Abstract Humans cannot synthesize ascorbic acid (AsA), making dietary intake from fruits and vegetables crucial. Kiwifruit is a rich source of AsA, however, the genetic and regulatory basis for its accumulation is not fully elucidated. This study integrates quantitative trait locus (QTL) mapping, transcriptomics, and functional characterization to uncover a novel regulatory role of AaAGPase, encoding an ADP-glucose pyrophosphorylase, in determining AsA levels in kiwifruit. A significant QTL was mapped on chr10 in a tetraploid A. arguta biparental population, explaining 11.46% of the variation in AsA levels. Based on gene annotation and expression differences between high- and low-AsA genotypes, AGPase, was identified as a negative regulator. Our results indicated that a promoter variation in AGPase affects both AsA and starch levels. Overexpression of AaAGPase in transiently transformed fruit and transgenic kiwifruit plants led to reduced AsA levels via downregulation of key genes in the L-galactose pathway while increasing starch levels. Conversely, virus-induced gene silencing of AaAGPase resulted in increased AsA but decreased starch levels. Additionally, we identified the Golden 2-like transcription factor, AaGLK, which binds to and activates the AaAGPase promoter. Manipulating AaGLK expression resulted in variations in AsA levels by regulating AaAGPase expression. These findings reveal that the AaGLK-AaAGPase module negatively regulates AsA biosynthesis in kiwifruit. Additionally, we developed a molecular marker based on the sequence variation of AGPase promoter for marker assisted selection. In summary, our study revealed that AaAGPase, which is transcriptionally activated by AaGLK, plays a novel role in determining AsA levels through carbohydrates substrates competition.

Actinidia Lindl. represents the most economically and nutritionally valuable genus within Actinidiaceae. However, species identification and intraspecific discrimination in this genus remain to be fully clarified, in particular, the genetic relationships and fine-scale variations within section Leiocarpae are still poorly understood. To address this, we assembled and annotated the chloroplast genomes of eight accessions, representing four species in this section: A. arguta, A. melanandra, A. kolomikta, and A. polygama. All eight chloroplast genomes exhibited remarkable conservation in structural organization and gene composition, with sizes varying between 156,661 and 157,614 bp and a uniform GC content of 37.20

Polyploidisation, creating redundant or diverged copies of the genome, is a major driving force in plant evolution, diversification and environmental adaptation, including for the kiwifruit genus (Actinidia Lindl.). We present a high-contiguity, haplotype-resolved genome assembly of the hexaploid Actinidia valvata rootstock cultivar 'Zhongmikangzhen No. 2' (ZK2), a novel rootstock and the first of its kind to receive Plant Variety Protection in China, exhibiting superior waterlogging tolerance. The assembly, ZK2, contains 174 chromosomes in 6 haplotypes (2n = 6x = 174), where > 82% of chromosomes per haplotype are telomere-capped at one or both ends, including three haplotypes achieved with 100% telomeric representation. In total, 212 055 protein-coding genes are predicted, or about 35 000 genes per haplotype on average. Our work on comparative genomics, including analyses of TE composition, collinearity, orthologs and phylogenies, strongly reveals an AABBBB subgenome structure derived from ancestral donors A. polygama (A1 and A2) and A. macrosperma (B1 to B4). Transcriptome analysis showed differential expressions between the homeologs under waterlogging stress, highlighting subgenome-specific regulatory dynamics. A key example involves an ethylene-response factor (ERF) gene: when the B1 copy of ERF was overexpressed in the kiwifruit transgenic lines, they illustrated an enhanced waterlogging resistance. In addition, this high-quality haplotype-resolved A. valvata genome assembly enables functional trait discovery, homeolog-aware genome-wide association studies, and targeted editing of beneficial alleles or homologous gene sets, supporting the breeding of resilient polyploid kiwifruit cultivars and rootstocks. This resource provides a foundation for future research in kiwifruit, particularly for rootstock-mediated crop improvement.

