Screening of UV ‐B‐induced genes from apple peels by SSH : possible involvement of MdCOP1 ‐mediated signaling cascade genes in anthocyanin accumulation

Suppression subtractive hybridization ( SSH ) was employed to identify candidate genes involved in red coloration in apple peel with the ultraviolet ( UV )‐B‐treated ‘Mutsu’. After reverse Northern blotting verification, nearly 80 clones were successfully sequenced. Large portions of the expressed sequence tags ( ESTs ) are well characterized anthocyanin biosynthesis‐related genes, such as chalcone synthase ( 11A5 ), flavonol synthase ( 12F3 ), anthocyanidin synthase ( 11H5 ) and UDP ‐glycosyl transferase ( 14A12 ) whose presence proved the success of SSH . Eight ESTs were selected for quantitative real‐time polymerase chain reaction analysis and their expressions were all elevated in ‘Induction’, further confirming the reliability of the SSH library. One EST , 11F4 ( CONSTITUTIVE PHOTOMORPHOGENIC 1 : COP1 ) with putative function in light signal relay was further analyzed in ‘Mutsu’ and ‘Tsugaru’, along with MdHY5 ( ELONGATED HYPOCOTYL 5 : the downstream target of COP1 ), MdMYB22 (a possible flavonol‐specific activator under the regulation of HY5 , belonging to the SG7 / PRODUCTION OF FLAVONOL GLYCOSIDES family) and MdMYBA . Results showed that MdCOP1 , MdHY5 , MdMYB22 and MdMYBA were all UV ‐B inducible genes and anthocyanin accumulation occurred after their increased expressions. Moreover, their expressions and anthocyanin content were enhanced under UV ‐B plus 17°C treatment. The presence of G box, a known consensus binding site of HY5 , in the MdMYBA promoter region implicated that it could be regulated by MdHY5 , which was verified by the result of the yeast one‐hybrid analysis. Our data suggested that UV ‐B irradiation would induce the utmost upstream light signaling factor, MdCOP1 , which activates MdHY5 signaling by binding to the promoter regions of MdMYBs , and finally leads to the red coloration of apple peels.