Biol 400F2016
Crowe
Class Data Assignment
Goals:
1) to familiarize you with the data collected in previous quarters and potentially give you
ideas of interesting results you want to follow up on this quarter.
2) to prompt you to consider important aspects of experimental design
3) to help you think about how best to present your data to make it easy to interpret for the
reader.
We will discuss the data in lecture on October 17, so please come prepared!
On the following pages you will find the results of a number of different experiments
performed by students in previous quarters of Bio 400. They are all focus on the role of the
histone acetyl transferase HAC1 in Arabidopsis stress responses.
For each figure:
1) Analyze the data and answer the associated questions
2) Consider what you liked/didn’t like about the way the data was presented. What would
you change?
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Biol 400F2016
Crowe

Fig. 1.
In order to characterize the role of HAC1 in plant stress, homozygous mutants
(Salk_080380) along with their CS60000 wild-type counterparts, were analyzed by RT-PCR
to measure HAC1 gene expression.
Leaf RNA was extracted and converted to cDNA. cDNA was amplified with gene-specific
primers to determine the relative amount of mRNA present in the leaf samples. Ubiquitin
(Ubq) primers were included as a control. HAC1 primers were used to detect the presence
of HAC1 cDNA. Samples were electrophoresed on an agarose gel and stained with ethidium
bromide to visualize the DNA (Fig. 1).
-RT WT M1 M2 M1
M1
M2 M2 WT WT
2
2
2
2
2
4
2
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2
4
Ubq Ubq Ubq Ubq HAC HAC HAC HAC HAC HAC
1
2
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10
11
Plant
l cDNA
Primer
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Fig 1. RT-PCR Analysis of HAC1 gene expression in Salk_122894 homozygous insertion
lines. RT-PCR analysis was performed on cDNA isolated from wild-type Arabidopsis CS60000
(lanes 3, 10, 11) or homozygous T-DNA mutants (lanes 4-9) were amplified with HAC1specific primers (lanes 6-11), or with Ubiquitin (Ubq) primers (lanes 2-5). A mock cDNA
synthesis reaction lacking reverse transcriptase (No RT control) was included as a negative
control (lane 2). Resulting PCR products were analyzed on a 1.5% agarose gel. PCR product
size was estimated by comparison to a 100 bp DNA molecular weight ladder (lanes 1 & 12).
What can you conclude from Fig. 1?
Why was it important to amplify mutant cDNAs with a ubiquitin primer?
What is the purpose of the –RT control in lane 2? If you saw a band there what would
that indicate?
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To determine whether loss of HAC1 had any effect on cold acclimation, wild-type and hac1
Arabidopsis were subjected to a 72 hour cold treatment at 3°C with a 16h light/8h dark
cycle or maintained at room temperature in the greenhouse with a similar light/dark cycle.
RNA was isolated from leaf samples and analyzed by RT-PCR as described for Fig. 1, except
additional primers were included to measure expression levels of the cold responsive
gene Cor15a and the C-repeat binding factor CBF1 (Fig. 2). CBF1 is a transcription factor
which itself is upregulated by cold, and has been shown to upregulate expression of
Cor15a.
What hypothesis was being
tested?
How do the size labels on the
gels (e.g. 351 bp) help you
interpret the data?
Do you think eIF4a was a
good choice for a control
gene for this experiment?
Why or why not?
What do you notice about the
pattern of HAC1 expression?
How does this help you
interpret the results for CBF1
and COR15a?
Figure 2. Role of HAC1 in cold gene regulation. Gel electrophoresis of
cDNA amplified with primers for the eukaryotic translation initiation
factor eIF4a, HAC1, and cold response genes CBF1 and COR15a. Wild
type and hac1 strains were exposed to 72 hours at indicated
temperatures. –RT and H20 are no reverse transcriptase and no template
negative controls, respectively. Expected sizes listed under gene names.
[Sp2012 AB Blue]
What tentative conclusions
can you make about the role
of HAC1 in COR (cold
regulated) gene expression?
Interestingly, another group followed up on these experiments and found some different
results as shown on the next page (Fig. 3).
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Figure 3. Role of HAC1 in cold gene regulation. RT-PCR and gel electrophoresis of cDNA
obtained from 4 wild types (WT) and 4 mutants (MT) and amplified with primers specific for
HAC1, CBF1, and COR15a. 100 bp was used for HAC1. 1kb ladder was used for CBF1 and
COR15a)[Yellow AC Sp2014]. NOTE: treatment was actually 4°C not -4°C.

