Julia Proctor

BIOL 400: AA
04/28/2015
Interactions Between Transcription Factor DREB1A/CBF3 and HAC1 in Response to Cold
Stress
Due to global climate change, we are seeing increasingly unpredictable weather, which
has a high impact on plant productivity in terms of crop yield and geographical distributions
(Ahmadizadeh 2014, Oakenfull 2013). Due to their sessile nature, plants are unable to escape
abiotic environmental stresses and must be able to acclimate to dropping temperatures in a
physiological, molecular, and cellular manner (Akhtar 2012, Oakenfull 2013). There are a variety
of mechanisms used to modify chromatin structure for transcriptional regulation in eukaryotic
organisms, such as acetylation, methylation, and glycosylation (Boycheva 2014). Acetylation in
particular is known for loosening chromatin coiling by adding an acetyl group to histones,
causing their charge to become less positive and thus repel the negatively charged DNA. HAC1
(also known as PCAT2) is a histone acetyltransferase protein in Arabidopsis thaliana,
homologous to p300/CBP in humans, is responsible for mediating DNA transformations to
upregulate or suppress transcription of stress-response genes (Bordoli 2001).
The DREB/CBF proteins belong to the AP2 family of transcription factors which bind to
DRE/CRT, a cis-acting dehydration responsive element/C-repeat motif, to regulate expression of
genes related to stress response and therefore play a key role in increasing stress tolerance in
plants (Akhtar 2012). Constitutive expression of DREB1B/CBF1 and DREB1A/CBF3 in
Arabidopsis has previously been shown to induce the expression of cold-response (COR) genes
to protect the plant from cold stress in non-acclimated plants (Ahmadizadeh 2014, Gilmour

2000). Having the ability to manipulate this protein family may serve as an alternative approach
to increasing crop yields in the future (Akhtar 2012).
H: Transcription factor DREB1A (also known as CBF3) will induce gene expression of COR
genes (specifically COR78) by activating histone acetyltransferase HAC1 in response to cold
stress in Arabidopsis thaliana.
Specific Aim 1: To determine if DREB1A activates HAC1 in Arabidopsis thaliana plants under
cold stress to promote expression of COR78
Arabidopsis thaliana wild-type (WT), CS60000, and mutant (MT), SALK_080380, plants will
be allowed to grow for ~20 days under standard greenhouse conditions. SALK_080380 has a TDNA insertion in both copies of the HAC1 gene. Twelve WT plants and twelve MT plants will
then be moved under a cardboard box with lamps on timers, which will alternate between light
and dark every 12 hours and serve as the only source of light. Two boxes will be set up in this
way; one remaining at room temperature (~26C) and another will be in the cold room (~4C).
When cold treatments were applied to Arabidopsis thaliana plants constitutively expressing
dreb1a/cbf3, protein expression was induced rapidly; transcript levels for COR78 increased
between one and four hours of cold treatment, then plateaued between eight and twelve hours of
cold treatment (Fowler 2005, Hajela 1990, Miura 2013). Three replicates of each WT and MT
Arabidopsis plants will go through each treatment (0, 2, 6, and 24 hours in the cold room). Three
WT and three MT plant will have remained in the Greenhouse with no exposure to the treatment
to serve as a control for how treatments might have affected expression levels. Once all have
incubated in the correct treatments over the 24 hour period, we will harvest rosette leaf tissue
from all 30 plants and use liquid nitrogen to freeze the samples. The following day, we will
isolate mRNA from the samples and use RT-PCR to analyze DREB1A/CBF3 and COR78

expression. Ubiquitin primers will be used to control for the amount of mRNA loaded into the
samples and reverse transcribed into cDNA, since ubiquitin is a highly expressed protein. I
predict that COR78 expression will increase as exposure to cold stress increases as DREB1A
activates HAC1.
Specific Aim 2: To determine if DREB1A is co-localized to the nucleus with HAC1 under cold
stress conditions in Arabidopsis thaliana
Arabidopsis thaliana wild-type (WT) plants will be genetically engineered to create GFP fusion
proteins with DREB1A and HAC1, respectively. One mutant will be DREB1A-GFP and HAC1.
The other mutant will be DREB1A and HAC1-GFP. The mutant Arabidopsis plants will be
allowed to grow for ~3 weeks in standard Greenhouse conditions. Then, twelve of the plants will
be put in a cold room (4C) and twelve of the plants will be left at room temperature (26C), for
varying amounts time treatments (0, 2, 6, and 24 hours) in triplicates. Three replicates of each
mutant plant will also be left in Greenhouse conditions to control for the effect of the treatment
on expression. Then we will use fluorescence microscopy to visualize the localization of
DREB1A-GFP and HAC1-GFP individually to see whether or not the GFP tagged proteins colocalize in the nucleus under cold stress. (This would signify HAC1 is in the nucleus to acetylate
histones to modify the chromatin structure and thus accessibility to transcribe and thus express
the gene). In extension, you could do a further study using yeast 2-hybrids of the two proteins to
see if they actually bind/interact with one another.
References:
Ahmadizadeh, M. "Bioinformatics Study of Transcription Factors Involved in Cold
Stress." Biozoojournals. Biharean Biologist, 7 July 2014. Web. 27 Apr. 2015.
Akhtar, M. "DREB1/CBF Transcription Factors: Their Structure, Function and Role in Abiotic

Stress Tolerance in Plants." Journal of Genetics. Indian Academy of Sciences, Dec. 2012.
Web.
Bordoli, L. "Plant Orthologs of P300/CBP: Conservation of a Core Domain in Metazoan
P300/CBP Acetyltransferase-related Proteins." Nucleic Acids Research 29.3 (2001): 58997. Oxford University Press. Web.
Boycheva, I. "Histone Acetyltransferases in Plant Development and Plasticity." Latest TOC RSS.
Current Genomics, 21 Oct. 2013. Web. 27 Apr. 2015.
Fowler, S. "Low Temperature Induction of Arabidopsis CBF1, 2, and 3 Is Gated by the
Circadian Clock." Plant Physiology 137.3 (2005): 961-68. Web.
Gilmour, S. "Overexpression of the Arabidopsis CBF3 Transcription Activator Mimics Multiple
Biochemical Changes Associated with Cold Acclimation." Plant Physiology, 2000. Web.
Hajela, R. "Molecular Cloning and Expression of Cor (Cold-Regulated) Genes in Arabidopsis
Thaliana." Plant Physiology 93.3 (1990): 1246-252. Web.
Hu, Y. "Jasmonate Regulates the INDUCER OF CBF EXPRESSION-C-REPEAT BINDING
FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in
Arabidopsis." The Plant Cell 25.8 (2013): 2907-924. Web.
Miura, K. Cold Signaling and Cold Response in Plants. International Journal of Molecular
Sciences 14.3 (2013): 5312-337. Web.
Oakenfull, R. "A C-Repeat Binding Factor Transcriptional Activator (CBF/DREB1) from
European Bilberry (Vaccinium Myrtillus) Induces Freezing Tolerance When Expressed in
Arabidopsis Thaliana." Ed. Sunghun Park. PLoS ONE 8.1 (2013): E54119. Web.