times, 5 min each, in chilled water).
Plants were dissected into various parts before they were
digested and bleached overnight in a 4:6 (v/v) perchloric
acid:hydrogen peroxide solution at 50°C (as described by
Pitcher and Daie, 1991). The label present in various plant
segments was counted as disintegrations per minute in a
scintillation counter.
Plant Materials
Sugar Feeding of Leaves
Sugar beet (Beta vulgaris L. cv mono-HyE4) seeds were
sterilized in 70% ethanol for 1 min and then were placed in
a 20% household bleach (Clorox) solution for 20 min. After
the seeds were washed three to four times in water, they
were placed in Petri dishes on filter paper that was wetted
with a basal culture medium (Sigma), which was the standard salts and additives of Murashige and Skoog (1962).
One week later, the seedlings were transferred to clear
plastic boxes (GA-7-Magenta box) to which was added 40
mL of the basal medium solidified with 0.8% agar alone
(control) or agar plus various concentrations of (90-300
mM) Glc or SUC.A set of plants was placed on osmotically
adjusted medium (with ethylene glycol to 300 mM) or
3-O-methyl Glc and monitored as osmotic controls. The
growth conditions were 14 h of light (high-pressure sodium lamps, 250 /LE m-' s-' at the plant level) and day/
night temperatures of 26/22"C. To determine age, emerging leaves (not longer than 5 mm) were dated. Unless
otherwise stated in a figure or table caption, leaf sets of the
same age (ranging from 10-14 d old within each treatment)
were sampled from 4-week-old plants. Leaves from each
treatment were pooled before they were frozen in liquid
nitrogen and stored at -80°C for further analysis. AI1
experiments were done as triplicates and repeated at least
twice. Each triplicate consisted of six to eight leaves
(pooled) obtained from three to four different plants.
These experiments were done to determine whether mature leaves from the culture-grown plants respond to sugars in a way similar to mature leaves from greenhouse/
soil-grown plants. Fully expanded mature leaves from
4-week-old culture-grown plants were harvested in the
middle of the light period (11 AM, maximum mRNA levels)
and were immediately placed in water, where the petioles
were cut again under water to avoid xylem embolism. The
petioles were then placed in sugar solutions (100 mM Glc or
mannitol as control) for 24 h under constant light. At least
five leaves (each from a different plant) were used for each
treatment. In the case of greenhouse-grown plants, discs
from fully expanded, mature leaves of 4-month-old plants
were placed for 24 h in 40 mM solutions (aerated) of mannitol (control), SUC,Glc, or Fru (data presented here are
from a separate set of experiments done entirely with
greenhouse-grown plants).
RNA lsolation and Blot Analysis
Total RNA was isolated as described by Chomczynski
and Sacchi (1987) and modified for sugar beet as described
by Harn et al. (1993). Briefly, 500 mg of frozen tissue were
ground in 5 mL of a solution containing 4 M guanidium
thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl,
and 0.1 M P-mercaptoethanol. Five milliliters of watersaturated phenol, 2 mL of 2 M sodium acetate, and 1 mL of
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Accelerated Leaf Development
ch1oroform:isoamyl alcohol (49:l) were sequentially added
to the homogenate, and the final suspension was centrifuged at l0,OOOg for 20 min. An equal volume of isopropano1 was added to the aqueous phase and RNA was precipitated at 10,OOOg for 20 min. Total RNA (15 p g ) was then
loaded onto each lane of a 1% agarose gel and fractionated
at 15 V overnight. The gel was then blotted onto a nylon
membrane (Hybond-N) soaked in 1OX SSC (3 M NaCl and
0.3 M sodium citrate, pH 7.0) and placed in a Vacugene
pump (Pharmacia). The membrane was baked at 80°C for
2 h and hybridized in a solution containing 0.25 mM
Na2HPO,*7H,O, pH 7.4, 1 mM EDTA, 1%casein, and 7%
SDS with a 32P-labeledcDNA for 20 h at 63°C. The cDNA
probes were sugar beet cyt-FBPase (Harn and Daie, 1992a),
a 0.52-kb insert from maize SPS (most conserved region
located at the middle of the sequence). The full-length SPS
cDNA was a gift from Calgene, Inc. (Davis, CA), the sequence of which was reported by Worrell et al. (1991); the
amaranth rbcL and rbcS by Berry et al. (1985); and the
radish rRNA by Delseny et al. (1983). Signal intensities
were quantified by densitometric scanning with a Laser
Scanning Densitometer (LKB, Uppsala, Sweden).
