The transcription factor AebHLH89 activates AeGMP1 transcription to regulate L-ascorbic acid accumulation in kiwifruit (Actinidia eriantha) revealed by genome-wide association study

Abstract

Actinidia eriantha, a kiwifruit species endemic to China, produces fruits with notable nutritional, medicinal, and economic value, particularly due to its high L-ascorbic acid (L-AsA) content. However, the regulatory mechanisms underlying L-AsA accumulation in its fruit remain poorly understood. This study meticulously measured L-AsA levels of fruits in 216 A. eriantha accessions from natural populations and performed a genome-wide association study, through which we identified significantly associated lead single nucleotide polymorphisms and insertion deletions, and characterized a key candidate gene AebHLH89, AePPR, AePP2Ab, and AePHL1 involved in the positive regulation of L-AsA accumulation. Functional experiments showed that overexpression of AebHLH89 significantly enhanced L-AsA accumulation, while its silencing via virus-induced gene silencing markedly decreased L-AsA levels. Yeast one-hybrid assay and dual-luciferase assay preliminarily revealed that AebHLH89 could bind to the AeGMP1 promoter and activates its transcription, thereby upregulating the L-AsA biosynthesis pathway and promoting L-AsA synthesis and accumulation. These findings provide valuable genetic resources for molecular marker-assisted breeding in kiwifruit and contribute to germplasm innovation. Simultaneously, the identification of key regulatory genes enhances our understanding of L-AsA metabolism and lays a theoretical foundation for the genetic improvement of kiwifruit.

References

[1] Huang HW, Liu YF . Natural hybridization, introgression breeding, and cultivar improvement in the genus Actinidia. Tree Genet Genomes. 2014; 10: 1113-22

[2] Xu XB, Liao GL, Huang CH, et al. Germplasm Resources of Actinidia eriantha. Beijing: Science Press; 2021

[3] Mandl J, Szarka A, Bánhegyi G . Vitamin C: update on physiology and pharmacology. Br J Pharmacol. 2009; 157: 1097-110

[4] Massip L, Garand C, Paquet ER, et al. Vitamin C restores healthy aging in a mouse model for Werner syndrome. FASEB J. 2010; 24: 158-72

[5] Aumailley L, Lebel M . The impact of vitamin C on different system models of Werner syndrome. Antioxid Redox Signal. 2021; 34: 856-74

[6] Smirnoff N. Ascorbic acid metabolism and functions: a comparison of plants and mammals. Free Radic Biol Med. 2018; 122: 116-29

[7] Zhang H, Xiang YL, He N, et al. Enhanced vitamin C production mediated by an ABA-induced PTP-like nucleotidase improves plant drought tolerance in Arabidopsis and maize. Mol Plant. 2020; 13: 760-76

[8] Liang JX, Xu MH, Yang BH, et al. Evolutionary analysis of ascorbate-glutathione cycle genes across green plants with lineage-specific profiling in grapevine (Vitis vinifera L.). Hortic Res. 2025; 13: uhaf247

[9] Su LY, Liu ZT, Chen PY, et al. Origin and evolution of signaling pathways responsible for ascorbic acid synthesis and catabolism during plant terrestrialization. Hortic Res. 2025; 12: uhaf184

[10] Dodd FL, Kennedy DO, Stevenson EJ, et al. Acute and chronic effects of multivitamin/mineral supplementation on objective and subjective energy measures. Nutr Metab (Lond). 2020; 17: 16

[11] Mellidou I, Kanellis AK . Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops. Front Chem. 2017; 5: 50

[12] Rowe S, Carr AC . Global vitamin C status and prevalence of deficiency: a cause for concern? Nutrients. 2020; 12: 2008

[13] Liao GL, Xu Q, Allan AC, et al. L-ascorbic acid metabolism and regulation in fruit crops. Plant Physiol. 2023; 192: 1684-95

[14] Liao GL, Chen L, He YQ, et al. Three metabolic pathways are responsible for the accumulation and maintenance of high AsA content in kiwifruit (Actinidia eriantha). BMC Genomics. 2021; 22: 13

[15] Liu XY, Wu RM, Bulley SM, et al. Kiwifruit MYBS1-like and GBF3 transcription factors influence L-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3. New Phytol. 2022; 234: 1782-800

