Tandem transcription factors PpNAC56 and PpNAC72 synergistically activate the transcription of PpPGF to regulate softening during peach fruit ripening

Tandem transcription factors PpNAC56 and PpNAC72 synergistically activate the transcription of PpPGF to regulate softening during peach fruit ripening | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tandem transcription factors PpNAC56 and PpNAC72 synergistically activate the transcription of PpPGF to regulate softening during peach fruit ripening Langlang Zhang, Xiaofei Wang, Kang Dong, Bin Tan, Xianbo Zheng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3627008/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Rapid softening after harvest is a significant challenge for peach production, as it results in fruit rotting and significantly reduces shelf life. In this study, we conducted an analysis that revealed the presence of two tandem genes, PpNAC56 and PpNAC72 , within the sr (slow ripening) locus, which are highly conserved in dicots. Additionally, we found that PpNAC56 is the orthologous gene of Non-ripening ( NOR ) in tomato. Transcript analysis demonstrated that both PpNAC56 and PpNAC72 are highly expressed in peach fruit, with their transcript levels up-regulated at the onset of peach fruit ripening. The yeast two-hybrid and bimolecular fluorescence complementation assays showed PpNAC56 interacts with PpNAC72 and this interaction is also detected in tomato and apple. Transient VIGS experiments showed that PpNAC56 and PpNAC72 positively regulate peach fruit softening. The Yeast one-hybrid, the dual luciferase assay and LUC bioluminescence imaging proved that PpNAC56 and PpNAC72 directly binds to the PpPGF promoters and activate its transcription. Notably, PpNAC56 and PpNAC72 together, have a higher levels of activation activity for PpPGF than the genes of PpNAC56 or PpNAC72 alone. In summary, our findings demonstrate that the tandem transcription factors PpNAC56 and PpNAC72 synergistically activate the transcription of PpPGF to regulate fruit softening during peach fruit ripening. Peach ripening NAC transcriptional regulation fruit softening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Message PpNAC56 and PpNAC72 directly binds to the PpPGF promoters, synergistically activate its transcription to regulate peach fruit softening. Introduction Peach [ Prunus persica (L.) Batsch] is a fruit crop of important economic importance, celebrated for its distinct flavor and nutrient content. However, the limited shelf life of peach fruits necessitates harvesting before their physiological ripening stage, resulting in inferior fruit quality (Eduardo et al. 2011 ). Regulation of fruit ripening is crucial not only for extending shelf life but also for strategically planning the marketing season. As a classical climacteric fruit, the ripening process of peach is influenced by various factors, with the plant hormone ethylene serving as a critical regulator. Ethylene exerts an effect on multiple physiological responses throughout peach fruit ripening (Tatsuki et al. 2007 ; Tatsuki et al. 2013 ; Monti et al. 2016 ). Furthermore, several transcription factors, including NAC , have been identified and functionally characterized as important regulators of fruit ripening (Lü et al. 2018; Gu et al. 2019 ; Wang et al. 2021 ; Dai et al. 2023 ; Cheng et al. 2023 ). The Non-ripening ( NOR ) gene, encoding a NAC transcription factor, serves as a pivotal regulator of fruit ripening in tomato. The loss-of-function mutant of SlNOR exhibited disrupted pigment accumulation, abnormal fruit softening and an extended storage life (Gao et al. 2020 ). Similarly, the silencing of NOR-like1 suppressed tomato fruit ripening and directly interacted with the promoters of several genes involved in tomato ripening processes, including ethylene biosynthesis, color change, and cell wall metabolism, thereby positively regulating their expression (Gao et al. 2018 ). Wang et al. ( 2022 ) reported that in climacteric melon, CmNAC-NOR directly induces the expression of CmACS5 , CmNCED3 , and CmZDS1 to promote fruit ripening. Additionally, MdNAC18.1 from apple has been identified as an indicator of apple harvest date, and fruit firmness at harvest and after harvest (Migicovsky et al. 2021 ). Transgenic introduction of Md NAC18.1 into the tomato nor mutant complemented the ripening deficiency observed in the nor mutant (Migicovsky et al. 2021 ). Furthermore, the NAC transcription factor RIPENING INDUCING FACTOR ( RIF ), which is an orthologue of NOR , plays a crucial role in the regulation of strawberry fruit ripening and knockout mutations of FvRIF result in a complete blockade of fruit ripening (Martin-Pizarro et al. 2021 ; Li et al. 2023 ). Moreover, FvRIF modulates anthocyanin biosynthesis and fruit softening by directly regulating the expression of related core genes (Li et al. 2023 ). Collectively, these studies demonstrate that NOR and its orthologue genes play vital roles in both climacteric and non‐climacteric fruit ripening. In peach, the identification of PpNAC56 (Prupe.4G187100), which shares homology with NOR, has revealed its significance as a key participant in the positive feedback loop of ethylene synthesis during peach fruit ripening. PpNAC56 exerts its influence by directly binding to the promoter regions of ethylene synthesis genes ACS and ACO , thereby triggering gene expression (Tatsuki et al. 2007 ; Tatsuki et al. 2013 ; Lü et al. 2018). Additionally, PpNAC56 also serves as an activator for flavor volatile biosynthesis and anthocyanin metabolism during peach fruit ripening (Zhou et al. 2015 ; Cao et al. 2021 ; Jin et al. 2022 ). These findings collectively underscore the significant roles played by PpNAC56 during peach fruit ripening. However, fruit ripening involves dramatic and complex changes beyond the generation of volatile chemicals and anthocyanin metabolism, and the precise contribution of PpNAC56 to other ripening-associated changes remain unclear. Furthermore, previous investigations have reported that PpNAC56 is located in the quantitative trait locus (QTL) qMD4.1 controlling maturity date (Pirona et al. 2013 ). In conjunction with PpNAC56 , another member of the NAC transcription factor, PpNAC72 (Prupe.4G186800), is also located in qMD4.1 locus and has been considered to be the candidate gene for maturity date (Nuñez -Lillo et al. 2015). Notably, a 9 bp insertion in the coding sequence of PpNAC72 has been consistently associated with the early-ripening phenotype (Pirona et al. 2013 ; Guo et al. 2020 ). However, the mechanisms by which PpNAC72 regulates fruit ripening remain unknown. Fruit softening is one of the most important physiological changes during peach fruit ripening. The rapid softening of harvested peaches makes them susceptible to rotting and significantly shortened their shelf life, thereby limiting potential industrial development (Qian et al. 2021 ). It has been reported that the peach fruit texture and flesh adhesion are closely related traits controlled by the F-M locus in linkage group 4 (Dettori et al. 2001 ; Dirlewanger et al. 2006 ; Ogundiwin et al. 2009 ). Additionally, two pectinase genes, namely Pp-endoPGM (PGM) and Pp-endoPGF (PGF), have been identified as key regulators of distinct fruit texture and flesh adhesion phenotypes across various peach accessions (Peace et al. 2005 ; Gu et al. 2016 ). The slow ripening (SR) trait is a mutation that inhibits the normal fruit ripening process in peach. This trait is controlled by a single sr gene, which has been mapped on linkage group 4 (G4), overlapping with the qMD4.1 locus (Eduardo et al. 2015 ; Meneses et al. 2016 ; Nuñez-Lillo et al. 2015 ). A deletion of 26.6 kb in the sr lotus has been showed to be co-localized with the SR trait/maturity date and the PpNAC72 located in the deletion sequence was identified as a candidate gene (Eduardo et al. 2015 ; Nuñez-Lillo et al. 2015 ). In this study, we conducted an analysis of the 26.6 kb deletion sequence and found that it contains not only the PpNAC72 gene but also the entire promoter sequence of PpNAC56 . The tandem arrangement of these two genes were is highly conserved in dicots. Furthermore, our findings demonstrate that both PpNAC56 and PpNAC72 play positive roles for fruit softening during ripening. Moreover, PpNAC56 physically interacted with PpNAC72 to form a protein heterodimer, resulting in a higher activity of PpPGF transcription. Materials and Methods Plant materials The peach cultivars used in this study were seven-year-old trees and maintained at the Fruit Tree Germplasm Repository of Henan Agricultural University (Henan Province, China). The fruit samples from the early-ripening variety ‘FeiYu’ and the late-ripening peach variety ‘JinQiuHongMi’ were collected about 7 days’ before or after full bloom (DAFB). The fruits were immediately frozen in liquid nitrogen and stored at -80°C. Three biological replicates, each consisting of five fruits from different trees, were used for the experiments. Nicotiana benthamiana seedlings were grown in pots under a 16/8 h photoperiod condition in a growth chamber at 25°C for four weeks, and the leaves were subsequently used for further tests. Methods Quantitative real-time PCR (qRT-PCR) Total RNA were extracted using the FastPure® Universal Plant Total RNA Isolation Kit (Vazyme, Jiangsu, China), followed by the synthesis of first-strand cDNAs using the HiScript®ⅢRT SuperMix for qPCR(+ gDNA wiper) (Vazyme, Jiangsu, China) according to the manufacturer’s instructions. qRT-PCR reactions were conducted in a total reaction volume of 10 µL, containing 5 µL of SYBR Green I Master Mix, 0.5 µM of each primer, and 200 ng of template cDNA by using the SYBR® Green PCR kit (Takara, China). Amplification was carried out on an ABI Prism 7500 FAST Sequence Detection System (Applied Biosystems, USA). The peach PpTEF2 (Prupe.4G138900) and tobacco β-tubulin (U91564) were selected as the reference gene (Tong et al. 2009 ; Schmidt and Delaney, 2010 ). The primer sequences used for qRT-PCR are listed in Table S1 . Three biological replicates were performed for each analysis. Gene isolation and promoter cloning The full-length Coding DNA Sequence (CDS) of the genes were isolated from the cDNA of peach cv. Huangshuimi using primers designed based on the reference sequences from peach genome version 2.0 ( https://phytozome-next.jgi.doe.gov/info/Ppersica_v2_1 ). Homologous genes used for phylogenetic analyses in other species were obtained from the NCBI database. Sequence alignments and phylogenic analyses were performed using MEGA7. Transient transformation in peach fruit The CDS fragments of PpNAC56 and PpNAC72 were cloned into the pSAK277 vectors to generate overexpression constructs using CloneExpress®ⅡOne Step Cloning Kit (Vazyme, China). Specifically, a 235-bp cDNA fragment of PpNAC56 and a 343-bp cDNA fragment of PpNAC72 were amplified and then inserted into the virus vector pTRV2 to generate the Virus-induced gene silencing (VIGS) constructs. The recombinant plasmids, along with empty