## The Roles of NAC Transcription Factors in Peach Fruit: Development, Ripening, and Quality Regulation ## Abstract NAC (NAM, ATAF1/2, and CUC2) transcription factors constitute one of the largest families of plant-specific transcriptional regulators, playing pivotal roles in diverse biological processes including fruit development, ripening, senescence, and stress responses. In peach (*Prunus persica*), NAC transcription factors have emerged as central regulators of fruit maturity, quality traits, and postharvest physiology. This review synthesizes current knowledge on the functional characterization of NAC transcription factors in peach fruit, covering their roles in anthocyanin biosynthesis, fruit texture determination, flavor volatile production, chlorophyll degradation, and maturity date regulation. We highlight key findings from genetic, transcriptomic, and epigenetic studies that have elucidated the molecular mechanisms by which PpNAC proteins orchestrate these processes. The review also discusses the regulatory networks involving NAC transcription factors and their downstream target genes, including those involved in cell wall metabolism, pigment biosynthesis, and aroma compound formation. Finally, we outline future research directions, including the application of CRISPR/Cas9 genome editing for functional validation and the potential for breeding improved peach cultivars through manipulation of NAC gene expression. ## Keywords NAC transcription factors; peach fruit; fruit ripening; anthocyanin biosynthesis; fruit quality; transcriptional regulation; *Prunus persica* ## 1. Introduction Peach (*Prunus persica* L. Batsch) is one of the most economically important stone fruits worldwide, valued for its nutritional content, flavor, and aesthetic appeal. The fruit ripening process in peach is a highly coordinated developmental program involving dramatic changes in color, texture, sugar content, aroma, and secondary metabolite accumulation [1]. Understanding the molecular mechanisms that govern these ripening-associated processes is essential for improving fruit quality, extending shelf life, and meeting consumer preferences. NAC transcription factors represent one of the largest families of plant-specific transcriptional regulators, with more than 100 members identified in the peach genome [2]. The name NAC is derived from three founding members: NAM (No Apical Meristem) from petunia, ATAF1/2 from *Arabidopsis thaliana*, and CUC2 (Cup-Shaped Cotyledon) from *Arabidopsis* [3]. These proteins are characterized by a highly conserved N-terminal NAC domain responsible for DNA binding and nuclear localization, while the C-terminal region exhibits high variability and functions as a transcriptional activation or repression domain [4]. In plants, NAC transcription factors have been implicated in a wide range of biological processes, including shoot apical meristem development, lateral root formation, leaf senescence, stress responses, and fruit ripening [5]. The role of NAC transcription factors in fruit ripening was first established in tomato, where the NAC family member NON-RIPENING (NOR) was identified as a key regulator of the ripening process [6]. Since then, NAC transcription factors have been functionally characterized in various fruit species, including apple, banana, citrus, kiwifruit, and peach [7]. In peach, recent advances in genomics, transcriptomics, and functional genomics have significantly expanded our understanding of how NAC transcription factors regulate fruit development and ripening. This review aims to provide a comprehensive overview of the current knowledge on the roles of NAC transcription factors in peach fruit, with particular emphasis on their regulatory functions in anthocyanin biosynthesis, fruit texture and stone adhesion, flavor formation, chlorophyll degradation, and maturity date determination. ## 2. Genome-Wide Identification and Expression Analysis of NAC Transcription Factors in Peach The completion of the peach genome sequence provided a foundation for genome-wide identification of transcription factor families [8]. Subsequent bioinformatic analyses identified over 100 NAC family members in the peach genome, which were classified into distinct phylogenetic subgroups based on their sequence similarity to NAC proteins from *Arabidopsis* and other plant species [2]. Expression profiling across different peach tissues and developmental stages revealed that many *PpNAC* genes exhibit tissue-specific and developmentally regulated expression patterns. Li et al. [2] conducted a comprehensive identification of peach NAP (NAC-like, activated by APETALA3/PISTILLATA) transcription factor genes and characterized their expression patterns in vegetative and reproductive organs during development and senescence. Their study identified multiple *PpNAP* genes that showed differential expression in flowers, leaves, and fruits at various developmental stages. Several *PpNAP* genes displayed increased expression during fruit ripening and senescence, suggesting their involvement in these processes [2]. This foundational work established the framework for subsequent functional studies of NAC transcription factors in peach. ## 3. NAC Transcription Factors in the Regulation of Anthocyanin Biosynthesis in Peach Fruit Anthocyanins are important secondary metabolites that contribute to the red coloration of peach fruit skin and flesh, and are associated with health benefits due to their antioxidant properties. The molecular genetics of blood-fleshed peach, a variety