 How
do these
results
differ from
those seen
in Fig. 2?
 Is
there
anything
different
about the
experimental set-up that could help explain these differences?
Is there anything else you would like to see before making conclusions from
this figure?
Another group of students performed an experiment to determine whether hac1 plants
responded appropriately to a different abiotic stress: Heat stress. Wild-type and hac1
plants were subjected to 40°C for 2 hours or maintained at 23°C. Expression of the heat
shock transcription factor HsfB1 that is known to upregulate heat shock genes was
measured (Fig. 4).
Why were the data
normalized to ubiquitin
expression?
How do you “normalize”
expression levels?
What do these data
suggest about the role of
HAC1 in expression of
HsfB1?
Figure 4: Heat-stress response in A. thaliana. Wild-type and hac1
Arabidopsis were subjected to 40°C for 2 hours or maintained at 23°C. RTPCR was performed on RNA isolated from leaf tissue using primers directed
against the heat shock transcription factor Hsfb1 and the control gene
Ubiquitin. Bands were quantified using Image J software. The graph shows
Hsfb1 expression normalized to ubiquitin levels [Sp2012 AC red]
Another group focused
their interest on the role of
HAC1 in salt stress. For this experiment both wild-type and hac1 plants were subjected to
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either 60 mM or 150 mM NaCl over a 1 week period (Fig. 5). Leaves were harvested and
analyzed for expression of the Ada2b gene that had previously been implicated in
mediating salt stress responses. Ada2b is a co-activator and a subunit of the GCN5 histone
acetyl transferase complex.
Expression of ada2-b
Expression of ada2-b increased in salt-stressed hac-1
mutants
7
6
5
4
3
2
1
0
6.03
3.06
1.05
hac-1 no
H2O
1.46
1.35
1.11
wild type
no H2O
hac-1
60mM
[NaCl]
wild type
60mM
[NaCl]
hac-1
150mM
[NaCl]
wild type
150mM
[NaCl]
Figure 5: HAC1 and salt stress.
Wild-type and hac-1 mutant A.
thaliana were exposed to different
15ml salt treatments over a period of
one week. They were then examined
by RT-PCR for expression of ubiquitin
and ADA2-b and quantified using
Image J software. The graph shows
ADA2b levels normalized to ubiquitin.
Can you tell from these data whether the salt regimen performed in this
experiment invoked a salt stress response?
What possible conclusions can you draw from this figure?
How could you explain this result based on the function of Ada2b?
Another group explored the role of HAC1 in drought stress (Fig. 6). CBF4 is a
transcription factor related to the C-repeat binding factor proteins CBF1 examined in Figs 2
& 3. However, CBF4 is upregulated in response to drought, not cold. Constitutive
expression of CBF4 confers drought tolerance in Arabidopsis.
Do hac1 plants express CBF4
after 7 days of drought? How
can you tell?
When does expression of
CBF4 peak in the wild-type
plant? When does it peak in
hac1 plants? Knowing what
you do about histone acetyl
Fig. 6. HAC1 and drought stress. Wild-type and mutant
Transferases, what could
plants were subjected to increasing days of drought. RT-PCR explain this difference?
was performed using primers to the HAC1 gene, the droughtresponsive CBF4 gene and a constitutively expressed control
gene (eIF4A). Numbers indicate number of days of drought
and “W” or “M” indicate wild-type or mutants, respectively.
[ACRedSp2014]
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Lastly, one group explored the role of HAC1 in acute drought stress. They exposed plants
to severe dehydration. They then measured expression of RD20 and RAP2.4 , two different
drought-stress response genes.
Fig. 7. HAC1 and drought stress. mRNA expression of RD20 and RAP2.4 in the
leaf tissue of wild-type (CS_60000) and hac1 mutant (SALK_080380) plants exposed
to 0, 1, 2 and 5 hours of acute drought conditions were measured using RT-PCR and
analyzed using gel electrophoresis. Arrows are included as markers to help
approximate relative band sizes of the PCR products. All genes appeared at expected
product sizes.
What additional tissues might you choose for measuring drought-responsive gene
expression?
What is a possible conclusion about the role of HAC1 in RD20 expression?
What is a possible conclusion about the role of HAC1 in RAP2.4 expression?
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