Transcriptional Regulation of Triterpenes in Russet Apple Skin

Abstract

The reduced risk of major diseases associated with apple consumption can be partly attributed to pentacyclic triterpene waxes produced in the cuticle which protects the fruit. Severe cuticle damage triggers the formation of rough, brownish patches called russet over the fruit skin. Russeting is commercially perceived as a skin abnormality that degrades fruit quality, particularly its visual appeal. It is characterized by an increased suberin production and a switch in the triterpene profile of the cuticle. Waxy apple varieties are rich in ursolic and oleanolic acids synthesized by the MdOSC1 gene. Russeted apples express the MdOSC5 gene to abundantly produce betulinic acid and betulinic acid-caffeate triterpenes which are potent immunomodulatory agents. Six transcription factors (TFs) and a BAHD acyltransferase gene linked to suberin production that strongly correlated with russeting and lupane biosynthesis were identified in a previous study. This research aimed to understand the transcriptional control of triterpene biosynthesis. Four MYB TFs – MdMYB52, MdMYB66, MdMYB67, MdMYB93, two NAC homeologs – MdNAC74 and MdNAC142, MdBAHD, MdOSC1 and MdOSC5 were selected as the candidate genes. Phylogenetic analysis of a subclade of NAC transcription factors in Arabidopsis, apple, pear, potato and strawberry provided insights into their roles in regulating stress response, senescence, suberin deposition and cell death. Eleven heritage apple cultivars with different degrees of russeting were studied for the expression levels of candidate genes and their triterpene profiles. All genes except MdOSC1 and MdMYB52 were upregulated in russet apples. Ursolic acid, oleanolic acid, betulinic acid and betulinic acid-caffeates were differentially accumulated in waxy apples and russet apples. Functional analysis revealed strong activation of MdNAC74 and MdNAC142 by the suberin master regulator MdMYB93. MdOSC5 was directly activated by the MdMYB66. MdMYB52 and MdMYB67 appear to be involved in a positive feedback loop which could regulate betulinic acid production or cell wall modifications that come along with russeting. This research provides answers that will aid in the elucidation of the transcriptional pathways that underly russeting, suberization and the shift in triterpenes associated with it. It can be used to manage russeting and create visually appealing new apple cultivars with strong cuticles and increased stress resistance.