Genotypic variation in physiological, biochemical, and transcriptional responses to drought stress in spring barley at an early growth stage

Drought is a major abiotic stress limiting barley (<i>Hordeum vulgare</i> L.) productivity. We evaluated 17 spring barley genotypes at the early leaf development stage under controlled laboratory conditions with optimal and drought treatments, integrating physiological, biochemical, and molecular traits. Drought reduced relative water content (–1.3% to –3.2%), plant height (–14.7% to –29.6%), and dry biomass (–2.3% to –24.9%), while inducing strong proline accumulation (+23.6% to +454%) and pigment loss (chlorophyll a –10.1% to –79.5%; carotenoids –6.2% to –70.9%). Principal component and discriminant analyses identified plant height and chlorophyll <i>a</i> as the most reliable discriminators, whereas relative water content was less predictive of the species. Multivariate stratification separated tolerant (Argument, Exalis, Slaven, Malz, Valis), intermediate (Laudis 550, Tango, Kompakt, LG Belcanto, SK Levitus), and sensitive (Kangoo, LG Tosca, LG Flamenco, Karmel, Bojos, Nitran, Tadmor) groups of genotypes. Gene expression profiling of 12 genotypes revealed a modest induction of <i>HvABF2</i> (1.77-fold), moderate upregulation of <i>HvSOD1</i> (1.82-fold) and <i>HvAPX1</i> (2.28-fold), and the strongest response in <i>HvP5CS</i> (3.29-fold), which did not consistently correlate with tolerance. Tolerant genotypes combined growth stability, pigment retention, and moderate osmotic adjustment, whereas sensitive genotypes relied on excessive proline accumulation, resulting in severe pigment and growth penalties. Overall, drought tolerance in barley at the early growth stage emerged from the coordinated regulation of growth, photoprotection, and stress-gene activation, providing a foundation that can guide the selection of genotypes for subsequent validation under field conditions and future breeding programmes.