Download Michigan State University Plant Genomics Program

DDF1 and DDF2: 2 transcription
factor genes involved in salt stress
responses
Juyeon Park
PI: Dr. Shinhan Shiu
Mentor: Dr. Cheng Zou
Shiu Lab at Plant Biology
Laboratories
Michigan State University
Topic
• The topic is the activity of
genes, DDF1 and DDF2;
how their functions relate to
evolution when comparing A.
thaliana and A. lyrata.
Purpose
Hypothesis: RNA expression
levels for DDF1 and DDF2
should be higher when A.
thaliana is put under salt stress
for 6 hrs at 250mM. Also,
DDF1 levels should be higher
than DDF2 levels. (so there
should be little or no
expression of DDF1 and DDF2
when the seedlings are treated
with water) (Magome, The
Plant Journal, 2004)
A. lyrata
Introduction
• When the gene DDF1 or DDf2 is
overexpressed it causes the
reduction of gibberellic acid
biosynthesis, increased tolerance
to high levels of salt. DDF1 is
expressed in all tissues examined,
but most abundantly expressed in
upper stems, while DDF2 is most
abundant in rosette leaves and
stems. The phenotype that incurs
with the overexpression of DDF1
is a dwarfed and delayed
flowering, hence DDF1.
(Arabidopsis.org)
(Magome, The Plant Journal, 2004)
The functional divergence of DDF1
and DDF2
• DDF1 and DDF2 are recently duplicated AP2 family
transcription factors and are related to genes that are
involved in stress responses.
• So to understand why both DDF1 and DDF2 were
retained after duplication, we’re studying
- expression divergence
- phenotypic divergence
- regulatory target divergence
- species specific evolution
(Lehti-Shiu, 2007)
Why are both copies of duplicated
genes retained after duplication?
A. Subfunctionalization
B. Neofunctionalization
(Lehti-Shiu, 2007)
Why are both copies of duplicated
genes retained after duplication?
C. Dosage
D. Neutral Processes
(Lehti-Shiu, 2007)
DDF1 and DDF2 phylogenetic tree
• Phylogenetic tree of DDFs
and related AP2
transcription factors found
in the Arabidopsis genome.
The tree was constructed
from amino acid sequences
of the conserved AP2
domain by the neighborjoining method.
(Magome, The Plant Journal, 2004)
So why DDF1 and DDF2
We’re interested in the evolutionary aspect of A.
thaliana and A. lyrata. We picked DDF1 and DDF2
because we’re interested in gene duplication, so
there are several questions to be answered:
1. Why were they both copies kept if they seem to have similar
functions?
2. Why did DDF1 and DDF2 split from its common ancestor 25-40
mya?
3. What are the differences in DDF1 and DDF2 expression patterns?
4. What are the differences in DDF1 and DDF2 functions?
So why on A. thaliana and A. lyrata?
1. We want to know why certain genes were kept across this
divergence and why some were thrown out, and if they were
kept, how their functions were affected when comparing lyrata
to thaliana.
2. Even when we discover the differences between expressed
activity between species, how will we know it’s due to the gene
differences as opposed to species divergence. So we need to
compare DDF1 to DDF2, but also between lyrata vs. thaliana.
3. That’s why we’re using RT-PCR to find mRNA levels that are
being expressed at a specific time of stress, in this case salt. The
advantage of looking at their mRNA levels is that we’ll see how
much DDF1 and DDF2 are being expressed at that particular
time, under those conditions.
Whole genome duplication
• The mechanism of DDF1 and DDF2’s
doubling is called whole genome duplication.
• During that time, the whole genome is
duplicated, but only a subset of them are kept,
the rest of them lose 1 copy and become
psuedogenes. They degrade over time and
eventually don’t even look like genes anymore
and disappear.
Figure 1
(A.thaliana’s
genome)
• Whole genome duplication causes a level of
redunancy in the genome.
• The finding of large scale duplications can give
valuable hints on how the organization of genetic
material has evolved. Shown here is a summary of
such segmental duplications in A. thaliana.
(Munich Information Center for protein sequencing,
http://mips.gsf.de/)
Materials and Methods
• A. Thaliana (ecotype Columbia) was grown on 1% sucrose
MS Media plates for 14 days in a growth chamber with a long
day cycle.
• Then about 2 plates/condition were put under a 6 hr salt
treatment on the 14th day starting at 8:30am. (salt treatment
was started usually in between 8:30 and 9:30am, to make sure
the salt stress was given at the same period of Columbia’s
circadian rhythm)
• Salt stress was given to the plant by putting the 2 week old
seedlings on filter paper that was soaked with a 250mM NaCl
solution. The filter paper was continuously soaked for 6 hrs.