plasmid (pTRV1 and pTRV2) as control, were transformed into A. tumefaciens GV3101 and incubated at 28 ℃ until an OD 600 of 0.6. The bacteria were then suspended in infiltration buffer (10 mM MES + 10 mM MgCl 2 + 150 µM AS, pH5.6). For the VIGS experiment, the culture containing the recombinant plasmids pTRV1 and pTRV2 was mixed at a ratio of 1:1, and 500 µL of this culture was infiltrated into both sides of peach fruit cv. ‘YuNongMiXiang’ at the end of the S3 development stage, following the protocol described by Wang et. al ( 2022 ). A total of 100 peach fruits were infiltrated, and the fruits were harvested to detect gene expression levels and measure fruit firmness two weeks after the injection. Measurement of fruit firmness Peel firmness and flesh firmness were measured using a TA-XTPlusC (Stable Micro Systems, UK). The texture analyzer was set with the following parameters: test depth of 5 mm, pre-test speed of 1 mm·s − 1 , test speed of 1 mm·s − 1 and post-test speed of10 mm·s − 1 . A stainless-steel probe with a diameter of 5 mm was used for the measurements. Each side of the fruit was measured three times. Measurements were taken at 48-hour intervals, and 10 fruits of two sides were tested for each treatment. Yeast two-hybrid (Y2H) assay The coding sequences of PpNAC56 , SlNAC561 and MdNAC72 were cloned into pGBKT7, while the coding sequences of PpNAC72 , SlNAC721 and MdNAC56 were fused to pGADT7. The resulting AD and BD plasmids were co-transformed into the Y2H-gold yeast strain. P53 and 53-BD + AD were used as positive control and negative control, respectively. The interaction between the proteins was analyzed on SD/-Trp/-Leu (− TL) and SD/-Trp/-His/-Leu/-Ade (− THLA) media supplemented with X-α-gal medium (Clontech, Mountain View, CA, USA). Primers used for cloning in this study are listed in Table S1 . Bimolecular Fluorescence Complementation (BiFC) assay The full-length CDS sequence of PpNAC56 and PpNAC72 were cloned into the pNC-Ecn and pNC-Enn vectors, respectively. These constructs were then introduced into A. tumefaciens GV3101 (pSoup). After incubation, the bacteria were collected and resuspended in infiltration buffer to an OD 600 = 0.8. The suspensions of Ecn-PpNAC56 and Enn-PpNAC72 were mixed at a ratio of 1:1 (v:v) and infiltrated into tobacco leaves. The green fluorescent protein (GFP) signals were visualized using a confocal microscope (Nikon, Japan). Yeast one-hybrid (Y1H) assay The full-length CDS of PpNAC56 and PpNAC72 were cloned into pB42AD vector as prey. Different lengths of the promoter regions of PpPGF were cloned into pLacZi vector as bait using the SE Seamless Cloning and Assembly Kit (Beijing ZOMANBIO, China). The Y1H assay was performed following the protocol described in previous study (Zhang et al. 2022 ). The primer sequences used for the Y1H assay are provided in Table S1 . Dual-luciferase (LUC) reporter assays About 1.8 kb promoter regions of PpPGF were amplified and cloned into pGreen II 0800-LUC reporter vectors. The constructs of SAK277-PpNAC56 and SAK277-PpNAC72 under 35S promoter were used as effector. For the transient expression assay, the effector and reporter vectors were separately transformed into A.tumefaciens GV3101 and incubated. After collection, the bacteria were suspended with solution (10 mM MgCl 2 + 10 mM MES + 150 µM AS, pH5.6) to an OD 600 of 0.8. The effector and the reporter were mixed at ratios of 4:1 or 2:2:1 (effector1: effector2: reporter) and statically incubated for 3 h at room temperature. Then, the mixture was infiltrated into four-week-old tobacco leaves. The LUC and REN luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, China). The LUC signal intensity was also analyzed using the In-Vivo NightSHADE LB 985 imaging system (Berthold Technologies, Bad Wildbad, Germany). Three biological replicates with a mixture of 6 samples each replicates were performed. Statistical analysis The data were presented as means and standard errors calculated from at least three biological replicates. Statistical analysis was conducted using Student’s t-test by SPSS version 21. Statistical significance is indicated with single or double asterisks at P < 0.05 and < 0.01, respectively. Results The tandem genes PpNAC56 and PpNAC72 were located in maturity date locus and were highly conserved in dicots A 26.6 kb deletion in sr lotus has been previously reported to co-localized with the SR trait/maturity date, and within this deleted region, PpNAC72 has been identified as a candidate gene for this trait (Eduardo et al. 2015 ; Nuñez-Lillo et al. 2015 ). In this study, the 26.6 kb deletion sequence was further analyzed and found that the deletion sequence encompasses not only the PpNAC72 gene but also the entire promoter region of the PpNAC56 (Fig. 1 a). This finding posits the possibility that both PpNAC56 and PpNAC72 genes may be candidate genes for the SR trait/maturity date. To further determine the functional roles of PpNAC56 and PpNAC72 , an orthologous gene comparison was conducted across multiple species. The analysis showed that PpNAC56 is the ortholog of the NOR and Nor-like in tomato and CmNAC-NOR in melon (Fig. 1 b) (Gao et al. 2018 ; Wang et al. 2022 ). Additionally, the tandem arrangement analysis of the NAC56 and NAC72 across various taxa revealed that monocotyledons solely possess NAC56 , whereas dicotyledons harbor both NAC56 and NAC72 with variable intergenic intervals (Table 1 ). Remarkably, the dicotyledonous tandem configuration of NAC56 and NAC72 exhibits a notable evolutionary conservation (Table 1 ). These results suggest an integrated role of these tandemly arranged genes in modulating the peach fruit ripening. Table 1 The tandem arrangement and interval region between two tandem genes in different species Species NAC56 NAC72 Interval region(bp)* Monocot Pineapple Aco023481 - - Duckweed Spipo7G0000300 - - Banana GSMUA_Achr1T08860_001 - - Corn Zm00008a029116_T01 - - Rice Os07g37920 - - Dicot Peach Prupe.4G187100 Prupe.4G186800 19863 Tomato Solyc07g063420 Solyc07g063410 15338 Apple MD03G1222600 MD03G1222700 40382 Melon Mel03c016540 Mel03c016536 21342 Orange orange1.1g016791m orange1.1g019011m 25906 Columbine Aqcoe1G321300 Aqcoe1G321200 12758 Papaya evm.model.supercontig_80.94 evm.model.supercontig_80.93 17071 Strawberry mrna31150.1-v1.0-hybrid mrna31149.1-v1.0-hybrid 14541 Grapevine GSVIVT01014405001 GSVIVT01014403001 15039 Cotton Gorai.009G433100 Gorai.009G433200 75796 Gorai.002G073800 Gorai.002G073700 54058 Arabidopsis AT3G15510 AT3G15500 7422 AT1G52880 AT1G52890 6462 Poplar Potri.011G123500 Potri.011G123300 40246 Potri.001G404400 Potri.001G404100 39180 Carrot DCAR_009975 DCAR_009973 25977 DCAR_022480 DCAR_022479 9975 * The interval length between two tandem genes on one chromosome Transcript levels of PpNAC56 and PpNAC72 are increased at onset of peach fruit ripening To further investigate the roles of PpNAC56 and PpNAC72 in peach fruit development and ripening, the transcript levels of these two genes were assessed during the development in early-ripening ‘Feiyu’ and late-ripening ‘JinQiuHongMi’ peach varieties. The transcriptional profiles of PpNAC56 and PpNAC72 presented congruent patterns during fruit development in both early- and late- ripening cultivars (Fig. 2 a and 2 b). At the preliminary stages of fruit development (S1 and S2), transcript abundance for both genes was relatively low. Contrastingly, a significant increase of gene expression was observed concomitant with the initiation of ripening (S3), reaching a peak expression levels prior to a subsequent decline during the ripening phase (S4). Additionally, tissue-specific expression assessments revealed a higher expression levels of PpNAC56 and PpNAC72 in the fruit than in other tissues like stem, mature leaves and flowers (Fig. S1 ). These results suggest a pivotal involvement of PpNAC56 and PpNAC72 in regulation the ripening process of peach fruits. Furthermore, a distinct peak expression level was observed at the S1 to S2 transition in both early and late- ripening varieties. This expression peak indicates a potential role for these two genes not only in the ripening stages but also in the initial phases of fruit development. PpNAC56 physically interacted with PpNAC72 The evolutionary conservation of the tandem gene arrangement and their synchronized expression profiles during peach fruit development and ripening suggest a potential for direct interaction between PpNAC56 and PpNAC72. The Y2H assays substantiated this hypothesis, revealing an interaction between PpNAC56 and PpNAC72 (Fig. 3 a). The interaction was further confirmed in planta via BiFC assays in tobacco leaves (Fig. 3 b). Additionally, variations of a 9 base pair insertion/deletion (INDEL) in the PpNAC72 coding sequence have been associated with the maturation date of peach fruits. To delineate the impact of different PpNAC72 allelic variants on its interaction with PpNAC56, Y2H assays were employed. These assays demonstrated that the allele of PpNAC72 bearing the 9-base pair segment exhibited an enhanced interaction with PpNAC56 (Fig. 3 c). The highly conserved tandem arrangement of NAC56 and NAC72 in dicotyledonous species implies a functional interaction conserved between the two genes. Additional Y2H assays confirmed that the physical interaction was also exist in tomato and apple (Fig. 3 d), indicating this protein-protein interaction is likely preserved across various dicot plants. The implication of these findings is that PpNAC56 and PpNAC72 may operate synergistically in the regulatory network governing fruit ripening. PpNAC56 and PpNAC72 regulate peach fruit softening To obtain further understanding of the contributions of PpNAC56 and PpNAC72 to fruit ripening processes, VIGS was utilized to suppress expression of the two genes in peach fruits. Peach fruit transiently injected with TRV2- PpNAC56 or TRV2- PpNAC72 were harvested 15 days after infiltration, along with control fruits injected with the empty vector TRV2 (Fig. 4 a). Subsequent RT-qPCR analysis revealed a significant reduction in the transcript levels of PpNAC56 and PpNAC72 in the silenced fruits compared to the TRV2 controls (Fig. 4 b). Post-infiltration observations noted that both PpNAC56 and PpNAC72 -silenced fruits demonstrated a significant increase in peel and flesh firmness after 7 days of storage when contrasted with fruits injected with TRV2 (Fig. 4 c, 4 d). In addition, the expression of PpPGF , one of the PG genes, was markedly reduced in the silenced fruits than in TRV2 controls (Fig. 4 e). These results suggest that PpNAC56 and PpNAC72 are involved in promoting peach fruit softening through upregulation of PpPGF expression. PpNAC56 and PpNAC72 regulate fruit softening by directly binds to the PpPGF promoter and activates its transcription To validate the hypothesis that PpNAC56 and PpNAC72 modulate PpPGF expression, Y1H assays were conducted. Yeast cells co-transformed with the PpPGF promoter as bait and pADT7 as prey exhibited blue colonies, indicating self-activation of the PpPGF promoter (Fig. 5 