Abstract:【Objective】The kiwifruit (Actinidia chinensis), originating from China, 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 'Hongyang' through open pollination, demonstrates resilience to such environmental influences. This study aims to elucidate the molecular mechanisms underlying the red coloration of 'Pingyuanhong' kiwifruit, providing 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, grafted onto Actinidia delicious seedling rootstock, and planted in rows spaced 3.0 meters apart with a plant spacing of 1.5 meters. Both cultivars were cultivated under identical soil conditions and management practices at the Wanxi Kiwifruit Research Institute, Huoqiu County, Anhui Province. From May to September 2020, fruit samples were collected every 30 days using random sampling principles, with each sample comprising 10 fruits 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) at 30, 60, 90, 120, and 150 days after full bloom (DAFB). Dynamic changes and increments in anthocyanin accumulation during fruit development were analyzed. Simultaneously, 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). This analysis aimed to identify differentially expressed genes (DEGs) across various periods and discover key genes involved in promoting anthocyanin synthesis in kiwifruit. The identified key genes were subsequently validated using real-time quantitative PCR (RT-qPCR).【Result】The results revealed that no anthocyanin was detected at 30 and 60 DAFB in either 'Pingyuanhong' or 'Hongyang' kiwifruit. Subsequently, at 90, 120, and 150 DAFB, the anthocyanin content in 'Pingyuanhong' kiwifruit was 3.34, 2.15, and 3.97 times higher than that in 'Hongyang' kiwifruit, respectively, and the anthocyanin mainly existed as cyanidin 3-O-galactoside, a letter amount of the cyanidin 3-O-glucoside was detected only at 90 DAFB in fruit of ‘Pingyuanhong’ kiwifruit. The significant elevation in anthocyanin content in 'Pingyuanhong' compared to 'Hongyang' kiwifruit was attributed not only to increased anthocyanin synthesis but also to a notable reduction in its degradation rate within 'Pingyuanhong' fruit.Transcriptomic analysis identified 2230 differentially expressed genes (DEGs) and six differentially expressed transcription factors of the MYB family at 90, 120, and 150 DAFB in both 'Pingyuanhong' and 'Hongyang' kiwifruit. 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' kiwifruit, were identified in the anthocyanin metabolic pathway. Validation results obtained through RT-qPCR were in concordance with the findings from transcriptomic analysis.【Conclusion】The anthocyanin form mainly existed in the form of cyanidin 3-O-galactoside in the fruit of ‘Pingyuanhong’ and ‘Hongyang’ kiwifruit, and the anthocyanin content of ‘Pingyuanhong’ was 3.34, 2.15 and 3.97 times that of ‘Hongyang’ kiwifruit at 90, 120 and 150 DAFB, respectively. The up-regulated expression of 4-coumarate-CoA ligase (4CL) and dihydroflavone alcohol-4-reductase (DFR) genes had promoted the anthocyanin synthesis in the fruit of ‘Pingyuanhong’ kiwifruit. The six transcription factors of MYB family were involved in the anabolism of anthocyanin in fruit of ‘Pingyuanhong’ kiwifruit.
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