TY - JOUR

T1 - Accumulation of Anthocyanin in the Aleurone of Barley Grains by Targeted Restoration of the MYC2 Gene

AU - Egorova, Anastasiya A.

AU - Zykova, Tatyana E.

AU - Hertig, Christian W.

AU - Hoffie, Iris

AU - Morozov, Sergey V.

AU - Chernyak, Elena I.

AU - Rogachev, Artem D.

AU - Korotkova, Anna M.

AU - Vikhorev, Alexander V.

AU - Vasiliev, Gennady V.

AU - Shoeva, Olesya Y.

AU - Kumlehn, Jochen

AU - Gerasimova, Sophia V.

AU - Khlestkina, Elena K.

N1 - Barley hybrids and resultant lines were grown using resources of the Greenhouse Core Facility supported by ICG, project number FWNR-2022-0017. This research was funded by the Russian Science Foundation, grant number 21-66-00012.

PY - 2024/12

Y1 - 2024/12

N2 - Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented barley, a single nucleotide insertion in this gene causes a frameshift mutation with a premature stop codon. It was hypothesized that restoring the MYC2 reading frame could activate anthocyanin accumulation in the aleurone. Using a targeted mutagenesis approach in the present study, the reading frame of MYC2 was restored in the non-pigmented cultivar Golden Promise. Genetic constructs harboring cas9 and gRNA expression units were developed, pre-validated in protoplasts, and then functional MYC2 alleles were generated at the plant level via Agrobacterium-mediated transformation. Anthocyanin accumulation in the aleurone layer of grains from these mutants was confirmed through microscopy and chemical analysis. The expression of anthocyanin biosynthesis genes was analyzed, revealing that the restoration of MYC2 led to increased transcript levels of F3H and ANS genes. These results confirm the critical role of the MYC2 transcription factor in the blue aleurone trait and provide a biotechnological solution for enriching barley grain with anthocyanins.

AB - Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented barley, a single nucleotide insertion in this gene causes a frameshift mutation with a premature stop codon. It was hypothesized that restoring the MYC2 reading frame could activate anthocyanin accumulation in the aleurone. Using a targeted mutagenesis approach in the present study, the reading frame of MYC2 was restored in the non-pigmented cultivar Golden Promise. Genetic constructs harboring cas9 and gRNA expression units were developed, pre-validated in protoplasts, and then functional MYC2 alleles were generated at the plant level via Agrobacterium-mediated transformation. Anthocyanin accumulation in the aleurone layer of grains from these mutants was confirmed through microscopy and chemical analysis. The expression of anthocyanin biosynthesis genes was analyzed, revealing that the restoration of MYC2 led to increased transcript levels of F3H and ANS genes. These results confirm the critical role of the MYC2 transcription factor in the blue aleurone trait and provide a biotechnological solution for enriching barley grain with anthocyanins.

KW - Cas9/gRNA

KW - flavonoid biosynthesis

KW - genome editing

KW - grain color

KW - nutritional value

KW - pigmented barley

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85211921002&origin=inward&txGid=81a44cb89704acd8a0a430a1c827490c

UR - https://www.mendeley.com/catalogue/af91e197-9615-3bf1-8240-65117d8135f1/

U2 - 10.3390/ijms252312705

DO - 10.3390/ijms252312705

M3 - Article

C2 - 39684416

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 23

M1 - 12705

ER -