[16] Liao GL, Lin LT, Liu YS, et al. Light-response gene L-galactose dehydrogenase positively contributes to ascorbic acid in Actinidia eriantha. Hortic Plant J. 2025

[17] Zou Q, Bao T, Yu L, et al. The regulatory module MdCPCL-MdILR3L mediates the synthesis of ascorbic acid and anthocyanin in apple. Plant Biotechnol J. 2025; 23: 1101-17

[18] Wang Y, Wu Y, Wang X, et al. Genome wide association analysis identifies candidate genes for fruit quality and yield in Actinidia eriantha. J Integr Agric. 2024; 23: 1929-39

[19] Bournonville C, Mori K, Deslous P, et al. Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase. Plant Cell. 2023; 35: 2615-34

[20] Liu XY, Bulley SM, Varkonyi-Gasic E, et al. Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress. Plant Physiol. 2023; 192: 982-99

[21] Liu XY, Li YC, Zhang XZ, et al. AcABI5a integrates abscisic acid signaling to developmentally modulate fruit ascorbic acid biosynthesis in kiwifruit. Hortic Res. 2025; 12: uhaf111

[22] Xu X, Huang BW, Fang X, et al. SlMYB99-mediated auxin and abscisic acid antagonistically regulate ascorbic acids biosynthesis in tomato. New Phytol. 2023; 239: 949-63

[23] Zhao JT, Sauvage C, Zhao JH, et al. Meta-analysis of genome-wide association studies provides insights into genetic control of tomato flavor. Nat Commun. 2019; 10: 10

[24] Zhang SW, Wu SQ, Jia ZQ, et al. Exploring the influence of a single-nucleotide mutation in EIN4 on tomato fruit firmness diversity through fruit pericarp microstructure. Plant Biotech J. 2024; 22: 2379-94

[25] Zhang MY, Xue C, Hu HJ, et al. Genome-wide association studies provide insights into the genetic determination of fruit traits of pear. Nat Commun. 2021; 12: 12

[26] Anees M, Zhu H, Umer MJ, et al. Identification of an aux/IAA regulator for flesh firmness using combined GWAS and bulked segregant RNA-Seq analysis in watermelon. Hortic Plant J. 2024; 10: 1198-213

[27] Hu L, Xu T, Cai Y, et al. Identifying candidate genes for grape (Vitis vinifera L.) fruit firmness through genome-wide association studies. J Agric Food Chem. 2025; 73: 8413-25

[28] Zhang HM, Lyu XJ, Sun ZY, et al. GWAS identifies a molecular marker cluster associated with monoterpenoids in grapes. Hortic Res. 2025; 12: uhaf144

[29] Chen L, Jia DF, Liu YS, et al. Genome-wide association studies provide molecular insights into the genetic determination of the fruit shape-related traits of Actinidia eriantha. Horticulturae. 2025; 11: 538

[30] Chen L, Liu YS, Gao H, et al. A model for selecting kiwifruit (Actinidia eriantha) germplasm resources with excellent fruit quality. Foods. 2024; 13: 4014

[31] Liao GL, Huang CH, Jia DF, et al. A high-quality genome of Actinidia eriantha provides new insight into ascorbic acid regulation. J Integr Agric. 2023; 22: 3244-55

[32] Yu C, Yan M, Dong H, et al. Maize bHLH55 functions positively in salt tolerance through modulation of AsA biosynthesis by directly regulating GDP-mannose pathway genes. Plant Sci. 2021; 302: 110676

[33] Li JF, Wang BX, Liu Y, et al. Progress of research in functions of PPR proteins in growth and development of rice. Plant Genet Resour. 2022; 23: 358-67

[34] Konert G, Trotta A, Kouvonen P, et al. Protein phosphatase 2A (PP2A) regulatory subunit B’γ interacts with cytoplasmic ACONITASE 3 and modulates the abundance of AOX1A and AOX1D in Arabidopsis thaliana. New Phytol. 2015; 205: 1250-63

[35] Wang Z, Zheng Z, Song L, et al. Functional characterization of Arabidopsis PHL4 in plant response to phosphate starvation. Front Plant Sci. 2018; 9: 1432

[36] Liu W, Zhao X, Li Y, et al. Genome-wide association study reveals the genetic architecture and key drought and yield related genes in common vetch (Vicia sativa L.). Plant Biotechnol J. 2025; 24: 1082-97

