1. Effect of Environmental Conditions on
Flowering Time in A. thaliana, Poppies, and
Tomatoes
By Tiffany Zhu
Tiffany Zhu, Grade
Mentor: Dr. Amy Litt
Facility: Pfizer Plant Research Lab at the New York Botanical Gardens

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Abstract:
Phenotypic plasticity is a new field that has only recently been seriously explored. It is
the study of the impact of environmental changes on the genomes of organisms.This leads to
traits that are not normally expressed in those organisms. There is also the phenomenon called
genetic imprinting in which those traits can get passed down to the next generation depending on
whether the imprinting is on genes from the egg or sperm. This new area of research indicates
that scientists may be wrong about how only genetics play a role in different phenotypes. Also
known as epigenetics, this research examines how different phenotypes result from
environmental influences. Epigenetics concentrates on regulation of gene expression of
phenotypes which are not directly caused by genetic variation. Though this has only recently
been explored, the idea of epigenetics has been in place for a long time. Lamarck was one of the
scientists that studied what was known as the inheritance of acquired characteristics.
Paper:
Epigenetics is a new field that has only recently been seriously explored. This field
studies inherited changes in phenotype that do not cause changes in genotype and concentrates
on the regulation of gene expression of phenotypes which are not directly caused by genetic
variation. It is a form of plasticity. Phenotypic plasticity is the ability of an organism to adapt to
the changing environment around them without changes in the DNA sequence. This can lead to
traits that are not normally expressed in those organisms. Sometimes those traits will then get
passed down to the next generation through a phenomenon called genetic imprinting. Genetic
imprinting occurs when a change on the gene like an added methyl group (which changes how

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the gene is regulated by turning it off) is passed down through either the egg or the sperm. The
research indicates that scientists may be wrong about how only the sequence of the DNA is
inherited.
A lot of general research has been done and new information is spreading. For instance,
responses of plants to environmental signals are caused by processes regulating gene expression
(Schlichting and Smith 2002). Sometimes these responses can change but other times selection
molds responses to produce predictable patterns of gene expression (Schlichting and Smith
2002). Another is that variation in plasticity can affect fitness levels of the plant (Callahan,
Dhanoolal, Ungerer 2005). Selection favored early flowering time after a prolonged
winter.(Callahan, Dhanoolal, Ungerer 2005).
In 2007, a new concept called genetic imprinting was discovered. This phenomenon
involves the inheritance of epigenetic markings on genes through either the sperm or the egg
(Stewart-Barlow 2007). After this breakthrough, new research followed. In one study, Brassica
rapa’s evolutionary response to drought and other weather changes resulted in earlier flowering
time. This is epigenetic because the drought may have changed the methylation of the chromatin
and changed the regulation of the genes (Franks, Sim, Weis 2007). With further research, it has
been discovered that DNA methylation plays a big role in epigenetics. In a study involving the
use of 5-azacytidine to demethylate A. thaliana, Bossdorf et al (2010) showed that the fitness
level and growth of the plant was reduced and flowering time was delayed. The variations
between the different phenotypes were only weakly related to the similarities between the
genotypes. This meant that any phenotypic differences found had nothing to do with the
genotypes themselves. This proved that natural epigenetic variation was independent of genetic
variation. This also demonstrated that plasticity resulted from epigenetic changes.

