1. Salicylates – plant hormones and painkillers
Salicylic Acid
Acetylsalicylic Acid
ASPIRIN
Named for Spiraea, the former name for meadowsweet
Named for white willow Salix alba
Photo credits: Geaugagrrl; Sten Porse

2. How do salicylates work in plants?
SA’s effects in plants and animals are unrelated
Expression of pathogen-induced genes
Increased synthesis of SA
Signal (e.g. Pathogen, UV light, developmental cues)
DEFENSE
Stress responses
Developmental responses
In plants SA’s main effect is to alter patterns of gene expression

3. History of salicylate research
In 1990 SA was shown to be an endogenous signal in defense responses
Hippocrates wrote about the use of willow to relieve pain ~ 2400 years ago
Untreated side
In 1987 endogenous SA was shown to be responsible for heat production in Arum lily flowers
SA treated
In 1979 White showed that pre-treatment of a leaf with aspirin or SA conferred resistance to tobacco mosaic virus (TMV)
National Library of Medicine; Raskin, I. (1992). Salicylate, a new plant hormone. Plant Physiol. 99:

4. Lecture outline Synthesis, conjugation and transport
Perception and signaling
Salicylates in whole-plant processes
Image credit: Prof. Dr. Otto Wilhelm Thomé Flora von Deutschland, Österreich und der Schweiz 1885, Gera, Germany

5. There are two pathways for SA synthesis, via ICS and PAL
Cytoplasm
ca. 5% total
Chloroplast
ca. 95% total
The major route for the biosynthesis of SA (up to 95% of total) takes place in the chloroplast. One of the key enzymes is ICS, isochorismate synthase
PAL
ICS
A small amount of SA is derived from cinnamic acid produced by PAL in the cytoplasm
Reprinted with permission from Chen, Z., Zheng, Z., Huang, J., Lai, Z. and Fan, B. (2009). Biosynthesis of salicylic acid in plants. Plant Signaling & Behavior. 4: ; see also Reprinted from Métraux, J.-P. (2002). Recent breakthroughs in the study of salicylic acid biosynthesis. Trends in Plant Science 7: with permission from Elsevier. .

6. In many plants ICS1 is upregulated by pathogen exposure
Expression of ICS1 increases upon exposure to a pathogen
PR1 is an SA-induced pathogen responsive gene
In the top experiment, -Erysiphe on the left is without pathogen, +Erysiphe on the right is with pathogen. The experiment was done in triplicate and shows results from each independent experiment three separate lanes.
On the lower panel, hpi is hours post infection. Expresison of two genes, ICS1 (biosynthesis of SA) and PR1 (response to SA) are shown as solid and white bars respectively.
Reprinted by permission from Macmillan Publishers Ltd. Wildermuth, M.C., Dewdney, J., Wu, G., and Ausubel, F.M. (2001). Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414: copyright 2001.

7. Regulation of SA accumulation
Other stress (e.g. UV light)
Bacterial pathogen
Pathogen effectors
SA synthesis is induced by pathogens or stress. Genetic studies have identified some of the signals that transduce pathogen perception to SA synthesis
Fungal or oomycete pathogen
ICS1 SA

8. Perception and signalingSA PR
SA induces an enormous transcriptional response
PR genes are pathogenesis related genes, and are involved in defense responses
Ward, E.R., Uknes, S.J., Williams, S.C., Dincher, S.S., Wiederhold, D.L., Alexander, D.C., Ahl-Goy, P., Metraux, J.P. and Ryals, J.A. (1991). Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell. 3:

9. NPR1 is a major activator of SA-mediated responses
Wild-type
npr1 (aka nim1)
NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1) is necessary for defense responses and at the core of SA signal transduction
The plant on the right is supporting a lot of pathogen growth, which appears as a white fuzz on the leaf surfaces (the disease caused by this pathogen is called downy mildew).
Plants were treated with SA, then three days later challenged with a fungal pathogen (Hyaloperonospora arabidopsidis)
Delaney, T.P., Friedrich, L., and Ryals, J.A. (1995). Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proc. Natl. Acad. Sci. USA 92: copyright 1995 National Academy of Sciences USA.

10. NPR1 oligomerizes via redox-sensitive cysteinesHS SH SS
Reduction
Oxidization
SA accumulation contributes to a reducing environment, which causes multimeric NPR1 to monomerize
NPR1 SA SA SA
NPR1
Then panel on the right shows confocal images of NPR-GFP fluorescence in mesophyll cells, with GFP fluorescence shown in the green channel and differential interference contrast in the red channel.
Monomeric NPR1 is imported into the nucleus (here it is GFP-labeled)
Kinkema, M., Fan, W., and Dong, X. (2000). Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell 12:

11. NPR1, NPR3 and NPR4 have recently been reported as SA receptors
In mid-2012, two groups reported that members of the NPR family serve as SA receptors
The two groups used different assays to characterize SA binding, leading to different conclusions
NPR3/4 bind SA and regulate NPR1
NPR1 is regulated directly by SA binding
and / or
Fu, Z.Q., Yan, S., Saleh, A., Wang, W., Ruble, J., Oka, N., Mohan, R., Spoel, S.H., Zheng, N. and Dong, X. (2012) NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486: 228–232.