[37] Jiang Q, Shen Y, Wu L, et al. Genomic signatures of local adaptation to precipitation and solar radiation in kiwifruit. Plant Divers. 2025; 47: 733-45

[38] McCallum J, Laing W, Bulley S, et al. Molecular characterisation of a supergene conditioning super-high vitamin C in kiwifruit hybrids. Plants-Basel. 2019; 8: 237

[39] Conklin PL, Norris SR, Wheeler GL, et al. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci USA. 1999; 96: 4198-203

[40] Cronje C, George GM, Fernie AR, et al. Manipulation of L-ascorbic acid biosynthesis pathways in Solanum lycopersicum: elevated GDP-mannose pyrophosphorylase activity enhances L-ascorbate levels in red fruit. Planta. 2012; 235: 553-64

[41] Xue CC, Xu JY, Wang C, et al. Molecular cloning and functional characterization of a soybean GmGMP1 gene reveals its involvement in ascorbic acid biosynthesis and multiple abiotic stress tolerance in transgenic plants. J Integr Agric. 2017; 17: 539-53

[42] Dai M, Xue Q, McCray T, et al. The PP6 phosphatase regulates ABI5 phosphorylation and abscisic acid signaling in Arabidopsis. Plant Cell. 2013; 25: 517-34

[43] Konert G, Rahikainen M, Trotta A, et al. Subunits B’γ and B’ζ of protein phosphatase 2A regulate photo-oxidative stress responses and growth in Arabidopsis thaliana. Plant Cell Environ. 2015; 38: 2641-51

[44] Kang SG, Price J, Lin PC, et al. The arabidopsis bZIP1 transcription factor is involved in sugar signaling, protein networking, and DNA binding. Mol Plant. 2010; 3: 361-73

[45] Zhou J, Hu Q, Xiao X, et al. Mechanism of phosphate sensing and signaling revealed by rice SPX1-PHR2 complex structure. Nat Commun. 2021; 12: 12

[46] Liao G, Zhong M, Jiang Z, et al. Genome-wide association studies provide insights into the genetic determination of flower and leaf traits of Actinidia eriantha. Front Plant Sci. 2021; 12: 730890

[47] Vashishtha K, Gaud C, Andrews S, et al. Librarian: a quality control tool to analyse sequencing library compositions. F1000Res. 2022; 11: 1122

[48] Yang Y, Huang L, Xu C, et al. Chromosome-scale genome assembly of areca palm (Areca catechu). Mol Ecol Resour. 2021; 21: 2504-19

[49] McCormick RF, Truong SK, Mullet JE . RIG: recalibration and interrelation of genomic sequence data with the GATK. G3 (Bethesda). 2015; 5: 655-65

[50] Ghosh S, Bollinedi H, Krishnan SG, et al. Dissection of the genetic basis for total γ-Oryzanol and its components in whole grain brown rice through genome-wide association study. J Agric Food Chem. 2025; 73: 12765-75

[51] Shi R, Cao Y, Yang T, et al. Genome-wide association study reveals the genetic basis of crude fiber components in Brassica napus L. shoots at stem elongation stage. J Agric Food Chem. 2024; 72: 16530-40

[52] Yang J, Lee SH, Goddard ME, et al. GCTA: a tool for genome-wide complex trait analysis. Am J Hum Genet. 2011; 88: 76-82

[53] Xu XB, Huang CH, Qu XY, et al. A new easy peeling Actinidia eriantha cultivar ‘Ganmi 6’. Acta Hortic Sin. 2015; 42: 2539-40

[54] Chen L, Gao Z, Mao J, et al. Transcriptome analysis of starch degradation in postharvest kiwifruit treated with different temperatures. Acta Agric Univ Jiangxiensis. 2023; 45: 591-604

[55] Fernandez-Pozo N, Rosli HG, Martin GB, et al. The SGN VIGS tool: user-friendly software to design virus-induced gene silencing (VIGS) constructs for functional genomics. Mol Plant. 2015; 8: 486-8

[56] Li YQ, Gao H, Jia DF, et al. AGPS and BAM genes dramatically improve ’Ganlv 1′ (Actinidia eriantha) fruit quality through starch metabolism during fruit development. Sci Hortic. 2024; 329: 113004