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

1
Stress Responses in Arabidopsis thaliana wrky65
Knockout Plants Julie Bullinga1, Anthony
Conteno2, Diane Bassham21Marshalltown Community
Schools, Marshalltown, Iowa 501582 Genetics,
Development and Cell Biology, Iowa State
University, Ames, Iowa 50011
ABSTRACT 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.
Figure 2 Autophagosomes Formation after Sucrose
Starvation 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. WT root tip
B. Atg18 root tip
C. wrky65-1 root tip
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 up-regulated 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 cells 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).
D. Number of Autophagasomes per Cell in
Arabidopsis Root Tips 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.

• 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.
• Sucrose stress showed a mild phenotype change in
  wrky65 mutants, which is supported by previous
  testing.
• 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.

REFERENCES Contento AL, Kim SJ, Bassham DC.
Transcriptome profiling of the response of
Arabidopsis suspension culture cells to Suc
starvation. Plant Physiol. 2004
Aug135(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 May5(5)199-206.
Review.  Ulker B, Somssich IE. WRKY
transcription factors from DNA binding towards
biological function. Curr Opin Plant Biol. 2004
Oct7(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 May42(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 Jan143(1)291-9. Epub 2006
Nov10.
CELL COMPONENTS
(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.   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.

• 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.
• 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.

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. PH.D. candidates Phan Quang Nguyen,
Yimo Liu, and Sang Jin Kim for their friendly
support.