In kiwifruit (Actinidia arguta), fruit coloration is typically accompanied by ripening; however, the intrinsic connection between these two processes remains unclear. In this study, we found that ethylene and auxin accelerated and suppressed fruit coloration and ripening, respectively, in A. arguta. ETHYLENE INSENSITIVE 3-LIKE 2 (AaEIL2) and ETHYLENE RESPONSIVE FACTOR (AaERF059) were mined and identified using yeast two-hybrid library screening and transcriptome analysis. AaEIL2, specifically induced to high expression by ethylene, was confirmed as a transcription factor that positively regulates coloration and ripening by targeting AaLDOX (leucoanthocyanidin dioxygenase involved in anthocyanin biosynthesis) and AaPG18 (polygalacturonase involved in cell wall degradation) based on its subcellular localization in Arabidopsis protoplasts, stable genetic transformation in transgenic tomato, and yeast one-hybrid and luciferase activity assays. AaERF059 also responds to ethylene and regulates ethylene-/auxin-mediated fruit coloration and ripening by targeting the downstream genes AaACS2 (ACC synthase, which is involved in ethylene biosynthesis) and AaGH3 (Gretchen-Hagen 3, which is involved in the auxin pathway). Overall, AaEIL2 and AaERF059 regulate ethylene-and auxin-mediated fruit coloration and ripening by maintaining a dynamic balance in a positive and negative regulatory manner. Our results not only identified key genes but also established an intrinsic connection between fruit coloration and ripening in A. arguta.

Background Anthocyanin accumulation in plant tissues requires coordinated regulation of both biosynthesis and transport. In Actinidia arguta, these pigments determine visual quality while enhancing nutritional value and antioxidant capacity, yet the mechanisms underlying the coordinated regulation of anthocyanin synthesis and transport remain poorly understood. Results In this study, we identified AaMYB114, an R2R3-MYB transcription factor, from a skin-color-associated co-expression module in Actinidia arguta. Transient overexpression of AaMYB114 in fruit peel significantly enhanced red pigmentation and anthocyanin content. Yeast one-hybrid (Y1H) and dual-luciferase assays demonstrated that AaMYB114 directly binds to and activates the promoters of key biosynthetic genes AaPAL, AaCHI, and AaF3H. Notably, AaMYB114 also directly targets the promoter of AaGST, a glutathione S-transferase essential for anthocyanin transport into the vacuole. Co-overexpression of AaMYB114 and AaGST synergistically intensified red coloration beyond either gene alone, while chemical inhibition of GST activity with ethacrynic acid suppressed pigmentation and attenuated the effect of AaMYB114 overexpression. Conclusions These findings establish AaMYB114 as a dual-function master regulator that simultaneously orchestrates anthocyanin biosynthesis and transport, providing both theoretical insights into pigment regulation and practical targets for molecular breeding of red-skinned kiwifruit cultivars.

Currently, the methods for identifying agro-products origin usually focus on a single algorithm and overemphasize predictive performance at the expense of interpretability, which greatly hampers the development of accurate and effective traceability methods as well as insights into the intrinsic mechanisms of the models. Therefore, using kiwifruit from six regions in China, this study combines hyperspectral imaging (HSI) technology with ensemble learning methods to construct a kiwifruit origin traceability model and utilizes explainable artificial intelligence (AI) to hierarchically and holistically interpret the optimal stacking ensemble model, which aims to improve the traceability accuracy while providing an in-depth understanding of the logic behind origin prediction using spectral features. The results revealed the stacking ensemble model, with Support Vector Machine (SVM), K-Nearest Neighbor (KNN), and Random Forest (RF) as base-learners, and Logistic Regression (LR) as the meta-learner, demonstrated the best discrimination performance with 95.24% accuracy, 95.79% precision, 95.24% recall, and 95.19% F1-score, the classification accuracy of whose was improved by 2.38% than that of the best single model. Furthermore, SHapley Additive exPlanations (SHAP) analysis elucidated the decision mechanism of the optimal traceability model by quantifying the contribution of spectral features, with wavelengths of 730 nm (SHAP 0.59) and 893 nm (SHAP 0.56) being dominant for origin prediction under the kiwifruit sample conditions of this experiment. These findings serve as a significant scientific groundwork for further optimizing the application of spectral techniques in kiwifruit traceability systems, and provide a perspective for applying this technology to distinguish the geographical origin of other agro-products.

