Metabolites and proteins involved in systemic resistance

Local infections by pathogens cause resistance in distal tissues and protects them against subsequent pathogen attacks. The resistance in the whole plant is known as systemic acquired resistance (SAR) (Vlot et al. 2021). The resistance in the distal tissue requires mobile signal molecules which are generated at the local infection site and transported to uninfected distal tissues either through the plant body itself or via the air as volatiles (Kachroo and Kachroo 2020; Shine et al.

Symplastic transport of AZI1 to the phloem

Movement of small proteins or metabolites to systemic tissues occurs often via the phloem (Dinant and Lemoine 2010). Uploading of AZI1 to the phloem occurs via the symplastic transport (Lim et al. 2016) and the protein reaches the phloem via sorting signals which direct it from the outer plastid membrane to the endoplasmic reticulum and plasmodesmata which transverse the cell wall and join the adjacent cells. Being a lipid-binding and membrane-bound protein, AZI1 appears to travel to the phloem companion cells via direct membrane–membrane contact sites, which have been identified at the outer plastid membrane, the endoplasmatic reticulum and the plasma membrane. In case of lipid transfer proteins such as AZI1 these contact sites also allow exchange of the bound lipids (Breuers et al. 2011; Wang and Benning 2012; Helle et al. 2013, Cecchini et al. 2015). Lim et al. (2016) demonstrated that two plasmodesmata-localized proteins regulated SAR function in both, signalling and plasmodesmata gating of AZI1. While PLASMODESMATA LOCALIZING PROTEIN1 (PDLP1) interacts with AZI1, is required for endoplasmatic reticulum-specific localization of AZI1, and contributes to the intracellular portioning of the protein, PDLP5, which impairs plasmodesmata permeability and thus transport of AZA to the neighboring cell. PDLP1 interacts with PDLP5 which regulates the symplastic transport and plasmodesmata gating (Lee et al. 2011; Lim et al. 2016). PDLP5 knockout mutants increase and overexpressor lines restrict general plasmodesmata permeability (Lee et al. 2011; Wang et al. 2013). Importantly, the pdlp1 mutants contained reduced AZI1-GFP protein levels, although the azi1-gfp transcript levels were not affected (Lim et al. 2016). This suggest that PDLP1 affect the stability of AZI1. Furthermore, in the pdlp1 mutant, AZI1 was primarily localized to the outer plastid membrane, whereas in wild-type plants, the majority of the protein is located in extraplastidic compartments. The studies by Lim et al. (2016) highlight the importance of PDLP1 for AZI1 stability and its traveling from the plastids to the plasmodesmata.

Jung et al. (2009) showed that AZI1 is important for generating vascular sap that confers disease resistance. AZA and petioles exudates failed to induce systemic immunity in azi1 plants. Pathogen-induced exudates from azi1 were inactive when applied to wild-type plants. Therefore, AZI1 modulates production and/or translocation of a mobile signal(s) during SAR. The AZI1 target in the vascular sap is unknown so far.

AZI1 gene activation and abiotic stress

AZI1 expression is stimulated by exogenously applied AZA (Jung et al. 2009). Whether stimulation of the endogenous AZA levels due to lipid peroxidation under stress in planta is a prerequisite for the AZI1 expression is not known.

The best studied biological stimulus for AZI1 activation comes from Arabidopsis leaf infiltration assays with Pseudomonas syringae (cf. Arabidopsis eFP Browser). Similarly effective is the bacterial effector flg22. Late stimulation of AZI1 expression was also observed after co-cultivation of Arabidopsis seedlings with Hyaloperonospora arabidopidis (cf. Arabidopsis eFP Browser). Relatively little is known about the role of AZI1 for other pathogenic or beneficial plant–microbe interactions, including pathogenic fungi, nematodes, insects, mycorrhizal fungi and beneficial endophytes, although some of them can produce AZA. For instance, AZA is produced by P. syringae (Javvadi et al. 2018) or the root colonizing endophytic fungus Piriformospora indica (Kundu et al. 2022), however, whether microbe-synthesized AZA activates AZI1 in plants or participates in defense priming in plants, is not known.