Leaf Photosynthesis and Chl Determination
A portable Li-Cor (Lincoln, NE) LI-6200 was used to
measure leaf photosynthesis rates. Chl was determined
after the tissue was homogenized in chilled 80% acetone.
The homogenate was centrifuged, and Chl content was
determined as described by Arnon (1949).
1649
desalted extracts were incubated for 10 min at 27°C. The
reaction was stopped by adding an equal volume of 30%
KOH, followed by boiling for 10 min. Suc was quantified
by the anthrone reaction method (Ashwell, 1957).
Suc synthase activity was assayed as described by More11
and Copeland (1985) with some modifications. The assay
mixture added to aliquots of the desalted extract contained
50 mM Mops-NaOH (pH 7.5), 15 mM Fru, 15 mM MgCl,,
and 15 mM uridine diphosphoglucose. The mixture was
incubated at 30°C for 30 min, after which the reaction was
stopped by adding an equal volume of 30% KOH. Rubisco
activity was measured by incorporation of I4C from sodium bicarbonate into acid-stable material as described by
Ghosh et al. (1989) and modified by Dreesmann et al.
(1994).Invertase activity was determined by the method of
Claussen et al. (1986).
Carbohydrate Analysis
Starch content was determined using the method described by Galtier et al. (1993).Frozen tissue was ground in
1 M HCIO,. After the sample was centrifuged, the pellet
was extracted in 80% acetone and resuspended in water.
The suspension was boiled for 60 min and incubated in 50
mM sodium acetate buffer (pH 4.6) containing 3 units of
amylase and 60 units of amyloglucosidae for another 60
min at 50°C. Glc was measured using a kit (Sigma). Suc was
quantitated by the anthron method (Ashwell, 1957).
RESULTS
Enzyme Activity
Expression of Some Genes Was Enhanced in
Sugar-Grown Plants
Frozen tissue was homogenized in cold extraction buffer
(50 mM Mops-NaOH [pH 7.5],1 mM EDTA, 15 mM MgCl,,
2.5 mM DTT, 0.1% Triton X-100, 2% polyvinylpolypyrolidone, 1 mM PMSF). After the sample was centrifuged at
l0,OOOg for 5 min, the supernatant was used to determine
enzyme activity and sugar content. Protein content was
determined by the method of Bradford (1976) using the
Bio-Rad protein assay kit and BSA as the standard.
Activities of the cyt-FBPase and chl-FBPase were assayed
according to the method of Kelly et al. (1982) as modified
by Harn and Daie (1992b). The production of Fru-6-P was
coupled to the reduction of NADP using phosphoglucose
isomerase and Glc-6-P dehydrogenase. For the cyt-FBPase,
the assay mixture contained 100 mM imidazole (pH 7.0), 5
mM MgCl,, 0.3 mM NADP, and 0.3 mM Fru-1,6-bisphosphate. The assay mixture for the chl-FBPase contained 100
mM Tris-HC1 (pH 8.8), 10 mM MgCl,, 0.6 mM Fru-1,6bisphosphate, 0.3 mM NADP, and 0.5 mM EDTA.
To determine SPS activity, crude extracts were desalted
on a Sephadex G-25 column equilibrated with extraction
buffer minus Triton X-100, polyvinylpolypyrolidone, and
PMSF. Activity was then determined as described by Huber et al. (1991). The "selective" (substrate-limiting) assay
mixture contained 6 mM Fru-6-P, 24 mM Glc-6-P, 20 mM
uridine diphosphoglucose, and 20 mM Pi. The "nonselective" (substrate-saturating) assay contained 20, 80, and 20
mM of the first three ingredients and no Pi. Aliquots of the
RNA blot analysis of rbcL, rbcS, cyt-FBPase, SPS, and 18s
rRNA are presented in Figure 1. The transcript level of 18s
rRNA remained unchanged (less than 10% variations) under a11 treatments, ruling out nonspecific effects on gene
expression. Steady-state transcript levels of rbcL, rbcS, and
cyt-FBPase increased in the presence of 90 mM Glc (20,40,
and 90% increase, respectively), 300 mM Glc (30, 62, and
118% increase, respectively), 90 mM (21, 48, and 92% increase, respectively) SUC,and 150 mM (37, 71, and 129%
increase, respectively). The response of the rbcL gene was
less dramatic than that of rbcS and cyt-FBPase genes. Sugars did not have any effect on SPS transcript level, suggesting the gene-specific nature of the response. A lack of
transcriptional control of SPS is consistent with strong
evidence indicating that SPS regulation and its coordination with photosynthesis occur mainly at the biochemical
level, i.e. fine control (Stitt and Quick, 1989) and coarse
control (posttranslational modification) of SPS protein (Huber and Huber, 1992).