Kiwifruit (Actinidia spp.) is an economically important fruit valued for its sensory and nutritional properties. This study characterized the metabolomic composition of four kiwifruit species (A. arguta, A. chinensis, A. deliciosa, and A. eriantha) using volatile profiling and widely targeted metabolomics. A total of 234 volatile organic compounds were detected, with esters as the dominant class. Quantitative differences in flavor-active compounds were observed among species. Widely targeted metabolomics identified 1184 metabolites. Metabolic profiles were significantly influenced by genetic background. A. arguta exhibited a flavonoid rich profile, for instance, quercetagetin-7-O-glucoside was exclusively found in A. arguta cultivars and validated as a biomarker for species identification. Six compounds (catechin, esculin, naringenin, quercetin, scopolin, and taxifolin) were validated by HPLC-MS/MS and have been associated with traditional Chinese medical applications, including alleviating irritability, promoting diuresis, and anti-inflammatory effects. This study provides a framework for breeding kiwifruit cultivars optimized for sensory appeal and health-promoting functions.

Actinidia arguta is a newly emerged, commercially cultivated Actinidia species. A. arguta has a beautiful appearance and is rich in anthocyanin, and is thus highly welcomed by consumers. However, the mechanism of anthocyanin regulation in A. arguta remains unclear. In this study, we assembled the nearly complete genome of the first red A. arguta cultivar, ‘Tianyuanhong’, with an N50 of 21 Mb. Comparative genome analysis revealed a role of the expansion/contraction of gene families in the species-specific trait formation of A. arguta. Through verification of transient overexpression and stable transformation, RNA-seq analysis revealed a key bHLH transcription factor, AaBEE1, which negatively regulates anthocyanin biosynthesis. DAP-seq analysis combined with Y1H, EMSA, Chip-qPCR and LUC suggested that AaBEE1 binds to the G-box of the AaLDOX promoter and suppresses its expression. Overall, we assembled the genome of A. arguta and clarified its AaBEE1-AaLDOX module-mediated molecular mechanism of anthocyanin regulation.

Waterlogging stress is one of the greatest environmental threats to kiwifruit growth and development. ERF-VII proteins have been demonstrated to play pivotal roles in regulating plant tolerance to waterlogging. Nevertheless, the genome-wide role of ERF-VII in kiwifruit waterlogging stress tolerance remains unclear. Here, we report the function and regulatory network of an ERF-VII transcription factor located to the nucleus, AvERF73, in kiwifruit waterlogging tolerance. Overexpression of AvERF73 in Arabidopsis thaliana and A. chinensis cv. Hongyang enhanced waterlogging tolerance in transgenic plants. Furthermore, we performed transcriptome analysis (RNA-seq) and DNA affinity purification sequencing (DAP-seq) to explore the regulatory mechanism of AvERF73. RNA-seq coupled with DAP-seq showed that AvERF73 might directly activate AcNAC022 involved in the “cellular response to hypoxia” process and AcHMGS1 involved in the mevalonate pathway to respond to waterlogging, which were also confirmed by a dual-luciferase reporter assay. Based on our results, we propose a putative working model for controlling waterlogging tolerance by AvERF73 in kiwifruit.