characterized by intense red pigmentation in the fruit flesh, revealed a critical role for NAC transcription factors in activating anthocyanin biosynthesis [1]. Zhou et al. [1] demonstrated that a NAC transcription factor, designated PpNAC1, directly activates the transcription of *PpMYB10.1*, a key MYB transcription factor that regulates the anthocyanin biosynthetic pathway in blood-fleshed peach. Through a combination of genetic mapping, transcriptome analysis, and functional assays, the authors showed that PpNAC1 binds to the promoter of *PpMYB10.1* and activates its expression, leading to the upregulation of anthocyanin biosynthetic genes such as *PpUFGT* (UDP-glucose:flavonoid 3-O-glucosyltransferase). The activation of this regulatory cascade results in the accumulation of anthocyanins specifically in the fruit flesh of blood-fleshed peach varieties [1]. This study provided the first direct evidence for the involvement of NAC transcription factors in anthocyanin regulation in peach and established a NAC-MYB-biosynthetic gene regulatory module. More recently, Dai et al. [9] identified another NAC transcription factor, PpNAP4, that promotes chlorophyll degradation and anthocyanin synthesis in the skin of peach fruit. Their work demonstrated that PpNAP4 directly targets genes involved in chlorophyll catabolism, including *PpNYC1* (NON-YELLOW COLORING1) and *PpPAO* (PHEOPHORBIDE A OXYGENASE), as well as anthocyanin biosynthetic genes. The dual function of PpNAP4 in promoting both chlorophyll degradation and anthocyanin accumulation highlights the coordinated regulation of pigmentation changes during peach fruit ripening. Furthermore, the authors showed that PpNAP4 physically interacts with other regulatory proteins to fine-tune the expression of its target genes [9]. This finding expands our understanding of the complex regulatory networks that control fruit coloration in peach. ## 4. NAC Transcription Factors Control Fruit Texture and Stone Adhesion Fruit texture is a critical quality attribute that influences consumer preference, postharvest handling, and shelf life. In peach, fruit texture traits such as firmness, melting behavior, and stone adhesion (freestone versus clingstone) are determined by the activity of cell wall-modifying enzymes. Recent research has uncovered a direct link between NAC transcription factors and the regulation of cell wall metabolism in peach fruit. Zhao et al. [10] demonstrated that PpNAC transcription factors regulate *ENDOPOLYGALACTURONASE* (*PpPG*) alleles and thereby control peach fruit texture and stone adhesion. Using a combination of genetic analyses, DNA binding assays, and transgenic approaches, the authors identified specific PpNAC proteins that bind to the promoter regions of *PpPG* genes and modulate their expression. The differential expression of *PpPG* alleles, regulated by PpNAC transcription factors, was shown to determine the extent of cell wall degradation in the fruit mesocarp and the abscission zone between the flesh and the stone. This study provided mechanistic insights into the regulation of fruit texture in peach and demonstrated that allelic variation in PpNAC binding sites contributes to the diversity of texture phenotypes observed among peach cultivars [10]. The findings have significant implications for peach breeding programs aimed at developing varieties with desirable texture characteristics and improved postharvest performance. ## 5. Regulation of Fruit Flavor and Aroma by NAC Transcription Factors The flavor of peach fruit is determined by a complex mixture of sugars, organic acids, and volatile organic compounds, with volatile esters being major contributors to the characteristic peach aroma. The biosynthesis of flavor-related volatile compounds is tightly regulated during fruit ripening, and transcription factors play central roles in this process. Cao et al. [7] investigated the transcriptional and epigenetic regulation of fruit flavor ester biosynthesis and revealed the involvement of NAC transcription factors in this process. Their study, conducted primarily in tomato but with comparative analyses in peach, demonstrated that NAC transcription factors directly regulate the expression of alcohol acyltransferase (*AAT*) genes, which catalyze the final step in ester volatile biosynthesis. The authors found that NAC transcription factors can recruit chromatin remodeling complexes to modulate the epigenetic landscape at target gene loci, thereby influencing the timing and intensity of volatile production during fruit ripening [7]. The epigenetic component of this regulation adds an additional layer of complexity to the control of flavor biosynthesis and suggests that NAC transcription factors may serve as integrators of developmental and environmental signals in the regulation of fruit quality traits. In peach specifically, Zhang et al. [11] discovered that two adjacent NAC transcription factors, designated PpNAC1 and PpNAC2, coordinately regulate fruit maturity date and flavor. Through quantitative trait locus (QTL) mapping and functional characterization, the authors showed that these two NAC genes are located in a genomic region associated with variation in harvest date among peach varieties. PpNAC1 and PpNAC2 activate downstream genes involved in ethylene biosynthesis and signaling, cell wall metabolism, and volatile production, thereby integrating the timing of ripening with the development of flavor characteristics. Natural variation in the expression levels of these NAC genes was associated with differences in