b). Consequently, the promoter region of PpPGF was dissected into four distinct fragments (Fig. 5 a). The assay results showed that the yeast cells co-transformed with PpNAC56 and fragment P2-baits or with PpNAC72 and fragment P4-baits turned blue (Fig. 5 b), which suggest that PpNAC56 and PpNAC72 regulate PpPGF expression by interacting with separate regions within the PpPGF promoter. Additionally, LUC reporter assays and LUC bioluminescence imaging were also performed to further verify this regulatory mechanism. A significant enhancement of the LUC/REN ratio was recorded in the presence of SAK-PpNAP56 or SAK-PpNAP72 (Fig. 5 d). Notably, simultaneous co-expression of both effectors (SAK-PpNAC56 and SAK-PpNAC72) with the reporter vector resulted in an even greater increase in the LUC/REN ratio compared to the individual effector (Fig. 5 d). These results were supported by visualization of the LUC fluorescence (Fig. 5 e). Collectively, these findings demonstrate that PpNAC56 and PpNAC72 controls peach fruit softening by directly binding to the PpPGF promoter and activating the transcription of PpPGF . Moreover, the evidence suggests a synergistic interaction between PpNAC56 and PpNAC72 in facilitating this transcriptional activation. Discussion The NAC (NAM, ATAF and CUC) is a plant-specific protein family and several members have been identified as key regulators of fruit ripening (Olsen et al. 2005 ; Forlani et al. 2021 ; Liu et al. 2022 ;). It has been reported that NAC proteins function through the formation of homodimers and/or heterodimers in plants (Puranik et al. 2012 ). Zhou et al ( 2015 ) demonstrated that one of NAC transcription factors, BL, can interact with PpNAC1 (also known as PpNAC56) and the heterodimer of BL and PpNAC1, rather than BL alone, activated the transcription of PpMYB10.1 , leading to anthocyanin pigmentation. Additionally, PpNAC1 and PpNAC2 were observed to form homodimers or heterodimers, activating PpAAT1 expression and synergistically regulating the content of volatile esters in peach fruit (Cao, 2019 ). In our study, we discovered that PpNAC56 physically interacts with PpNAC72, forming a protein heterodimer. This interaction leads to higher activity of the PpPGF promoter, which plays a regulatory role in fruit softening during peach fruit ripening. Taken together, these findings suggest that PpNAC56 serves as a central regulator modulating various metabolic pathways during peach fruit ripening either independently or through the forming of homodimers and/or heterodimers with other NAC genes. Phylogenetic analyses of PpNAC56 and PpNAC72, in conjunction with their respective homologous genes, reveals that these two tandemly arrayed genes are highly conserved in dicotyledonous species. Accordingly, the physically interaction between NAC56 and NAC72 were also verified in tomato and apple (Fig. 3 d), which suggested a conserved biological role of NAC56 and NAC72 in the modulation of fruit ripening processes. Extensive research has delineated the involvement of NAC56 homolog in fruit ripening. However, reports on NAC72 and its corresponding homologs remain scant. Notably, Wei et al. ( 2023 ) demonstrated that in apple, phosphorylation of MdNAC72 mediated by MdMAPK3 exerts influence over the softening of apple fruit during storage. Moreover, the overexpression of AdNAC72 in kiwifruit not only enhances AdMsrB1 expression, but also increases free Met and ACC content and ethylene production rates (Fu et al. 2021 ). In our study, both PpNAC56 and PpNAC72 have regulatory effects on the softening of peach fruit. All these results suggest the NAC72 may participate in regulation of fruit ripening, thereby indicate the evolutionary conservation of the functional roles of PpNAC56 and PpNAC72. Previous research has reported that PpNAC72 is a prime candidate gene implicated in the regulation of maturity date located on Chr. 4 (Pirona et al. 2013 ). A 9 bp insertion/deletion (INDEL) within the coding sequence of PpNAC72 has been correlated with phenotypic variation in maturity date, where early ripening individuals exhibit the 9 bp insertion while late ripening ones lack this segment. However, the explicit function and genetic variation of PpNAC72 in regulating developmental and ripening processes remained unclear. In present study, phenotypic and molecular characterizations of PpNAC72 -knockdown transgenic peach fruits elucidated that PpNAC72 plays a key role in accelerating the ripening of peach fruits by regulating fruit softening. Additionally, our findings suggest that the presence of the 9 bp insertion within PpNAC72 could potentiate its interaction with PpNAC56, thereby potentially contributing to the manifestation of an earlier ripening phenotype. In addition to PpNAC72 , PpNAC56 is likewise posited as a key regulatory gene for peach maturity date, because of its significant contributions to fruit ripening. Here, our investigation supports the notion that both the PpNAC56 and PpNAC72 are viable candidate genes for the SR trait/maturity date. The peach fruit developmental and ripening encompasses distinct growth phases: the first exponential growth phase (S1), the onset of pit hardening (S2), the second exponential growth phase (S3), and ripening (S4) (Tonutti et al. 1991 ). Notably, the span of the S2 phase varies according to the cultivar, being shorter in early ripening varieties and longer in late ripening ones (Bonghi et al. 2011 ). In this study, both PpNAC56 and PpNAC72 exhibited a high expression levels during the S3 and S4 phases which suggested their critical roles in peach fruit ripening. Intriguingly, our data reveal expression peaks for PpNAC56 and PpNAC72 at the S1 to S2 transition in both early and late ripening varieties. This transitional juncture marks a deceleration in fruit growth and precedes the lignification of the endocarp, an event synonymous with the onset of pit hardening which persists until the terminus of the S2 stage (Dardick et al. 2010 ). The increasing expression of PpNAC56 and PpNAC72 at the onset of S2 stage indicated the potential roles in influencing peach fruit development and consequently modulating the maturity date of the peach fruit. Declarations Funding This work was supported by the National Natural Science Foundation of China (32002014), the National Key Research and Development Program of China (2019YFD1000104), and the Special Fund for Henan Agriculture Research System (S2014-11-G02). Author information Authors and Affiliations College of Horticulture, Henan Agricultural University, Zhengzhou 450002, China Langlang Zhang, Xiaofei Wang, Kang Dong, Bin Tan, Xianbo Zheng, XiaYe, Wei Wang, Jun Cheng, Jiancan Feng Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Xiaofei Wang, Langlang Zhang and Kang Dong. The first draft of the manuscript was written by Langlang Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Corresponding author Correspondence to Jun Cheng and Jiancan Feng Competing Interests The authors have no relevant financial or non-financial interests to disclose. References Bonghi C, Trainotti L, Botton A, Tadiello A, Rasori A, Ziliotto F, Zaffalon V, Casadoro G, Ramina A (2011) A microarray approach to identify genes involved in seed-pericarp cross-talk and development in peach. BMC Plant Biol 11:107. https://doi.org/10.1186/1471-2229-11-107 Cao XM (2019) Metabolism and Regulation of volatile esters in peach fruit. Dissertation, Zhejiang University. https://doi.org/10.27461/d.cnki.gzjdx.2019.001945 Cao XM, Wei CY, Duan WY, Gao Y, Kuang JF, Liu MC, Chen KS, Klee H, Zhang B (2021) Transcriptional and epigenetic analysis reveals that NAC transcription factors regulate fruit flavor ester biosynthesis. 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Front Genet 12:671300. https://doi.org/10.3389/fgene.2021.671300 Monti LL, Bustamante CA, Osorio S, Gabilondo J, Borsani J, Lauxmann MA, Maulión E, Valentini G, Budde CO, Fernie AR, Lara MV, Drincovich MF (2016) Metabolic profiling of a range of peach fruit varieties reveals high metabolic diversity and commonalities and differences during ripening. Food Chemistry 190:879-888. https://doi.org/10.1016/j.foodchem.2015.06.043 Nuñez-Lillo G, Cifuentes-Esquivel A, Troggio M, Micheletti D, Infante R, Campos-Vargas R, Orellana A, Blanco-Herrera F, Meneses C (2015) Identification of candidate genes associated with mealiness and maturity date in peach [Prunus persica (L.) Batsch] using QTL analysis and deep sequencing. Tree Genet Genomes 11:86. https://doi.org/10.1007/s11295-015-0911-9 Ogundiwin EA, Peace CP, Gradziel TM, Parfitt DE, Bliss FA, Crisosto CH (2009) A fruit quality gene map of Prunus. BMC Genomics 10:587. https://doi.org/10.1186/1471-2164-10-587 Olsen AN, Ernst HA, Leggio LL, Skriver K (2005) NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci 10:79-87. https://doi.org/10.1016/j.tplants.2004.12.010 Peace C, Crisosto C, Gradziel T (2005) Endopolygalacturonase: a candidate gene for freestone and melting flesh in peach. Molecular Breeding 16:21-31. https://doi.org/10.1007/s11032-005-0828-3 Pirona R, Eduardo I, Pacheco I, Linge CDS, Miculan M, Verde I, Tartarini S, Dondini L, Pea G, Bassi D, Rossini L (2013) Fine mapping and identification of a candidate gene for a major locus controlling maturity date in peach. BMC Plant Biol 13:166. https://doi.org/10.1186/1471-2229-13-166 Puranik S, Sahu PP, Srivastava PS, Prasad M (2012) NAC proteins: regulation and role in stress tolerance. Trends in Plant Science 17:369-381. https://doi.org/10.1016/j.tplants.2012.02.004 Qian M, Xu Z, Zhang ZH, Li Q, Yan XY, Liu HK, Han MY, Li FR, Zheng JC, Zhang D, Zhao CP (2021) The downregulation of PpPG21 and PpPG22 influences peach fruit texture and softening. Planta 254:22. https://doi.org/10.1007/s00425-021-03673-6 Schmidt GW, Delaney SK (2010) Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet 283:233-241. https://doi.org/10.1007/s00438-010-0511-1 Tatsuki M, Haji T, Yamaguchi M (2007) The peach 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, is required for fruit softening. Springer Dordrecht 227-228. https://doi.org/10.1007/978-1-4020-6014-4_48 Tatsuki M, Nakajima N, Fujii H, Shimada T, Nakano M, Hayashi K, Hayama H, Yoshioka H, Nakamura Y (2013) Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch). Journal of Experimental Botany 64(4):1049-1059. https://doi.org/10.1093/jxb/ers381 Tong ZG, Gao ZH, Wang F, Zhou J, Zhang Z (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10:71. https://doi.org/10.1186/1471-2199-10-71 Tonutti P, Casson P, Ramina A (1991) Ethylene biosynthesis during peach fruit development. J Am Soc Hortic Sci 116:274-279. https://doi.org/10.21273/jashs.116.2.274 Wang JF, Tian SW, Yu YT, Ren Y, Guo SG, Zhang J. Li MY, Zhang HY, Gong GY, Wang M, Xu Y (2022) Natural variation in the NAC transcription factor NONRIPENING contributes to melon fruit ripening. Journal of Integrative Plant Biology 64(7):1448-1461. https://doi.org/10.1111/jipb.13278 Wang W, Liu SL, Cheng X, Cui ZG, Jiang YB, Zheng XB, Tan B, Cheng J, Ye X, Li JD, Li ZQ, Zhang LL, Wang XB, Zhang HP, Lian XD, Feng JC (2022) Ethylene and polyamines form a negative feedback loop to regulate peach fruit ripening via the transcription factor PpeERF113 by regulating the expression of PpePAO1. Postharvest Biol Tec 190:111958. https://doi.org/10.1016/j.postharvbio.2022.111958 Wang XB, Pan L, Wang Y, Meng JR, Deng L, Niu L, Liu H, Ding YF, Yao JL, Nieuwenhuizen NJ, Ampomah-Dwamena C, Lu ZH, Cui GC, Wang ZQ, Zeng WF (2021) PpIAA1 and PpERF4 form a positive feedback loop to regulate peach fruit ripening by integrating auxin and ethylene signals. Plant Science 313:111084. https://doi.org/10.1016/j.plantsci.2021.111084 Wei Y, Liu Z, Lv TX, Xu YX, Wei YJ, Liu WT, Liu L, Wang A, Li T (2023) Ethylene enhances MdMAPK3-mediated phosphorylation of MdNAC72 to promote apple fruit softening. Plant Cell 35(8):2887-2909. https://doi.org/10.1093/plcell/koad122 Zhang ST, Fu MM, Li ZQ, Li JW, Hai LF, Chen CY, Zheng XB, Tan B, Li JD, Cheng J, Wang W, Zhang LL, Ye X, Feng JC (2022) VvEIL2 and VvEIL4 regulate ethylene synthesis and carotenoid metabolism during senescence of grape rachis. Postharvest Biology and Technology 187:111853. https://doi.org/10.1016/j.postharvbio.2022.111853 Zhou H, Kui LW, Wang H, Gu C, Dare AP, Espley RV, He H, Allan AC, Han Y (2015) Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant J 82:105-121. https://doi.org/10.1111/tpj.12792 Supplementary Files FigS1.jpg Fig. S1 Transcript levels of PpNAC56 and PpNAC72 in different tissues of peach.S1-5, ML, BF and F represented stem, mature leaf, Bloom flower and Fruit, respectively. TableS1.xlsx Table S1 Primer sequences used in this study. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 21 Nov, 2023 Reviewers invited by journal 21 Nov, 2023 Editor invited by journal 17 Nov, 2023 Editor assigned by journal 16 Nov, 2023 First submitted to journal 16 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3627008","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":252053738,"identity":"5e4bc33e-b0b3-48f3-93bd-af8b0e299c29","order_by":0,"name":"Langlang Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBACPnYGNhDNw9jMfAwmaIBXCxszRIsccztbGmlajNn7ecyI1cL+7MHPHbWJvc083x583GGX2MDevE2CoeYOPlvSDXvPHE+c2cy73XDmmeTEBp5jZRIMx57h03JMgrftWOLGZt5t0rxtzIkNEjlmEowNh/FoYWyT/AvUsv8wzzPpv231iQ3ybwhpYWYDGl5jzNjMwybN2HYYaAsPIS1sbNKybQfkGJvZzCR7244bt/GkFVskHMOthZ+9/Znk27Y6Hsb+w88kfrZVy/azH95440MNbi1QgKQAHE0JhDQwMNQRVjIKRsEoGAUjFwAAAIxMeCqdUJwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6510-2401","institution":"Henan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Langlang","middleName":"","lastName":"Zhang","suffix":""},{"id":252053739,"identity":"babf1fda-d96f-452e-a757-6fc73050b1c3","order_by":1,"name":"Xiaofei Wang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"Wang","suffix":""},{"id":252053740,"identity":"c77ce191-9745-4c44-a5ae-127d23860040","order_by":2,"name":"Kang Dong","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Dong","suffix":""},{"id":252053741,"identity":"0aa7d4bf-55fa-4bb7-8ecd-54164288ed99","order_by":3,"name":"Bin Tan","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Tan","suffix":""},{"id":252053742,"identity":"470c99b6-94de-41d0-ae84-2b707bb4700c","order_by":4,"name":"Xianbo Zheng","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xianbo","middleName":"","lastName":"Zheng","suffix":""},{"id":252053743,"identity":"41f5a865-ae55-4dd9-897c-c54f2d386f2c","order_by":5,"name":"Xia Ye","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Ye","suffix":""},{"id":252053744,"identity":"4f6911de-5136-4148-b7a0-248c014e8c90","order_by":6,"name":"Wei Wang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":252053745,"identity":"095fcc63-0cef-4463-8207-4c6513996ca0","order_by":7,"name":"Jun Cheng","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Cheng","suffix":""},{"id":252053746,"identity":"2d4ee709-2c34-45d1-bac4-91aae6903140","order_by":8,"name":"Jiancan Feng","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiancan","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2023-11-17 17:44:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3627008/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3627008/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":47080053,"identity":"d047ff8e-bc3f-45e7-b738-e536ce74ced8","added_by":"auto","created_at":"2023-11-25 23:49:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138296,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe tandem genes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC56\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC72\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e located in maturity date locus and were highly conserved in dicots\u003c/strong\u003e. (a) The tandem genes \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e located in the 26.6 kb deletion sequence in \u003cem\u003eSR\u003c/em\u003e lotus. (b) Phylogenetic analyses of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e with their homologous genes in other species.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/70f38edcc0c95dcb83b84151.png"},{"id":47080054,"identity":"7f3a910e-db37-4dca-a5dc-3f49e7b843b4","added_by":"auto","created_at":"2023-11-25 23:49:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":150171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscript levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC56\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC72\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e during peach fruit development\u003c/strong\u003e \u003cstrong\u003eand ripening. \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eTranscript levels of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e in early-ripening peach cultivar ‘FeiYu’. (b) Transcript levels of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e in late-ripening peach cultivar ‘JinQiuHongMi’. The fruit development stage was divided into four periods (S1, S2, S3 and S4; identified with black lines,) according to the days after full bloom (DAFB). The minus days represented the days before full bloom.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/3236e9efa13d6c83764b21aa.png"},{"id":47080903,"identity":"dfd8c7ed-7fff-4b5f-aa72-f6d4942548b2","added_by":"auto","created_at":"2023-11-25 23:57:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":300289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePpNAC56 physically interacted with PpNAC72. \u003c/strong\u003e(a) Y2H analyses confirmed the interaction between PpNAC56 and PpNAC72. The pGBKT7-53 + pGADT7-RecT is a positive control, and pGBKT7-53 + pGADT7 is a negative control. (b) BiFC assay of the interaction between PpNAC56 and PpNAC72. ECN-PpNAC56+ENN is used as a negative control. (c) Y2H analyses confirmed the interaction abilities of between PpNAC56 and PpNAC72 (The PpNAC72 with a 9-bp deletion was identified with star). (d) Y2H analyses confirmed the interaction between the two tandem genes of NAC56 and NAC72 in apple and tomato.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/bb1c50200d9fc711ecdc3215.jpg"},{"id":47080902,"identity":"e7bff06a-df6d-40be-8012-612a3ae7572b","added_by":"auto","created_at":"2023-11-25 23:57:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":178016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC56\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e PpNAC72\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e delayed peach fruit softening. \u003c/strong\u003e(a) Appearance of peach fruits injected with TRV2, TRV2-\u003cem\u003ePpNAC56\u003c/em\u003eand TRV2-\u003cem\u003ePpNAC72\u003c/em\u003e. The red circles indicate the injected sites. (b) Relative expression of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003edetected by RT-qPCR in the transient fruits. (c, d) The peel and flesh firmness of the silencing fruits at different days after harvest. Ten fruits were used for each treatment and two injected sides of each fruit were test. (e) Relative expression of \u003cem\u003ePpPGF \u003c/em\u003ein the transient fruits. Data are the means ± SD. The asterisks indicate statistically significant differences between the TRV2 and the silencing fruits, as determined by Student’s t-test (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/99b1c579fcd4c312ae960b62.jpg"},{"id":47080059,"identity":"44e86be5-d822-4bf2-87d3-b238ba04087d","added_by":"auto","created_at":"2023-11-25 23:49:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":475433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePpNAC56 and PpNAC72 directly binds to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpPGF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromoter and activates its transcription. \u003c/strong\u003e(a) Schematic diagrams of four distinct \u003cem\u003ePpPGF\u003c/em\u003e promoter fragments. (b) The growth and colour of yeast cells co-transformed with prey (PpNAC56-pADT7 or PpNAC72-pADT7) and reporter construct containing different fragment of \u003cem\u003ePpPGF\u003c/em\u003e promoter, on the medium containing aureobasidin A and X-gal. (c) Schematic diagrams of reporter and effector vectors. (d, e) Dual LUC activation assay and LUC bioluminescence imaging in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves co-transformed with the reporter driven by \u003cem\u003epro-PpPGF\u003c/em\u003eand the two effectors, SAK-PpNAC56 and SAK-PpNAC72, alone or together. pGreenII 62-SK was used a negative control. Values are the means ± SD. The multiple comparisons were subjected to ANOVA using Duncan test, statistically significant differences (p \u0026lt; 0.05) were indicated by diverse lowercase.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/f3f2e012b7e5f37253707060.jpg"},{"id":47081887,"identity":"722fb68d-c246-4471-a1ee-df41c6047002","added_by":"auto","created_at":"2023-11-26 00:05:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1327739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/34fc3180-ca62-4f29-99f8-c0d04c9edfa3.pdf"},{"id":47080057,"identity":"cf522f62-7842-4aed-8e0b-1ff066267afa","added_by":"auto","created_at":"2023-11-25 23:49:42","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":84340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Transcript levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC56\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePpNAC72\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in different tissues of peach.\u003c/strong\u003eS1-5, ML, BF and F represented stem, mature leaf, Bloom flower and Fruit, respectively.\u003c/p\u003e","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/693eb9e5ad392ec3c8e18c5c.jpg"},{"id":47080058,"identity":"77e31597-859e-4545-abe8-b49cdcc9e7c5","added_by":"auto","created_at":"2023-11-25 23:49:43","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1 Primer sequences used in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3627008/v1/8bd238a71813fda96a6151a9.xlsx"}],"financialInterests":"","formattedTitle":"Tandem transcription factors PpNAC56 and PpNAC72 synergistically activate the transcription of PpPGF to regulate softening during peach fruit ripening","fulltext":[{"header":"Key Message","content":"\u003cp\u003ePpNAC56 and PpNAC72 directly binds to the \u003cem\u003ePpPGF\u003c/em\u003e promoters, synergistically activate its transcription to regulate peach fruit softening.