Center for Integrated Animal Genomics

1. Research Experience in Molecular Biotechnology & Genomics • Summer 2010 • Plant Science Institute Center for Integrated Animal Genomics Autophagy is associated with ER stress in Arabidopsis thaliana Junmarie Soto1,2, Yimo Liu1,3 and Diane C. Bassham1,3 1 Department of Genetics; Development and Cell Biology; Iowa State University; Ames, IA; 2 Universidad de Puerto Rico Recinto de Humacao; Humacao, PR; 3 Interdepartmental Genetics Program ABSTRACT Autophagy is a protein degradation process in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Previous research showed that nutrient starvation, pathogen infection, oxidative stress, leaf senescence and salt and osmotic stress induce autophagy in plants. Other studies performed in yeast show that Endoplasmic Reticulum (ER) stress can induce autophagy. In this study we tested whether ER stress can induce autophagy in plants. We used 2mM monodansylcadaverine (MDC) DTT treatment to induce ER stress in Arabidopsis thaliana seedlings. We used MDC staining and GFP-AtATG8e plants to observe if autophagy was induced. Our results show that 2mM DTT can induce autophagy. Figure 4. RT-PCR analysis of AtATG18a, AtATG9 and 18s rRNA expression RT-PCR was used to test the expression of AtATG18a, AtATG9 and 18s rRNA genes during control and ER stress conditions. WHAT IS AUTOPHAGY? Figure 1. 2mM DTT induce autophagy  (A) One-week-old WT seedlings were transferred to control MS liquid media or to MS liquid media containing 0.16 M NaCl or 2mM DTT for 4–6 h followed by MDC staining, and observed using fluorescence microscopy. Scale bar = 50 μm. (B) One-week-old GFP-AtATG8e seedlings were transferred to control MS liquid media or to MS liquid media containing 0.16 M NaCl or 2mM DTT for 4–6 h followed by fluorescence microscopy. (C) The number of MDC-stained/GFP-AtATG8e autophagosomes per root section was counted in control conditions and after DTT treatment. (D) One week old GFP-AtATG8e seedlings were transferred to control MS liquid media or MS liquid media containing Conc A treatment or MS liquid media containing DTT or MS liquid media containing DTT plus Conc A treatment for 12 h and observed using a fluorescence microscope. CONCLUSION In this study we confirmed that 2mM DTT can cause ER stress in Arabidopsis plants, and that this ER stress can induce autophagy in wild type and GFP-AtATG8e. We also discovered that KO bzip28, OX bzip28, KO S1P, and OX S1P mutants have constitutive autophagy. RESEARCH QUESTION • Does ER stress induce autophagy in plants? RESULTS REFERENCES [1]Bassham DC. Plant autophagy – more than a starvation response. Curr Opin Plant Biol 2007; 10:587-93. [2]Bassham DC. Function and regulation of macroautophagy in plants. Biochimica et Biophysica Acta 2009; 1793:1397-1403 [3]Contento AL, Xiong Y, Bassham DC. Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein. Plant J 2005; 42:598-608. [4]Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK, Lippincott-Schwartz J. Mitochondria Supply Membranes for Autophagosome Biogenesis during Starvation. Cell 2010; 141:656-67. [5]Liu JX, Srivastava R, Howell SH. An Endoplasmic Reticulum Stress Response in Arabidopsis is Mediated by Proteolytic Processing and Nuclear Relocation of a Membrane-Associated Transcription Factor, bzip28. The Plant Cell 2007; 19:4111-19. [6]Liu Y, Xiong Y, Bassham DC. Autophagy is required for tolerance of drought and salt stress in plants. Autophagy 2009; 5:7, 954-63. [7]Xiong Y, Contento AL, Bassham DC. AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. The Plant Journal 2005; 42:535-46. [8]Xiong Y, Contento AL, Nguyen PQ, Bassham DC. Degradation of Oxidized Proteins by Autophagy during Oxidative stress in Arabidopsis. Plant Physiology 2007; 143:291-99. [9]Yorimitsu T, Nair U, Yang Z, Klionsky DJ. Endoplasmic Reticulum Stress Triggers Autophagy. Journal of Biological Chemistry 2006; 281:40, 30299-304. Figure 2. KO bzip28 and OX bzip28 have constitutive autophagy (A) One-week-old KO bzip28 seedlings were transferred to control MS liquid media and to MS liquid media plus 2mM DTT for 4–6 h followed by MDC staining and observed using fluorescence microscopy. Scale bar = 50 μm. (B) One-week-old OX bzip28 seedlings were transferred to control MS liquid media and to MS liquid media plus 2mM DTT for 4–6 h followed by MDC staining and observed using fluorescence microscopy. Figure 3. KO S1P and OX S1P have constitutive autophagy (A) One-week-old KO S1P seedlings were transferred to control MS liquid media and to MS liquid media plus 2mM DTT for 4–6 h followed by MDC staining and observed using fluorescence microscopy. Scale bar = 50 μm. (B) One-week-old OX S1P seedlings were transferred to control MS liquid media and to MS liquid media plus 2mM DTT for 4–6 h followed by MDC staining and observed using fluorescence microscopy. ACKNOWLEDGEMENTS   I thank my faculty mentor, Dr. Diane C. Bassham and my other research mentor Yimo Liu, Plant Science Institute, for their help. I also thank the NSF-REU program and Dr. Max F. Rothschild, Director, for their support. Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371