The seedlings were then incubated at 22 degrees Celsius for
the 6 hrs.
Materials and Methods-continued
Figure 2
Materials and Methods-continued
• Along with the NaCl treatment to 2 plates of seedlings, 2
plates of seedlings were also treated with water as a type of
control to make sure that we see what the 'normal' level of
RNA would be under no stress conditions for DDF1 and
DDF2.
• Since even moving the plants between the filter paper and the
plate to the 50mL reaction tube could cause some form of
wounding to the small seedlings, to eliminate the chances of
RNA levels changing because of those minor stresses, we are
treating the seedlings with water on filter paper, which
shouldn't be harmful to them, but they would still undergo the
same procedures that the NaCl treated seedlings go through.
Results
• In order to figure out which
annealing temperatures to
use, ran PCR with A.
thaliana (Columbia ecotype)
with Actin, DDF1 and
DDF2 primers at 55 and 60
degrees. Then used agarose
gel electrophoresis.
• In the end, decided to use
touchdown PCR with 60
degrees to 50 degrees (20
cycles to 20 cycles)
Results
• Before, actin primers
didn’t give any results,
but now only actin had
results, so decided to
order new primers.
Results of 2nd RNA extraction
• RNA on a gel. First lane
is RNA that was
extracted from water
treated seedlings. Third
lane is RNA that was
extracted from 250mM
NaCl treated seedlings.
• Both are intact.
Results of 2nd RNA extraction
• We didn’t know whether
to treat the RNA with
DNAse or not. So we
treated half of the RNA
from water treated
seedlings with DNAse
and kept half as is.
Reverse transcripted
both into cDNA.
Results
• In order to figure out what
combination of DDF2
primers to use, we took the
control cDNA and the
cDNA reverse transcripted
from DNAse treated RNA
and ran it on a 1.5% agarose
gel with a DDF2 primer
with 285 basepairs and a
DDF2 primer with 238 base
pairs.
Results of 3rd RNA extraction
• Water treated RNA,
250mM NaCl treated
RNA. Both are intact. 3
bands are visible.
Results of 3rd RNA extraction
• The first 4 are
DDF1 primer with
338 bp. The next 4
are DDF2 with
285 bp.
Results of 4th RNA extraction
• Extracted RNA again,
this time with a gradient.
Water, 250mM NaCl,
1M NaCl.
• The 1M NaCl treated
RNA is too degraded.
We know now not to
use this concentration.
Results of 4th RNA extraction
• used only the
water treated
cDNA and
250mM NaCl
cDNA for PCR.
• No results for
Actin or DDF1.
suspect
contamination.
Results of primer testing
• PCR with DDF1,
DDF2, actin primers
respectively, with A.
thaliana (columbia
ecotype) template,
then a DDF1
designed plasmid.
No results for Actin.
Results of 4th RNA extraction
1- actin, water treated cDNA
template
2- actin, 250mM NaCl cDNA
template
3- actin negative control
4- DDF1, water treated cDNA
template
5- DDF1, 250mM NaCl treated
cDNA template
6- DDF1 negative control
7- DDF2, water treated cDNA
template
8- DDF2, 250mM NaCl treated
cDNA template
9- DDF2 negative control
Conclusions
• Based on the results, DDF1 and DDF2 are
expressed more under salt stress and there are
greater differences of expression between
cDNA from water treated seedlings and cDNA
from salt treated seedlings in DDF1 than
DDF2.
Further Research
1. More gradients will be needed to find
optimal NaCl conditions. Seedlings can be
treated with 500mM NaCl and 750mM NaCl
to create a more detailed gradient. Then 2
more replicates will be needed for each
concentration. 3 replicates total will be
needed as evidence for each salt condition.
2. Other abiotic stresses can be tested in the
same way: cold, drought, and heat.
Acknowledgements
•
•
•
•
Dr. Shinhan Shiu
Dr. Melissa Lehti-Shiu
Dr. Cheng Zou
Program Coordinators
References
• Magome H, Oda K, (2004) dwarf and delayed-flowering 1, a
novel Arabidopsis nutant deficient in gibberellin biosynthesis
because of overexpression of a putative AP2 transcription
factor, The Plant Journal, 720-729
• Blanc G, Hokamp K, Wolfe, K. (2003) A Recent Polyploidy
superimposed on Older Large-scale Duplications in the
Arabidopsis Genome, Genome Research, 137- 142
• Verslues P, et.al. (2005) Methods and concepts in quantifying
resistance to drought, salt and freezing, abiotic stresses that
affect plant water status, The Plant Journal, 523-539
• Clauss M, Koch, M, (2006) Poorly known relatives of
Arabidopsis thaliana, Trends in Plant Science, 449-459
Questions?