For the genes that did respond, 300 mM Suc was not
optimal. Transcript levels were not as high as those in
plants grown at lower Suc concentrations (90 or 150 mM).
Concentration-dependent gene expression has been observed for other systems, such as potato SUCsynthase,
ADP-Glc pyrophosphorylase, and granule-bound starch
synthase genes (Maas et al., 1990; Muller-Rober et al., 1990;
Van der Steege et al., 1992).
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Kovtun and Dale
1650
c
0
Sucrose
Glucose
90
300
90
150
300
rbcl
rbcS
cyt-FBPase
SPS 4H H»
ISSrRNA
Figure 1. Total RNA blot analysis of genes encoding four carbon
metabolism enzymes (cyt-FBPase, rbcS, rbcl, and SPS). Samples
were taken from 4-week-old, culture-grown sugar beet seedlings.
Numbers at the top are sugar concentrations (in mM) in the agar
media. 18S rRNA was used as an internal control. C, Control (media
without sugar and osmoticum).
Furthermore, 3-O-methyl Glc, a nonmetabolized analog
of Glc, was ineffective in terms of altering the patterns of
gene expression (Fig. 2). Since 3-O-methyl Glc had little
effect on gene expression in our hands and by others
(Krapp et al., 1993; Jang and Sheen, 1994), it appeared that
some sugar metabolite (or only sugars that can be altered
by metabolism) would have served as the direct molecular
signal in the signal's transduction pathway. Jang and Sheen
(1994) recently showed that sugars that serve as substrate
for a hexose kinase are direct signals that mediate control
of transcription of photosynthetic genes (in that case repression). Based on their observations, they proposed a
role for hexose kinase as both a sensor and the transmitter
of the signal.
Ineffectiveness of 300 mM 3-O-methyl Glc (which can be
considered as an osmoticum) suggested the lack of an
osmotic effect on gene expression in the system. In addition, we know from our previous work (Harn and Daie,
1992b; Dreesmann et al., 1994) that the transcript levels of
cyt-FBPase, Rubisco (both genes), and SPS genes in sugar
beet remain unchanged under water-stress conditions
(moderate but long term, 4-5 d). Lack of osmotic effects on
sugar-responsive genes have also been concluded by several other research groups (Krapp et al., 1993; Sheen, 1994).
Nonetheless, initial experiments were done with osmoticum control (300 mM), which confirmed negligible effects
of the osmotic potential of the media.
Plants Grew Faster on Sugar-Containing Media
Plant Physiol. Vol. 108, 1995
sugar. Leaves of sugar-grown seedlings were darker green
and thicker than control plants. In general, a positive correlation existed between biomass production and sugar
concentration in the media; maximal shoot and root growth
(2- to 3-fold) occurred with either 300 mM Glc or 150 mM
Sue (optimal Sue concentration). Growth parameters of
plants grown on osmoticum only (osmotic controls) were
not significantly different from those in controls (without
osmoticum and sugar), again ruling out nonspecific osmotic effects. In addition, sugar-grown plants had 2- to
3-fold higher total protein and total RNA, Chl content, and
leaf photosynthesis rates, whereas osmotic control plants
did not (Table II).
Whereas internal Sue and Glc levels increased 1.5- to
3-fold, leaf starch did not change in response to sugar in the
media (Table III). Moreover, concentrations of both Sue and
Glc increased in the leaves irrespective of the carbon source
in the media. Increased leaf Glc content in the presence of
Sue is assumed to have been due to invertase activity
(which remained high in sugar-grown plants; see below).
