Persian walnut is a drought-sensitive species with considerable genetic variation in the photosynthesis and water
use efficiency of its populations, which is largely unexplored. Here, we aimed to elucidate changes in the efficiency of photosynthesis and water content using a diverse panel of 60 walnut families which were submitted to a
progressive drought for 24 days, followed by two weeks of re-watering. Severe water-withholding reduced leaf
relative water content (RWC) by 20%, net photosynthetic rate (Pn) by 50%, stomatal conductance (gs) by 60%,
intercellular CO2 concentration (Ci) by 30%, and transpiration rate (Tr) by 50%, but improved water use efficiency (WUE) by 25%. Severe water-withholding also inhibited photosystem II functionality as indicated by
reduced quantum yield of intersystem electron transport (φEo) and transfer of electrons per reaction center (ET0/
RC), also enhanced accumulation of QA (VJ) resulted in the reduction of the photosynthetic performance (PIABS)
and maximal quantum yield of PSII (FV/FM); while elevated quantum yield of energy dissipation (φDo), energy
fluxes for absorption (ABS/RC) and dissipated energy flux (DI0/RC) in walnut families. Cluster analysis classified
families into three main groups (tolerant, moderately tolerant, and sensitive), with the tolerant group from dry
climates exhibiting lesser alterations in assessed parameters than the other groups. Multivariate analysis of
phenotypic data demonstrated that RWC and biophysical parameters related to the chlorophyll fluorescence such
as FV/FM, φEo, φDo, PIABS, ABS/RC, ET0/RC, and DI0/RC represent fast, robust and non-destructive biomarkers for
walnut performance under drought stress. Finally, phenotype-environment association analysis showed significant correlation of some photosynthetic traits with geoclimatic factors, suggesting a key role of climate and
geography in the adaptation of walnut to its habitat conditions.