Anthocyanin accumulation and related anabolic key genes in fruit of Pingyuanhong kiwifruit (Actinidia chinensis)

Abstract:【Objective】The kiwifruit (Actinidia chinensis) exhibits a diverse array of pulp colors during its developmental stages, ranging from light green, red to purple. The variance in red pulp coloration primarily arises from differences in anthocyanin and proanthocyanidin levels. Hongyang kiwifruit, renowned for its red fruit core, faces challenges of discoloration or lightening under high temperatures or low elevation conditions. Conversely, Pingyuanhong kiwifruit, a novel cultivar derived from the open pollination population of Hongyang, demonstrates resilience to such environmental influences. This study aimed to elucidate the molecular mechanisms underlying the red coloration of Pingyuanhong kiwifruit and provide insights for breeding new cultivars resilient to environmental variations, particularly suitable for low elevation or high-temperature regions.【Methods】The experimental materials consisted of fruits harvested from 5-year-old Hongyang and Pingyuanhong kiwifruit trees on A. delicious seedling rootstock. Both cultivars were cultivated under identical soil conditions and management practicesat the Wanxi Kiwifruit Research Institute, Huoqiu County, Anhui Province. From May to September 2020, the fruit samples were collected every 30 days using random sampling principles, each sample consisted of 10 fruits and replicated three times. Upon sampling, fruits were wrapped in damp gauze, stored in ice boxes, and transported to the Provincial Key Laboratory of Horticultural Crop Quality Biology, Anhui Agricultural University. The fruits were then peeled, mashed, and thoroughly mixed before being flash- frozen with liquid nitrogen and stored at -80 ℃ until further analysis. Anthocyanin content in the fruits of Pingyuanhong and Hongyang kiwifruit was assessed using high-performance liquid chromatography (HPLC) on 30, 60, 90, 120, and 150 days after full bloom (DAFB). The dynamic changes and increments in anthocyanin accumulation during fruit development were analyzed. Simultaneously, the total RNA was extracted from fruit samples at these five stages for transcriptomic sequencing using an Illumina HiSeqTM 2000 next- generation sequencer at BGI (BGI Co., Ltd., Shenzhen, Guangdong, China). The identified key genes were subsequently validated using real-time quantitative PCR (RT-qPCR).【Results】No anthocyanin was detected on 30 and 60 DAFB in either Pingyuanhong or Hongyang fruits. Subsequently, the anthocyanin contents in Pingyuanhong fruits on 90, 120, and 150 DAFB were 3.34, 2.15, and 3.97 times higher than those in Hongyang fruits, respectively, and the anthocyanin mainly existed as cyanidin 3-O-galactoside, only a little amount of the cyanidin 3-O-glucoside was detected on 90 DAFB in the fruits of Pingyuanhong fruits. The significant elevation in anthocyanin content in Pingyuanhong compared with Hongyang fruits was attributed not only to increased anthocyanin synthesis but also to a notable reduction in degradation rate within Pingyuanhong fruits. The transcriptomic analysis identified 2230 differentially expressed genes (DEGs) and six differentially expressed transcription factors of the MYB family on 90, 120, and 150 DAFB in both Pingyuanhong and Hongyang fruits. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these DEGs were primarily associated with functions and metabolic pathways related to anthocyanin synthesis, transportation, and storage. Two key DEGs, Acc23647 (Acc4CL) and Acc23632 (AccDFR), consistent with the observed changes in anthocyanin content in Pingyuanhong fruits, were identified in the anthocyanin metabolic pathway. The validation results obtained through RT-qPCR were in concordance with the findings from transcriptomic analysis.【Conclusion】The anthocyanin forms mainly existed in the form of cyanidin 3-O- galactoside in the fruits of Pingyuanhong and Hongyang kiwifruit, and the anthocyanin content of Pingyuanhong fruits was hier than those of Hongyang fruits. The up-regulated expression of 4-coumarate-CoA ligase (4CL) and dihydroflavone alcohol-4- reductase (DFR) genes would promote the anthocyanin synthesis in the fruits of Pingyuanhong kiwifruit. The six transcription factors of MYB family would be involved in the anabolism of anthocyanin in the fruits of Pingyuanhong kiwifruit.