Also, Glc-grown plants had higher Sue levels compared to
controls, suggesting either an increased capacity for de
novo Sue synthesis (confirmed by higher cyt-FBPase and
SPS activities; Table IV) or the possibility for conversion of
excess Glc to Sue. Collectively, these observations, along
with that of enhanced gene expression, prompted us to
further study and characterize the culture-grown system in
the context of leaf carbon metabolism, leaf development,
and sink-to-source transition.
Activity of Some Enzymes Was Higher in
Sugar-Grown Plants
The activity of Rubisco, cyt-FBPase, and chl-FBPase remained unchanged in the presence of sugars, but SPS
activity under both nonselective and selective assay conditions increased 44 and 90%, respectively (Table IV). On a
leaf area basis, the activities of all measured enzymes were
significantly greater in sugar-grown plants than in controls, which is a reflection of the overall increase in leaf
protein content (except SPS, which showed an increase in
specific activity as well). Noteworthy is the increase in the
MG
rbcS
Cyt-FBPase
Figure 2. Total RNA blot analysis of rfacS and cyt-FBPase genes.
Samples were taken from 4-week-old, culture-grown sugar beet seedlings. C, Control; S, 150 mM Sue; C, 300 mM Glc; and MG, 300 mM
3-O-methyl Glc.
Table I presents data on growth parameters of seedlings
grown for 4 weeks on media containing
various levels of
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Accelerated Leaf Development
1651
Table 1. Crowth parameters o f sugar beet plants grown on culture media for 4 weeks
Data are the means 2
SE
of three replicates. Numbers in parentheses are percentages of control.
Sugar
Biomass
Shoot
Root
49 t- 6 (100)
46 i 6 (100)
3.4 2 0.4 (100)
119 2 24 (243)
124 t- 18 (253)
110 2 23 (239)
114 t- 25 (250)
9.1 t 2.0 (268)
10.0 2 3.0 (288)
81 t 14(165)
130 t 15 (266)
75 t 14 (163)
119?20(261)
5.8 i 0.7 (171)
1 1 .O i 2.0 (332)
64 i 15 (131)
60 t- 15 (130)
4.4 2 0.8 (1 29)
mg dry wt
mM
Control
SUC
90
150
Clc
90
300
Osmotic control
300
SPS activity without any apparent increase in its transcript
(Fig. 1). Activation of SPS was possibly related to higher
levels of the SPS activator, Glc-6-P (as a result of higher leaf
portion at the bottom was still importing Suc (consistent
with sink status).
Sugar-induced differences in vein accumulation (and
presumably Suc loading) and leaf transition were not very
obvious in older (13 d old) leaves (Fig. 3A, right), suggesting that given sufficient time (presumably to build up their
interna1 machinery) leaves on sugar-free media eventually
caught up with those grown on sugar. Apparently, regardless of the presence of externa1 carbon, 2-week-old leaves
would have attained source status. Further evidence in
support of an advanced developmental stage came from
two additional observations: (a) the site of accumulation of
Suc in the veins of sugar-grown leaves (transitional and
source) was likely to have been in the phloem tissue, because when leaves grown on 300 mM Glc were treated with
labeled Suc in the dark (Fig. 38), substantial amounts of
label accumulated in their veins (entry into the transpiration stream would have been minimal in the dark, and
washing the leaf after uptake would have removed most of
the label from the apoplastic space, including xylem), and
(b) in the sugar-grown leaves, the activity of Suc synthase,
an enzyme of higher activity in sink than in source leaves
(Claussen et al., 1985; Nguyen-Quoc et al., 1990), was significantly lower than those in the control plants (Table V).
Although it is reasonable to assume that label was in the
phloem tissue, the presence in the sieve elements can be
ascertained only by microautoradiography.
hexose), and other posttranslational modifications of the
SPS protein.
