CsAKT1 is a key gene for the CeO2 nanoparticle's improved cucumber salt tolerance: a validation from CRISPR-Cas9 lines

Salinity is one of the main factors limiting the crop growth and yield. The application of nanomaterials such as cerium oxide nanoparticles (nanoceria) improves salt tolerance in many plant species. The known mechanisms of the nano-improved crop salt tolerance mostly occur at the physiological and hormonal levels but not at the gene level. The lack of proper methods to precisely investigate the key genes involved in the nano-improved crop salt tolerance is one of the main reasons for this. We found that both the leaf and root applications of PNC (polyacrylic acid-coated nanoceria) improved the cucumber salt tolerance, showing higher shoot and root dry weight and better photosynthetic performance than the control plants. Under salinity stress, PNC-treated cucumber plants showed a significant lower malondialdehyde (MDA) content of leaf (46.7% and 13.3% for leaf and root application of PNC, respectively) and root (31.4% and 19.9% for leaf and root application of PNC, respectively) and reactive oxygen species (ROS) levels such as the H2O2 content of leaf (46.6% and 22.0% for leaf and root application of PNC, respectively) and root (50.0% and 29.9% for leaf and root application of PNC, respectively). Further experiments showed that under the salinity stress, compared with control plants, PNC-treated cucumber plants had significantly higher leaf (216.1% and 59.5% for leaf and root application of PNC, respectively) and root (148.2% and 71.8% for leaf and root application of PNC, respectively) K+ content. The RNA seq showed that under the salinity stress, modulation on AKT1 is more responsible for the PNC improved K+ maintenance in cucumber. Further, the CsAKT1 depletion experiment (CRISPR-Cas9 lines) confirmed that under salinity stress, CsAKT1 plays an important role in the potassium uptake in cucumber plants treated with PNC. Our results showed that foliar PNC delivery enabled stronger cucumber salt tolerance than the root application associated with a better maintained K+/Na+ ratio. CRISPR-Cas9 lines further confirmed that CsAKT1 is a key gene involved in the PNC-improved cucumber salt tolerance. Our work not only adds more knowledge to the mechanisms underlying nano-improved plant salt tolerance but also suggests a CRISPR-Cas9 approach to study the key genes involved in it.