PbrNAC34a- PbrMYB3/65- PbrACO2 cascade plays a role in citrate difference between the pericarp and cortex tissues of pear (P. bretschneideri Rehd.) fruit.

Citrate is critical to the flavor of horticultural fruit and governed by ACO. However, the specific ACO and its upstream regulators involved in citrate metabolism during pear (Pyrus spp.) fruit development remained uncharacterized. This study identified and characterized six PbrACOs from the Pyrus bretschneideri Rehd. genome. Comprehensive analyses of citrate levels, cyt/mitACO activities, and PbrACOs expression profiles in the pericarp and cortex tissues of developing 'Yali' and 'Dangshansuli' fruits revealed PbrACO2 as a candidate gene. Subsequently, PbrACO2 was confirmed as a mitochondrial aconitase catalyzing citrate-to-isocitrate conversion in vitro and in vivo. Analysis of differentially expressed transcription factors (TFs) and cis-acting elements in the PbrACO2 promoter identified nuclear PbrMYB3 and PbrMYB65, derived from whole genome duplication/segmental duplication, as candidate upstream regulators. These MYB TFs, without direct relationship, bound, as monomers, to the same two MYB-binding sites in the PbrACO2 promoter to activate its transcription, thereby promoting citrate isomerization in pear and tomato. Further investigation revealed that PbrMYB3 and PbrMYB65 are transcriptionally regulated by PbrNAC34a. Given their tissue-dependent expression profiles, the PbrNAC34a-PbrMYB3/65-PbrACO2 cascade partially accounts for citrate differences between pear fruit pericarp and cortex tissues. These findings enhance understanding of citrate accumulation in Rosaceae fruit and provide genetic resources for pear breeding.