A new insight into the contribution of putrescine to defense in Arabidopsis thaliana

ABSTRACT

Polyamines are known to accumulate in response to stress. Compelling evidence indicate a protective role for polyamines during defense. However, signaling pathways underlying polyamine functions have not been fully elucidated. We recently found that the polyamine putrescine (Put) accumulates during effector triggered immunity (ETI). Treatment with Put triggered local and systemic transcriptional reprogramming partly overlapping with systemic acquired resistance (SAR) responses. In addition, Put treatment led to local salicylic acid (SA) accumulation and systemic defenses against virulent bacteria. Consistent with this, we found that Put signaling is mainly ROS dependent and partly compromised by ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) and NONEXPRESSOR of PR GENES1 (NPR1) loss-of-function mutations. Here, we propose a preliminary model by which putrescine contributes to local and systemic defenses in Arabidopsis thaliana.

Polyamines are small aliphatic amines ubiquitous in living cells. In nature, the most abundant polyamines are putrescine (Put), spermidine (Spd) and spermine (Spm). Polyamines, and Put in particular, accumulate in response to various stresses. In general, increases in polyamine levels have been associated with abiotic and biotic stress resistance.^1,2 Arginine decarboxylation catalyzed by arginine decarboxylase (ADC) is a limiting step for Put biosynthesis in Arabidopsis thaliana (hereafter Arabidopsis).³ Arabidopsis carries two genes encoding arginine decarboxylase (ADC1 and ADC2), which exhibit differential expression regulation in response to different stresses.^4–7 Put content is also conditioned by the activity of copper-containing amine oxidases (CuAO), which oxidize the primary amino groups of polyamines generating the corresponding aldehydes, H₂O₂ and NH₄^+.8–11 ROS production through polyamine oxidation has been proposed to underlie many polyamine functions, including defense signaling.⁹ Despite the many studies about the biological significance of polyamine accumulation during stress, polyamine signaling pathways remain elusive.

Recent work by Liu et al.¹² provides a new insight into polyamine signaling through the identification of genes transcriptionally responsive to different polyamines, some of which carry identical charges (diamines Put and Cad; triamine Spd; tetraamines Spm and tSpm). Twelve-day-old Arabidopsis seedlings were treated with 100 μM of each polyamine for 1 h. Global gene expression analyses indicated that these compounds, rather than being mere stress marker metabolites, elicit stress signaling including biotic responses. Among the polyamines tested, Put exhibited the largest number of differentially expressed genes. Many deregulated genes were common between polyamines, although quantitative differences were evidenced. Importantly, no correlation was found between transcriptional responses and polyamine charge. Most polyamine responsive genes were related to stress, defense, elicitation and hypoxia. We hypothesized that H₂O₂, a common product of polyamine oxidation, might underlie the similar transcriptional responses observed. Indeed, 2´,7´-dichlorofluorescein diacetate (DCFDA) and 3–3´-diaminobenzidine (DAB) staining evidenced ROS production in roots and leaves of Arabidopsis plants treated with different polyamines. To further investigate the importance of H₂O₂ in polyamine signaling, we focused on Put, which level increases during defense.¹³ Co-treatment of Put (100 μM) with the H₂O₂ scavenger dimethylthiourea (5 mM) suppressed most transcriptional responses to the polyamine. We concluded that Put elicits ROS-dependent transcriptional reprogramming. Interestingly, transcriptional responses elicited by Put were partly compromised in ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) and NONEXPRESSOR of PR GENES1 (NPR1) loss-of-function mutants, which pointed to the requirement of a functional EDS1, salicylic acid (SA) and NPR1-dependent pathway for full expression of polyamine responses. Consistent with this, Put triggered SA accumulation in local but not distal leaves, whereas SA or its analogue benzothiadiazole S-methyl ester (BTH) had no obvious effects on polyamine levels.

