Exogenous auxin regulates the growth and development of peach fruit at the expansion stage by mediating multiple-hormone signaling

Background Fruit expansion stage is crucial to fruit yield and quality formation, and auxin plays a significant role by mediating multi-hormone signals during fruit expansion. However, till now, it is still unclear of the molecular regulatory network during auxin-mediated peach fruit expansion. Results Here, exogenous NAA application markedly increased IAA content and drastically decreased ABA content at the fruit expansion stage. Correspondingly, NAA mainly induced the auxin biosynthesis gene (1 PpYUCCA) and early auxin-responsive genes (7PpIAA, 3 PpGH3, and 14 PpSAUR); while NAA down-regulated ABA biosynthesis genes (2 PpNCED, 1 PpABA3, and 1 PpAAO3). In addition, many DEGs involved in other plant hormone biosynthesis and signal transduction were significantly enriched after NAA treatment, including 7 JA, 7 CTK, 6 ETH, and 3 GA. Furthermore, we also found that NAA treatment down-regulated most of genes involved in the growth and development of peach fruit, including the cell wall metabolism-related genes (PpEG), sucrose metabolism-related genes (PpSPS), phenylalanine metabolism-related genes (PpPAL, Pp4CL, and PpHCT), and transcription factors (PpNAC, PpMADS-box, PpDof, PpSBP, and PpHB). Conclusion Overall, NAA treatment at the fruit expansion stage could inhibit some metabolism processes involved in the related genes in the growth and development of peach fruit by regulating multiple-hormone signaling networks. These results help reveal the short-term regulatory mechanism of auxin at the fruit expansion stage and provide new insights into the multi-hormone cascade regulatory network of fruit growth and development. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04514-2.


Background
Peach [Prunus persica (L.) Batsch] is generally classified as a climacteric fruit, popular among consumers because of its beautiful appearance, delicate flesh aromatic flavor, and high nutritional value [1].Fruit expansion stage of peach is the key stage of fruit yield and quality formation, which is a complex and genetically programmed process that results in a whole set of physiological, biochemical, and structural changes in the color, flavor, aroma, texture, and nutritional value [2,3].
Auxin is the first plant hormone to be discovered, which influences numerous facets of plant development and growth, notably cell division, elongation, and distinctions.crucial essential [4], apical dominance [5], root architecture development [6], the induction of blooming [7], and fruit development [8].During flower development, auxin can serve as an alternative signal replacing pollination and fertilization to trigger fruit-setting [9]; during the young fruit stage, auxin promotes the expansion of fruit by promoting the elongation and enlargement of cells [10]; and during fruit ripening stage, auxin has more different and complex effects among different species, which promotes or inhibits fruit ripening and softening.In general, auxin is regarded as a negative regulator of fruit ripening.For example, in grape, auxin treatment delayed ripening onset and delayed harvest by inhibiting cell expansion [11].In tomato, exogenous auxin delayed fruit ripening and strongly down-regulated the expression patterns of many genes related to carotenoid metabolism, cell degradation, and energy metabolism [12].However, auxin promotes maturation by inducing ethylene release in some climacteric fruit.In peach, the release of ethylene increased, the firmness of fruit decreased, and the expression levels of PpACSI, PpACO1, PpPG, and PpPME were significantly increased after exogenous auxin treatment [13,14].Similarly, in apple, applying naphthaleneacetic acid (NAA) increased fruit ethylene production and stimulated the expression of MdACS1, MdACO1, and MdERS1 genes [15].In addition, different concentrations and treatment stages of auxin application also had different regulatory effects.In banana, treatment with 100 μM IAA delayed banana fruit ripening and inhibited ethylene production, but 57.1 μM IAA treatment showed the opposite result [16,17].In apple, NAA treatment failed to induce ethylene biosynthesis at 55 or 85 days after full bloom (DAFB), but strongly induced ethylene production at 115 DAFB [18].In peach, auxin significantly induced ethylene production and promoted fruit softening and the induction of ethylene by auxin increased with treatment concentration at mature stage [13], however, at 30 days after full blossom, low concentrations of NAA (0.25 mM) promoted peach fruit maturity, while high concentrations of NAA (0.5 mM, 1 mM, 2 mM) inhibited fruit maturity and even caused fruit deformation and abscission [19].Therefore, it is necessary to accurately verify the dosage and treatment period of auxin.
Plant hormones do not act in isolation in the process of plant growth, development, maturity, and senescence.On the contrary, many endogenous and exogenous plant hormones, such as auxin (AUX), ethylene (ETH), abscisic acid (ABA), and gibberellic acid (GA), operate together or against one another to govern plant growth and development [3].In peach, PpIAA1 and PpERF4 form a positive feedback loop to regulate peach fruit ripening by integrating auxin and ethylene signals [14].Similarly, in papaya, an auxin response factor CpARF2 interacted with an important ethylene signal transcription factor CpEIL1, thus increasing the ripening-associated genes CpACS1, CpACO1, CpXTH12, and CpPE51 [20].Previous studies suggest that there is crosstalk between ABA and ethylene and that ABA regulates fruit ripening via ethylene signal.In climacteric fruits, ABA can affect ethylene production by regulating the expression of genes related to ethylene synthesis, such as ACS and ACO; while ethylene also regulates the expression of the ABA biosynthesis gene PpNCED through PpERF3 [21][22][23].In addition, ABA, brassinosteroids, and jasmonic acid can also promote ripening in climacteric fruits, where they modulate the concentration of ACO and/or ACS transcripts to enhance ethylene levels [24].Thus, exploring the crosstalk among plant hormones regulating fruit growth and development seems to be of great significance.
Although previous studies have shown that auxin plays significant roles in the growth and development of climacteric fruit through mediating multi-hormone signals.However, the molecular mechanisms and regulatory networks of auxin-induced fruit growth and development at the fruit expansion stage are still unclear.Therefore, auxin treatments were applied to peach at the fruit expansion stage.Firstly, HPLC-MS and GC-MS were used to determine the hormonal changes after auxin treatment, Then, transcriptomic analysis was used to develop a multi-hormone regulatory network in response to auxin, Finally, the differentially expressed genes (DEGs) analysis revealed the auxin-mediated regulation mode of sucrose metabolism, cell wall metabolism, phenylalanine metabolism, and transcription factors.These results provide new insights into the auxin-mediated multi-hormone regulatory network at peach fruit expansion stage.