Vein Accumulation and SUC Loading Was Advanced in
Sugar-Crown Plants
Autoradiograms of 7- and 13-d-old leaves (fourth and
third true leaves, respectively) that had been treated with
['4C]Suc in light or dark are shown in Figure 3, A and B,
respectively. When incubated with labeled SUC,7-d-old
control leaves behaved as typical sink leaves. (High sink
demand and label was located mostly in the mesophyll
cells instead of being in minor veins [Fig. 3A, left]. In
contrast, plants grown on either 150 mM Suc or 300 mM Glc
showed characteristics that are indicative of developmentally advanced leaves [Fig. 3A, left].) These characteristics
include a decline in leaf sink demand (weaker importer of
carbon), accumulation and/or loading of Suc in minor
veins, and directional (tip to bottom/basipetal) development of vein-loading capacity and leaf transition-progressive termination of import (Turgeon, 1989; Pitcher and
Daie, 1991). Note that the leaf grown on 300 mM Glc is a
relatively advanced transitional leaf approaching fullsource status, because whereas the tip had ceased to import
labeled Suc (termination of sink status at the tip), a small
Table II. Characteristics o f sugar beet plants grown on culture media for 4 weeks
Data are the means tSugar
SE
of three replicates. Numbers in parentheses are percentages of control.
Protein
Chl
Photosvnthesis
ymol m-z
pg cm-'
mM
Control
Total RNA
5~~
260 2 20 (100)
9 2 1 (100)
17 2 l ( l O 0 )
2.7 2 1 .O (100)
420 t- 40 (165)
500 2 30 (195)
15 t 3 (167)
18 2 2 (200)
32 t- 3 (190)
38 2 4 (234)
5.2 t- 0.5 (1 92)
6.4 i 0.7 (237)
330 t- 40 (128)
650 i 60 (252)
10 2 2 (111)
21 i 3 (233)
24 t- 2 (139)
51 t- 4 (300)
3.6 i 0.7 (1 33)
6.0 i 0.8 (222)
310 2 30 (121)
10 i 2 (111)
18 t- 2 (107)
2.8 2 1 .O (1 04)
SUC
90
150
Clc
90
300
Osmotic control
300
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
1652
Plant Physiol. Vol. 108, 1995
Kovtun and Daie
Table 111. Sugar content in leaves o f sugar beet plants grown on
culture media for 4 weeks
rbcS and cyt-FBPase Genes Were Not Sugar Repressed in
Detached Mature Leaves of Culture-Grown Plants
Data are the means 2 SE of three replicates. Numbers in parentheses are percentages of control.
When detached, mature leaves from culture-grown
plants were fed with Glc for 24 h, they behaved differently
than those from soil-grown plants. In these plants, no
significant repression of rbcS and cyt-FBPase genes was
observed (Fig. 5). We know, however, from our extensive
previous work with greenhouse-grown plants that rbcS and
cyt-FBPase are strongly repressed when mature leaves are
treated with sugars (Fig. 6; J.S. Lee and J. Daie, unpublished results). (Also see Cheng et al., 1992; Krapp et al.,
1993, for similar results on other carbon metabolism genes.)
Sunar
mM
Glc
Starch
SUC
nmol cm-'
mg cm-'
Control
suc
29 t 3 (1 00)
46 i 5 (1 00)
16.8 t 1.2 (1 00)
90
150
53 i 6 (1 86)
51 i 7 (180)
73 2 8 (1 57)
81 i 9 (176)
15.0 i 0.7 (90)
41 ? 6 (145)
90 2 21 (310)
66 2 8 (143)
122 2 15 (243)
Glc
90
300
15.2
?
0.8 (91)
DISCUSSION
At various stages of plant phenology, specific metabolic
processes must be satisfied and maintained. Sugars are a
prime source of carbon skeleton for a host of other important molecules, including ATP. If sugar levels increase at a
specific stage of development, selective changes in enzyme
activity and/or the expression of their corresponding
genes may occur. Under such conditions, key developmental processes may be altered or accelerated. For example,
Friend et al. (1984) showed that Brassica campestris flowered
earlier and more profusely if grown on culture media
containing 80 mM SUC.Earlier and enhanced flowering
occurred whether plants were maintained under autotrophic (light) or heterotrophic (dark) conditions. In our culture-grown system, the increase in transcript level, enzyme
activity, leaf photosynthesis, and other characteristics suggested that, in addition to serving as molecular signals,
sugars play important physiological roles in advancing leaf
transition and the development of export capacity.