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePeach [\u003cem\u003ePrunus persica\u003c/em\u003e (L.) Batsch] is a fruit crop of important economic importance, celebrated for its distinct flavor and nutrient content. However, the limited shelf life of peach fruits necessitates harvesting before their physiological ripening stage, resulting in inferior fruit quality (Eduardo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Regulation of fruit ripening is crucial not only for extending shelf life but also for strategically planning the marketing season. As a classical climacteric fruit, the ripening process of peach is influenced by various factors, with the plant hormone ethylene serving as a critical regulator. Ethylene exerts an effect on multiple physiological responses throughout peach fruit ripening (Tatsuki et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tatsuki et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Monti et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, several transcription factors, including \u003cem\u003eNAC\u003c/em\u003e, have been identified and functionally characterized as important regulators of fruit ripening (L\u0026uuml; et al. 2018; Gu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eNon-ripening\u003c/em\u003e (\u003cem\u003eNOR\u003c/em\u003e) gene, encoding a \u003cem\u003eNAC\u003c/em\u003e transcription factor, serves as a pivotal regulator of fruit ripening in tomato. The loss-of-function mutant of \u003cem\u003eSlNOR\u003c/em\u003e exhibited disrupted pigment accumulation, abnormal fruit softening and an extended storage life (Gao et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, the silencing of \u003cem\u003eNOR-like1\u003c/em\u003e suppressed tomato fruit ripening and directly interacted with the promoters of several genes involved in tomato ripening processes, including ethylene biosynthesis, color change, and cell wall metabolism, thereby positively regulating their expression (Gao et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Wang et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that in climacteric melon, CmNAC-NOR directly induces the expression of \u003cem\u003eCmACS5\u003c/em\u003e, \u003cem\u003eCmNCED3\u003c/em\u003e, and \u003cem\u003eCmZDS1\u003c/em\u003e to promote fruit ripening. Additionally, \u003cem\u003eMdNAC18.1\u003c/em\u003e from apple has been identified as an indicator of apple harvest date, and fruit firmness at harvest and after harvest (Migicovsky et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Transgenic introduction of Md\u003cem\u003eNAC18.1\u003c/em\u003e into the tomato \u003cem\u003enor\u003c/em\u003e mutant complemented the ripening deficiency observed in the \u003cem\u003enor\u003c/em\u003e mutant (Migicovsky et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, the NAC transcription factor \u003cem\u003eRIPENING INDUCING FACTOR\u003c/em\u003e (\u003cem\u003eRIF\u003c/em\u003e), which is an orthologue of \u003cem\u003eNOR\u003c/em\u003e, plays a crucial role in the regulation of strawberry fruit ripening and knockout mutations of \u003cem\u003eFvRIF\u003c/em\u003e result in a complete blockade of fruit ripening (Martin-Pizarro et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, \u003cem\u003eFvRIF\u003c/em\u003e modulates anthocyanin biosynthesis and fruit softening by directly regulating the expression of related core genes (Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Collectively, these studies demonstrate that \u003cem\u003eNOR\u003c/em\u003e and its orthologue genes play vital roles in both climacteric and non‐climacteric fruit ripening.\u003c/p\u003e \u003cp\u003eIn peach, the identification of \u003cem\u003ePpNAC56\u003c/em\u003e (Prupe.4G187100), which shares homology with NOR, has revealed its significance as a key participant in the positive feedback loop of ethylene synthesis during peach fruit ripening. PpNAC56 exerts its influence by directly binding to the promoter regions of ethylene synthesis genes \u003cem\u003eACS\u003c/em\u003e and \u003cem\u003eACO\u003c/em\u003e, thereby triggering gene expression (Tatsuki et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tatsuki et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; L\u0026uuml; et al. 2018). Additionally, \u003cem\u003ePpNAC56\u003c/em\u003e also serves as an activator for flavor volatile biosynthesis and anthocyanin metabolism during peach fruit ripening (Zhou et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These findings collectively underscore the significant roles played by \u003cem\u003ePpNAC56\u003c/em\u003e during peach fruit ripening. However, fruit ripening involves dramatic and complex changes beyond the generation of volatile chemicals and anthocyanin metabolism, and the precise contribution of \u003cem\u003ePpNAC56\u003c/em\u003e to other ripening-associated changes remain unclear. Furthermore, previous investigations have reported that \u003cem\u003ePpNAC56\u003c/em\u003e is located in the quantitative trait locus (QTL) \u003cem\u003eqMD4.1\u003c/em\u003e controlling maturity date (Pirona et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In conjunction with \u003cem\u003ePpNAC56\u003c/em\u003e, another member of the NAC transcription factor, \u003cem\u003ePpNAC72\u003c/em\u003e (Prupe.4G186800), is also located in \u003cem\u003eqMD4.1\u003c/em\u003e locus and has been considered to be the candidate gene for maturity date (Nu\u0026ntilde;ez -Lillo et al. 2015). Notably, a 9 bp insertion in the coding sequence of PpNAC72 has been consistently associated with the early-ripening phenotype (Pirona et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the mechanisms by which PpNAC72 regulates fruit ripening remain unknown.\u003c/p\u003e \u003cp\u003eFruit softening is one of the most important physiological changes during peach fruit ripening. The rapid softening of harvested peaches makes them susceptible to rotting and significantly shortened their shelf life, thereby limiting potential industrial development (Qian et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It has been reported that the peach fruit texture and flesh adhesion are closely related traits controlled by the \u003cem\u003eF-M\u003c/em\u003e locus in linkage group 4 (Dettori et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Dirlewanger et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ogundiwin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, two pectinase genes, namely Pp-endoPGM (PGM) and Pp-endoPGF (PGF), have been identified as key regulators of distinct fruit texture and flesh adhesion phenotypes across various peach accessions (Peace et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe slow ripening (SR) trait is a mutation that inhibits the normal fruit ripening process in peach. This trait is controlled by a single \u003cem\u003esr\u003c/em\u003e gene, which has been mapped on linkage group 4 (G4), overlapping with the \u003cem\u003eqMD4.1\u003c/em\u003e locus (Eduardo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Meneses et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Nu\u0026ntilde;ez-Lillo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A deletion of 26.6 kb in the \u003cem\u003esr\u003c/em\u003e lotus has been showed to be co-localized with the SR trait/maturity date and the \u003cem\u003ePpNAC72\u003c/em\u003e located in the deletion sequence was identified as a candidate gene (Eduardo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nu\u0026ntilde;ez-Lillo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, we conducted an analysis of the 26.6 kb deletion sequence and found that it contains not only the \u003cem\u003ePpNAC72\u003c/em\u003e gene but also the entire promoter sequence of \u003cem\u003ePpNAC56\u003c/em\u003e. The tandem arrangement of these two genes were is highly conserved in dicots. Furthermore, our findings demonstrate that both \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e play positive roles for fruit softening during ripening. Moreover, PpNAC56 physically interacted with PpNAC72 to form a protein heterodimer, resulting in a higher activity of \u003cem\u003ePpPGF\u003c/em\u003e transcription.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eThe peach cultivars used in this study were seven-year-old trees and maintained at the Fruit Tree Germplasm Repository of Henan Agricultural University (Henan Province, China). The fruit samples from the early-ripening variety \u0026lsquo;FeiYu\u0026rsquo; and the late-ripening peach variety \u0026lsquo;JinQiuHongMi\u0026rsquo; were collected about 7 days\u0026rsquo; before or after full bloom (DAFB). The fruits were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C. Three biological replicates, each consisting of five fruits from different trees, were used for the experiments. \u003cem\u003eNicotiana benthamiana\u003c/em\u003e seedlings were grown in pots under a 16/8 h photoperiod condition in a growth chamber at 25\u0026deg;C for four weeks, and the leaves were subsequently used for further tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003e Total RNA were extracted using the FastPure\u0026reg; Universal Plant Total RNA Isolation Kit (Vazyme, Jiangsu, China), followed by the synthesis of first-strand cDNAs using the HiScript\u0026reg;ⅢRT SuperMix for qPCR(+\u0026thinsp;gDNA wiper) (Vazyme, Jiangsu, China) according to the manufacturer\u0026rsquo;s instructions. qRT-PCR reactions were conducted in a total reaction volume of 10 \u0026micro;L, containing 5 \u0026micro;L of SYBR Green I Master Mix, 0.5 \u0026micro;M of each primer, and 200 ng of template cDNA by using the SYBR\u0026reg; Green PCR kit (Takara, China). Amplification was carried out on an ABI Prism 7500 FAST Sequence Detection System (Applied Biosystems, USA). The peach PpTEF2 (Prupe.4G138900) and tobacco β-tubulin (U91564) were selected as the reference gene (Tong et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Schmidt and Delaney, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The primer sequences used for qRT-PCR are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Three biological replicates were performed for each analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eGene isolation and promoter cloning\u003c/h2\u003e \u003cp\u003eThe full-length Coding DNA Sequence (CDS) of the genes were isolated from the cDNA of peach cv. Huangshuimi using primers designed based on the reference sequences from peach genome version 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/info/Ppersica_v2_1\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/info/Ppersica_v2_1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Homologous genes used for phylogenetic analyses in other species were obtained from the NCBI database. Sequence alignments and phylogenic analyses were performed using MEGA7.