Functional Analysis of LTS-PYL in Modulating Plant Drought Responses

Drought severely limits plant productivity, and understanding its regulatory mechanisms remains essential. Here, we characterize Lipid Transport Superfamily-Polyketide cyclase/dehydrase (<i>LTS-PYL</i>), a PYR/PYL/RCAR-domain gene, using Arabidopsis overexpression and CRISPR-Cas9 genome-edited lines to elucidate its role in drought adaptation. <i>LTS-PYL</i> overexpression enhanced early seedling growth, increasing root length (RL) by 40% and 31%, whereas genome-edited lines exhibited severe defects, including 42%, 28% reductions in fresh weight and 63%, 50% decreases in root length relative to WT-T. Under drought stress, overexpression lines displayed strong growth and reproductive resilience, with shoot length (SL) increased by up to 80%, silique length (Sil L) by 61%, and seed number doubled compared with WT-T. In contrast, genome-edited lines showed marked reductions in these traits, confirming their drought sensitivity. <i>LTS-PYL</i> overexpression strongly suppressed oxidative stress, reducing H₂O₂ by 74% and 68% and O₂^-· by 39% and 38%, while increasing relative water content (RWC) by 42% and 39%. Genome-edited lines exhibited elevated (H₂O₂, O₂^-·) and up to 33% lower RWC. Antioxidant capacity was also strengthened in overexpression plants, with catalase (CAT) and peroxidase (POD) activities increasing by 138%, 168% and 62%, 148%, and malondialdehyde (MDA) and electrolyte leakage (EL) reduced by 23%, 37%, relative to WT-T. Conversely, genome-edited lines showed weakened antioxidant defenses and higher membrane damage. Transcriptionally, overexpression activated drought-responsive genes, elevating <i>LTS-PYL</i> (604%, 472%), <i>DREB2A</i> (227%, 200%), and ABA levels (48%, 34%), whereas genome-edited lines showed strongly reduced expression and ABA decreases of 66%, 62%. Additionally, <i>LTS-PYL</i> enhanced osmotic adjustment, increasing proline (58%, 53%), sugars (37%, 46%), and sucrose (111%, 100%), while limiting chlorophyll (Chl) loss to 9%, 20%. Genome-edited lines exhibited reduced osmolytes and severe chlorophyll decline. Overall, <i>LTS-PYL</i> acts as a strong positive regulator of drought tolerance, integrating ABA signaling, osmotic adjustment, ROS detoxification, and transcriptional activation.