Drought-induced flavonoid biosynthesis and chalcone synthase upregulation reflect cultivar-specific responses in Lotus corniculatus

This study investigated drought-induced morphological, physiological and molecular responses in three Lotus corniculatus L. (birdsfoot trefoil, BFT) cultivars (‘Gelsvis’, ‘Baco’, and ‘Izis’) under progressive water deficit conditions (100% field capacity (FC) (control), 50% FC (moderate) and 30% FC (severe)). Expression levels of chalcone synthase (CHS) gene family members ( LcCHS3 , LcCHS4 , LcCHS10 , LcCHS11 , LcCHS12 ) and flavonoid biosynthetic genes ( LcIFS2 , LcFLS , and LcF3’H ) were analyzed by qRT-PCR. The analysis of data revealed that drought significantly reduced plant height, bunch diameter, leaf dimensions, root length, leaf and branch number, chlorophyll content, and biomass traits across all cultivars. In contrast, water limitation triggered a metabolic shift towards enhanced secondary metabolite biosynthesis, leading to significant accumulation of quercetin and kaempferol derivatives, isoflavones, isoflavanes, and phenylpropanoid acids. The expression levels of genes encoding key flavonoid biosynthetic enzymes were compared among the cultivars. All five CHS paralogs were strongly induced at 50% and 30% FC. Transcriptional responses were cultivar-specific: ‘Gelsvis’ showed the strongest LcF3’H induction, ‘Izis’ the strongest LcIFS2 and LcFLS induction, and ‘Baco’ the largest relative LcCHS4 increase but down-regulated LcFLS and LcF3’H under severe stress, in all cultivars accompanied by metabolite enrichment. The morphological and biomass decline under drought was broadly comparable across cultivars, with branch number being the one trait ‘Gelsvis’ significantly preserved. Multivariate analyses showed opposite, treatment-driven trajectories of growth traits and flavonoid pathway components under drought. This study demonstrates cultivar-specific responses of L. corniculatus to water-deficit conditions and identifies chalcone synthase upregulation as closely associated with the observed flavonoid accumulation under water limitation. ‘Gelsvis’ combined the strongest LcF3’H induction with the highest kaempferol-derivative accumulation under severe stress, but did not maintain growth or biomass better than ‘Baco’ and ‘Izis’, making it a candidate genetic resource for breeding forage legumes with strong flavonol-branch responsiveness under water limitation. As classical physiological indicators of drought tolerance (e.g., relative water content, gas exchange, oxidative damage, antioxidant enzyme activity) were not directly measured, this reflects transcriptional and metabolic stress responsiveness rather than confirmed drought tolerance. Because all traits were measured once, after 21 days of stress, in three biological replicates and without functional validation of individual genes, these associations should be regarded as exploratory.