Research Experience in Molecular Biotechnology & Genomics
Summer 2010
Center for Integrated Animal Genomics
Plant Science Institute
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
Control
DTT
A
MDC
B
GFPAtATG8e
WHAT IS AUTOPHAGY?
Without DTT
GFP-AtATG8e
DIC (3D)
D
• Does ER stress induce autophagy in plants?
RESULTS
Control
100
18a
80
60
40
20
9
0
GFP-8 GFP-8 +dtt
WT
WT + dtt
With DTT
GFP-AtATG8e
DIC (3D)
- Con
A
18s
Figure 4. RT-PCR analysis of AtATG18a, AtATG9 and 18s
rRNA expression
+ Con
A
RT-PCR was used to test the expression of AtATG18a,
AtATG9 and 18s rRNA genes during control and ER stress
conditions.
(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.
DTT
Control
DTT
A
A
B
B
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.
DTT
120
Figure 1. 2mM DTT induce autophagy
RESEARCH QUESTION
Control
C Quantification of Autophagosomes
Autophagosomes/root section
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 GFPAtATG8e plants to observe if autophagy was
induced. Our results show that 2mM DTT can
induce autophagy.
NaCl
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.
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.
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; 19:411119.
[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; 143:29199.
[9]Yorimitsu T, Nair U, Yang Z, Klionsky DJ. Endoplasmic Reticulum Stress
Triggers Autophagy. Journal of Biological Chemistry 2006; 281:40, 30299-304.
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