Sugar uptake and starch consumption
in Brassica rapa
Surya Bhupatiraju, Zaroug Jaleel, and Whitney Hagins*
Department of Science, AP Biology
Lexington High School, 251 Waltham Street, Lexington, MA 02421
*Correspondence: [email protected]
Abstract
Plants have numerous mechanisms for obtaining
sugar; normally, in the absence of sunlight, plants
will use starch granules for ATP production. This
experiment was performed to test how plants take
up sugars from their environment without sunlight.
It was hypothesized that plants, when deprived of
sunlight, and provided with no sugar, would rely
solely on their starch granules. However, when
provided with sugar, the plants should use sugar
uptake mechanisms rather than their reserves.
When the plants were provided with light, it was
hypothesized that plants would photosynthesize
normally, neither relying on starch nor outside
sugar. To test this discs were cut out from 7-9 day
old Brassica rapa plants. The discs were then placed
in solutions that had different molarities of sucrose
or glucose and half the solutions were subjected
to constant light while the other half were placed
in zero light. Starch amounts were measured using
an IKI solution and a color scale. The data showed
that sugar uptake increased linearly with molarity.
These results also clearly suggest that the plants
must have taken up the required sugars from
their environment to undergo cellular respiration
for ATP synthesis. On the other hand, the discs
that were placed in the light were able to undergo
photosynthesis and as a result, these plants were
able to acquire the necessary sugars. Therefore,
the results remain consistent with the hypothesis.
Introduction
Autotrophic organisms in the Plantae kingdom produce
their own food by using sunlight as their energy source.
However, what happens if this source of light is cut
off ? Where do the plants turn to in order to satiate their
daily food requirements? All plants and animals have
stores of starch, which consist of numerous glucose
molecules glycosidically linked together to form chains
of monosaccharides used to make polysaccharides.
In plants, this polysaccharide is called starch. When
glucose molecules are linked with  - 1, 4 linkages,
the resulting linear molecule is called amylose. When
glucose molecules are linked with  - 1, 6 linkages,
this highly branched molecule is called amylopectin.
Starch consists of approximately 20-25% amylose, and
approximately 75-80% amylopectin. These complex
polysaccharides contain glucose and sucrose, which
are the prime subjects of this experiment. By isolating
plants in different environments, this experiment aims
to investigate how plants use the starch reserves in their
cells versus free glucose or sucrose in their immediate
environment.
Two of the main processes involved in energy
production in plants are photosynthesis and cellular
respiration. Photosynthesis is the process that plants
use to obtain sugars by using CO2, H2O, and light energy
(photons). There are two parts to photosynthesis: the
light reactions and the Calvin cycle (dark reactions). In
the “light reactions”, energy from incoming photons
is used to reduce an electron accepter called NADP+
into NADPH. In the process, a pair of electrons and
a hydrogen nucleus (H+) is added. Water is also split
in the process, which is the source of the atmospheric
O2 that is released. The light reactions also produce a
small of ATP by phosphorylation. In the Calvin cycle,
the sugars are produced. First, carbon dioxide is fixed
from the plants’ environments, where the NADPH
and ATP produced in the light reactions can reduce
CO2 to carbohydrates. Note how this process creates
little ATP for the rest of the cell to use. In addition,
the result of the Calvin-Benson cycle is not glucose
but G3P (glycerate 3-phosphate). Approximately 5
out of 6 of these intermediate sugars are recycled into
the Calvin-Benson cycle to produce RuBP (ribulose 1,
5-bisphosphate), while the rest are condensed to hexose
phosphates. These sugars can later become glucose,
which is further processed to become energy for the
organism.
During cellular respiration, glucose that was either
produced by photosynthesis or ingested is converted
into ATP that the cell uses to carry out various cellular
processes. Cellular respiration actually consists of
three parts: glycolysis, the Krebs cycle, and oxidative
phosphorylation. In short, both glycolysis and the
Krebs cycle reduce NAD+ to NADH, which are used
as electron carriers in oxidative phosphorylation. With
substrate-level phosphorylation, these two processes
Bhupatiraju, Jaleel, and Hagins
also produce small amounts of ATP. In addition, the
Krebs cycle also produces CO2, which is a by-product of
cellular respiration. Finally, in oxidative phosphorylation,
the electrons from NADH are put through the electron
transport chain, creating a proton gradient between the
inter-membrane space and the mitochondrial matrix.