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransient transformation in peach fruit\u003c/h2\u003e \u003cp\u003eThe CDS fragments of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e were cloned into the pSAK277 vectors to generate overexpression constructs using CloneExpress\u0026reg;ⅡOne Step Cloning Kit (Vazyme, China). Specifically, a 235-bp cDNA fragment of \u003cem\u003ePpNAC56\u003c/em\u003e and a 343-bp cDNA fragment of \u003cem\u003ePpNAC72\u003c/em\u003e were amplified and then inserted into the virus vector pTRV2 to generate the Virus-induced gene silencing (VIGS) constructs. The recombinant plasmids, along with empty plasmid (pTRV1 and pTRV2) as control, were transformed into A. \u003cem\u003etumefaciens\u003c/em\u003e GV3101 and incubated at 28 ℃ until an OD\u003csub\u003e600\u003c/sub\u003e of 0.6. The bacteria were then suspended in infiltration buffer (10 mM MES\u0026thinsp;+\u0026thinsp;10 mM MgCl\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;150 \u0026micro;M AS, pH5.6). For the VIGS experiment, the culture containing the recombinant plasmids pTRV1 and pTRV2 was mixed at a ratio of 1:1, and 500 \u0026micro;L of this culture was infiltrated into both sides of peach fruit cv. \u0026lsquo;YuNongMiXiang\u0026rsquo; at the end of the S3 development stage, following the protocol described by Wang et. al (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A total of 100 peach fruits were infiltrated, and the fruits were harvested to detect gene expression levels and measure fruit firmness two weeks after the injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of fruit firmness\u003c/h2\u003e \u003cp\u003ePeel firmness and flesh firmness were measured using a TA-XTPlusC (Stable Micro Systems, UK). The texture analyzer was set with the following parameters: test depth of 5 mm, pre-test speed of 1 mm\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, test speed of 1 mm\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and post-test speed of10 mm\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A stainless-steel probe with a diameter of 5 mm was used for the measurements. Each side of the fruit was measured three times. Measurements were taken at 48-hour intervals, and 10 fruits of two sides were tested for each treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid (Y2H) assay\u003c/h2\u003e \u003cp\u003eThe coding sequences of \u003cem\u003ePpNAC56\u003c/em\u003e, \u003cem\u003eSlNAC561\u003c/em\u003e and \u003cem\u003eMdNAC72\u003c/em\u003e were cloned into pGBKT7, while the coding sequences of \u003cem\u003ePpNAC72\u003c/em\u003e, \u003cem\u003eSlNAC721\u003c/em\u003e and \u003cem\u003eMdNAC56\u003c/em\u003e were fused to pGADT7. The resulting AD and BD plasmids were co-transformed into the Y2H-gold yeast strain. P53 and 53-BD\u0026thinsp;+\u0026thinsp;AD were used as positive control and negative control, respectively. The interaction between the proteins was analyzed on SD/-Trp/-Leu (\u0026minus;\u0026thinsp;TL) and SD/-Trp/-His/-Leu/-Ade (\u0026minus;\u0026thinsp;THLA) media supplemented with X-α-gal medium (Clontech, Mountain View, CA, USA). Primers used for cloning in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular Fluorescence Complementation (BiFC) assay\u003c/h2\u003e \u003cp\u003eThe full-length CDS sequence of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e were cloned into the pNC-Ecn and pNC-Enn vectors, respectively. These constructs were then introduced into A. \u003cem\u003etumefaciens\u003c/em\u003e GV3101 (pSoup). After incubation, the bacteria were collected and resuspended in infiltration buffer to an OD \u003csub\u003e600\u003c/sub\u003e = 0.8. The suspensions of Ecn-PpNAC56 and Enn-PpNAC72 were mixed at a ratio of 1:1 (v:v) and infiltrated into tobacco leaves. The green fluorescent protein (GFP) signals were visualized using a confocal microscope (Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eYeast one-hybrid (Y1H) assay\u003c/h2\u003e \u003cp\u003eThe full-length CDS of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e were cloned into pB42AD vector as prey. Different lengths of the promoter regions of \u003cem\u003ePpPGF\u003c/em\u003e were cloned into pLacZi vector as bait using the SE Seamless Cloning and Assembly Kit (Beijing ZOMANBIO, China). The Y1H assay was performed following the protocol described in previous study (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The primer sequences used for the Y1H assay are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase (LUC) reporter assays\u003c/h2\u003e \u003cp\u003eAbout 1.8 kb promoter regions of \u003cem\u003ePpPGF\u003c/em\u003e were amplified and cloned into pGreen II 0800-LUC reporter vectors. The constructs of SAK277-PpNAC56 and SAK277-PpNAC72 under 35S promoter were used as effector. For the transient expression assay, the effector and reporter vectors were separately transformed into \u003cem\u003eA.tumefaciens\u003c/em\u003e GV3101 and incubated. After collection, the bacteria were suspended with solution (10 mM MgCl\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;10 mM MES\u0026thinsp;+\u0026thinsp;150 \u0026micro;M AS, pH5.6) to an OD\u003csub\u003e600\u003c/sub\u003e of 0.8. The effector and the reporter were mixed at ratios of 4:1 or 2:2:1 (effector1: effector2: reporter) and statically incubated for 3 h at room temperature. Then, the mixture was infiltrated into four-week-old tobacco leaves. The LUC and REN luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Vazyme, China). The LUC signal intensity was also analyzed using the In-Vivo NightSHADE LB 985 imaging system (Berthold Technologies, Bad Wildbad, Germany). Three biological replicates with a mixture of 6 samples each replicates were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were presented as means and standard errors calculated from at least three biological replicates. Statistical analysis was conducted using Student\u0026rsquo;s t-test by SPSS version 21. Statistical significance is indicated with single or double asterisks at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u0026lt;\u0026thinsp;0.01, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe tandem genes\u003c/b\u003e \u003cb\u003ePpNAC56\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePpNAC72\u003c/b\u003e \u003cb\u003ewere located in maturity date locus and were highly conserved in dicots\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA 26.6 kb deletion in \u003cem\u003esr\u003c/em\u003e lotus has been previously reported to co-localized with the SR trait/maturity date, and within this deleted region, \u003cem\u003ePpNAC72\u003c/em\u003e has been identified as a candidate gene for this trait (Eduardo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nu\u0026ntilde;ez-Lillo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, the 26.6 kb deletion sequence was further analyzed and found that the deletion sequence encompasses not only the \u003cem\u003ePpNAC72\u003c/em\u003e gene but also the entire promoter region of the \u003cem\u003ePpNAC56\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This finding posits the possibility that both \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e genes may be candidate genes for the SR trait/maturity date.\u003c/p\u003e \u003cp\u003eTo further determine the functional roles of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e, an orthologous gene comparison was conducted across multiple species. The analysis showed that \u003cem\u003ePpNAC56\u003c/em\u003e is the ortholog of the \u003cem\u003eNOR\u003c/em\u003e and \u003cem\u003eNor-like\u003c/em\u003e in tomato and \u003cem\u003eCmNAC-NOR\u003c/em\u003e in melon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) (Gao et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, the tandem arrangement analysis of the \u003cem\u003eNAC56\u003c/em\u003e and \u003cem\u003eNAC72\u003c/em\u003e across various taxa revealed that monocotyledons solely possess \u003cem\u003eNAC56\u003c/em\u003e, whereas dicotyledons harbor both \u003cem\u003eNAC56\u003c/em\u003e and \u003cem\u003eNAC72\u003c/em\u003e with variable intergenic intervals (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Remarkably, the dicotyledonous tandem configuration of \u003cem\u003eNAC56\u003c/em\u003e and \u003cem\u003eNAC72\u003c/em\u003e exhibits a notable evolutionary conservation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggest an integrated role of these tandemly arranged genes in modulating the peach fruit ripening.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe tandem arrangement and interval region between two tandem genes in different species\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNAC56\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNAC72\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInterval region(bp)*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eMonocot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePineapple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAco023481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDuckweed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSpipo7G0000300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBanana\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGSMUA_Achr1T08860_001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eZm00008a029116_T01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOs07g37920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"16\" rowspan=\"17\"\u003e \u003cp\u003eDicot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrupe.4G187100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrupe.4G186800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19863\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTomato\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSolyc07g063420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSolyc07g063410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15338\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMD03G1222600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMD03G1222700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40382\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMelon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMel03c016540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMel03c016536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21342\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eorange1.1g016791m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eorange1.1g019011m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25906\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColumbine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAqcoe1G321300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAqcoe1G321200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12758\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePapaya\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eevm.model.supercontig_80.