LsMybW-encoding R2R3-MYB transcription factor is responsible for a shift from black to white in lettuce seed.

KEY MESSAGE: We identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars. Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black-in its wild ancestor-to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F2 population derived from a cross between "ShinanoPower" (white) and "Escort" (black). The white seed trait was controlled by a single recessive locus (48.055-50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of "ShinanoPower" and "Escort." Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativa Myb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli.

P450 (cytochrome P450) enzymes catalyse the mono-oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7-ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7-ethoxycoumarin, was de-ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7-ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2-hydroxylation. CYP2A5 catalysed 2- and 4-hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7-beta-hydroxy-DHEA and two P450 species produced a 2-alpha-hydroxy-DHEA. Four P450 species catalysed flavanone 2- and 4-hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high-throughput screening for drug discovery.