By chemiosmosis, (H+) molecules pass through ATP
synthase to produce ATP. For every glucose molecule
that is “processed” by the cell, approximately 36-38
ATP molecules are produced from both substrate-level
and oxidative phosphorylation.
Plants carry out both of these processes, and
therefore is an excellent model organism to examine
these reactions. The plants that were used for this
experiment were Wisconsin Fast PlantsTM. Specifically,
the Rapid-Cycling Brassica rapa species was used, a
common plant genetically modified to complete their
life cycle within 21 days. This made it possible to sample
leaves almost a week after planting them as there would
be enough leaf tissue available for experimentation.
This project seeks to discover whether small
leaf discs will use free glucose from either their
environment or their starch reserves under “light” or
“dark” conditions. One way to induce this selective
uptake is to sever leaf tissue from the plant itself. This
was done by punching out holes from leaves using a
straw, and putting leaves into separate groups. Each
group had different concentrations of sucrose and
glucose in this case. Additionally, some were in the dark,
while others were under continuous light. These discs
were kept in their respective environments for about
48 hours, at which point, they would first be boiled in
alcohol and water (to eliminate chlorophyll), and then
tested for starch content, by being dipped in an iodinepotassium-iodide (IKI) solution. The IKI solution then
stains the amylopectin and amylose, which can be used
to determine the relative abundance of starch.
The topic of sugar uptake was studied in 1981
by David A. Reinhart and Robert J. Thomas, both of
whom worked on an experiment involving the uptake
of 14C-sucrose into plants. For their experiment, they
treated mature leaves of the plant Polytrichum commune
with 14C-sucrose. By using radioactive tracers in the
sugar, they were able to trace the locations to which the
sucrose was going; they called these depositories “sinks”.
Their research showed that sucrose accumulated in
the growing stem apex, young leaves, bud initials, and
underground axes. Reinhart’s and Thomas’ project deals
more with where the sucrose goes rather than what
happened to starch granules in the chloroplasts; our
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project has a greater emphasis on the starch content in the
plants, and its relationship to sucrose and glucose uptake.
In another experiment, done in 1983, Robert A. Saftner
and Roger E. Wyse probed the relationship between plant
hormones and the amount of sugar uptake by sugar beet
root tissue discs. Using several different plant hormones,
either alone or in various combinations, a variety of results
were discovered. Of particular interest is the fact that none
of these hormones or any combinations of them had any
notable effect on the passive mechanism of sucrose uptake.
Saftner, Jaleh Daie, and Wyse performed another
experiment in 1982 that has even more relevance to our
project. They explored the possibility of active versus
passive transport of sucrose in plant cells. They found
that the active sucrose uptake showed a dependence
on external sucrose; when the external concentration
reached 20 M sucrose, the active transport mechanism
appeared to approach saturation. With passive transport,
the sucrose uptake rate in unplasmolyzed tissue showed
a linear dependence with external sucrose concentration.
Something interesting to note is that both of Wyse’s projects
involve sugar beet root and they used discs cut out from the
roots. It is also interesting to note that these projects were
all done with sucrose, and not any other sugar. In 1978,
Roger Wyse independently worked on a project where he
dealt strictly with the uptake rates of sucrose, fructose, and
glucose, and how two or more of the sugars could inhibit
the uptake rates of other sugars. He found that while
fructose and glucose displayed typical saturation kinetics,
they both showed a markedly lower uptake rate than the
uptake rate of sucrose, especially at higher concentrations.
Additionally, it was shown that glucose uptake rates actually
had a strong inhibitory effect on the uptake rates of sucrose
and fructose, while the latter had little effect on the uptake
rates of glucose. The sucrose uptake rates were largely
linear for concentrations between 0.5 and 500 M. The
other two both showed a “leveling off ” of the uptake rates
as concentrations increased; fructose starts to level off at 6
M and glucose starts to level off at 0.75M. These results
show that sucrose is actually more usable by plant cells than
glucose or fructose, both of which are monosaccharides.