Transcriptome analysis and biological pathways identification during peach fruit development
The growth and development of peach fruit presented a typical double-sigmoidal curve, which included four stages, namely, the first exponential growth stage (20DPA), the pit hardening stage (50DPA), the second exponential growth stage (70DPA), and mature stage (90DPA).In this study, the peach flesh samples of four developmental stages were selected for dynamic transcriptome analysis, and each sample consisted of three independent replicates.After read filtering, 79.86 Gb clean reads were obtained from the 12 samples, with an average of 6.66 Gb clean reads per sample, of which 80.82%~87.03%clean reads were uniquely mapped to the peach reference genome (Supplementary Table S2).Moreover, fragments per kilobase of transcript per million mapped reads (FPKM) density distribution profiles were created to depict the gene expression profile of each sample (Fig. 1A).According to the results of Pearson correlation coefficient analysis and principal component analysis (Fig. 1B and C), we observed significant differences between the four stages and high reproducibility between the three biological replicates of the samples.These data demonstrated that the considerable quality of transcriptome sequencing could be used for further analysis.
In this study, three comparison groups (DPA20 vs. DPA50, DPA20 vs. DPA70, and DPA20 vs. DPA90) were selected, which produced 7032, 6885, and 7656 DEGs, respectively (Fig. 1D and Supplementary Table S3).Further, the DEGs were annotated using the GO and KEGG databases.In the DPA50 vs. DPA20 comparison group, 3893 DEGs were enriched to 913 GO terms, and 1654 DEGs were enriched to 347 KEGG pathway terms.In the DPA70 vs. DPA20 comparison group, 3746 DEGs were enriched to 853 GO terms, and 1349 DEGs were enriched to 341 KEGG pathway terms.In the DPA90 vs. DPA20 comparison group, 4180 DEGs were enriched to 928 GO terms, and 1601 DEGs were enriched to 351 KEGG pathway terms.Among these terms, the top 50 GO terms and the top 20 KEGG pathway terms were selected and displayed in Fig. 2A and 2B.Particularly, in the DPA70 vs. DPA20 and DPA90 vs. DPA20 comparison groups, the DEGs were significantly enriched in Plant hormone signal transduction (ko04075) and Flavonoid biosynthesis (ko00941).These results indicated that 70DPA might be a critical period for peach fruit to enter the ripening process, with significant changes in hormone levels and color during this period.

