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Stress Responses in Arabidopsis thaliana wrky65 Knockout Plants
Julie Bullinga1, Anthony Conteno2, Diane Bassham2
1Marshalltown
Community Schools, Marshalltown, Iowa 50158
2 Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011
WRKY transcription factors are believed to be involved in regulation of plant
response to nutrient stress, pathogen infection, senescence, and trichome
development. We are interested in studying the role of WRKY65 transcription
factors. Three individuals of an Arabidopsis thaliana wrky65 knockout line
were observed during sucrose starvation, salt stress, and oxidative stress
environments for phenotype changes. The plants’ autophagic response to
sucrose starvation was also studied by comparing the number of
autophagosomes produced per cell in the root. We found wrky65 mutants had
more autophagosomes in response to sucrose starvation; however they appear
to experience greater levels of chlorosis and necrosis during stress tests. This
leads us to question whether the increased autophagy in wrky64 knockout
plants is significant, since we would expect more autophagosomes to prevent
cell damage caused from stress. The increased number of autophagosomes in
wrky65 suggests this gene may not necessarily be involved in autophagosome
formation, but may instead be involved in transport of the autophagosome to
the vacuole, and as a result more are produced.
INTRODUCTION
WRKY proteins are a family of transcription factors with a binding preference
for W-box enhancer elements on DNA. The W-box enhancer increases or
decreases the rate of RNA polymerase assembly (Eulgem et al., 2000).
WRKY transcription factors are suggested to be involved in the regulation of
genes required for plant pathogen defense, senescence, and trichome
development pathways. Transcription of WRKY genes is known to be upregulated in response to abiotic stresses (such as nutrient starvation,
wounding, drought and temperature changes), pathogen infection, and
senescence (Ulker and Somssich, 2004).
Stress conditions, such as nutrient starvation, also induce the process of
autophagy in plant cells. Autophagy is a method for the cell to recycle foreign
and damaged or excess cell components. An autophagosome is a double
membrane structure that forms around portions of the cytoplasm. The
autophagosome travels to a vacuole or lysosome where it fuses and releases
the captured cell components into the organelle. Enzymes in the vacuole or
lysosome degrade these components for re-use by the cell. It is thought that
the increase in autophagy during starvation is a mechanism for extending the
cell’s life by providing it with recycled nutrients (Xiong et al., 2005).
Autophagy is also known to increase during oxidative and salt stresses.
(Xiong et al., 2007).
Figure 1: Stress Responses of wrky65 knockout plants
Figure 2: Autophagosomes Formation after Sucrose Starvation
7 day old seedlings of Arabidopsis wild-type (WT) plants maintaining gene function,
Atg18a RNAi plants that are effectively autophagy knockouts, and progeny from three individuals of a wrky65
knockout (1, 2, and 3) were transferred from minus sucrose plates to stress plates. Plants were then observed for
phenotypic changes.
Seedling plants on minus sucrose plates were placed in the dark for 4 days to ensure plant
did not create sugar via photosynthesis. Seedlings where then stained with MDC and then
viewed with a UV fluorescence microscope with a DAPI filter in the Bessey Microscopy
Facility.
A. Sucrose Stress
Seedlings were transferred to minus sucrose plates and wrapped with
aluminum foil to prevent photosynthesis. wrky65 mutants demonstrated a
mild phenotype. wrky65-2 mutant underwent mild chlorosis, compared to
WT. Atg18a RNAi mutant showed greater level of chlorosis. Chlorosis
was expected in Atg18a RNAi because it is a phenotypic indication of an
autophagy knockout. Previous results lab showed wrky65 mutants
undergoing mild chlorosis with wrky65-3 having the most yellowing
followed by wrky65-1 (Contento, personal communication). Images above
show Day15 of the stress test.
B. Oxidative Stress:
Seedlings were transferred to methylviologen (MV) plate. Methylviologen is
known to increase oxidative free radicals by interfering with electron
transport chain of photosynthesis. wrky65 mutants demonstrated more
oxidative stress than WT or Atg18a RNAi. The leaves on all plant appeared
to turn under and experienced some level of necrosis. WT plants showed
mild level of necrosis with some chlorosis. Atg18a RNAi plants displayed
more necrosis and chlorosis than WT. Chlorosis in Atg18a RNAi plants was
milder compared to chlorosis in wrky65-1 and wrky65-2. Approximately half
of the wrky65-1 and wrky65-2 died due to chlorosis and rest experienced
more severe necrosis. wrky65-3 appeared to be affected mainly by
necrosis. These findings supported previous results showing phenotype
change in all wrky65 mutants (Contento, personal communication).
