Salicylic acid and plant defense

Overview:  Induced Salicylic Acid Accumulation and Response
The phytohormone salicylic acid, 2-hydroxybenzoate, has long been known to accumulate in plants in response to diverse pathogens including viruses, bacteria, and fungal biotrophs and to be required for both local and systemic resistance responses.  Because phytohormones control critical biological processes, the concentration and locale of active phytohormone is tightly regulated, with both synthesis and modification of the phytohormone playing important roles (Wildermuth, COPB 2006). Compared with other phytohormones, such as the growth regulator indole acetic acid (IAA), our understanding of the synthesis, chemical modification, and regulation of SA, as well as its receptor(s), specific mode(s) of action, and impacted genes and processes is quite limited.  Here at UC Berkeley, my laboratory has built on my previous finding that the bulk of pathogen-induced SA is synthesized from chorismate via isochoristmate synthase 1 (ICS1) in Arabidopsis (Wildermuth et al. Nature 2001) to (i) explore the transcriptional impact of SA and the regulatory circuitry of its response and (ii) identify biochemical control points impacting SA accumulation and activity.  

Ultimately, our research may facilitate crop tolerance of (a)biotic stress, our understanding of the mechanisms of action of aspirin (active agent:  salicylate) in humans, and the impact and integration of stress hormones with those mediating growth and development.

SA-impacted genes, processes, and circuitry of response
Arabidopsis defense responses mediated by SA are important in limiting the extent of powdery mildew growth (Dewdney et al. Plant Journal 2000; Wildermuth et al. Nature 2001).  To define the transcriptional impact of SA in this system, replicated global expression profiling was performed and two novel statistical methods developed in collaboration with Terry Speed (UC Berkeley, Statistics) were employed.  The multivariate empirical Bayes statistic was designed for the analysis of replicated time series expression data and takes into account replication, moderation, and the relationship between samples over time (Tai and Speed, Annals of Statistics 2006; Chandran et al. Plant Physiology 2009).  The innovative adaptive model building procedure for cis-acting regulatory element identification uses multivariate time series expression data and the relative distance between cis-acting regulatory elements to identify individual and interacting motifs of significance within and across principal components (Zhang, Wildermuth, and Speed, Annals of Applied Statistics 2008).  

We found SA synthesis via ICS1 has an extensive transcriptional impact, altering the expression of ~4% of profiled genes, and identified genes with distinct and previously unresolved patterns of SA-impacted or SA-independent temporal expression (Chandran et al. Plant Physiology 2009; Zhang, Wildermuth, and Speed, Annals of Applied Statistics 2008).  This allowed us to identify known and novel SA-impacted processes and process components as well as to elucidate the regulatory circuitry of the host response.  For example, cis-acting regulatory elements bound by ethylene responsive factors (ERFS) were associated with cross-talk between SA and ethylene/jasmonic acid (JA) signaling pathways in powdery mildew, consistent with findings for the tomato ERF Pti4 (Gu et al., Plant Cell 2002) and AtERF1 (unpublished).  Furthermore, the enhanced sensitivity achieved using replicated time series data with these powerful statistical methods allowed us to identify regulators with altered expression in whole leaves that were previously below selection thresholds.  For instance, we identified PUX2, a plant ubiquitin regulatory X domain-containing protein, as an SA-induced novel regulator of powdery mildew resistance, with pux2 mutants exhibiting enhanced resistance to powdery mildew (Chandran et al., Plant Physiology 2009).  

In contrast to the extensive transcriptional response associated with the robust activation of SA synthesis and response in the later stages of a powdery mildew infection (above), a distinct and more limited transcriptional response is associated with low levels of SA.  To investigate regulators and processes responding to low levels of SA, as well as SA perception, my laboratory has developed a primary root inhibition assay (Jones and Wildermuth).   The associated phenotype is independent of NPR1 allowing us to identify components of SA signaling, and response that are independent of the master regulator NPR1.