Transcriptome analysis and biological pathways identification in response to auxin treatment
To examine the effect of auxin on peach fruit growth and development, transcriptome sequencing of peach fruit samples treated with NAA and NPA was performed.After removing reads containing adapter, reads containing Nbase, and low-quality reads, these two samples generated 5.86 and 6.92 Gb clean reads, respectively (Supplementary Table S2).After NAA treatment, 867 DEGs (30.60%) were up-regulated, 1966 DEGs (69.40%) were down-regulated; while after NPA treatment, 1139 DEGs (55.29%) were up-regulated, and 921 DEGs (44.71%) were down-regulated (Supplementary Table S3).GO databases were divided into three major functional categories: molecular function (MF), cellular component (CC), and biological process (BP).In the DPA70_N vs. DPA70 comparison group, a total of 1535 DEGs were enriched to 465 GO terms, including 168 BP, 40 CC, and 254 MF; in the DPA70_P vs. DPA70, a total of 1081 DEGs were enriched to 455 GO terms, including 163 BP, 45 CC, and 244 MF.Furthermore, the KEGG enrichment results showed that 520 DEGs were enriched to 316 KEGG pathways in the DPA70_N vs. DPA70 comparison group, and 430 DEGs were enriched to 295 KEGG pathways in the DPA70_P vs. DPA70 comparison group.Interestingly, 18 DEGs were significantly enriched in response to auxin (GO: 0009733) and 62 DEGs were significantly enriched in Plant hormone signal transformation (ko04075) in the DPA70_N vs. DPA70 comparison group; while 36 DEGs were significantly enriched in Plant hormone signal transformation (ko04075) in the DPA70_P vs. DPA70 comparison group (Fig. 3).The analysis implied that auxin can regulate the growth and development of peach fruit at the fruit expansion stage by mediating multi-hormone signaling.

Mult-hormone biosynthesis and signaling genes in response to auxin treatment
The growth and development of peach fruits are co-regulated by multiple hormones.In order to understand the response of multiple hormones to auxin during the peach fruit expansion period, six plant hormones were identified in this exploration, including 3-indoleacetic acid (IAA), abscisic acid (ABA), methyl jasmonate (MeJA), ethylene (ETH), gibberellin (GA), and transzeatin (ZT).Among them, ethylene (ETH) is not produced at the fruit expansion stage.Compared with the control group, NAA treatment significantly increased the content of IAA and reduced the content of ABA; While NPA treatment significantly reduced the content of IAA and ZT (Supplementary Fig. S1).

Cell wall metabolism in response to auxin treatment
The fruit cell wall is a complex structure composed of pectin, cellulose, hemicellulose, and glycoprotein.The change in cell wall structure and composition is essential for fruit ripening and softening.In this study, 13 DEGs involved in cell wall metabolism were enriched after auxin treatment (Supplementary Table S7

Sucrose metabolism in response to auxin treatment
The soluble sugars in peach fruit are mainly sucrose, glucose, fructose, and sorbitol, and the sucrose content is the highest.Sucrose phosphate synthase (SPS) is a pivotal enzyme that catalyzes sucrose formation, which converts UDP: uridine diphosphate glucose and fructose-6-phosphate into sucrose-6-phosphate and other converts In addition, fructokinase (FK) and hexokinase (HK) play a significant role in sucrose metabolism, which can catalyze the formation of glucose-6-phosphate and fructose-6-phosphate from glucose and fructose.In this study, 9 DEGs of the sucrose metabolism pathway were identified (Fig. S2 and Supplementary Table S8), including 3 PpSPS, 3 PpSUS, 1 PpINV, 1 PpFK, and 1 PpHK.Interestingly, 3 PpSUS, 1 PpFK, 1 PpHK, and 1 PpINV were significantly up-regulated and 1 PpSPS was significantly down-regulated after NAA and NPA treatments.

