CRISPR/Cas9 knockout of PtaAGL16 improves post-drought recovery and shoot vitality in hybrid poplar

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.