Biochemical control over SA accumulation and activity
To examine biochemical control points impacting SA accumulation and activity, my laboratory first focused on the AtICS1 (aka. EDS16, SID2) enzyme.  We established that Arabidopsis ICS1 acts as a monofunctional isochorismate synthase and not a bifunctional salicylic acid synthase (i.e. bacterial enzyme Irp9) and that it is localized to the plastid stroma where chorismate is synthesized (Strawn et al., Journal of Biological Chemistry 2006).  We further determined that ICS1 operates near equilibrium with an affinity for chorismate that would allow it to compete effectively with other pathogen-induced chorismate-utilizing enzymes such as AtASA1 and AtCM1 that channel flux to indolic compounds and phenylpropanoids, respectively.  Unlike those enzymes whose activities are allosterically modulated by the aromatic amino acids, there is no evidence for allosteric or other post-translational control over ICS1 activity.  Therefore, ICS1 transcription likely drives flux to SA and modification of SA likely exerts fine control over its activity.

Phytohormone modification via glycosylation, methylation, and amino acid conjugation can alter the activity, stability, and transport of the phytohormone, profoundly impacting phytohormone function and metabolism (Wildermuth, Current Opinion in Plant Biology 2006).  Though phytohormone amino acid conjugation of auxin and jasmonate by GH3 family enzymes play critical roles in either repressing (e.g. IAA) or activating (e.g. JA) the expression of downstream responses, little was known about a possible role for amino acid conjugation of SA.  In collaboration with Roger Innes (University of Indiana) we identified a novel regulator of SA metabolism, the GH3 acyl adenylate thioester-forming enzyme GH3.12 referred to as PBS3 (Nobuta et al., Plant Physiology 2007).  We found T-DNA and EMS mutants in PBS3 are compromised in total SA accumulation, the induction of the SA-dependent marker gene PR1, and resistance to pathogens with exogenous SA application rescuing these phenotypes.  Similar findings were independently reported by the groups of Jean Greenberg and Ramesh Raina.
To define the function of PBS3, my laboratory performed detailed biochemical and kinetic analyses.  We showed PBS3 acts as a 4-substituted benzoic acid amino acid synthetase in vitro, with SA competitively and reversibly inhibiting this activity (Okrent et al., Journal of Biological Chemistry 2009).  Notably, this is the first report of competitive inhibition of a GH3 enzyme and presents a novel mechanism that could be used to rapidly and reversibly fine-tune phytohormone activity mediated by amino acid conjugation.  Taken together with our previous results, this led us to hypothesize that PBS3 acts upstream of SA biosynthesis to prime (or activate) its synthesis, with PBS3 activity inhibited by SA once sufficient SA has accumulated.  Mechanistically, how does PBS3 act to limit SA accumulation and downstream gene expression?  Ongoing work in the laboratory is exploring this question using two complementary approaches:  (i) small molecule profiling to detect potential in planta substrates or products of PBS3 and (ii) systematic evaluation of the impact of the pbs3 mutation on SA synthesis and downstream response using a system developed in my laboratory to uncouple these responses.

Broader Impacts of Research on Salicylic Acid
A detailed knowledge of SA metabolism and response informs our understanding of disease outcomes in an agricultural context.  This knowledge can be used to investigate the function of agrochemicals and their in planta metabolites, as we did in collaboration with John Casida’s group at UC Berkeley (Ford et al. PNAS 2010), or to better predict disease (and yield) outcomes as environmental conditions change (i.e. with climate change).

In addition, salicylic acid regulation and function in plants can also inform our understanding of the wonder drug aspirin (acetylsalicylic acid) which is converted to SA in vivo.  Similar to its role in inhibiting prostaglandin synthesis in mammals thus reducing inflammatory responses, SA in plants inhibits synthesis of a prostaglandin-like compound, the hormone jasmonic acid.  Furthermore, SA in plants can facilitate apoptosis and impact cell division, functions which could be particularly relevant to a role for aspirin in cancer prevention.

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