In the present study changes in growth parameters, leaf
photosynthesis rates, biochemical characteristics, and transcript levels were well correlated. We have reported similar
developmental changes in the leaves of soil-grown sugar
beet plants (Harn et al., 1993). Moreover, the concurrent
increase in SPS activity and decline in Suc synthase activity
indicated that leaf transition in the culture-grown plants
was similar to that in severa1 other plants (Giaquinta, 1978;
Claussen et al., 1985; Walker and Huber, 1989; NguyenQuoc et al., 1990). Together, the data suggested that leaf
development and sink-to-source transition in the culture-
SUC Export Capacity and Sink-to-Source Transition
Was Also Accelerated
A key experiment to provide additional evidence for
advanced leaf development (transition from net importer
to net exporter) was to demonstrate whether sugar-grown
leaves would have developed the capacity to export
['4C]Suc out of leaves earlier than controls. To answer this
question, labeled Suc was applied to fourth true leaves
ranging in age from 5 to 11 d (after emergence), and the
arrival of label was monitored in the petiole and other sink
tissues, including younger leaves, stems, and roots (Fig. 4).
Regardless of the growing conditions, leaves younger than
7 d did not export substantial amounts of label. However,
7-d-old leaves grown on sugar exported nearly 50% of the
label, compared to about 10% by controls. The difference
between export capacity of controls and sugar-grown
leaves became smaller in older leaves so that 9-d-old leaves
of either plant type exported 35 to 50% of label out of the
leaf. There was no difference in export capacity of 11-d-old
leaves of either type (about 65%), indicating that control
leaves had reached comparable export capacity to that of
the sugar-grown leaves. The fact that the youngest and the
oldest leaves of both control and sugar-grown plants exported about equal amounts of labeled Suc further supported the assumption for different developmental stages,
at least during a certain period (leaves younger than 11 d
and older than 5 d).
Table IV. Enzyme activity in the leaves of sugar beet plants grown on culture media for 4 weeks
Data are the means i SE of three replicates. Numbers in parentheses are percentages of control.
Sugar
FBPase
Rubisco
SPS
chl
C\'t
nmol min-
f7lM
' mg-'
Nonselective
Selective
Activation
State
%
protein
Control
suc
324 2 25 (100)
44 t 5 (100)
46
6 (100)
16 t 3 (100)
5.8 2 0.7 (100)
36
90
150
318 i 32 (98)
327 t 36 (101)
48 i 6 (105)
43 2 4 (98)
48 i 6 (105)
47 ? 7 (102)
15 f 2 (95)
17 2 2 (105)
6.8 t 1 .O (1 17)
7.8 i 1.1 (1 35)
45
46
308 2 22 (95)
3 3 6 k 18 (103)
45 i 6 (104)
35 2 5 (89)-
48-? 5 (104)
41 ? 5 (89)
18 t 3 (113)
23 2 4 (144)
6.8
1 1 .o ? 2.0 (1 90)
38
48
?
Glc
90
300
- I - -
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Copyright © 1995 American Society of Plant Biologists. All rights reserved.
?
1 .O (1 17)
Accelerated Leaf Development
lit
t
Sucrose (ISO mM)
*
Glucose (300 mM)
Glucose (300 mM)
Figure 3. Autoradiograms of leaves from culture-grown sugar beet
seedlings grown for 4 weeks in the presence of 0 to 300 mM Sue or
Glc. Left and right panels are 7- and 13-d-old leaves, respectively.
Seedling stems were placed in 10 mM labeled Sue solutions for 6 h
under light conditions (A) or in the dark (B). Same-age leaves were
used as described in "Materials and Methods."
grown plants were fundamentally similar to processes that
occur in developing leaves of soil-grown plants.
Based on work with a culture-grown, albino tobacco
system, Turgeon (1989) concluded that preprogrammed,
developmental processes (i.e. leaf transition) override metabolic conditions in the leaves. However, in our culture-
1653
grown plants, higher leaf carbon balance was correlated
with an accelerated pace of sink-to-source transition, suggesting that metabolic conditions (elevated leaf sugar content) may override some aspects of leaf development. In
addition to the present data, other evidence suggests that
metabolic conditions can override some types of regulation
(e.g. light-dependent expression of rbcS [Sheen, 1990] and
chl-FBPase [Kossmann et al., 1992]). Chourey and Taliercio
(1994) recently concluded that metabolic regulatory controls override the normal controls of tissue and cell specificity for the expression of two Sue synthase genes in
maize.
