Identification of Lycopene epsilon cyclase (LCYE) gene mutants to potentially increase β-carotene content in durum wheat (Triticum turgidum L.ssp. durum) through TILLING.

Abstract

Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-carotene levels. Durum wheat is a tetraploid species that has two LCYE genes (LCYE-A and LCYE-B) associated to the A and B genomes. The objective of this work was to produce durum wheat LCYE mutants through EMS to potentially increase β-carotene content. The LCYE point mutations created with EMS were identified using a Kronos TILLING (Targeting Induced Local Lesion IN Genomes) mutant population. Specific primers that amplified exons 3 through 10 of the LCYE genes were designed and validated. To simplify the TILLING procedure, fragments were digested with CJE (Celery Juice Extract) and visualized on 2% agarose gels. 6X mutant pools were identified, which showed cleavage products and then made into 2X pools to identify mutant individuals. LCYE mutants were then sequenced and evaluated with BLOSUM62, SIFT and PSSM algorithms. Mutants with substitutions W437*, P334L and G368R in LCYE-A and P405L, G352R and T393I in LCYE-B predicted to affect protein function were selected. Substitution W437* increased β-carotene in 75% and overall total carotenoids content in leaves of the mutant 2426 (A1 mutant line), but no significant differences relative to the control were found in grains through HPLC. Finally, the increased levels of β-carotene on leaves have potential applications to improving plant resistance under contaminated environmental conditions.