Our decision to pursue this project stemmed
mostly from a project done by Dr. Paul Williams using
Brassica rapa. Williams’ variable of interest was the glucose
in the plant when each plant is exposed to different
amounts of light. His setup was similar to ours’, in that
he also controlled the molarity of the sucrose and glucose
solutions. However, he used screens to simulate varying
degrees of light, and investigated how the sugar was taken
up. Our hypothesis has multiple components. For the discs
Bhupatiraju, Jaleel, and Hagins
that are placed in the dark, it is hypothesized that the
leaf discs will use more of their starch reserves as the
concentration of sugar increases regardless of whether
or not it is glucose or sucrose. Thus, as the sugar
concentrations increase, the discs should appear darker.
For the discs that are placed in the light, they should
all photosynthesize normally, and the concentration of
sugar in their immediate environment should not really
have a significant effect on the starch consumption of
the leaf discs. Finally, it is hypothesized that the discs
that are placed in sucrose should follow similar, if not
the exact same patterns as the ones placed in glucose.
Materials and Methods
Brassica rapa seeds were grown for 7-9 days under
continuous fluorescent light. The variation in time was
due to the amount of leaf tissue used. Plastic straws
were used to punch out 96 leaf discs from the leaf
tissue. Three leaf discs were placed into film canister
lids, and the lids were arranged in groups of 8. Into
4 of the lids, 2mls of the following concentrations of
glucose were added to each lid: 0M, 0.25M, 0.50M, and
0.75M. The same procedure was done for the sucrose
solutions in the remaining 4 lids. Water saturated
paper towels were placed on 4 different black plastic
boxes with clear lids; half were labeled “light” and
half “dark”. The two “dark” boxes were placed in a
cardboard box, covered with clear plastic wrap, a black
trash cover, and kept in the dark for 48 hours. The two
“light” boxes were covered with clear plastic wrap and
placed under fluorescent light for 48 hours. Leaf discs
from each experimental condition were then placed
into beakers containing 15mls of water. The beakers
were boiled until the color from the chlorophyll was
removed (generally >2 minutes). To ensure that all the
chlorophyll was extracted from the leaf discs, they were
then transferred to 10mls of ethanol and incubated for
10-15 minutes. Each leaf disc was then carefully stained
in a 25% iodine solution (to test for the levels of starch),
laid on plastic wrap and grouped by sugar, light/dark,
and molar concentration. After a 10 minute incubation
at room temperature, the color of the leaf discs were
compared to the color scale.
Page 3 of 5
was completely white). Our results indicate that all leaf
discs that were placed in the light used low amounts of
starch from their reserves. The only irregularity that was
observed was that the discs that were placed in 0.25M
glucose used more starch than the other “light” ones.
Other than this concentration, the plants in the light
had a darkness ranging from 8.133 to 8.533, with an
average darkness of 8.40133. However, the dark plants
exhibited a very distinct pattern of starch consumption.
It was found that as the molarity of sugars surrounding
the discs increased, the discs used more of the sugar,
and consequently, less of their starch. During trial
1, no “light” data was available, due to procedural
difficulties(Table 1). In our next trial, the chlorophyll
wasn’t properly removed in many of the samples, thus
making it difficult to measure how dark they were(Table
2). In trial 3 we discovered that if the plants were left
to sit in ethanol, the chlorophyll would be lost more
efficiently, and this led to improved results(Table 3). We
performed 2 more trials, using this modified protocol,
and got more robust data (Tables 4 and 5). The averages
of all 5 trials is shown in Table 6.