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The purpose of this study is to examine if altering temperature and light conditions will
cause an epigenetic change in the flowering time of Solanum lycopersicum cv[cultivar]Micro-
Toms, Papaver somniferum, and Arabidopsis thaliana (Col-0).
Materials/Method:
Five plants were grown per species for four conditions (high light, low light, 15°C, and
22°C) yielding 60 plants. The high light condition was created in the 22°C chamber with a clear
filter over the plants. The low light condition was created in the same 22°C chamber with a dark
filter over the plants. The growth chambers mimicked the 15°C and 22°C conditions so plants
were just placed in there without filters. Seeds were germinated in 2 inch pots and a cover was
placed over the tray to maintain humidity so that the seedlings would not dry out. Gradually we
removed the cover so that the seedlings would develop their cuticles. Germination day was noted
as was flowering time. Basic data collected consisted of plant height, number of fruit for
tomatoes and number of pods for A. thaliana. All plants in each group were numbered to keep
track of genetic or epigenetic changes in each individual separately. Seeds were collected once
the plants were mature or when the fruit was mature in the case of tomatoes. This process was
carried out for two more generations.
DNA was extracted from leaf samples when the third generation plants started to flower.
The DNEasy Plant Maxi Kit was used. Leaf samples were grinded with a pestle in liquid
nitrogen and the ground tissue was transferred into a 50 mL collection tube containing 5 mL of
the preheated Buffer AP1 and 10 µL of RNase A. The solution was vortexed until no tissue
clumps were visible and was then incubated in the bead bath for 10 minutes at 65°C. The tube
was inverted 2-3 times during incubation. 1.8 mL of Buffer AP2 was added and mixed before
incubation in an ice bath for 10 minutes. The tube was then centrifuged at 3000 xg or about 3750

5. 5
rpm for 5 minutes. After that the supernatant was poured into the QIAshredder maxi spin column
which is in a 50 mL tube. The tube was centrifuged a second time at 3000 xg for 5 minutes and
the supernatant was then transferred carefully into a new 50 mL tube. The volume(s) in the tube
was multiplied by 1.5 and that amount of Buffer AP3/E was added in. It was vortexed quickly
before being transferred into the DNeasy Maxi spin column which is also in a 50 mL tube. The
tube was centrifuged a third time for 5 minutes. The supernatant was discarded and then 12 mL
of Buffer AW was added before the tube was centrifuged for 10 minutes at the same speed. After
that the supernatant was discarded and the spin column was transferred to a new 50 mL tube. 1.0
mL of Buffer AE was used for elution. The tube was incubated at room temperature (15 - 25°C)
for 5 minutes and was then centrifuged for 5 minutes at same speed. Samples were stored in the
freezer.
After extracting the DNA we used the Nanodrop 1000 to check the concentrations of the
DNA. After this we set up restriction digests.We were hoping to use the methylation sensitive
enzyme Sma1 and the non-methylation sensitive enzyme Xma1 to see if there was any
methylation. The products of the digest were run on a 1% agarose gel with 1kb and 100bp
ladders and stained with ethidium bromide to see the bands. The gel was put on a
transilluminator equipped with a camera to record the image. We then used a bisulfite
sequencing technique with the Epitect Bisulfite Kit when the digests didn’t work. We decided to
concentrate on a gene called FLOWERING LOCUS C. Only the A. thaliana DNA was used since
it was cost and time efficient. The procedure was as follows. There were four tubes, one for each
A. thaliana group (i.e. high light, low light, etc). All four reactions contained a constant amount
of Bisulfite Mix (85 µL) and of the DNA Protect Buffer (35 µL). The total amount of DNA and
of the RNase-free water added to those two had to equal 140 µL. Then these reactions were

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placed into the thermal cycler which vacillated in temperature for thermal DNA denaturation and
sulfonation and deamination of cytosines.The tubes were centrifuged and 560 µL of Buffer BL
was added. Each sample was transferred to an EpiTect spin column to be centrifuged at
maximum speed for 1 minute. The flow-through was discarded and then 500 µL of Buffer BW
was added before being centrifuged at maximum speed again for 1 minute. The flow-through
was discarded and then this same step with Buffer BW was repeated again. Afterwards the
columns were transferred to new 2 mL collection tubes and centrifuged at maximum speed for 1
minute again. The columns were left open in 1.5 mL microcentrifuge tubes and incubated for 5
minutes at 56°C. 20 µL of Buffer EB was added onto the center of each membrane. The DNA
was eluted by centrifugation for 1 minute at a speed of about 15,000 x g (12,000 rpm). The end
product was sent to the Yale Science Hill DNA Sequencing Facility. The sequences were
trimmed and aligned to identify sites that might have been methylated.
Results:
The digests did not work as the DNA ended up being uncut. The bisulfite sequencing was
successful because we got good sequences however there was no methylation found. Table 1
below is a summary of the basic data collected for the first generation. It contains the average
and standard deviation (to show variability) of each trait. Plant height was calculated in
millimeters while flowering time was calculated in days. For plant height the average of the
maximum heights obtained by the plants before they were discarded was used. The number of
seed pods refers to A. thaliana and the number of fruit refers to tomato. To obtain the maximum
average possible for the seed pods and fruit, the day with the maximum number of seed pods and