C. Salt Stress:
Seedlings were transferred to 160mM sodium chloride plates. All
plants underwent chlorosis when placed on the salt plates. Most
plants showed significant loss of color by day 5 and most plants
had lost all pigment by day 8. There was variation in rate of
chlorosis for all plant types. The greatest difference occurred in
wrky65 mutants with a couple plants still having a little pigment
on day 20. These findings contradict previous results that
showed WT plants retained green pigment longer than other
plant types (Contento, personal communication). Images above
show Day 11 plates because all plants, except a few wrky65
mutants, had undergone total chlorosis by Day 15.
(Contento)
WRKY65 is a member of the WRKY family whose role is unknown. A microarray
study showed the transcription of WRKY65 is increased by sucrose starvation
at 24 and 48 hour time points (Contento et al., 2004). We are interested in
whether WRKY 65 is involved in autophagy, since sucrose starvation also
induces autophagy. Autophagy activity can be observed on the organismal
level in Arabidopsis thaliana using observation of phenotypic changes. These
include responses common with increased sensitivity to abiotic stress
conditions, such as increased chlorosis and necrosis. Autophagy can be
observed at the cellular level by staining with the autophagosome-specific
fluorescent dye monodansylcadaverine (MDC) and microscopy to image the
autophagosomes.
CONCLUSIONS
• Salt stress testing was not conclusive. Previous testing showed wrky65 knockouts were more
sensitive to salt stress than WT. Our results do not confirm this.
• wrky65 mutants were more sensitive to oxidative stress. This also supported previous findings.
All wrky65 mutants showed increased necrosis and wrky65-1 and wrky65-2 also showed
increase in chlorosis.
• Wrky65-1 showed a significant increase (p-value = 0.0026 ) in number of autophagosomes as
compared to WT, confirming previous observations. wrky65-2 and wrky65-3 showed no
significant increase in formation of autophagosomes.
C. wrky65-1 root tip
Autophagosomes per cell during Suc starvation
7
6
5
4
3
2
1
0
WT
W65-K
W65-Pi
W65-Xi
Atg18-RNAi
wrky65 mutants showed an increase in the number of autophagosomes compared to WT. A
t-test showed the WRKY65 pi mutant produced a statistically significant (p-value = 0.0026)
increase in number of autophagosomes compared to WT. Atg18a RNAi mutants showed a
significant (p-value = 0.0040) decrease in number of autophagosomes compared to WT.
REFERENCES
Contento AL, Kim SJ, Bassham DC. Transcriptome profiling of the response of Arabidopsis suspension culture cells to Suc starvation.
Plant Physiol. 2004 Aug;135(4):2330-47. Epub 2004 Aug 13.
Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000
May;5(5):199-206. Review.
Ulker B, Somssich IE. WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol. 2004
Oct;7(5):491-8. Review.
Xiong Y, Contento AL, Bassham DC. AtATG18a is required for the formation of autophagosomes during nutrient stress and
senescence in Arabidopsis thaliana.
Plant J. 2005 May;42(4):535-46.
Xiong Y, Contento AL, Nguyen, PQ, Bassham, DC. Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in
Arabidopsis. Plant Physiol. 2007 Jan;143:(1):291-9. Epub 2006 Nov10.
CONTINUING and SUGGESTED FUTURE RESEARCH
• Oxidative protein recovery experiment will be repeated using a different standard curve than we
recently tried since our sample readings fell below the standard curve and proteins expressed will
be visualized with Western Blot techniques.
• Rate of root growth is being tested on 100mM and 200mV salt stress plates.
To analyze the function of WRKY65, a wrky65 knockout mutant was tested for
responses to sucrose starvation, oxidative stress, and salt stress. The plants
were compared to wild type and ATG18 RNAi, an effective autophagy knockout,
at the organismal level for phenotype change and at the cellular level for
autophagy activity during sucrose starvation.
B. Atg18 root tip
D. Number of Autophagasomes per Cell in Arabidopsis Root Tips
• Sucrose stress showed a mild phenotype change in wrky65 mutants, which is supported by
previous testing.
CELL COMPONENTS
A. WT root tip
# ATGs
ABSTRACT
ACKNOWLEDGEMENTS
The National Science Foundation for funding the Research Experience for Teachers (RET) program.
Dr. Adah Leshem-Ackerman and RET program for providing this internship opportunity.
Dr. Diane Bassham for opening her lab to this internship program and her oversight.
Dr. Anthony Contento for his teaching and allowing a teacher to participate in his research.
• Phenotype for salt stress may be repeated to determine if a larger sample will reveal greater
differences between the plants.
• Repetition of the autophagosome formation after sucrose starvation test using larger sample size
to produce more statistically robust data.
PH.D. candidates Phan Quang Nguyen, Yimo Liu, and Sang Jin Kim for their friendly support.