Table 1: Trial 1
Glucose
Light
Dark
Sucrose
Light
Dark
Table 2: Trial 2
Glucose
Light
Dark
Sucrose
Light
Dark
Results
For each experimental condition, a total of 5 trials were
run, and the results were recorded. For all figures the
numbers indicate the relative level of darkness of the
leaf disc after being stained with iodine: a result of
“10” indicates nearly complete darkness and a measure
of “1” indicates that no staining was visible (leaf disc
Bhupatiraju, Jaleel, and Hagins
Glucose
Sucrose
Light
Light
Page 4 of 5
Glucose
Light
Dark
Sucrose
Light
Dark
Dark
Dark
Table 6: Calculated averages of all 5 experimental trials
for all conditions of glucose or sucrose under “light”
or “dark” environments
Discussion
Glucose
Light
Dark
Sucrose
Light
Dark
Glucose
Light
Dark
Sucrose
Light
Dark
For the leaf discs that were placed in the light, all of
them used low amounts of starch from their reserves.
This is because the plants were able to photosynthesize
under the light, and there would be no need to use the
starch reserves. However, the dark plants exhibited a
very distinct pattern of starch consumption. It was
found that as the molarity of sugars surrounding the
discs increased, the discs used more of the sugar, and
consequently, less of their starch. The reason for this
is clear; with more sugar available, the leaf disc will
be able to use more of the free sugars. What this also
shows is that the plant can use its own starch reserves
in conjunction with sugar uptake mechanisms when
deprived of sunlight. Additionally, the results support
the fact that as the molarity of the sugar concentration
increases, the amount of starch used decreases nearly
linearly. In fact, the correlation between darkness of
leaf disc and the molarity of the glucose concentrations
has a correlation coefficient of 0.985001, while the
correlation between darkness of leaf discs and the
sucrose concentrations has a correlation coefficient
of 0.996817; these results strongly support a linear
correlation. The experiment performed did not show
any statistically significant difference between the
uptake rates of sucrose versus glucose, although a
subtle change was observed in the data after averages
were taken.
Experiments done by Dr. Roger Wyse showed
that sucrose uptake rates were considerably faster than
the uptake rates of both glucose and fructose. This
would imply that if a leaf disc were isolated in a sucrose
solution, the plant would take up sucrose faster, use less
starch, and therefore be darker in color. On average, for
Bhupatiraju, Jaleel, and Hagins
the discs exposed to the same molarities of sugar, there
was more starch left in the plants exposed to sucrose.
These results are consistent with previous research
which showed that sucrose uptake rates were faster than
glucose uptake rates, and therefore the disc would be able
to use less of its starch granules. The data suggests that
occasionally, plants will still use starch even when placed
in 48 hours of continuous light. It should be expected that
the plants exposed to light should have a darkness index
close to 9 - 10. However, their darkness reached as low as
7.756. This suggests that plants may be forced to dip into
their starch reserves when photosynthetic availability is
low. What can be inferred is that the plants are constantly
in an equilibrium of producing extra glucose and using
starch from its reserves. Further experimentation may
be done to show how and when plants use starch, even
when under 48 hours of fluorescent light. Another idea
for further experimentation is investigating how plants
use both photosynthesis and sugar uptake in conjunction
with one another to optimize their growth.
It is important to acknowledge the existence of
errors in the procedural and data collection processes.
For example in the first trial, the “light” containers were
not covered, and as a result, all the water evaporated.
Therefore, a significant part of data was lost in the first
trial. Another important problem that came up was with
the issue of removing the chlorophyll. At first, the leaf
discs were heated in an ethanol solution on a hot plate
and it was found this method did not completely destroy
the chlorophyll. After a while, the ethanol evaporates
very quickly, and if the discs are left too long to boil,
they would be singed to a crisp, and if not left long
enough, they would retain their pigment. Therefore, it
was decided that instead of letting the ethanol boil, it
would be better to let the leaf discs sit in the ethanol
solution for 10 minutes, which turned out to work quite
well. Our later data is more complete, reflecting our new
procedural changes.
Based on the results that were obtained, this
experiment affirms the hypothesis presented. When
plants are provided with sugar and are kept in the light,
both the plant’s starch granules and the free sugar outside
the leaf are more or less distractions to the plant’s normal
functions. However, when the plant is placed in the dark
and without a source of sunlight, it will use the free sugar
in its environment, and the higher the concentration of
sugar, the less of its own starch it will use. With these
results, it has been shown that plants have multiple
mechanisms for maintaining their homeostasis and have
a variety of options when in dire need of nutrients.
Page 5 of 5
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