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fruit was used for the average. Pods for poppies were not counted because poppies usually
produce only one flower and thus only one pod with seeds. Table 2 below Table 1 is a summary
of the basic data that was collected for the third generation.
Arabidopsis thaliana Poppy Tomato
high
light
low
light
15°C 22°C high
light
low light 15°C 22°C high
light
low
light
15°C 22°C
plant ht
(mm)
230.78 ±
29.1
106.7 ±
60.43
270.16 ±
15.05
243.54 ±
16.77
222.2 ±
46.33
150.26 ±
79.87
207.68 ±
89.54
53.46 ±
19.93
59.62 ±
2.95
98.56
± 8.0
56.32 ±
5.58
59.4 ±
4.19
fl time
(days)
23 ± 0 27.8 ±
19.7
43.8 ±
5.26
24 ± 0 63.4 ±
13.69
67 ±
36.76
75.8 ±
8.49
39.2 ±
1.1
26.6 ±
3.71
52.6 ±
3.7
37.8±
4.09
40.2
± 5.02
seed
pod/
fruit#
225 ±
58.7
28.6 ±
22.94
235.4 ±
24.37
290.6 ±
50.85
n/a n/a n/a n/a 16.4 ±
3.58
2 ±
1.87
4.6 ±
2.07
4.4 ±
1.34
Table 1: Here we can see that there was a lot of variability in each group for each species. This
means that there were a lot of differences between the groups as expected. There is also a trend
in the flowering time which can be seen here. For example, the low light tomato plants had a
higher average plant height than the high light plants but they flowered later. The 15°C plants
had a higher average plant height than the 22°C plants but they also flowered later. This suggests
that the relationship between plant height and flowering time is inversely proportional. The
higher the plants grow, the later they flower.
Arabidopsis thaliana Poppy Tomato
high
light
low
light
15°C 22°C high
light
low
light
15°C 22°C high
light
low
light
15°C 22°C
plant ht
(mm)
315.6 ±
26.65
153.6 ±
86.07
242.6 ±
152.68
357.8 ±
29.37
161.4 ±
34.18
150.6 ±
75.04
346.2 ±
119.66
207.6 ±
31.29
103.6 ±
11.3
154.4 ±
9.1
100.4 ±
8.56
110.6
±
8.65
fl time
(days)
38 ±
4.47
45.4 ±
5.81
61 ±
14.82
23.8 ±
3.7
39.8 ±
8.14
78.8±
49.27
98.8 ±
2.68
26.8 ±
13.97
37.2 ±
3.83
92.6 ±
4.77
79 ±
4.8
60.4 ±
3.58
seed
pod/
fruit #
99 ±
42.77
9 ± 11 103.2 ±
119.51
122.8 ±
41.12
n/a n/a n/a n/a 5.2 ±
2.49
4.6 ±
2.7
4.4 ±
3.51
7.6 ±
1.14

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Table 2: The trend from the first generation does not continue in the third generation which is
due to many issues that occurred. There is no longer an inversely proportional relationship; there
is a directly proportional relationship in the case of A. thaliana and no distinguishable
relationship in the other two species.
Errors occurred in the sequencing which are shown below. We expected to see T’s in
place of C’s if there was methylation however we only found changes in sequences that were not
changes from C’s to T’s. Those changes were probably PCR errors. A22 (A. thaliana in the 22°C
condition) and AH (A. thaliana in the high light condition) became the control groups because
those represented standard lab conditions. The nitrogenous bases are highlighted in color. The
ones that are not highlighted indicate where changes occurred. The sequences were aligned in a
program called MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). BioEdit was used to look
at the sequences.
A22
A15
AH
AL
A22
A15
AH
AL
A22
A15
AH
AL
A22
A15
AH
AL
Ruler in Inches Ruler in Inches
Ruler in Inches Ruler in Inches

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The relationship between average flowering times for each group of the poppies and tomatoes
corresponded with those of Arabidopsis and since Arabidopsis was the only one used for the
sequencing it makes sense to show only the results of Arabidopsis. Below, the boxplots for the
first generation Arabidopsis are shown in Figure 1. The boxplots for the third generation
Arabidopsis are shown in Figure 2.
Figure 1: AL had a wide range of flowering times but on average it flowered later than AH
which ended up with the same flowering time for all five plants. A15 flowered later than A22
which also ended up with the same flowering time for all five plants.