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eevm.model.supercontig_80.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17071\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrawberry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emrna31150.1-v1.0-hybrid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emrna31149.1-v1.0-hybrid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14541\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrapevine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGSVIVT01014405001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGSVIVT01014403001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCotton\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorai.009G433100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGorai.009G433200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75796\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorai.002G073800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGorai.002G073700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54058\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArabidopsis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAT3G15510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAT3G15500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7422\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAT1G52880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAT1G52890\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6462\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePoplar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePotri.011G123500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePotri.011G123300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40246\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePotri.001G404400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePotri.001G404100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarrot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDCAR_009975\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDCAR_009973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25977\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDCAR_022480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDCAR_022479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9975\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*\u003cb\u003eThe interval length between two tandem genes on one chromosome\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscript levels of\u003c/b\u003e \u003cb\u003ePpNAC56\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePpNAC72\u003c/b\u003e \u003cb\u003eare increased at onset of peach fruit ripening\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the roles of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e in peach fruit development and ripening, the transcript levels of these two genes were assessed during the development in early-ripening \u0026lsquo;Feiyu\u0026rsquo; and late-ripening \u0026lsquo;JinQiuHongMi\u0026rsquo; peach varieties. The transcriptional profiles of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e presented congruent patterns during fruit development in both early- and late- ripening cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). At the preliminary stages of fruit development (S1 and S2), transcript abundance for both genes was relatively low. Contrastingly, a significant increase of gene expression was observed concomitant with the initiation of ripening (S3), reaching a peak expression levels prior to a subsequent decline during the ripening phase (S4). Additionally, tissue-specific expression assessments revealed a higher expression levels of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e in the fruit than in other tissues like stem, mature leaves and flowers (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results suggest a pivotal involvement of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e in regulation the ripening process of peach fruits. Furthermore, a distinct peak expression level was observed at the S1 to S2 transition in both early and late- ripening varieties. This expression peak indicates a potential role for these two genes not only in the ripening stages but also in the initial phases of fruit development.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePpNAC56 physically interacted with PpNAC72\u003c/h2\u003e \u003cp\u003eThe evolutionary conservation of the tandem gene arrangement and their synchronized expression profiles during peach fruit development and ripening suggest a potential for direct interaction between PpNAC56 and PpNAC72. The Y2H assays substantiated this hypothesis, revealing an interaction between PpNAC56 and PpNAC72 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The interaction was further confirmed in planta via BiFC assays in tobacco leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Additionally, variations of a 9 base pair insertion/deletion (INDEL) in the PpNAC72 coding sequence have been associated with the maturation date of peach fruits. To delineate the impact of different PpNAC72 allelic variants on its interaction with PpNAC56, Y2H assays were employed. These assays demonstrated that the allele of PpNAC72 bearing the 9-base pair segment exhibited an enhanced interaction with PpNAC56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The highly conserved tandem arrangement of NAC56 and NAC72 in dicotyledonous species implies a functional interaction conserved between the two genes. Additional Y2H assays confirmed that the physical interaction was also exist in tomato and apple (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), indicating this protein-protein interaction is likely preserved across various dicot plants. The implication of these findings is that \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e may operate synergistically in the regulatory network governing fruit ripening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePpNAC56 and PpNAC72 regulate peach fruit softening\u003c/h2\u003e \u003cp\u003eTo obtain further understanding of the contributions of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e to fruit ripening processes, VIGS was utilized to suppress expression of the two genes in peach fruits. Peach fruit transiently injected with TRV2-\u003cem\u003ePpNAC56\u003c/em\u003e or TRV2-\u003cem\u003ePpNAC72\u003c/em\u003e were harvested 15 days after infiltration, along with control fruits injected with the empty vector TRV2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Subsequent RT-qPCR analysis revealed a significant reduction in the transcript levels of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e in the silenced fruits compared to the TRV2 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Post-infiltration observations noted that both \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e-silenced fruits demonstrated a significant increase in peel and flesh firmness after 7 days of storage when contrasted with fruits injected with TRV2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In addition, the expression of \u003cem\u003ePpPGF\u003c/em\u003e, one of the PG genes, was markedly reduced in the silenced fruits than in TRV2 controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These results suggest that PpNAC56 and PpNAC72 are involved in promoting peach fruit softening through upregulation of \u003cem\u003ePpPGF\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePpNAC56 and PpNAC72 regulate fruit softening by directly binds to the\u003c/b\u003e \u003cb\u003ePpPGF\u003c/b\u003e \u003cb\u003epromoter and activates its transcription\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo validate the hypothesis that PpNAC56 and PpNAC72 modulate \u003cem\u003ePpPGF\u003c/em\u003e expression, Y1H assays were conducted. Yeast cells co-transformed with the \u003cem\u003ePpPGF\u003c/em\u003e promoter as bait and pADT7 as prey exhibited blue colonies, indicating self-activation of the \u003cem\u003ePpPGF\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Consequently, the promoter region of \u003cem\u003ePpPGF\u003c/em\u003e was dissected into four distinct fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The assay results showed that the yeast cells co-transformed with PpNAC56 and fragment P2-baits or with PpNAC72 and fragment P4-baits turned blue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), which suggest that PpNAC56 and PpNAC72 regulate \u003cem\u003ePpPGF\u003c/em\u003e expression by interacting with separate regions within the \u003cem\u003ePpPGF\u003c/em\u003e promoter. Additionally, LUC reporter assays and LUC bioluminescence imaging were also performed to further verify this regulatory mechanism. A significant enhancement of the LUC/REN ratio was recorded in the presence of SAK-PpNAP56 or SAK-PpNAP72 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Notably, simultaneous co-expression of both effectors (SAK-PpNAC56 and SAK-PpNAC72) with the reporter vector resulted in an even greater increase in the LUC/REN ratio compared to the individual effector (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These results were supported by visualization of the LUC fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Collectively, these findings demonstrate that PpNAC56 and PpNAC72 controls peach fruit softening by directly binding to the \u003cem\u003ePpPGF\u003c/em\u003e promoter and activating the transcription of \u003cem\u003ePpPGF\u003c/em\u003e. Moreover, the evidence suggests a synergistic interaction between PpNAC56 and PpNAC72 in facilitating this transcriptional activation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe NAC (NAM, ATAF and CUC) is a plant-specific protein family and several members have been identified as key regulators of fruit ripening (Olsen et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Forlani et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e;). It has been reported that NAC proteins function through the formation of homodimers and/or heterodimers in plants (Puranik et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Zhou et al (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) demonstrated that one of NAC transcription factors, BL, can interact with PpNAC1 (also known as PpNAC56) and the heterodimer of BL and PpNAC1, rather than BL alone, activated the transcription of \u003cem\u003ePpMYB10.1\u003c/em\u003e, leading to anthocyanin pigmentation. Additionally, PpNAC1 and PpNAC2 were observed to form homodimers or heterodimers, activating \u003cem\u003ePpAAT1\u003c/em\u003e expression and synergistically regulating the content of volatile esters in peach fruit (Cao, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, we discovered that PpNAC56 physically interacts with PpNAC72, forming a protein heterodimer. This interaction leads to higher activity of the \u003cem\u003ePpPGF\u003c/em\u003e promoter, which plays a regulatory role in fruit softening during peach fruit ripening. Taken together, these findings suggest that \u003cem\u003ePpNAC56\u003c/em\u003e serves as a central regulator modulating various metabolic pathways during peach fruit ripening either independently or through the forming of homodimers and/or heterodimers with other NAC genes.