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CsAKT1 is a key gene involved in PNC improved cucumber salt tolerance.

The NAC transcription factor is a type of plant-specific transcription factor that can regulate plant salt tolerance, but the underlying mechanism is unclear in grafted vegetables. H2O2 and ABA in pumpkin rootstocks can be transported to cucumber scion leaves, promoting stomatal closure to improve salt tolerance of grafted cucumbers. Despite these observations, the regulatory mechanism is unknown. Here, our research revealed that CmoNAC1 is a key transcription factor that regulates H2O2 and ABA signaling in pumpkin roots under salt stress. The function of CmoNAC1 was analyzed using root transformation and RNA-seq, and we found that pumpkin CmoNAC1 promoted the production of H2O2 and ABA via CmoRBOHD1 and CmoNCED6, respectively, and regulated K+/Na+ homeostasis via CmoAKT1;2, CmoHKT1;1 and CmoSOS1 to improve salt tolerance of grafted cucumbers. Root knockout of CmoNAC1 resulted in a significant decrease in H2O2 (52.9% and 32.1%)/ABA (21.8% and 42.7%) content and K+/Na+ ratio (81.5% and 56.3%) in leaf and roots of grafted cucumber, respectively, while overexpression showed the opposite effect. The root transformation experiment showed that CmoNCED6 could improve salt tolerance of grafted cucumbers by regulating ABA production and K+/Na+ homeostasis under salt stress. Finally, we found that CmoNAC1 bound to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2 and CmoHKT1;1 using yeast one-hybrid, luciferase, and electrophoretic mobility shift assays. In conclusion, pumpkin CmoNAC1 not only binds to the promoters of CmoRBOHD1 and CmoNCED6 to regulate the production of H2O2 and ABA signals in roots, but also binds to the promoters of CmoAKT1;2 and CmoHKT1;1 to increase the K+/Na+ ratio, thus improving salt tolerance of grafted cucumbers.

Background Salinity is one of the main factors limiting crop growth and yield. Applications of nanomaterials such as cerium oxide nanoparticles (nanoceria) improved salt tolerance in many plant species. The known mechanisms of nano-improved crop salt tolerance are mostly at the physiological and hormonal but not the gene level. Lack of proper methods to precisely investigate the key genes involved in nano-improved crop salt tolerance is one of the main reasons. Results We found that both leaf and root application of PNC (poly acrylic acid coated nanoceria) improved cucumber salt tolerance, showing higher shoot and root dry weight, and better photosynthetic performance than control plants. Under salinity stress, PNC treated cucumber plants showed 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) level such as 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 salinity stress, compared with control plants, PNC treated cucumber plants had significant 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. RNA seq showed that under salinity stress, the modulation on AKT1 is more responsible for the PNC improved K+ maintenance in cucumber. Further, CsAKT1 depletion experiment (CRISPR-Cas9 lines) confirmed that under salinity stress, CsAKT1 plays an important role in potassium uptake in cucumber plants treated with PNC. Conclusions Our results showed that foliar PNC delivery enabled stronger cucumber salt tolerance than the root application is associated with the better maintained K+/Na+ ratio. CRISPR-Cas9 lines further confirmed that CsAKT1 is a key gene involved in 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.

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