Evidence obtained in our laboratory with 4-month-old,
greenhouse-grown sugar beet plants (Fig. 6; J.S. Lee and J.
Daie, unpublished results) indicates that when mature
leaves are exposed to sugar solutions some genes encoding
carbon metabolism enzymes are repressed significantly
and rapidly (as early as 4 h). In such sugar-feeding experiments, repression was most pronounced for rbcS and cytFBPase and moderate for rbcL, and SPS expression was not
affected. Those results were consistent with other work in
which sugar repression of various photosynthetic genes
was reported (Sheen, 1990; Cheng et al., 1992; Criqui et al.,
1992; Harter et al., 1993; Krapp et al., 1993). To our surprise,
results of this study with culture-grown plants revealed
that the expression of rbcS and cyt-FBPase genes was
strongly enhanced in sugar-grown plants. Furthermore,
rbcL expression was moderately enhanced and expression
of SPS gene was unaltered. Therefore, the sugar response
was gene specific, whether it was in the direction of repression or enhancement. It is interesting that there was also
consistency in the magnitude of the response in the two
systems. It is likely that the actual in situ (compartmental)
100
J
>,
IGlucose
CDControl
'
-28
70 ~ni
§ 60 S?
-24
8
o
50-
-20 b
40
-16
__^
^s
o
^
C/3
Data are the means ± SE of three replicates. Numbers in parentheses are percentages of control.
| 30-
Sugar
Sue Synthase
Invertase
-8
LJJ
100
§
.,.?
P
o
& 20 -
Table V. Enzyme activity in the leaves of sugar beet plants grown
on culture media for 4 weeks
40
4J
5
7
-4
0
9
11
Leaf age (days)
mM
Control
Sue
90
150
Glc
90
300
nmol mlrT ' mg~ ' protein
7.6 ± 1.1 (100)
6.2 ± 1.0(82)
4.6 ± 0.6 (60)
8.6 ± 1.3 (113)
2.5 ± 0.1 (33)
Figure 4. Export of labeled Sue out of leaves by 4-week-old, culturegrown sugar beet seedlings grown with or without 300 mM Glc in the
agar media. Labeled Sue (10 mM) was applied to same-age, abraded
22.4 ± 0.3 (55)
attached leaves. Translocation out of the leaf was allowed to con24.6 ± 0.9 (60)
tinue for up to 10 h under light conditions. After the plant was
washed, label was measured in all plant segments. Export was cal35.2 ± 2.2 (86)
culated from label in the petiole, stem, and roots as a percentage of
29.4 ± 1.3 (72)
Downloaded from on July 31, 2017the
- Published
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total labelby
taken
up by the leaf.
40.8 ± 3.8(100)
Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Kovtun and Dale
1654
Initial
24h feeding
rbcS
cyt-FBPase
Figure 5. Total RNA blot analysis of rbcS and cyt-FBPase genes.
Plants were grown with (C) or without (C) Glc in the media for
several weeks. Fully mature, exporting leaves ( 1 3 d old) from each
treatment were cut and either frozen immediately (Initial) or exposed
for 24 h to a solution that contained 100 HIM Glc (24 h feeding) under
constant light conditions.
Plant Physiol. Vol. 108, 1995
higher cab transcript levels in Arabidopsis seedlings when
Sue was included in the media. However, cab transcript
levels declined in the presence of sugar in mature plants
and in the protoplasts (transient expression system) (Sheen,
1990; Criqui et al., 1992; Krapp et al., 1993). These results
indicate that DNA sequences (both positive- and negativeresponse elements) are not the sole determinant of transcription regulation. In fact, protein-protein interactions
can influence the regulatory function by causing conformational changes in a DNA-binding protein. Under specific
physiological conditions, a differential effect of a molecular
signal (e.g. sugar) may be manifested in opposite directions
because of a different ratio between frans-acting factors,
which would then result in stimulation or repression of
gene expression.
