A melon ( Cucumis melo ) homologue of REPRESSOR OF PHOTOSYNTHETIC GENES prevents chloroplast differentiation in the fruit flesh

ABSTRACT Fruit flesh color in melon can be orange, green or white, depending on the accumulation of the orange carotenoid β-carotene or / and green chlorophylls. The dominant allele of Green flesh ( Gf ) causes orange melons, but in the absence of this allele the flesh of ripe melon can be white or green depending on the White flesh ( Wf ) locus, being white dominant over green. The identity of Wf has remained unclear despite several candidates have been proposed. Here we identified Wf by fine mapping of a segregating population derived from the white-fleshed variety Piel de Sapo (PS, gf gf / Wf Wf ) and the orange-fleshed Védrantais (VED, Gf Gf / wf wf ). Wf corresponds to the gene MELO3C003098 , herein referred to as CmRPGE1 as it encodes a fruit-specific homologue of REPRESSOR OF PHOTOSYNTHETIC GENES (RPGE) microproteins. Similar to RPGE homologues from other plants, overexpression of the PS allele ( CmRPGE1 PS ) caused a pale green leaf phenotype in Nicotiana benthamiana and Arabidopsis thaliana . By contrast, a 10-nucleotide deletion in the VED allele ( CmRPGE1 VED ) resulted in a loss of RPGE function. The active CmRPGE1 PS microprotein interacts with a fruit-localized melon homologue of ARABIDOPSIS PSEUDO-RESPONSE REGULATOR2 (APRR2), a GARP family transcription factor. Binding of CmRPGE1 PS retains the melon APRR2 homologue in the cytosol, hence preventing the regulation of target genes involved in chloroplast biogenesis. In green fruit cultivars, the non-functional CmRPGE1 VED allele allows APRR2 to perform its function, leading to chloroplast development and consequently a green flesh phenotype. SIGNIFICANT STATEMENT Fruit color is a critical quality trait that directly influences consumer preference and market value in melon. In this study, we demonstrate that the White flesh (Wf) gene, a major determinant of melon fruit flesh color, encodes a fruit-specific homologue of REPRESSOR OF PHOTOSYNTHETIC GENES (RPGE) microproteins. The encoded microprotein, CmRPGE1, interacts with melon homologues of ARABIDOPSIS PSEUDO-RESPONSE REGULATOR2 (APRR2), a GARP family transcription factor. In white flesh melons, the CmAPRR2.2 paralog is sequestered in the cytoplasm upon interaction with CmRPGE1, thereby preventing the expression of target genes involved in chlorophyll accumulation. Conversely, the truncated version of CmRPGE1 present in green-fleshed melons fails to interact with CmAPRR2.2 and to prevent its nuclear localization and function, allowing chloroplasts differentiation.