Auxin regulates the growth and development of peach fruit at the expansion stage by mediating multiple-hormone signaling
In this study, we identified 160 DEGs after NAA treatment, of which 89 DEGs were involved in the plant hormone biosynthesis and signal transduction, 10 DEGs were involved in the sucrose metabolism, 7 DEGs were involved in the cell wall metabolism, 4 DEGs were involved in the phenylalanine metabolism, and 50 DEGs were involved in the transcription factors (Fig. 7).After NAA treatment, 40 DEGs involved in the plant hormone biosynthesis and signal transduction were significantly up-regulated, 5 IAA, 9 ABA, 2 JA, 3 ETH, and 1 GA, which were positively correlated with the changes in the IAA content; while 49 DEGs involved in the plant hormone biosynthesis and signal transduction were significantly down-regulated, 14 AUX, 18 ABA, 5 JA, 7 CTK, 3 ETH, and 2 GA, which were negatively correlated with the changes in the IAA content.Interestingly, NAA significantly up-regulated most of the auxin biosynthesis and signal transduction genes and down-regulated most of the auxin biosynthesis and signal transduction genes, which is consistent with the changes in the IAA and ABA content after NAA treatment.In addition, NAA treatment down-regulated most of genes involved in the growth and development of peach fruit, the cell wall metabolism-related genes (PpEG), sucrose metabolismrelated genes (PpSPS), phenylalanine metabolism-related genes (PpPAL, Pp4CL, and PpHCT), and transcription factors (PpNAC, PpMADS-box, PpDof, PpSBP, and PpHB), which were negatively correlated with the changes in the IAA content.Therefore, we speculated that NAA treatment at the fruit expansion stage could inhibit the growth and development of peach fruit by regulating multiple-hormone signaling networks.

qRT-PCR validation of DEGs from RNA-seq
To confirm the accuracy and reproducibility of the expression profiles obtained from the RNA-seq data, 16 DEGs were chosen randomly for qRT-PCR analysis after NAA and NPA treatments (Supplementary Table S1 and Fig. 8).Those genes were involved in auxin biosynthesis and signal transduction (PpYUCCA , PpIAA6, PpIAA26, PpARF18, and PpGH3), ABA biosynthesis and signal transduction (PpNCED), phenylalanine metabolism (PpCCoAMT, PpCYP98A2), sucrose metabolism (PpSPS3, PpFK4), cell wall metabolism (PpXTH, PpEG), and transcription factor (PpSPL3, PpSPL12A, PpAGL103, and PpNAC9).For the 16 candidate genes tested, the results of qRT-PCR agreed with the changes in transcript abundance expression profiles by RNA-seq, indicating the accuracy and reliability of the RNA-seq data.

Identification and expression analysis of auxin-related genes
Auxin is an important Plant hormone, which plays an important regulatory role in all stages of plant growth and development.In this study, a total of 87 DEGs involved in auxin biosynthesis and signal transduction were identified from transcriptome data, including 6 DEGS (6 PpYUCCA ) related to auxin biosynthesis and 81 DEGs (5 PpAUX1, 3 PpTIR1, 18 PpIAA, 15 PpARF, 6 PpGH3, and 34 PpSAUR ) related to auxin signal transduction.YUCCA is a rate-limiting enzyme gene involved in the auxin biosynthesis pathway, and overexpression of the YUCCA gene promoted auxin synthesis in potato [25].In this study, the expression levels of PpYUCCA varied at different developmental stages of peach fruit, 1 PpYUCCA was highly expressed at the young fruit stage, 2 PpYUCCA at the young fruit and pit hardening stage, 2 at the young fruit, expansion, and mature stage, which might participate in the synthesis of auxin at different periods.ARF and Aux/IAA are important proteins in the auxin signaling transduction pathway [26].In this study, 3 PpARF and 1 PpIAA were highly expressed at the young fruit stage, 2 PpIAA at the pit hardening stage, 1 PpIAA at the young fruit and pit hardening stage, 1 PpARF at the expansion stage, 1 PpARF and 3 PpIAAat the expansion, and mature stage, indicating that these auxin-related genes are involved in the specific stages of peach fruit growth and development.Moreover, with the growth and development of the peach fruit, 3 PpARF and 1 PpIAA showed an upward trend, which might promote fruit ripening; while 2 PpARF and 3 PpIAA showed a downward trend, which might inhibit fruit ripening.Overall, these results indicated that members of the PpARF and PpIAA families had their specific functions during plant growth and development, but also shared redundant functions with other members of the family [27].