The physiological and/or developmental context for the
response in the two mature leaf systems we had studied
(greenhouse-grown versus culture-grown plants) was
likely to have been very different. Therefore, one might
speculate that some sugar-responsive genes have regulatory sequences that exhibit dual functions (presumably in
conjunction with other factors). Such interpretations are
not, however, possible with our current data. Furthermore,
answering such questions is hindered: in addition to limited structural knowledge of sugar-responsive elements for
many carbon metabolism genes, it is unlikely that a universal sugar-responsive elements is responsible for a wide
spectrum of sugar-modulated gene expression in higher
plants.
Several conclusions emerged from the present data. First,
response to sugars was gene and signal specific (only metabolizable sugars were effective). Second, in the context of
end-product regulation, key enzymes of photosynthesis
and Sue synthesis are differentially regulated, which may
or may not include regulation at the transcription level.
Third, depending on the developmental and/or physiological stage of the leaf and/or the plant (sink versus source or
greenhouse versus culture-grown), sugars may exert different effects on the expression of the same gene. Fourth,
enhanced gene expression in the presence of sugars was
sugar concentrations are different and thus lead to differential expression of the same gene in a concentrationdependent manner as described by Salanoubat and Belliard
(1989) and Van der Steege et al. (1992).
The lack of sugar repression in mature leaves of culturegrown plants (Fig. 5) indicated that the well-documented
model of down-regulation of carbon metabolism genes by
sugars (Fig. 6; Sheen, 1994) was not operative in these
plants. If the sugar-induced regulation of transcription is,
in fact, missing in the sugar-grown plants, then enhanced
gene expression in these plants was most likely due to
advanced leaf development. Related observations have
been made in potato plants; Glc repressed the rbcS gene
only in mature but not in immature leaves (Krapp et al.,
1993), suggesting that sugar repression is operative in specific cell types and/or during specific developmental
stages. It remains to be unequivocally determined whether
these observations were a direct function of sugar concentrations (metabolic conditions/sugar signaling), related to
developmental programming, or both. Lest we overlook
100
key relevant phenomenon, reports of related observations
90
are offered in the following.
_
80
o
Depending on the developmental stage of the plant or
~
70
leaf, sugars can modify the level and production of transO
60
acting proteins that bind to distinct regulatory sequences
•f£
50
on genes encoding key enzymes, leading to the induction
40
or repression of gene expression. Some upstream regulatory sequences possess versatile regulation functions, in
H
30
that a single gene product can both repress and enhance
20
transcription of the same gene. For example, some tran10
scriptional regulators are known to stimulate or repress
0
expression of the same gene depending on the physiological conditions of the cell (Miesfeld et al., 1987; Sakai et al.,
Figure 6. Changes in the level of rbcS (solid bars) and cyt-FBPase
1988; Diamond et al., 1990). McKendree and Fed (1992)
(open bars) transcripts in mature leaves of 4-month-old, greenhousehave also shown that only under tissue culture conditions
grown plants. Leaf discs were incubated in solutions containing
was a G-box element of the Arabidopsis adh promoter
either 40 mM sugar or mannitol (control) for 24 h under constant light
conditions.
functionally important. Brusslan
and Tobin
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from(1992)
on Julyreported
31, 2017 - Published
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*
Copyright © 1995 American Society of Plant Biologists. All rights reserved.
Accelerated Leaf Development
likely t o have been due t o accelerated leaf development.
Collectively, t h e d a t a suggested that addition of a s u g a r t o
t h e culture m e d i a caused accelerated leaf growth, development, and transition from sink t o source. The observed
accelerated pace of growth and development was manifested a t physiological, biochemical, and molecular levels.
Together w i t h other data obtained in o u r laboratory and b y
other investigators (mostly f r o m nonplant systems), it is
suggested that, d e p e n d i n g on t h e physiological and / o r
developmental status of the plant, sugars may regulate
expression of t h e same gene i n different directions.
ACKNOWLEDCMENTS
We wish to thank Alexander Kovtun for technical assistance.
We also thank Professor Ray Evert (Botany Department, University of Wisconsin-Madison) and Professor Robert Turgeon (Section
of Plant Biology, Cornell University) for stimulating discussions
and helpful suggestions in the preparation of the manuscript.
Received March 17, 1995; accepted April 27, 1995.
Copyright Clearance Center: 0032-0889/95/108/1647/10.
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