Besides biotic stress, expression profiles demonstrate that the AZI11 mRNA level responds also to abiotic stress, however functional analyses are often missing. Xu et al. (2011) showed that the AZI1 transcript level, as well as that of its paralog EARLI1 (Zhang and Schäppi 2007), increases after exposure of Arabidopsis seedlings to cold. The increase of the AZI1 mRNA level was slow, since more than 6 h at 4 °C was required for the induction. The mRNA level declined to basal levels when the plants were transferred back to room temperatures. Overexpression of AZI1 resulted in reduced electrolyte leakage during freezing damage, while AZI1 knockdown and knockout lines showed increased tendencies in cellular damage after freezing treatment. When Saccharomyces cerevisiae cells were transformed with AZI1 under the control of GAL1 promoter, the survival rate of yeast cells harbouring AZI1 increased after freezing treatment. This demonstrates that AZI1 might be multifunctional and associated with cold tolerance of Arabidopsis (Xu et al. 2011). The involvement of AZI1 in cold stress adaptation is further supported by expression profiling of mutants manipulated in cold stress-acclimation genes. Similar results were obtained for Thelunsiella salsuginea (Wong et al. 2006).

The ICE-CBF-COR (Inducer of CBF Expression—C-repeat Binding Factor—Cold Regulated) signalling pathway is an important regulator for cold-stress acclimation (Gusain et al. 2023). CBF overexpressors show increased cold tolerance and high levels of AZI1 gene expression (Wong et al. 2006). Likewise, DEHYDRATION-RESPONSIVE-ELEMENT-BINDING PROTEIN1 (DREB1) genes are induced by cold stress, and overexpression of DREB1 induced strong expression of other stress-responsive genes, resulting in increased tolerance to high-salt and freezing stresses (Ito et al. 2006). Among the genes which are up-regulated in the DREB1 overexpressor lines after exposure to cold stress is AZI1 (Maruyama et al. 2004).

Besides cold, AZI1 is involved in salinity stress tolerance. Pitzschke et al. (2014b) showed that azi1 mutants are hypersensitive to salt. At 150 mM salt stress, only 7% of the azi1 mutant seeds, 70% of wild-type seeds, and 90% of the seeds of AZI1 overexpressor lines germinated. Furthermore, AZI1 overexpressors thrived better than the azi1 mutants under high salt conditions. Another example provides mutants in with the salt stress signalling gene ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) was manipulated. The overexpressor lines performed better under salt stress and this was associated with the higher expression levels of AZI1 and EARLI1 (Davletova et al. 2005). Furthermore, exposure of the salt tolerant xero-halophyte Haloxylon salicornicum to salt stress resulted lower stearic acid and palmitic acid levels. Panda et al. (2021) speculated that breakage of lipid membranes might lead to higher accumulation of AZA. In conclusion, AZA is also involved in abiotic stress tolerance in various plant species, such as cold (Davletova et al. 2005) and salt tolerance (Atkinson et al. 2013). When Arabidopsis seedlings are exposed to simultaneous biotic and abiotic stresses, AZI1 was down-regulated in leaves and conferred drought susceptibility when overexpressed (Atkinson et al. 2013). More functional analyses are required to understand the role of AZI1 in abiotic stress responses.

Conclusion and open questions

The AZA/AZI1 pathway is involved in both biotic and abiotic stress responses in plants, and a comparative analysis of both stimuli might be helpful to throw more light on the molecular mechanism of systemic resistance. AZA accumulates in response to lipid peroxidation and Zöller et al. (2012) showed that lipid peroxidation is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species during the interaction of Arabidopsis with P. syringae, i.e. biotic stress. 1O2 was identified as the major cause of lipid oxidation under basal conditions, while LOX2- and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection (Zöller et al. 2012). It remains to be determined, whether all AZI1-mediated biotic and abiotic stress responses are linked to AZA and lipid peroxidation in the plastids. Barely anything is known about the role of AZA in AZI1-dependent abiotic stress responses and whether these responses are restricted to local tissues or operate systemically. Systemic signal propagation induced by abiotic stresses might be agriculturally important, e.g. for crop plants with roots in cold soil and aerial parts exposed to extreme heat. Finally, the role of the secreted AZI1 in the apoplast or at the plasma membrane for systemic immune responses and local abiotic stress responses has not yet been studied. This is particularly interesting since other members of the HyPRP family which are found in the apoplast, participate in abiotic stress responses (Saikia et al. 2020).

