Pyramiding of mutations in lycopene ε-cyclase and β-hydroxylase 1 increases β-carotene content and modifies carotenoid metabolism in durum wheat

Carotenoids are essential pigments in plants, playing critical roles in photosynthesis, photoprotection, and stress tolerance, particularly under environmental conditions such as high light intensity and drought. To enhance β-carotene content in durum wheat (Triticum durum Desf.), a TILLING approach was used to generate null mutants for the lycopene ε-cyclase (LCYE) and β-hydroxylases 1 (HYD1) genes, which are key players in carotenoid biosynthesis. Homozygous mutants for both genes were obtained by crossing single homeoallelic mutant lines, resulting in three distinct mutant lines (LxH_1, LxH_2, LxH_3). Carotenoid metabolism and antioxidant-related genes expression were analyzed during seed ripening, revealing significantly reduced expression of LCYE and HYD1, while violaxanthin de-epoxidase (VDE) gene was upregulated at later stages. The mutant lines also showed significantly higher β-carotene accumulation in seeds, with an increase of up to 245 % compared to the control, while lutein content was reduced by over 99 %. In leaves, β-carotene levels remained unchanged, but zeaxanthin and violaxanthin accumulated at significantly higher levels compared to the control plants. Chlorophyll content was reduced in the mutant leaves, leading to altered chlorophyll a/b ratios and an overall decrease in total carotenoid levels. Although photosynthetic efficiency was lower in the mutants, gas exchange parameters remained unaffected, suggesting that primary carbon assimilation was not severely compromised. Phenotypic analysis revealed a reduction in plant height, spike length, and spikelet number; however, key yield traits were largely preserved. Notably, the mutant lines exhibited albinism under cold acclimation conditions, a phenotype absent in the control plants, likely due to the crucial role of lutein in photoprotection at low temperatures. These findings demonstrate that the pyramiding of mutations in LCYE and HYD1 effectively alters carotenoid composition, impacts photosynthesis-related traits, and influences plant responses to environmental stresses. This study provides valuable insights for breeding programs aimed at enhancing carotenoid content in wheat, with potential applications in improving both nutritional quality and stress resilience in cereal crops.