Mult-hormone in response to auxin at the fruit expansion stage
Peach fruit growth and development is a complex process controlled and integrated by multiple hormone signals, with some hormones acting as activators and others as inhibitors [2].Numerous studies have shown that auxin regulated fruit growth and development through interaction with other hormones [28].In this study, NAA treatment significantly increased the content of IAA and reduced the content of ABA; While NPA treatment significantly reduced the content of IAA and ZT.It's worth noting that ethylene was not produced at the fruit expansion stage after NAA treatment.
Auxin has an important regulatory role in the growth and development of peach fruit.After NAA treatment, an auxin synthesis gene PpYUCCA (Prupe.5G074800)increased 32 times, indicating that NAA treatment stimulated the accumulation of auxin by inducing the expression level of PpYUCCA genes.Meanwhile, 24 early auxin-responsive gene families, including 7PpIAA, 3 PpGH3, and 14 PpSAUR , were rapidly induced and up-regulated by NAA treatment, consistent with the previous conclusions [29].In addition, 4 PpARF and 3 PpAUX1 were down-regulated in response to NAA treatment, indicating a negative feedback regulation of NAA biosynthesis.
ABA is a potent accumulation hormone in mature fruits, essential in regulating the ripening process in climacteric and non-climacteric fruits [30].With the fruit ripening of fruit, the expression of ABA biosynthesis and signal-related genes increased, and PpNCED1 and PpNCED2 promoted ripening and senescent by regulating ABA synthesis [31].In addition, ABA can promote fruit ripening by increasing the expression levels of ethylene synthesis genes (PpACO1 and PpACS1) and auxin signaling genes [32].In this study, NAA treatment significantly affected the gene expression abundance in ABA biosynthesis and signaling pathways.As the ratelimiting enzymes for ABA biosynthesis, 2 PpNCED, 1 PpABA3, and 1 PpAAO3 were down-regulated by NAA treatment, which inhibited the ABA accumulation.And in the ABA signal transduction pathway, 14 DEGs were down-regulated and 9 DEGs were up-regulated, indicating that these genes in the ABA signal transduction pathway had different responses to NAA, but most of them were inhibited.
Ethylene is usually recognized as a trigger of the ripening process in climacteric fruits [33,34].Two systems influence the creation of ethylene in climacteric fruit: system 1 is only in charge of producing low basal ethylene quantities at the pre-climacteric stage, and system 2 is in control of producing a large amount of ethylene at the climacteric stage and controlling the ripening process through self-catalysis.Previous studies have found that a high concentration of auxin can stimulate the synthesis of system 2 ethylene through its inductive action on the expression of PpACS1 in MF peaches during postripening; in contrast, the low concentrations of auxin due to the low expression of PpYUC11 suppress PpACS1 expression and ethylene production in SH peaches during post-ripening [13,35,36].In this study, we found that ethylene was not produced at the fruit expansion stage after NAA treatment.In the ethylene biosynthesis pathway, two PpSAMS (Prupe.2G022600,Prupe.3G004000) were down-regulated; and in ethylene signal transduction pathway, two crucial rate-limiting enzymes, PpACS and PpACO did not change significantly.Therefore, we speculated that ethylene was not the primary regulatory hormone of peach fruit at the early fruit growth stage, and the dual effect of NAA on peach fruit ripening might be related to the changes in ethylene content.
In addition, we found that NAA treatment also promoted GA biosynthesis-related genes but inhibited JA and CTK biosynthetic-related genes.N-1-naphthylphthalamic acid (NPA) is an auxin polar transport inhibitor, and 68 DEGs of the plant hormone biosynthesis and signal transduction were significantly enriched after NPA treatment, including 25 AUX, 17 ABA, 9 JA, 8 CTK, 6 ETH, and 3 GA.However, we found that the regulation pattern of NPA was not completely antagonistic with that of auxin, and the specific molecular mechanism remains to be further studied.In conclusion, auxin application mainly triggered IAA biosynthesis and inhibited ABA biosynthesis of peach at the fruit expansion stage, suggesting a complex involvement of auxin and its interaction with phytohormones during the growth and development of peach fruit.