The initiation of the AZA/AZI1 signaling at plastids needs to be investigated in more details. AZA is present in roots and shoots, but the plastids and the intraorganellar membrane structure as the site of lipid peroxidation differ substantially in two types of plastids. In both organs, AZI1 has been shown to be associated at least in part with plastids. In particular, in the aerial tissue, HyPRPs are mainly found in epidermal cells. Their plastids play key roles in defense against microbes (cf. Banday et al. 2022). Investigating the role of root plastids for the generation of AZA and the AZA/AZI1 interaction is important for unravelling the function of the signalling compounds in roots. Furthermore, AZI1 and EARLI1 expression is strongly down-regulated in roots upon colonisation by fungi (Banday et al. 2022), whereas this was not observed for other HyPRP genes. This raises the question whether AZI1/EARLI1 might have also other functions in roots, e.g., by controlling root colonisation or entry of fungal hyphae into the roots. Banday et al. (2022) have already demonstrated that HyPRPs regulate the interaction with the plant growth-promoting rhizobacteria Pseudomonas simiae WCS417 in the roots to influence colonization, root system architecture, and/or biomass. Further studies are required to understand the differences in the regulation of these HyPRP genes, as well as function and signalling of the proteins in roots and shoots upon pathogenic and beneficial microbial attacks.

Disruption of galactolipids in the plastid membranes by lipid peroxidation generates breakdown products including the oxo-acid AZA which protrude to the aqueous phase. In particular, during membrane repair, this promotes AZA release from the membrane and mobility to neighbouring membranes, either alone or complexed by AZI1. The role of AZI1 for the movement of AZA between membranes requires further attention. Whether membrane disruption during the oxidative process plays a role for AZA movement, should be investigated.

Based on the current knowledge about the regulation of the pathway, localisation of AZI1 in or at plastids and its trafficking to other cellular membranes are early events that proceed the activation of the systemic movement of the priming signal of AZA/AZI1 to distal tissue (cf. Cecchini et al. 2021). Plastid targeting of AZI1 is promoted by MAPK3/6, which is activated by biotic (Pitzschke et al. 2009) and abiotic stresses (e.g. Li et al. 2014). Besides MAPK3/6 activation, biotic and abiotic stresses also generate ROS (Takata et al. 2020; Rodriguez et al. 2010), which–in turn—promote plastid-association of AZI1 via MAPK3/6 signaling, but also lipid peroxidation in plastids which generates AZA. It would be interesting to know how the AZI1 and AZA generation is coordinated (Fig. 2).

Fig. 2
figure 2

A model highlighting the open questions (yellow) during early events required for the generation of AZA and AZI1 in response to biotic and abiotic stress. For details, cf. text

Full size image

A main question centers around the long-distance transportation of AZA or the AZA signal to systemic tissue. If AZA can travel root-, but not shootwards, one has to postulate different mechanisms for the propagation of the information from the roots to the shoots and from the shoots to the roots. AZA is not water soluble and it is well known that membrane contact sites between plastid envelopes, endoplasmatic reticulum, plasma membrane and membrane material at the plasmodesmata are the sites of exchange of small molecules, including AZA (Andersson et al. 2007; Toulmay and Prinz 2011; Li et al.

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Open Access funding enabled and organized by Projekt DEAL. Funding was received from the Deutsche Forschungsgemeinschaft (CRC1127, project ID: 239748522 to R.O.) PR was supported by the Indian Council of Agricultural Research, New Delhi, India, and the International Max-Planck Research School (Chemical Ecology, Jena).

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  1. Ralf Oelmüller

    Present address: Max-Planck-Institute for Chemical Ecology, Hans-Knöll-Straße 8, D-07745, Jena, Germany

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  1. Matthias Schleiden Institute, Plant Physiology, Friedrich-Schiller University Jena, Dornburger Str. 159, D-07743, Jena, Germany

    Y. N. Priya Reddy & Ralf Oelmüller

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Priya Reddy, Y.N., Oelmüller, R. Lipid peroxidation and stress-induced signalling molecules in systemic resistance mediated by azelaic acid/AZELAIC ACID INDUCED1: signal initiation and propagation. Physiol Mol Biol Plants 30, 305–316 (2024). https://doi.org/10.1007/s12298-024-01420-1

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