\u003c/p\u003e \u003cp\u003ePhylogenetic analyses of PpNAC56 and PpNAC72, in conjunction with their respective homologous genes, reveals that these two tandemly arrayed genes are highly conserved in dicotyledonous species. Accordingly, the physically interaction between NAC56 and NAC72 were also verified in tomato and apple (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), which suggested a conserved biological role of \u003cem\u003eNAC56\u003c/em\u003e and \u003cem\u003eNAC72\u003c/em\u003e in the modulation of fruit ripening processes. Extensive research has delineated the involvement of NAC56 homolog in fruit ripening. However, reports on NAC72 and its corresponding homologs remain scant. Notably, Wei et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that in apple, phosphorylation of MdNAC72 mediated by MdMAPK3 exerts influence over the softening of apple fruit during storage. Moreover, the overexpression of \u003cem\u003eAdNAC72\u003c/em\u003e in kiwifruit not only enhances \u003cem\u003eAdMsrB1\u003c/em\u003e expression, but also increases free Met and ACC content and ethylene production rates (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In our study, both \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e have regulatory effects on the softening of peach fruit. All these results suggest the NAC72 may participate in regulation of fruit ripening, thereby indicate the evolutionary conservation of the functional roles of PpNAC56 and PpNAC72.\u003c/p\u003e \u003cp\u003ePrevious research has reported that \u003cem\u003ePpNAC72\u003c/em\u003e is a prime candidate gene implicated in the regulation of maturity date located on Chr. 4 (Pirona et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A 9 bp insertion/deletion (INDEL) within the coding sequence of PpNAC72 has been correlated with phenotypic variation in maturity date, where early ripening individuals exhibit the 9 bp insertion while late ripening ones lack this segment. However, the explicit function and genetic variation of PpNAC72 in regulating developmental and ripening processes remained unclear. In present study, phenotypic and molecular characterizations of \u003cem\u003ePpNAC72\u003c/em\u003e-knockdown transgenic peach fruits elucidated that \u003cem\u003ePpNAC72\u003c/em\u003e plays a key role in accelerating the ripening of peach fruits by regulating fruit softening. Additionally, our findings suggest that the presence of the 9 bp insertion within PpNAC72 could potentiate its interaction with PpNAC56, thereby potentially contributing to the manifestation of an earlier ripening phenotype.\u003c/p\u003e \u003cp\u003eIn addition to \u003cem\u003ePpNAC72\u003c/em\u003e, \u003cem\u003ePpNAC56\u003c/em\u003e is likewise posited as a key regulatory gene for peach maturity date, because of its significant contributions to fruit ripening. Here, our investigation supports the notion that both the \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e are viable candidate genes for the SR trait/maturity date. The peach fruit developmental and ripening encompasses distinct growth phases: the first exponential growth phase (S1), the onset of pit hardening (S2), the second exponential growth phase (S3), and ripening (S4) (Tonutti et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Notably, the span of the S2 phase varies according to the cultivar, being shorter in early ripening varieties and longer in late ripening ones (Bonghi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this study, both \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e exhibited a high expression levels during the S3 and S4 phases which suggested their critical roles in peach fruit ripening. Intriguingly, our data reveal expression peaks for \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e at the S1 to S2 transition in both early and late ripening varieties. This transitional juncture marks a deceleration in fruit growth and precedes the lignification of the endocarp, an event synonymous with the onset of pit hardening which persists until the terminus of the S2 stage (Dardick et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The increasing expression of \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e at the onset of S2 stage indicated the potential roles in influencing peach fruit development and consequently modulating the maturity date of the peach fruit.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32002014), the National Key Research and Development Program of China (2019YFD1000104), and the Special Fund for Henan Agriculture Research System (S2014-11-G02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollege of Horticulture, Henan Agricultural University, Zhengzhou 450002, China\u003c/p\u003e\n\u003cp\u003eLanglang Zhang, Xiaofei Wang, Kang Dong, Bin Tan, Xianbo Zheng, XiaYe, Wei Wang, Jun Cheng, Jiancan Feng\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Xiaofei Wang, Langlang Zhang and Kang Dong. The first draft of the manuscript was written by Langlang Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Jun Cheng and Jiancan Feng\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBonghi C, Trainotti L, Botton A, Tadiello A, Rasori A, Ziliotto F, Zaffalon V, Casadoro G, Ramina A (2011) A microarray approach to identify genes involved in seed-pericarp cross-talk and development in peach. BMC Plant Biol 11:107. https://doi.org/10.1186/1471-2229-11-107\u003c/li\u003e\n\u003cli\u003eCao XM (2019) Metabolism and Regulation of volatile esters in peach fruit. Dissertation, Zhejiang University. https://doi.org/10.27461/d.cnki.gzjdx.2019.001945\u003c/li\u003e\n\u003cli\u003eCao XM, Wei CY, Duan WY, Gao Y, Kuang JF, Liu MC, Chen KS, Klee H, Zhang B (2021) Transcriptional and epigenetic analysis reveals that NAC transcription factors regulate fruit flavor ester biosynthesis. Plant J 106:785-800. https://doi.org/10.1111/tpj.15200\u003c/li\u003e\n\u003cli\u003eCheng X, Cui ZG, Jiang YB, Chen Y, Tan B, Cheng J, Zhang LL, Ye X, Wang XB, Zhang HP, Lian XD, Li JD, Li ZQ, Zheng XB, Feng JC (2023) PpeERF115 regulates peach fruit ripening by increasing polyamine turnover through up-regulation of genes involved in polyamine synthesis and catabolism. Postharvest Biology and Technology 204:112432. https://doi.org/10.1016/j.postharvbio.2023.112432\u003c/li\u003e\n\u003cli\u003eDai JY, Xu Z, Xu YT, Fang ZH, Shah K, Kang T, Wu HX, Zhang D, Xing LB, Ma JJ, Liu HK, Hu YN, Zhao CP (2023) A novel NAC transcription factor, PpNAP6, is involved in peach ripening by activating ethylene synthesis. Postharvest Biology and Technology 201:112363. https://doi.org/10.1016/j.postharvbio.2023.112363\u003c/li\u003e\n\u003cli\u003eDardick CD, Callahan AM, Chiozzotto R, Schaffer RJ, Piagnani MC, Scorza R (2010) Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence. BMC Biol 8:13. https://doi.org/10.1186/1741-7007-8-13\u003c/li\u003e\n\u003cli\u003eDettori MT, Quarta R, Verde I (2001) A peach linkage map integrating RFLPs, SSRs, RAPDs, and morphological markers. 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Plant J 82:105-121. https://doi.org/10.1111/tpj.12792\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Peach, ripening, NAC, transcriptional regulation, fruit softening","lastPublishedDoi":"10.21203/rs.3.rs-3627008/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3627008/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRapid softening after harvest is a significant challenge for peach production, as it results in fruit rotting and significantly reduces shelf life. In this study, we conducted an analysis that revealed the presence of two tandem genes, \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e, within the \u003cem\u003esr\u003c/em\u003e(slow ripening) locus, which are highly conserved in dicots. Additionally, we found that \u003cem\u003ePpNAC56\u003c/em\u003e is the orthologous gene of \u003cem\u003eNon-ripening\u003c/em\u003e (\u003cem\u003eNOR\u003c/em\u003e) in tomato. Transcript analysis demonstrated that both \u003cem\u003ePpNAC56 \u003c/em\u003eand \u003cem\u003ePpNAC72\u003c/em\u003e are highly expressed in peach fruit, with their transcript levels up-regulated at the onset of peach fruit ripening. The yeast two-hybrid and bimolecular fluorescence complementation assays showed PpNAC56 interacts with PpNAC72 and this interaction is also detected in tomato and apple. Transient VIGS experiments showed that \u003cem\u003ePpNAC56\u003c/em\u003e and \u003cem\u003ePpNAC72\u003c/em\u003e positively regulate peach fruit softening. The Yeast one-hybrid, the dual luciferase assay and LUC bioluminescence imaging proved that PpNAC56 and PpNAC72 directly binds to the \u003cem\u003ePpPGF\u003c/em\u003e promoters and activate its transcription. Notably, PpNAC56 and PpNAC72 together, have a higher levels of activation activity for \u003cem\u003ePpPGF\u003c/em\u003e than the genes of PpNAC56 or PpNAC72 alone. In summary, our findings demonstrate that the tandem transcription factors \u003cem\u003ePpNAC56\u003c/em\u003e and\u003cem\u003e PpNAC72\u003c/em\u003e synergistically activate the transcription of \u003cem\u003ePpPGF\u003c/em\u003e to regulate fruit softening during peach fruit ripening.\u003c/p\u003e","manuscriptTitle":"Tandem transcription factors PpNAC56 and PpNAC72 synergistically activate the transcription of PpPGF to regulate softening during peach fruit ripening","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-25 23:49:38","doi":"10.21203/rs.3.rs-3627008/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-11-21T06:38:41+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-11-21T06:33:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Molecular Biology","date":"2023-11-18T02:06:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-16T14:24:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Molecular Biology","date":"2023-11-16T08:27:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-molecular-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plan","sideBox":"Learn more about [Plant Molecular Biology](https://www.springer.com/journal/11103)","snPcode":"11103","submissionUrl":"https://submission.nature.com/new-submission/11103/3","title":"Plant Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c4d2abce-70de-488c-acf9-1ea6498ac7e2","owner":[],"postedDate":"November 25th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-02-19T14:11:32+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-25 23:49:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3627008","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3627008","identity":"rs-3627008","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}