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 neighbor-joining 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.

• 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/)

Figure 1(A.thaliana’s genome)

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 extraction1- actin, water treated cDNA tem

plate2- actin, 250mM NaCl cDNA te

mplate3- actin negative control4- DDF1, water treated cDNA te

mplate5- DDF1, 250mM NaCl treated c

DNA template6- DDF1 negative control7- DDF2, water treated cDNA te

mplate8- DDF2, 250mM NaCl treated c

DNA template9- 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?