maturity date and flavor profiles across diverse peach germplasm [11]. This study is particularly significant because it links the regulation of ripening timing with flavor quality, two traits that are often inversely correlated in breeding programs. ## 6. NAC Transcription Factors in Fruit Maturity Date Regulation The timing of fruit maturity is an important agronomic trait that affects harvest scheduling, market availability, and fruit quality. The identification of genetic factors controlling maturity date has been a major goal in peach breeding. Zhang et al. [11] provided compelling evidence that two adjacent NAC transcription factors are key determinants of fruit maturity date in peach. Their study combined genetic mapping in a segregating population with functional analysis to demonstrate that allelic variation at the *PpNAC1-PpNAC2* locus accounts for a significant proportion of the phenotypic variation in harvest date. The regulatory mechanism involves the direct activation of ethylene biosynthetic genes by PpNAC1 and PpNAC2, which in turn triggers the ripening program. The expression levels of these NAC transcription factors are themselves regulated by both genetic and environmental factors, providing a mechanism for the integration of internal developmental cues with external signals such as temperature [11]. This discovery opens up possibilities for marker-assisted selection of peach varieties with optimal maturity dates and flavor quality, and provides a foundation for understanding the evolutionary diversification of ripening behaviors within the *Prunus* genus. ## 7. Regulatory Networks and Future Perspectives The accumulated evidence from the studies reviewed above paints a picture of NAC transcription factors as central hubs in the regulatory networks that control multiple aspects of peach fruit development and ripening. These transcription factors operate at different levels of the regulatory hierarchy: some function as master regulators that control major developmental transitions, while others act as more specialized modulators of specific metabolic pathways. The functional diversity of NAC proteins is achieved through several mechanisms, including differential expression patterns, protein-protein interactions, post-translational modifications, and variations in DNA binding specificity. Looking forward, several research directions merit further investigation. First, the application of CRISPR/Cas9 genome editing technology for functional validation of candidate *PpNAC* genes in peach would provide definitive evidence for their roles in fruit development and ripening. The successful application of genome editing in tomato for manipulating ripening regulatory genes [12] suggests that similar approaches could be adapted for peach, although challenges related to transformation efficiency and regeneration remain. Second, the integration of multi-omics data, including transcriptomics, epigenomics, and metabolomics, will be essential for constructing comprehensive regulatory networks that capture the complexity of NAC transcription factor function. The study by Cao et al. [7] demonstrated the importance of epigenetic regulation in NAC-mediated control of fruit flavor biosynthesis, and future studies should explore how chromatin modifications influence NAC transcription factor activity and target gene accessibility in peach fruit. Third, natural variation in *PpNAC* genes and their regulatory sequences represents a valuable resource for peach improvement. Identifying favorable alleles associated with desirable fruit quality traits and incorporating them into breeding programs through marker-assisted selection or genomic selection could accelerate the development of superior peach cultivars. Finally, the potential cross-talk between NAC transcription factors and other signaling pathways, including those mediated by hormones (ethylene, auxin, abscisic acid) and environmental factors (temperature, light), warrants further investigation. Understanding how NAC transcription factors integrate multiple signals to coordinate fruit development and ripening will be crucial for developing strategies to improve fruit quality under changing environmental conditions. ## 8. Conclusion NAC transcription factors have emerged as key regulators of fruit development, ripening, and quality in peach. Research over the past decade has revealed their involvement in anthocyanin biosynthesis, fruit texture determination, flavor volatile production, chlorophyll degradation, and maturity date regulation. The discovery of specific NAC proteins and their downstream target genes has provided mechanistic insights into the molecular control of these important agronomic traits. The continued elucidation of NAC transcription factor functions and regulatory networks will not only advance our fundamental understanding of fruit biology but also provide practical tools for the genetic improvement of peach and related stone fruit species. ## References [1] Zhou, H., Lin-Wang, K., Wang, H., Gu, C., Dare, A.P., Espley, R.V., He, H., Allan, A.C., & Han, Y. (2015). Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. *The Plant Journal*, 82(1), 105-121. [2] Li, F., Li, J., et al. (2016). Identification of Peach NAP Transcription Factor Genes and Characterization of their Expression in Vegetative and Reproductive Organs during Development and Senescence. *Frontiers in Plant Science*, 7, 147. [3] Souer, E., van Houwelingen, A., Kloos, D., Mol, J., & Koes, R. (1996). 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