The C-repeat binding factors (CBFs) gene is essential for plants’ cold response, which could not only be induced by the inducer of CBF expression (ICE) genes but also activated the expression of the cold-regulated (COR) gene, thereby participating in the ICE-CBF-COR cold response pathway. However, this gene family and its functions in Actinidia arguta remain unclear. In this study, whole-genome identification and functional analysis of CBF family members in A. arguta were performed. Eighteen CBF genes, which were located on four chromosomes and had five tandem repeats, were identified. The proteins encoded by the genes were predicted to be located in the nucleus and cytoplasm. The results of the promoter cis-acting element analysis revealed light response elements, low-temperature response elements, and hormone (methyl jasmonate, gibberellin, salicylic acid, etc.) response elements. We analyzed collinearity with other kiwifruit genomes, and, interestingly, the number of CBF family members differed across geographic locations of A. arguta. RT-qPCR revealed that the expression of the CBF gene family differed under low-temperature treatment; specifically, we observed differences in the expression of all the genes. Based on phylogenetic relationships and RT-qPCR analysis, the expression of AaCBF4.1 (AaCBF4) was found to be highly upregulated, and the function of this gene in cold resistance was further verified via overexpression in transgenic Arabidopsis. AaCBF4-overexpressing plants showed higher tolerance to cold stress, showing a higher germination rate, higher chlorophyll content and lower relative electrolyte leakage. In addition, compared with the wild-type Arabidopsis, the overexpressing plants exhibited significantly reduced oxidative damage due to the reduction in reactive oxygen species production under cold stress. Therefore, AaCBF4 plays an important role in improving the cold resistance of Actinidia arguta and can be further used to develop kiwifruit germplasm resources with strong cold resistance.

Low temperatures severely threaten the growth and development of kiwifruit. Research has demonstrated that proteins belonging to the 14-3-3 family play a pivotal regulatory function in the ability of plants to resist stress. However, this specific roles of the genes in kiwifruit cold tolerance remain unclear. It had been identified that beta-amylase gene, AaBAM3.1, exhibits a positive regulatory effect on kiwifruit's tolerance to low temperature. In our research, we obtained the Actinidia arguta 14-3-3 gene general regulatory factor 1 (AaGRF1) from yeast one- hybrid (Y1H) screening library of the AaBAM3.1 promoter; the expression level of AaGRF1 was enhanced by low- temperature stress. Subcellular localization, Y1H and dual-LUC assay indicated that the AaGRF1 protein resides within the nucleus and possesses the ability to interact with the AaBAM3.1 promoter. Moreover, we also studied the role of AaGRF1 gene in cold resistance of kiwifruit. When AaGRF1 was overexpressed in kiwifruit, the transgenic plants exhibited enhanced cold tolerance. The level of antioxidants and soluble sugars in these plants were elevated compared to wild-type (WT) lines. RNA-seq of the transgenic and WT lines revealed that AaGRF1 might interact with genes in the 'ascorbate-glutathione' and 'starch and sucrose' pathways, thereby enhancing the cold resistance of kiwifruit. In summary, we hypothesize that the 14-3-3 gene AaGRF1 may positively modulate the cold resistance in kiwifruit by accumulating more antioxidants and soluble sugars.

Actinidia arguta has become popular with consumers recently because of its edible and colorful fruit skin. The 3D spatial organization of its genome plays a key role in the formation of various biological traits. However, the function of 3D genome reorganization during fruit skin color formation is poorly understood in A. arguta. In this study we constructed the 3D genome of the red-skinned A. arguta cultivar 'Zhonghongbei' (ZHB) and the green-skinned cultivar 'Zhonglvbei' (ZLB), and performed chromatin structure comparisons between them at compartment, topologically associating domain (TAD), and loop levels. Global compartment comparisons at whole 3D genome level between red-skinned and green-skinned A. arguta showed that A-B compartment transition specifically occurred in chromosome 7 and chromosome 16, based on which all genes within 3 Mb upstream and downstream of A-B compartment transition were retrieved to construct a four-way Venn diagram, which showed that AaCBP60B-like, encoding calmodulin-binding protein 60 B-like, is the key candidate gene negatively correlating with fruit color. Exogenous calcium chloride treatments enhancing AaCBP60B-like expression to repress anthocyanin biosynthesis proved a negative role of AaCBP60B-like in anthocyanin biosynthesis. Overexpression and virus-induced gene silencing assays of AaCBP60B-like revealed the inhibition of anthocyanin biosynthesis derived from differential expression of AaCBP60B-like resulting from a 346-bp InDel variation located at the AaCBP60B-like promoter resulting in activity differences in red- and green-skinned A. arguta. ATAC-seq results proved that the 346-bp InDel variation affects 3D genome organization. Our study provides the first 3D chromosome organization in red- and green-skinned A. arguta, based on which a candidate gene, AaCBP60B-like, involved in anthocyanin regulation is identified.