Auxin inhibits some metabolism processes in fruit development by regulating cell wall, sucrose, and phenylpropanoid metabolisms
Fruit ripening and softening are accompanied by the degradation of pectin, cellulose, hemicellulose, and other cell wall substances, resulting from the interaction of various cell wall structure enzymes [37,38].In this process, polygalacturonase (PG), pectin esterase (PE), β-galactosidase (β-GAL), and pectate lyases (PL) are involved in the depolymerization of pectin; xyloglucan:xyloglucosyl transferase (XET/XTH), and Endo-1,4-β-D-glucanase (EG) are involved in the depolymerization of cellulose or hemicellulose.Polygalacturonase (PG) is an important enzyme involved in pectin degradation, which can be divided into endo-Polygalacturonase (Endo-PG) and exo-Polygalacturonase (Exo-PG).Studies have found that Endo-PG is a candidate gene for controlling the softening of peach fruit [39].However, in this study, NAA treatment did not significantly affect PpPG genes at the fruit expansion stage, which may be due to the specific expression of PpPG genes during fruit ripening and softening.Endo-1,4-β-D-glucanase (EG) is a key enzyme that catalyzes the hydrolysis of cellulose, and the enzyme activity of EG is closely related to the fruit ripening of white pear [40].Interestingly, the expression levels of 4 PpEG were significantly reduced after NAA treatment.Furthermore, the PpPE, PpPL, and PpXTH genes showed negative and positive regulation by NAA treatment, suggesting that these genes reflected diverse regulatory mechanisms in response to auxin.All in all, these findings indicated that NAA treatment inhibited the degradation of pectin and cellulose by directly controlling the expression of these softening-related genes.
Sucrose, glucose, fructose, and sorbitol are the primary sugar in peach fruit, which are not only essential to fleshy fruit growth and development but also central to fruit quality [41].Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in regulating sucrose synthesis.In this study, PpSPS1, PpSPS2, and PpSPS3 showed an upward trend with the growth and development of peach fruit.After NAA treatment, PpSPS3 were significantly down-regulated, suggesting that NAA treatment inhibited sucrose synthesis.Sucrose synthesis (SUS) is a multigene family, and 6 PpSUS genes were identified in peach [42], 5 VvSUS genes in grape [43], 11 MdSUS genes in apple [44], and 30 PbSUS genes in pear [45].SUS hasdual activity, SS I have the activity of sucrose degradation, and SS II has the activity of sucrose biosynthesis.In our study, different members of the PpSUS family had certain temporal expression specificity, and 3 PpSUS were significantly up-regulated after NAA treatment, which might exercise different functions in sucrose metabolism.In addition, invertase (INV) can irreversibly catalyze the hydrolysis of sucrose to glucose and fructose; and fructokinase (FK) and hexokinase (HK) can catalyze glucose and fructose to glucose-6-phosphate and fructose-6-phosphate [46].In this study, NAA treatment up-regulated the expression of 1 PpFK, 1 PpHK, and 1 PpINV.Similarly, previous studies showed that sucrose accumulation in peach fruit involved the coordinated interaction of genes related to sucrose cleavage, resynthesis, and transport [47].
The growth and development of fruit are regulated by many transcription factors, which can be divided into positive and negative regulatory factors [52].In tomato, NAC transcription factor NOR-like1 regulated genes involved in ethylene synthesis, chlorophyll degradation, carotenoid accumulation, and fruit softening and promote fruit ripening [53].In papaya, a MADXbox CpAGL18 activated the promoters of CpACS1 and CpSAUR32 and regulated fruit ripening via ethyleneauxin crosstalk [54].In banana, Dof transcription factor MaDof23 inhibited fruit ripening, while tomato transcription factor SlDof1 promoted fruit softening [55].In this study, 197 TFs were identified, including 63 HB, 61 NAC, 33 MADS-box, 23 Dof, and 17 SBP, which regulated different stages of fruit development.For instance, 47 TFs in Sub Class 2 showed an upward trend, which might be positive regulatory factors of fruit growth;77 TFs in Sub Class 6 and Class 7 showed a downward trend, which might be negative regulatory factors of fruit growth.In addition, 39 TFs (4 Dof, 11 HB, 4 MADS-box, 16 NAC, and 4 SBP) were down-regulated, and only 11 TFs (2 Dof, 3 HB, 2 MADS-box, and 4 NAC) were upregulated after NAA treatment, indicating that NAA mainly inhibits fruit ripening.

