Characterization and analysis of the promoter region of Monodehydroascorbate reductase 4 (CpMDAR4) in papaya

The aim of this research was to study the effects of cis-element differences between the X, Y and Yh alleles on the expression of CpMDAR4, a potential candidate gene for sex differentiation in papaya, using a transcriptional reporter system in a model species Arabidopsis thaliana. Possible effects of a retrotransposon insertion in the Y and Yh alleles on the transcription and expression of CpMDAR4 alleles in papaya flowers were also examined. When comparing promoters and cis-regulatory elements among genes in the non-recombining region of the sex chromosomes, paired genes exhibited differences. Our results showed that differences in the promoter sequences of the CpMDAR4 alleles drove the expression of a reporter gene to different flower tissues in Arabidopsis. β-glucuronidase staining analysis of T2 and T3 lines for constructs containing 5’ deletions of native Y and Yh allele promoters showed the loss of specific expression of the reporter gene in the anthers, confirming the existence and location of cis-regulatory element POLLEN1LELAT52. The expression analysis of CpMDAR4 alleles in papaya flowers also showed that all alleles are actively expressed in different flower tissues, with the existence of a shorter truncated isoform, with unknown function, for the Y and Yh alleles due to an LTR-RT insertion in the Y and Yh chromosomes. The observed expression patterns in Arabidopsis thaliana flowers and the expression patterns of CpMDAR4 alleles in papaya flowers, suggest that MDAR4 might have a role on development of reproductive organs in papaya, and that it constitutes an important candidate for sex differentiation. To evaluate tissue-specific expression of CpMDAR4 alleles in papaya flowers, total RNA was extracted from small flower buds (< 1 mm), reproductive organs of pre-meiotic (1-6 mm) and post-meiotic (7-12 mm) flower buds (including aborted pistils or “pistillode”), mature flowers (15-20 mm) and gametophytes of mature ‘AU9’ female flowers and ‘AU9’ male flowers.