Fruit quality traits play an important role in consumption of kiwiberry (Actinidia arguta). The genetic basis of fruit quality traits in this woody, perennial and dioecious fruit crop remains largely unknown. This study aimed to identify the underlying genetic basis of fruit quality traits in A. arguta, using a single nucleotide polymorphism (SNP) genetic linkage map previously developed in a tetraploid F1 population of ‘Ruby-3’ × ‘KuiLv-M’. The F1 population was phenotyped over three years (2020–2022) for fruit quality traits, including skin color, flesh color, fruit weight, fruit diameter, total soluble solids, fruit longitudinal diameter and fruit shape index. A total of nine QTLs were detected for five traits, explaining 10%–32% of the trait variation. For fruit color, the support interval of a major QTL on LG9 contained an MYB transcription factor MYB110, which was previously demonstrated to control color regulation in kiwifruit, thus suggesting that the MYB110 is the candidate gene for fruit color in kiwiberry. The linked marker for fruit color was validated in an F1 population and 25 kiwiberry cultivars. In conclusion, the knowledge obtained through the QTL mapping is applicable to improve the efficiency and cost-effectiveness in kiwiberry breeding.

[Objective]This experiment aimed to investigate the genetic trends of fruit-related traits in the F1 generation of Actinidia chinensis 'Jintao' with different paternal parents,understand the influ-ence of different paternal genotypes on the sex ratio,single fruit weight,soluble solids content(SSC),and flesh color of the F1 generation and try to screen out potential superior paternal parents.[Methods]Using A.chinensis 'Jintao' as the maternal parent and 10 different male A.chinensis genotypes as pater-nal parents,10 hybrid combinations were established.The sex ratio of the F1 generation was recorded,and fruit weight,soluble solids content(SSC),and flesh color were measured to analyze genetic trends and differences.Gray relational analysis(GRA)was applied for a comprehensive evaluation of fruit traits across the 10 hybrid combinations.At the harvest stage,10 fruits were randomly sampled from each plant to measure single fruit weight and post-ripening SSC,while flesh color was visually de-scribed using the RHS color chart.Data were processed and statistically analyzed using Excel 2019,and GRA was also performed.SPSS 25.0 was used to calculate means,standard deviations,coefficients of variation(CV),chi-square values,and significance of differences.[Results]Among the 10 hybrid combinations,8 exhibited male-biased sex ratio in the F1 generation,while 2 showed female-biased ra-tios.However,the differences between the male and female amount were not significant,comforming to a 1∶1 sex ratio.The average single fruit weight of the F1 generation ranged from 59.95 g to 79.84 g,all smaller than that of the maternal parent(82 g),with coefficient of variations(CV)of 18.75％-26.00％.The top three combinations for average single fruit weight were I-3,I-8,and I-2(79.84 g,79.34 g,and 74.51 g respectively),significantly or marginally bigger than the others.The combination I-10 had the smallest average single fruit weight,only 59.95 g,significantly smaller than the most others.The maximum single fruit weight ranged from 85.42 g to 141.52 g among the 10 hybrid combinations,with a proportion exceeding the maternal parent ranged from 7.14％to 34.42％.Among them,I-8,I-3,and I-2 had the highest proportion of offspring surpassing the maternal parent(34.42％,34.29％,and 30.77％,respectively).The average SSC of the F1 generation ranged from 12.9％to 16.1％,with CV of 9.14％-15.25％.Only three combinations surpassed the maternal parent in average SSC,while the other seven were lower.The combinations I-2,I-9,and I-5 had the highest average SSC(16.1％,15.9％,and 15.7％respectively),significantly higher than the others.The combination I-10 had the lowest average