Auxin-mediated multi-hormone to inhibit some metabolism processes in the development of peach fruit at the expansion stage
Peach [Prunus persica (L.) Batsch] is a typical representative of climacteric fruit, the growth and development of fruit is controlled and integrated by multiple hormone signals.Some studies have verified that auxin played significant roles in the ripening and softening of climacteric fruit through mediating multi-hormone signals.
For example, the application of auxin at the later stage of fruit development can promote peach fruit ripening and softening by inducing ethylene production [13,36].However, the molecular mechanisms and regulatory networks of auxin-induced fruit growth and development at the fruit expansion stage are still unclear.Our work displays the important role of NAA at the expansion stage of peach fruit and outlines the molecular mechanisms and signaling pathways (Fig. 9).In our study, we found that NAA treatment at the fruit expansion stage inhibited the growth and development of peach fruit by coordinating the content of IAA, ABA, and other hormones, but ethylene was not produced at this stage.Therefore, we speculated that ethylene was not the primary regulatory hormone of peach fruit at the early fruit growth stage, and the dual effect of NAA on the growth and development of peach fruit might be related to the changes in ethylene content.The application of auxin at the early stage of fruit development can delay peach fruit ripening, similar to the inhibitory effect of auxin on non-climacteric fruits; and the application of auxin at the later stage of fruit development can promote fruit ripening by inducing ethylene production.All in all, NAA treatment at the fruit expansion stage could inhibit some metabolism processes involved in the related genes in the growth and development of peach fruit by regulating multiple-hormone signaling networks.These results help reveal the short-term regulatory mechanism of auxin at the fruit expansion stage and provide new insights into the multi-hormone cascade regulatory network of fruit growth and development.

Conclusion
At the fruit expansion stage, NAA treatment significantly increased the content of IAA and reduced the content of ABA by coordinating the plant hormone biosynthesis and signal transduction genes.Furthermore, NAA treatment down-regulated most of genes involved in the growth and development of peach fruit, including the cell wall metabolism-related genes (PpEG), sucrose metabolismrelated genes (PpSPS), phenylalanine metabolism-related genes (PpPAL, Pp4CL, and PpHCT), and transcription factors (PpNAC, PpMADS-box, PpDof, PpSBP, and Fig. 9 The putative molecular network of auxin regulates the development of peach by mediating multiple-hormone signaling at the fruit expansion stage PpHB).Overall, we found that NAA treatment at the fruit expansion stage could inhibit some metabolism processes involved in the related genes in the growth and development of peach fruit by mediating multiple-hormone signaling networks.

Plant materials and hormone treatments
Five-year-old 'Xiaobaifeng' peach trees were selected as the experimental material.The plant materials were grown under common field conditions at the National Peach Germplasm Repository in Nanjing, Jiangsu province, China (our partners, we have a long-term relationship).According to the growth and development curve of peach, fruit samples were collected at four stages: young fruit (20 days post anthesis, 20DPA), pit hardening (50 days post anthesis, 50DPA), expansion (70 days post anthesis, 70DPA), and mature stage (90 days post anthesis, 90DPA).Based on the results of preliminary experiments and horticultural practices [56], the fruits were soaked in 0.2 M 1-Naphthaleneacetic acid (NAA, with 1% Tween-80) and 0.2 M N-1-naphthylphthalamic acid (NPA, an auxin inhibitor, with 1% Tween-80) for 1 min at 70DPA, and control (CK) fruits were treated with clean water with 1% Tween-80 solution.The fruit samples were collected 24 h after treatment and contained three biological replicates.All of the fruit samples were separated into skin and flesh, and the flesh samples were immediately frozen in liquid nitrogen and stored at -80℃.Part of the flesh samples was used for hormone quantification analysis, and the other part was used for RNA-seq experiment.
In addition, ethylene production was measured using a gas chromatograph [57].Firstly, six peach fruits were placed in an airtight container for 2 h at 25 ± 0.5 °C.Then 1 mL of headspace gas was withdrawn from the chamber and injected into a gas chromatograph (Agilent 7890A, CA, USA), using helium as the carrier gas.Detector and injector temperatures were 220 ℃.Each measurement included three independent biological replicates.

RNA extraction, cDNA library construction, and transcriptome sequencing
According to the manufacturer's instructions, total RNA was extracted from peach flesh using the RNAprep Pure Plant Kit (TianGen, Beijing, China).The concentration of RNA was measured by an Agilent 2100 Bioanalyzer (Agilent, USA), and the quality of RNA was checked by 1.5% agarose gel electrophoresis.Then, mRNA purification and cDNA library construction were performed with the Ultra II RNA Library Prep Kit for Illumina (NEB, USA).Finally, the qualified cDNA library was sequenced using an Illumina Novaseq6000 (Novogene Technology Co., Ltd, Beijing, China).

