2010 Volume 79 Issue 3 Pages 263-274
The carotenoid metabolism enzyme zeaxanthin epoxidase (ZEP) is a key regulator of carotenoid accumulation in Citrus fruit. The expression level of the gene encoding this enzyme, ZEP, influences varietal differences in the conversion of zeaxanthin to violaxanthin during fruit development. To determine how this gene is regulated, we investigated the structure of its alleles and analyzed allele-specific ZEP expression in heterozygous Citrus fruit. Four independent genomic sequences were isolated from a BAC library of Satsuma mandarin (Citrus unshiu Marc.). All ZEP gene sequences in Satsuma mandarin consisted of 16 exons and 15 introns. Genetic analyses of hybrid populations and analyses of sequence variations identified that two of the sequences were alleles from the ZEP-1/-2 locus, and others were from different locus/loci, ZEP-3/-4, for which allelism has not been confirmed. The ZEP-4 allele was expressed in young-stage fruit, but not in mature-stage fruit, while ZEP-1/-2 alleles were expressed as fruit matured. Our results showed that the newly identified isoform, ZEP-4, did not contribute to the accumulation of violaxanthin during fruit maturation. Expression levels of ZEP-1/-2 alleles in the fruit of three heterozygous Citrus cultivars were compared using allele-specific RT-PCR. Transcripts of ZEP-1 alleles were more abundant than those of ZEP-2 alleles in all three cultivars. Sequence diversity among the 5' UTRs of ZEP alleles was also analyzed. The implications of sequence diversity with respect to diversity of the expression and phylogenetic relationships among ZEP genes in Citrus are discussed.