SSC,only 12.9％.The maximum SSC across the 10 combinations ranged from 16.9％to 20.1％,with 8.33％-63.46％exceeding the maternal parent.The combinations I-2,I-5,and I-9 had the highest pro-portions(63.46％,50.00％,and 46.51％,respectively).The flesh color of F1 generation showed a segre-gation of yellow,yellowish green,and light green,with yellow flesh being the most prevalent(41.18％-84.21％),while yellowish green and light green proportions were nearly equal.The combination I-3 did not produce light green flesh phenotype.By assigning values to different flesh colors,the top three com-binations in descending order were I-7,I-9,and I-3.The gray correlation analysis of three traits re-vealed the following order of correlation and weight coefficients:SSC＞single fruit weight＞flesh col-or.The weighted relational degrees of 10 combinations ranked as:I-9＞I-3＞I-2＞I-7＞I-8＞I-5＞I-1＞I-4＞I-6＞I-10.[Conclusion]The F1 generation exhibited extensive segregation in single fruit weight and SSC,with an overall trend toward smaller fruit size and lower SSC.The differential analysis re-vealed significant variations in these traits among the F1 generation of different hybrid combinations,suggesting that the observed differences might be attributed to the influence of the paternal parent.The flesh color also displayed distinct segregation,further indicating the paternal contribution to this trait.Based on the mean values of related traits and the proportion of F1 generation exceeding the maternal parent,it could be inferred that the paternal lines of I-8,I-3,and I-2 would possess potential for breed-ing large-fruit cultivars,while I-2,I-5,and I-9 would be more likely to produce offspring with high SSC.Furthermore,the paternal parents of combinations I-7,I-9,and I-3 may breed cultivars with yel-low-flesh.The comprehensive evaluation results demonstrated that the paternal lines of combination I-9,I-3,and I-2 seems to be more likely to breed new kiwifruit varieties with large fruit size,high soluble solids content,and yellow flesh.

Kiwifruit has extremely high nutritional value, but its rootstock strongly influences the physiology and metabolism of its scion fruit. Here, we evaluated the influence of a new kiwifruit rootstock, Actinidia valvata ′Zhongmikangzhen No. 2′, on the leaves and fruits of the scion A. deliciosa ′Zhongmi 2′. The leaf size, leaf mineral element contents, fruit size (single-fruit weight, longitudinal diameter and transverse diameter), and fruit quality traits (soluble sugar content, vitamin C content, total acidity and dry matter content) of ′Zhongmi 2′ grafted on ′Zhongmikangzhen No. 2′ (zk) and A. deliciosa seedling (zp) rootstock were measured at five fruit developmental stages (30, 60, 90, 120, and 150 days after flowering (DAF)). The zk fruits were significantly larger than those of zp and their leaves were also longer. The leaf length and width of the zk fruits was significantly greater than those of the zp fruits. The leaf contents of Cu, P and Mo significantly differed at 60, 120 and 120 DAF, respectively. The fruits of the two stock–scion combinations may presented different flavors. UPLC–MS/MS analysis of the fruit metabolome revealed 1697 differentially expressed metabolites. These metabolites, which were divided into multiple categories through KEGG analysis, indicated significant differences in fruit quality and resistance. Decanoic acid (decreased in zk-hard) and 9-oxononanoic acid (increased in zk-hard) may collectively affect fruit aroma. Moreover, α-linolenic acid and 12-oxo-phytodienoic acid (a precursor of jasmonic acid), both of which increase in zk-hard, contribute to increased nutritional value and are involved in the abiotic stress response, respectively. This study revealed the effects of different rootstock–scion combinations on fruit quality traits and leaf mineral elements and provides a basis for studying the mechanisms of rootstock–scion interactions.

