Conclusions and Future Directions
Although SO receives far less attention in the field of plant stress biology than other ROS such as hydrogen peroxide (H2O2) and superoxide (O2-), a growing body of evidence indicates that it plays a role in plant responses to diverse challenges, including biotic as well as abiotic stresses. Singlet oxygen can be generated in gaseous form by photosensitive phytoalexins at the plant surface, where it is able to act as a direct defense against insect herbivores and other biotic stressors. In addition, SO accumulates within plant cells in the cytosol, peroxisomes, nucleus, mitochondria, and especially in the chloroplasts. Numerous abiotic and biotic stresses perturb the cell’s photosynthetic machinery, promoting SO generation, and this positions SO well to sound the alarm and activate adaptive responses (Lu and Yao, 2018). SO can also be generated as the result of the enzymatic activities of lipoxygenases or other stress-responsive peroxidases (Chen et al., 2021; Dmitrieva et al., 2020). This highly reactive ROS is an important player in retrograde signaling and is known to reprogram nuclear gene expression through more than one distinct pathway, including sensing by EX1 at the grana margins and signaling via the β-carotene derivative β-CC in the grana core. SO also modulates phytohormone signaling and, via EX1, can activate programmed cell death. While SO signaling is known in some cases to promote plant adaptation to stress, certain necrotrophic plant pathogens appear to have co-opted this response to facilitate the infection, secreting SO-inducing phytotoxins that form lesions and create infection courts for the pathogen. In short, SO plays diverse and important roles in biotic stress responses, and its adaptive significance for the host plant and for the attacker varies among different interaction pairs. Unfortunately, SO accumulation, signaling, and outcomes for resistance or susceptibility have been characterized in very few biotic interactions so far. To better understand the roles of SO in plant biotic interactions, we propose that the biotic stress community has the following research needs:
  1. Improved methods for direct detection and quantification of SO in vivo . Several reviews cover the current options for SO detection in destructive assays and in situ detection (Dmitrieva et al. 2020; Prasad et al. 2018; You et al., 2018). While fluorescent sensors such as Singlet Oxygen Sensor Green (SOSG) are useful tools, uneven penetration of these exogenous sensors represent a significant challenge. SO-responsive reporter gene constructs overcome this issue, but have separate concerns about specificity. Therefore, there is a need for improved SO-specific in vivosensors, to characterize accumulation of this molecule in plant interactions with biotic stressors.
  2. Comprehensive identification of SO-responsive signaling pathways, and elucidation of the relationships among them . The relationships among the known components of SO signaling (e.g. EX1/EX2, β-CC, SAFE1, GUN4, and multiple transcription factors) are not yet well-understood. Moreover, studies suggest the existence of other yet-to-be-identified nodes in SO signaling (e.g. D’Alessandro et al., 2018). As a more comprehensive understanding of the SO response network emerges, it will enable a better understanding of how this network impacts biotic interactions.
  3. Characterization of the effects of different biotic stresses on SO accumulation and the different branches of SO signaling, including comparisons of compatible and incompatible biotic interactions . As yet, the role of SO has been examined in relatively few biotic interactions, and often in an indirect fashion. As a greater number of biotic interactions are considered from the perspective of SO, we anticipate that important commonalities and differences will emerge among interaction types. Within a particular combination of host plant and attacker species, it can be particularly informative to compare compatible and incompatible interactions, which are governed by known determinants of virulence/avirulence in the attacker and/or resistance or susceptibility factors in the host. These comparisons could help identify correlations between SO responses and the outcomes of the interaction.
  4. More precise tools to manipulate SO accumulation in vivo, including control of the timing, dosage, and localization of its generation . Because the consequences of SO accumulation likely vary depending upon the timing, dosage, and location of accumulation, tools are needed to manipulate these variables and assess their consequences. Mammalian cell lines have recently been engineered to express a genetically encoded photosensitizer targeted to specific subcellular compartments that can deliver different doses of SO depending upon light exposure (Liang et al. 2020). The development of similar sensors for use in plants could dramatically advance our understanding of SO signaling, and in particular, the role of different organelles in SO-mediated responses.
  5. Evidence on how promotion or attenuation of SO accumulation or SO signaling pathways, singly and in combination, impact the outcome of plant biotic interactions . For definitive evidence on the adaptive significance of particular signaling events, we ultimately rely on the ability to manipulate these events and test the phenotypic effects of enhancing or abrogating them. For example, null mutations in EX1 and EX2 have been invaluable in identifying the roles of these proteins in SO signaling. Therefore, identifying a more comprehensive set of signaling nodes (#2 above) and developing methods to enhance or inhibit them, in combination with methods to manipulate accumulation of SO itself (#4), are essential steps towards understanding the roles of SO in biotic stress.
In closing, SO signaling is an important emerging area of study in the field of plant stress biology, and advances in this area will likely identify novel mechanisms of plant adaptation to biotic attack in addition to environmental stress. Due to its close relationship with the photosynthetic machinery, SO also is an important linker between primary metabolism and defense. Understanding this linkage is critical to in order to leverage plant defense mechanisms for the protection of crop health and productivity of the face of increasing environmental stresses and changing pest pressures.