Poplar Response to Abiotic Stress An Integrated Morphological, Physiological, Biochemical, and Molecular Analysis of Populus alba L. under Salt and Cadmium Stress.

This thesis investigates physiological, molecular, and genome editing strategies to enhance abiotic stress tolerance in Populus alba L. “Villafranca.” First, long-term exposure (30 days) to different salinity levels (0–180 mM NaCl) revealed that plants maintained growth at 60 mM, despite early declines in stomatal conductance and photosynthesis. Higher salinity (120–180 mM) induced basal leaf chlorosis, biomass loss, and reduced flavonols, while Na⁺ preferentially accumulated in roots and basal tissues. Leaves preserved K⁺/Na⁺ balance and elevated Ca²⁺ with stress, consistent with signaling and selective uptake. Phenylpropanoid/flavonoid pathways and proline increased under stress, and vacuolar Na⁺/H⁺ antiporter (NHX) transcripts declined at all salt levels, indicating disruption of ion-homeostasis under prolonged salinity. Building on these insights, CRISPR/Cas9 editing targeted three salt-related genes, NAC13, NHX1, and HKT1;1, using efficient dual-gRNA designs and a CaMV 35S-driven Cas9, resulting in independent NAC13 and HKT1;1 lines with wild-type-like morphology and providing a tractable pipeline for functional validation of salt tolerance determinants. Finally, the role of MTP1 in cadmium resilience was tested in two CRISPR lines. Under 100 μM Cd for 46 days, a homozygous line (6.13; 4-nt deletion) and wild type maintained morphology and accumulated more Cd, upregulating root-biased detoxification (GSTs, peroxidases, SULTR3;1), whereas a heterozygous line (3.2; 23-nt deletion) was Cd-sensitive, with reduced photosynthesis and lower Cd accumulation. Collectively, the work highlights the sensitivity of Populus alba “Villafranca” to salt stress and provides CRISPR-edited lines as tools for future work. We also show that MTP1 helps sequester cadmium and protect leaves. Together, these findings support breeding strategies for salty or metal-contaminated soils.