Kiwifruit is a dioecious woody liana fruit tree, and the non-fruitfulness of male plants leads to a great deal of blindness in the selection of male plants in crossbreeding. In this study, we induced the development of male plant ovary by externally applying plant growth regulators (PGRs) and performed histological observation, phytohormone content determination and transcriptome analysis on the abortive ovary of the male kiwifruit (Con), the ovary of the female kiwifruit (Fem) and the PGR-induced developing ovary of the male kiwifruit (PT). Histological analysis showed that the Con ovary was devoid of ovules and the carpels were atrophied, the Fem ovary had ovules and the PT ovary was devoid of ovules, but the carpels developed normally and were not atrophied. Endogenous phytohormone content measurements displayed higher levels of trans-zeatin (tZT) in PT and Fem than Con, and lower levels of gibberellin (GA3) and abscisic acid (ABA) than Con. Transcriptome analysis revealed significant differences in many key genes in the cytokinin and auxin pathways, which were consistent with the results of phytohormone content measurements. Meanwhile, the genes related to carpel development, SPT (DTZ79_04g03580) and SK41 (DTZ79_19g04340), were highly expressed in PT, suggesting that they may play a key role in PGR-induced development of the ovary in male kiwifruit. These results provide information for elucidating the potential regulatory network of PGR-induced ovary development in male flowers and contribute to further identification of valuable target genes.

Red Actinidia arguta has recently become highly popular because of its red appearance resulting from anthocyanin accumulation, and has gradually become an important breeding direction. However, regulators involved in anthocyanin biosynthesis have not been fully characterized in A. arguta. Here, we demonstrated that a key R2R3-MYB transcription factor, AaMYB61-like, plays a crucial role in A. arguta anthocyanin biosynthesis. The RT-qPCR results revealed that transient overexpression of AaMYB61-like in A. arguta fruit at 90–100 DAFB significantly promoted anthocyanin biosynthesis, as did the gene expression levels of AaCHS, AaCHI, AaF3H, AaLDOX, and AaF3GT, whereas the result of VIGS revealed the opposite results in A. arguta fruit at 105–115 DAFB. A transcriptional activation assay indicated that AaMYB61-like exhibited transcriptional activation activity. Y1H and LUC assays revealed that AaMYB61-like activates the promoters of AaCHS, AaLDOX, and AaF3GT. In addition, AabHLH137 was found to be related to fruit color from the transcriptome data. We demonstrated that AaMYB61-like promotes anthocyanin biosynthesis by interacting with AabHLH137 via Y2H, BiFC, and Agrobacterium-mediated co-transformation. Our study not only reveals the functions of AaMYB61-like and AabHLH137 in anthocyanin regulation, but also broadly enriches color regulation theory, establishing a foundation for clarifying the molecular mechanism of fruit coloration in kiwifruit.

Actinidia arguta possesses different colors in the fruit skin and flesh, but the underlying mechanism has not yet been clarified. In this study, we conducted 36 samples RNA-seq to investigate the phenotypic expression of different fruit tissues (skin and flesh) in red and green A. arguta varieties during different coloring phases. GO and KEGG enrichment results of differentially expressed genes (DEGs) suggested that the red color of the skin and flesh was derived from anthocyanin transport and flesh softening, respectively. Weighted gene co-expression network analysis (WGCNA) revealed MEyellow and MEblack modules significantly correlated with skin and flesh coloration, and two genes, Glutathione S-transferases (AaGST) and β-galactosidases (AaBGAL), were identified as hub genes involved in different tissue-specific coloration. Transient overexpression in apples and kiwifruits confirmed the role of AaGST and AaBGAL in color formation. Our results preliminarily explore the mechanism of red color formation in different A. arguta fruit tissues and provide novel insights into red color formation.