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Figure 2: AH flowered earlier than AL by 8 days and had a shorter range which was about 10
days. AL meanwhile saw one of its plants flower in 38 days and so on until 54 days had passed.
The range for AL was 16 days. A15 also flowered later than A22 and had a longer range.
Discussion:
The basic data says that flowering time was affected which is not surprising considering
the different conditions. The project had been designed with four conditions and three species
because the four conditions affected each species differently. A. thaliana are known to adapt well
to cool climates and little light. Micro-toms and poppies are known to prefer warm climates and
a lot of light. The boxplots, though they only contain data from A. thaliana, represent the patterns
of the other two species too. AH and A22 flowered earlier than AL and A15 in both the first
generation and the third generation.

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Unfortunately, we do not have enough molecular data to support that an epigenetic
change occurred on flowering time. There are many reasons for this. During the growth of the
first generation plants, we encountered watering troubles and fluctuations in the temperature of
the 15°C growth chamber. Several plants died. Then there was a switch to 3 inch pots during the
growth of the second generation because the 2 inch pots seemed to be too small. By that time the
chamber was fixed. This meant that the plants had more room for growth and it would be
expected that plant growth would have increased and that perhaps flowering time would have
been shortened. However the Arabidopsis from the high light and low light conditions were
taken to my school to see if more generations could be squeezed in but those plants died and
several months were lost. During the growing period of the third generation there was an issue
with the soil and there were a lot of problems getting plants in the 15°C chamber to germinate so
a tablespoon of fertilizer to a full watering can had to be used once a week for two weeks to help.
This caused significant delays in flowering time as many young plants died and had to be
replanted.
When it came time to try out the digests, the digests did not work. We optimized the
digests with altering amounts of DNA, buffer, and enzyme. That seemed to help a bit but the
DNA was not cut properly. We performed a PCR with a control DNA sample to check if the
enzymes were working which they were. The DNA was then purified. We tried the digests again
and ran multiple gels. Despite our efforts, the digests were not successful. The bisulfite
sequencing was next. Unfortunately there was no additional methylation. The four groups were
identical except for areas of error (There were G’s where there was supposed to be A’s). We
were looking for T’s where there were supposed to be C’s as explained before, because
methylation occurs at cytosines. One reason for why there were no epigenetic changes could be

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that not enough generations were grown. Five generations were supposed to have been grown
but there wasn’t enough time. Another reason was that there were only five plants per species per
group. That is too small of a sample size to effectively get results and interpret data. As seen in
the tables, the standard deviation varied too much within a group. An example would be A15
plant height. The standard deviation was ± 15.05 for the first generation and ± 152.68 for the
third generation. Because of these reasons the data collected does not support nor refute my
hypothesis.

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Works Cited
1. Schlichting, C., and H. Smith. 2002. Phenotypic plasticity: linking molecular mechanisms
with evolutionary outcomes. Evolutionary Ecology 16: 189-211
2. Callahan, H., N. Dhanoolal, M. Ungerer. 2005. Plasticity genes and plasticity costs: a new
approach to using an Arabidopsis recombinant inbred population. New Phytologist 166: 129-
140.
3. Stewart-Barlow, K. A/Prof. 2007. Genetic Imprinting. The Australasian Genetics Resource
Book 6: 203-207.
4. Franks, S., S. Sim, and A. Weis. 2007. Rapid evolution of flowering time by an annual plant in
response to a climate fluctuation. PNAS 104: 1278-1282.
5. Bossdorf, O., D. Arcuri, C. Richards, M. Pigliucci. 2010. Experimental alteration of DNA
methylation affects the phenotypic plasticity of ecologically relevant traits in Arabidopsis
thaliana. Evolution Ecology 24: 541-553.