1. Stress Responses in Arabidopsis thaliana wrky65 Knockout Plants Julie Bullinga 1, Anthony Conteno 2, Diane Bassham 2 1 Marshalltown Community Schools, Marshalltown, Iowa 50158 2 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. 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. 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 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). 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 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. 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. Figure 1: Stress Responses of wrky65 knockout plants 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. 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. A. WT root tipB. Atg18 root tip C. wrky65-1 root tip 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. 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. 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. (Contento) CELL COMPONENTS