Transcriptome data analysis
For high-quality clean reads, reads were filtered by removing reads containing adapter, Nbase, and low-quality reads.The clean reads were mapped to the peach reference genome (GCF_000346465.2) using HISAT2.For each transcription region, fragments Per Kilobase Million (FPKM) were calculated to quantify its transcript abundance using RSEM software.Differentially expressed genes (DEGs) were analyzed using the DESeq2 R package.P-value≤0.05and |log2(Fold change)| ≥1 were set as the threshold for significantly differential expression.To obtain the biological function of DEGs, we performed enrichment analysis using Gene Ontology (GO) (http:// geneo ntolo gy.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https:// www.genome.jp/ kegg/) databases [58,59].DEGs mapped in GO and KEGG pathways with a P-value of ≤0.05 were considered significantly enriched.All transcriptome data analysis and picture drawing were performed using the online platform NovoMagic (https:// magic.novog ene.com/).

Validation of RNA-seq by qRT-PCR
To validate the accuracy of the RNA-Seq data, 16 DEGs were selected randomly for qRT-PCR verification.The PpTEF2 gene was used as an internal control for normalization, and the primers were shown in Supplementary Table S1.According to the manufacturer's instruction, quantitative real-time (qRT)-PCR was performed using Hieff ® qPCR SYBR ® Green Master Mix (Yeasen, Shanghai, China) with the Applied Biosystems 7500 Real-Time PCR System (ABI, USA).The amplification system consisted of 10 μL of Hieff ® qPCR SYBR ® Green Master Mix, 0.8 μL of upstream and downstream primer, 2.0 μL of cDNA, and 6.4 μL of ddH2O in a total volume of 20 μL.Each PCR assay was carried out by three biological replicates.The relative expression level was calculated with the formula 2 -ΔΔCT = normalized expression ratio.

Statistical analysis
The data processing and difference significance analysis were carried out using Excel 2019 and SPSS 22.0.Data were expressed as means ± SD.Comparisons between groups were performed by one-way ANOVA corrected with Tukey's multiple comparison tests at a significance level of P < 0.05.

Conclusions
At the fruit expansion stage, NAA treatment significantly increased the content of IAA and reduced the content of ABA by coordinating the plant hormone biosynthesis and signal transduction genes.Furthermore, NAA treatment down-regulated most of genes involved in the growth and development of peach fruit, including the cell wall metabolism-related genes (PpEG), sucrose metabolismrelated genes (PpSPS), phenylalanine metabolism-related genes (PpPAL, Pp4CL, and PpHCT), and transcription factors (PpNAC, PpMADS-box, PpDof, PpSBP, and PpHB).Overall, we found that NAA treatment at the fruit expansion stage could inhibit the growth and development of peach fruit by mediating multiple-hormone signaling networks.

Fig. 1
Fig. 1 Comparison of transcriptomes of different stages of peach fruit development.A Boxplot of gene expression distribution.The abscissa is the sample name, and the ordinate is log10 (FPKM).B Pearson correlation between samples.C Principal component analysis of samples.Different colors indicate different developmental stages.D Volcano map of other compare groups.Genes with significant differential expression are indicated by red dots (up-regulated), green dots (down-regulated), and gray dots (no significant change)

Fig. 2
Fig. 2 GO and KEGG enrichment analysis of the DEGs.A The GO terms of the top 50 significantly enriched pathways.The different colors are used to distinguish biological processes (BP), cellular components (CC), and molecular functions (MF).B The KEGG pathway terms of the top 20 significantly enriched pathways.The number of DEGs is demonstrated through the magnitude of the dot, and the color of the dot stipulates the severity of DEGs

Fig. 4
Fig. 4 Expression heatmap of auxin biosynthesis and signal transduction genes during peach fruit development.Blue indicated low expression, and red indicated high expression

Fig. 5
Fig. 5 Expression heatmap of six plant hormone biosynthesis and signaling genes in response to auxin treatment.Blue indicated low expression, and red indicated high expression

Fig. 6
Fig. 6 Screening of TFs during peach fruit development.A Gene expression heatmap of 197 TF.The color ranges from red to blue, indicating log 10 (FPKM) from large to small.B K-means cluster analysis of 197 TFs.All data are normalized using the z-score

Fig. 7
Fig. 7 Expression heatmap of 160 DEGs involved in the plant hormone biosynthesis and signaling transduction, sucrose metabolism, cell wall metabolism, phenylalanine metabolism, and transcription factors in response to auxin treatment.Blue indicated low expression, and red indicated high expression

Fig. 8
Fig. 8 Validation of RNA-seq by qRT-PCR.Comparisons between different treatments were performed by a one-way analysis of variance (ANOVA) test at a significance level of P<0.05.Different lowercase letters indicate a significant difference at the 5% level between different treatments