We previously found that Put accumulated in response to the purified PAMP flg22 and Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC mutant, which is defective in the type three secretor system.¹³ In the work by Liu et al.¹², we further studied whether effector triggered immunity (ETI) was also associated with changes in polyamine levels by inoculating with Pst DC3000 expressing the AvrRpm1 effector. Polyamine levels evidenced a significant accumulation of Put in local but not systemic leaves of wild-type plants inoculated with Pst DC3000 AvrRpm1. This response was also observed in eds1-2, sid2-1 and npr1-1 mutants. Importantly, Put also accumulated in pathogen-free assays using transgenic lines expressing estradiol-inducible AvrRpm1.¹² We concluded that local Put accumulation is part of the ETI metabolic response.

Having found that Put accumulates in local leaves during ETI and elicits local SA accumulation, we investigated whether Put responses were transmitted to systemic tissues. Transcriptional analyses of local leaves infiltrated with Put (500 µM) and systemic (untreated) leaves identified a strong overrepresentation of gene ontology (GO) terms related to defense, systemic acquired resistance (SAR) and response to SA in both tissues. To further investigate the potential contribution of Put to the establishment of SAR, local leaves were pre-inoculated with Put (500 μM) or mock (5 mM MES pH = 5.7) before inoculation of distal leaves with virulent Pst DC3000 bacteria. Bacteria growth quantitation¹⁴ indicated that Put pretreatment enhanced resistance in systemic tissues. This systemic response was compromised in eds1-2, sid2-1 and npr1-1 mutants, consistent with the requirement of an EDS1/SA/NPR1-dependent pathway for Put responses. Given the importance of H₂O₂ production for polyamine signaling, we finally tested the potential contribution of CuAOs to systemic defenses. In Arabidopsis, putrescine can be oxidized by CuAOs producing H₂O₂.¹ Among CuAOs, A. thaliana AMINE OXIDASE1 (ATAO1/CuAOβ) and CuAO1-3 (CuAO γ1, α3 and ζ) exhibit high affinity for Put.^11,15,16 Systemic resistance was determined through quantitation of Pst DC3000 bacteria growth in systemic (distal) leaves of plants locally pre-treated with Put (500 µM) or the avirulent SAR-inducing Pst DC3000 AvrRpm1. The analysis of Put and Pst DC3000 AvrRpm1-triggered systemic resistance in copper amine oxidase mutants (atao1-3, cuao1-3, cuao2-1 and cuao3-1) suggested that the different CuAOs additively contribute to Put-triggered systemic responses.

Overall, the work by Liu et al.¹² highlights the importance of polyamines in defense with a focus on Put. The work provides evidence for the involvement of a ROS/EDS1/SA/NPR1 dependent pathway for Put signaling, which involves local and systemic transcriptional reprogramming contributing to defense (Figure 1). A more detailed analysis of upstream and downstream events required for polyamine signaling, as well as the identification of signals transmitted from local to systemic tissues is required for a deeper understanding of polyamine functions in plants. Such fundamental studies might provide new leads for plant protection against disease.

Figure 1.

Proposed model for the contribution of put to defense. Inoculation with Pseudomonas syringae pv. tomato DC3000 expressing the AvrRPM1 effector (Pst DC3000 AvrRpm1) triggers PAMP-triggered immunity (PTI) and effector triggered immunity (ETI), which leads to increased ADC2 expression and Put accumulation in local tissues. Put is then oxidized by action of copper-containing amine oxidases, thus producing 4-aminobutanal, hydrogen peroxide (H₂O₂) and ammonia (NH₄⁺). The hydrogen peroxide produced by Put oxidation is required for the deregulation of polyamine responsive genes, which are also partly dependent on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) and NONEXPRESSOR of PR GENES1 (NPR1) defense components. Deregulated genes are related to defense, elicitation, stress responses and hypoxia. Put accumulation in local tissues triggers an unknown signal which is transmitted to distal leaves, leading to gene expression changes partly overlapping with SAR. This response is associated with enhanced disease resistance in systemic tissues against pathogenic bacteria (Pst DC3000)

Funding Statement

This work was supported by the BFU2017-87742-R grant of the Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia (Ministerio de Economía y Competitividad, Spain), the Agencia Estatal de Investigación (AEI, Spain) and the Fondo Europeo de Desarrollo Regional (FEDER).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.