NIN-like Protein7 is controlled by oxygen and nitric oxide and contributes to stress tolerance through PROTEOLYSIS6

Since the initial discovery of ethylene as a biologically active gaseous molecule in plants by Neljubov in 1901 (Kende, 1998), over a century of research has highlighted the importance of gas signaling for organisms to control molecular and physiological responses. Oxygen (O₂) and nitric oxide (NO) are two gases critical for both plant development and stress responses and are sensed through a PROTEOLYSIS6 (PRT6)-dependent pathway of proteolysis (Holdsworth and Gibbs, 2020). When O₂ and NO are plentiful, certain proteins sensitive to levels of these gasses are degraded through proteolysis. This occurs through O₂- and NO-dependent modifications that ultimately lead to ubiquitination and proteasomal degradation by the E3 ligase PRT6 (Licausi et al., 2011; Gibbs et al., 2014). However, when either the level of NO or O₂ drops (hypoxia) or when PRT6 is genetically removed, target proteins are no longer ubiquitinated, become stable, and can activate a wide variety of downstream responses that include slower vegetative growth and enhanced stress responses (Holdsworth and Gibbs, 2020). This PRT6-dependent proteolytic pathway therefore allows plants to couple the cellular levels of O₂ and NO into appropriate responses.

The specificity of protein substrates of this so-called “PRT6 N-degron pathway” partially depends on a conserved N-terminal amino acid sequence with a cysteine (which may be oxidized through O₂ or NO) in the second position. Since the identification of the first plant-specific substrates in 2011, a quest to identify additional substrates has ensued (Holdsworth and Gibbs, 2020). The NIN-like protein7 (NLP7) transcription factor is a principal activator of nitrate responses and controls nitrate reductase-mediated NO biosynthesis from nitrate (Marchive et al., 2013). In addition, NLP7 holds a cysteine in its second N-terminal amino acid sequence, making it a potential target of the PRT6 N-degron pathway.

In this issue of Plant Physiology, Castillo et al. (2021) examine if and how PRT6 controls NLP7 function and how these proteins together contribute to plant growth, nitrate metabolism, NO homeostasis, and multiple physiological responses. Because mutations in NLP7 and PRT6 affect the production and sensing of NO, respectively, the authors crossed prt6 and nlp7 Arabidopsis (Arabidopsis thaliana) single mutants to create prt6nlp7 double mutants. In addition, they produced NLP7 complementation lines in the nlp7 single and double mutant background in which the NLP7 expression level and/or NLP7 protein stability were enhanced relative to wild type. The authors observed that the typical slower vegetative growth in prt6 was further amplified in prt6nlp7, but that any type of NLP7 complementation restored growth back to wild-type levels. Moreover, the authors showed that NO levels were lower in the nlp7 mutant, higher in prt6, and that the double mutant showed even higher NO levels than prt6, suggesting that NLP7-controlled nitrate reductase is not the only driving source of NO in these lines. Surprisingly, NLP7 complementation could not restore the high NO levels detected in prt6-1nlp7-1 plants back to wild-type or even prt6 levels. Together these results suggest that while NLP7 complementation is functional to restore growth in the prt6nlp7 double mutant, the slower growth is unlikely to be caused by the higher endogenous NO levels but possibly by a reduction in PRT6-dependent NO sensing.

In theory, if a phenotype is caused by potentially stabilized NLP7 in the prt6 background, one would expect that removal of NLP7 in the prt6nlp7 double mutant would at least partially reverse this phenotype. However, similar to the slower growth in prt6, the authors typically observed the reverse when looking at multiple physiological responses, in which the prt6nlp7 double mutant further amplified the prt6 phenotype. By comparing publicly available datasets (Marchive et al., 2013; Gibbs et al., 2014), they identified common genes regulated by both NLP7 and PRT6 that included germination-, sucrose-, and abscisic acid (ABA)-driven transcriptional signatures. When they subsequently looked at germination rates under high sucrose and ABA, prt6 germinated more poorly than the wild type and nlp7, whereas prt6nlp7 germinated much more poorly compared to prt6. Similar responses were found for flooding tolerance, in which prt6 was more sensitive to flooding stress and became even more sensitive when NLP7 was also removed. Collectively, these results suggest that NLP7 is unlikely to be a PRT6 target but is essential for enhanced tolerance to sucrose, ABA, and submergence stress in prt6 mutants, but not when PRT6 is functional.

To test whether NLP7 is a true target of the PRT6 N-degron pathway, the authors crossed their NLP7 reporter lines to a prt6 mutant and subsequently assessed both NLP7 steady states and turnover rates. Surprisingly, no differences in NLP7 stability were found between prt6 and the wild-type background, suggesting that NLP7 is not directly controlled by the PRT6 N-degron pathway. However, they could show that NLP7 turnover depends on both O₂ and NO as the proteins became more stable as the levels of these gases declined. While such O₂- and NO-dependent protein dynamics are typical for substrates of the PRT6 N-degron pathway, additional evidence ruled out a PRT6-dependent pathway when looking at the dynamics of both N- and C-terminally GFP-tagged NLP7 reporter lines. NLP7 typically accumulates in the nucleus in response to nitrate (Marchive et al., 2013). This nuclear NLP7 localization was similar in both prt6 and the wild-type roots grown on nitrate-supplemented media and could be abolished by a treatment with NO. Collectively, these results highlight that while NLP7 proteostasis is regulated by NO and O₂ levels, it is not directly controlled through the PRT6 N-degron pathway.

The work of Castillo et al. (2021) identifies a functional interaction between the nitrate signaling transcription factor NLP7 and the E3 ligase PRT6 in which the removal of NLP7 typically exacerbates the deleterious effects in prt6. It would be interesting to uncover how NLP7 exactly contributes to prt6 phenotypes in general and under conditions where prt6 mutants display enhanced rather than reduced tolerance, as was previously shown for salt, drought, heat, and hypoxia stress tolerance (Riber et al., 2015; Vicente et al., 2017; Hartman et al., 2019). Finally, while NLP7 does not seem to be a direct target of PRT6-mediated turnover, it is still possible NLP7 is an indirect target of PRT6 that requires additional signals, such as hypoxia, to become stable. Still, it is exciting that NLP7 becomes more stable in response to both hypoxia and NO removal, suggesting there could still be unidentified NO and O₂ sensing mechanisms remaining to be discovered (Holdsworth, 2017).