The Transcriptional Regulation of Softening in Apple

Abstract

Fruit texture is a primary determinant of Malus x domestica (apple) fruit quality and is regulated during ripening by the hormone ethylene. In many cultivars loss of a crisp juicy texture is associated with fruit ripening, with overripe apples becoming soft and mealy. POLYGALACTURONASE1 (MdPG1) is an important enzyme for apple fruit ripening, suppression of which results in firmer apple fruit with improved cell to cell adhesion. Expression of MdPG1 has previously been reported to be up-regulated late in fruit development, in response to ethylene and cold and was consistently lower in firmer apple cultivars. In previous experiments conducted at Plant and Food Research (PFR) the apple cell wall modifying genes β-GALACTOSIDASE1 (MdβGAL1) and XYLOGLUCAN ENDOTRANSGLUCOSYLASE1 (MdXET1) were found to have similar expression patterns to MdPG1, suggesting that these genes may also be involved in apple fruit softening and, along with MdPG1, may be regulated by a common transcription factor pathway. As the regulation of cell wall modifying genes in apple has not previously been examined in depth, promoters were isolated for MdPG1, MdβGAL1 and MdXET1 from 'Royal Gala' and 'Granny Smith', which were found to contain putative ethylene and cold related cis-acting regulatory motifs. Two MdPG1 alleles were identified in 'Royal Gala' and one in 'Granny Smith', with SNPs in the promoter regions occurring in close proximity to an ethylene response factor binding motif and a cold response factor binding motif. Using a heterologous transient assay system, ethylene and cold regulated transcription factors were identified which activated expression of MdPG1, MdβGAL1 or MdXET1. APETALA2 DOMAIN38 (MdAP2D38), expression of which increased in cold stored apple fruit, activated the promoters of MdPG1, MdβGAL1 and MdXET1 and was itself activated by INDUCER OF CBF EXPRESSION (MdICE1). This suggests that these transcription factors may represent components of a common cold regulatory pathway. MdPG1 and MdXET1 were activated by EIN3- LIKE1 (MdEIL1), which is homologous to ETHYLENE INSENSITIVE3 (AtEIN3) in the ethylene signal transduction pathway in Arabidopsis. This activation was suppressed by the addition of EIN3-BINDING F BOX PROTEIN1 (MdEBF1), a homolog of EIN3-BINDING F BOX PROTEIN1 (AtEBF1) and EIN3- BINDING F BOX PROTEIN2 (AtEBF2) which in Arabidopsis degrade AtEIN3 in the absence of ethylene. Increases in MdPG1 and MdXET1 expression in ethylene treated fruit and in different cultivars over the course of postharvest softening was consistently preceded by MdβGAL1. This suggests that MdβGAL1 acts earlier in postharvest softening and is regulated by different ethylene responsive transcription factors. The promoters of the 'Royal Gala' and 'Granny Smith' MdPG1 alleles were shown to be differentially activated, with one allele from both 'Granny Smith' and 'Royal Gala' displaying increased activation by members of the cold response pathway, including MdAP2D38, and reduced sensitivity to ethylene relative to the second 'Royal Gala' allele. This suggests that differences in MdPG1 expression between cultivars, and the resultant effect on firmness, may be due to the differential activation of the promoters of these alleles by ethylene and cold.