12. In the nucleus, SA-activated NPR1 promotes transcription
NO SA
+ SA
-TGAs
-WRKYs
+TGAs
+WRKYs
In the absence of SA and activated NPR1, negative regulators repress defense genes
In the presence of SA and activated nuclear NPR1, positive regulators activate defense genes

13. NPR1 binds TGA transcription factors and promotes DNA binding
DNA binding of TGA2 is enhanced by NPR1
The Arabidopsis genome encodes 10 TGA factors. Some are positive and some negative regulators of defense genes
NPR1
+TGAs
NPR1
-TGAs
-TGAs
+TGAs
Bound
DNA
Yeast-two hybrid assay showing NPR1/ TGA interaction
+TGAs
Free
DNA
Zhang, Y., Fan, W., Kinkema, M., Li, X., and Dong, X. (1999). Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc. Natl. Acad. Sci. USA 96: ; Despres, C., DeLong, C., Glaze, S., Liu, E., and Fobert, P.R. (2000). The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell 12:

14. Signaling downstream of SA - summary
Questions remain
Questions remain ?
NPR1
NPR3/4
Activating histone marks ?
+WRKY
-WRKY
DNA recombination / repair?
Kesarwani, M., Yoo, J. and Dong, X. (2007). Genetic interactions of TGA transcription factors in the regulation of pathogenesis-related genes and disease resistance in Arabidopsis. Plant Physiol. 144:

15. Salicylates and whole plant physiology
Responses to pathogens
Recognition
Local and hypersensitive response
Systemic acquired resistance
Cross-talk
Other functions
Abiotic stress
Thermogenesis

16. Pathogen Triggered Immunity involves SA synthesis and response
1. PAMPs perceived by PRRs
PAD4 / EDS1 SA
ICS1
3. NPR1 mono-merization and nuclear import
NPR1
2. Activation of ICS1 and increased SA synthesis
NPR1

17. Effector Triggered Immunity involves SA synthesis and response
R protein SA
4. Recognition of effectors directly or indirectly by R proteins and increased SA synthesis
NPR1
NPR1

18. Salicylates are indispensible signals for local and systemic resistance
SA-deficient plants are deficient in local and hypersensitive responses (HR) and systemic acquired resistance (SAR) SA
Mobile signal HR

19. Local responses – hypersensitive responseSA
ROS accumulation
Cell death,
PR genes induced
Wall fortification
Pathogen perception triggers a local hypersensitive response that includes an oxidative burst, wall fortification, induction of PR genes and cell death

20. Systemic acquired resistance was described in TMV-infected tobacco
This plant was not pre-inoculated with TMV and shows large lesions upon TMV inoculation
This experiment demonstrates systemic signaling
This plant was pre-inoculated with TMV on the three bottom leaves. Seven days later two upper leaves were inoculated. The upper leaves show tiny lesions indicating an enhanced resistance response
Reprinted from Ross, A.F. (1961). Systemic acquired resistance induced by localized virus infections in plants. Virology 14: with permission from Elsevier.

21. Systemic resistance may involve multiple signalsSA
SAR
MeSA
AzA
SA is necessary in systemic tissue for SAR, but the nature of the mobile signal(s) is still up in the air
It is likely that multiple signals contribute to SAR
3PG
3PG XY
DIR1, lipids

22. Heat production in arum lilies involves SA synthesis
Blowflies on Helicodiceros muscivorus, the dead horse arum
Some arum lilies smell like rotting flesh, attracting flies and carrion-eating insects as pollinators. They produce heat to carry the odor and mimic a decaying corpse
There are some fun videos of arum lilies on the Teaching Tools YouTube channel (
Titan arum
(Amorphophallus titanum)
Photo credit: U.S. Botanic Garden

23. Ongoing studies - Regulation of defense transcriptome
What is the inter-relationship between different SA receptors?
How does cellular redox transmit SA signals, and vice versa?
NPR1
NO SA
+ SA
-TGAs
-WRKYs
+TGAs
+WRKYs
NPR1
NPR3
NPR4
SNI1
How does chromatin remodeling contribute to the regulation of SA-responsive genes?
What about the many other genes that affect SA signaling and response?

24. Ongoing studies- Systemic signals and signaling
SAR
Systemic acquired resistance – What other signals are involved? How much information is conveyed? How is resistance maintained for long periods? SA
Mobile signal(s)
Local response –
What are the respective roles of SA and redox in the hypersensitive response? HR