Blown out of the water: mutation in calcium transporter CAX1 provides anoxia tolerance in Arabidopsis Free

The increasing frequency and intensity of flooding events worldwide results in an urgent need for flood-resistant crops (Huang et al., 2022). To create this new generation of crops, we need to understand in detail how plants detect, respond, and eventually survive flooding in natural systems.

During flooding, light and oxygen availability drops dramatically while the gaseous hormone ethylene rapidly accumulates due to reduced gas diffusion under water. The perception of low oxygen and high ethylene levels by plants triggers the stabilization of group VII ethylene response factors (ERFVIIs), known to induce hypoxia-responsive genes and thereby improving plant survival (Hartman et al., 2019). Although the molecular components behind ERFVIIs-mediated flooding tolerance are well characterized, calcium signaling has also been proposed to regulate low oxygen stress responses, even though its exact role remains unclear (Huang et al., 2022).

In this issue of Plant Physiology, Yang et al. (2022) investigated the role of CAX genes (H⁺/Ca eXchangers) in regulating plant responses to low oxygen conditions in Arabidopsis (Arabidopsis thaliana). To reveal a potential role of these calcium transporters in low oxygen stress acclimation in plants, the authors carried out a thorough analysis of cax mutants using elegant physiological experiments as well as state-of-the-art-omics and calcium imaging techniques. Strikingly, cax1 mutants displayed an astonishing anoxia survival capacity compared to wild-type Col-0 plants. Using a GasPak anaerobic system, the authors showed that 80% of the cax1 plants fully recovered after anoxia stress compared to less than 5% of Col-0. The increased survival of cax1 mutants was also observed after complete submergence where cax1 mutants did not show visible symptoms of prolonged hypoxia stress. Interestingly, the enhanced survival in cax1 mutants was lost in CAX1 complementation lines (cax1::sCAX1 in the cax1 background), clearly showing that CAX1 mediates tolerance to anoxia and submergence.

Next, since anoxia stress is associated with the induction of reactive oxygen species, the authors wondered whether cax1 mutants are impaired in their anoxia-mediated stress responses. Yang et al. (2022) showed that cax1 mutants display lower malondialdehyde levels, used as a proxy for oxidative-stress-induced lipid peroxidation, and lower levels of hydrogen peroxide (H₂O₂) as compared to the control plants. Furthermore, trypan blue staining revealed that cax1 mutants had significantly lower cell death post anoxia. These results nicely show that cax1 mutants exhibit dampened anoxia-mediated stress responses, likely associated with the greater survival of the mutant compared to Col-0.

To identify the molecular processes involved, which could explain differences in survival between the sensitive lines (Col-0) and the tolerant line (cax1), Yang et al. (2022) performed a transcriptomic analysis on Col-0 and cax1 mutant leaves before, during, and after anoxic conditions. Strikingly, ∼50% of the genes induced in cax1 mutants pre-anoxia were induced only during anoxia or post-anoxia in Col-0, indicating a certain degree of priming for anoxia tolerance in cax1 mutants. In addition, 17 of the 49 known core hypoxia-regulated genes, usually targeted and induced by ERFVIIs, were expressed in cax1 even under aerobic conditions. These results point towards the role of CAX1 in the regulation of hypoxia-regulated genes independently of ERFVIIs (ERFVIIs being degraded in the presence of oxygen). Also, cax1 mutants showed specific modulation of genes associated with (photo)oxidative stress, defense responses, metabolic processes, growth-related processes, and ion transport; all of which were absent in Col-0. Yang et al. (2022) also performed a proteomic analysis highlighting very distinct proteome reprogramming in response to anoxia stress with only a slight overlap between Col-0 and cax1. While these data are still relatively complex to interpret, the authors provide interesting datasets and insights toward the role of calcium transporters in plant anoxia tolerance.

Finally, the authors investigated the relationship between changes in cytosolic calcium and anoxia tolerance. Since CAX genes primarily transport and sequestrate calcium ions (Ca²⁺) from the cytosol to the vacuole, cax1 mutants likely display abnormal distribution of Ca²⁺, which in turn influences anoxia responses. To test this, Yang et al. (2022) generated stable Col-0 and cax1 lines expressing the calcium biosensor GCaMP3, a GFP-based fluorescent protein featuring a Ca²⁺-sensitive calmodulin domain. Under normal conditions, Ca²⁺ dynamics were similar between Col-0 and cax1, while they strongly differed post anoxia. The altered Ca²⁺ homeostasis in cax1 mutants likely facilitates reoxygenation and thereby increases plant survival post anoxia. In addition, cax1 mutants displayed higher calcium levels in the meristem and newly-forming leaves compared to Col-0, while the opposite was observed in older leaves. This tissue specificity is intriguing and requires further investigation.

The blooming calcium research field indicates the involvement of calcium signals in a myriad of environmental responses in plants, such as temperature, plant–microbe interactions, plant–plant competition and now low oxygen responses (Aldon et al., 2018; Huang et al., 2022; Pantazopoulou et al., 2022; Yang et al., 2022). Overall, Yang et al. (2022) provides an in-depth mutant analysis and identified the calcium transporter CAX1 as a negative regulator of anoxia and submergence tolerance in Arabidopsis. While the exact molecular players behind the cax1-mediated anoxia tolerance are still unknown, the authors reveal a clear role for calcium signaling in plant responses to anoxic conditions.

Interestingly, the authors did not find a clear phenotype for cax1 in response to waterlogging stress, which is intriguing and surprising since recently published data show that hypoxia and ethylene downregulate CAX1 as well as other CAX genes in Arabidopsis roots, resulting in increased hypoxia tolerance similar to that of cax1 mutants (Liu et al., 2022; see https://utrecht-university.shinyapps.io/2022Liu2022/). It would be interesting to further investigate the role of CAX genes in Arabidopsis roots upon submergence and waterlogging in future studies. In addition, the improved anoxia survival in cax1 mutants has great implications for agriculture and could become an important engineering target for crops often suffering from flash floods.

Conflict of interest statement. None declared.

References