Auxin efflux carrier PsPIN4 identified through genome-wide analysis as vital factor of petal abscission

PIN-FORMED (PIN) proteins, which function as efflux transporters, play many crucial roles in the polar transportation of auxin within plants. In this study, the exogenous applications of auxin IAA and TIBA were found to significantly prolong and shorten the florescence of tree peony (Paeonia suffruticosa Andr.) flowers. This finding suggests that auxin has some regulatory influence in petal senescence and abscission. Further analysis revealed a total of 8 PsPINs distributed across three chromosomes, which could be categorized into two classes based on phylogenetic and structural analysis. PsPIN1, PsPIN2a-b, and PsPIN4 were separated into the “long” PIN category, while PsPIN5, PsPIN6a-b, and PsPIN8 belonged to the “short” one. Additionally, the cis-regulatory elements of PsPIN promoters were associated with plant development, phytohormones, and environmental stress. These genes displayed tissue-specific expression, and phosphorylation sites were abundant throughout the protein family. Notably, PsPIN4 displayed distinct and elevated expression levels in roots, leaves, and flower organs. Expression patterns among the abscission zone (AZ) and adjacent areas during various flowering stages and IAA treatment indicate that PsPIN4 likely influences the initiation of peony petal abscission. The PsPIN4 protein was observed to be co-localized on both the plasma membrane and the cell nucleus. The ectopic expression of PsPIN4 reversed the premature flower organs abscission in the Atpin4 and significantly protracted florescence when introduced to Col Arabidopsis. Our findings established a strong basis for further investigation of PIN gene biological functions, particularly concerning intrinsic relationship between PIN-mediated auxin polar


Introduction
Auxin is a ubiquitous plant hormone that establishes an intertissue concentration gradient through specialized polar transport to serve various crucial roles in the regulation of plant growth, development, and response to external cues (Enders and Strader, 2015).Auxin transporters can be divided into two groups based on function and location.The first group primarily facilitates intercellular auxin transportation and comprises transporters located on the plasma membrane such as AUXIN1/LIKE AUX1 (AUX/LAX), pin-formed (PIN), and ATP-binding cassette (ABC).The second group features transporters that regulate intracellular auxin homeostasis and are distributed within intracellular compartments, including several PINs with short hydrophilic loops, PIN-Like transporters (PILS), and WALLS ARE THIN 1 (WAT1) (Ranocha et al., 2013;Kuhn et al., 2017;Zhou and Luo, 2018).
The PIN protein family is expansive, with diverse members identified in over 30 plant species (Yue et al., 2015;Huang et al., 2020).Driven by evolution, sub-functionalization or new functionalization has led to significant modifications in protein structure (Mravec et al., 2009;Ditengou et al., 2018).Members of this family are crucial auxin transporters, making them vital components of various auxin-regulated processes such as organogenesis, development, tropism, and morphogenesis.To ensure precise auxin delivery, PIN proteins must be strategically distributed within a plant.Categorized as short or long, these proteins inhabit separate cellular spaces and serve distinct roles.Typically, long PIN proteins are polarized on the cell membrane to facilitate intercellular auxin transport.For example, auxin circulation in root tips is facilitated by the distinct polar localization patterns of Arabidopsis PIN1-3, PIN4, and PIN7 (Blilou et al., 2005;Paponov et al., 2005;Vieten et al., 2005).Additionally, during embryonic development, the PIN1 and PIN7 proteins orient themselves toward the suspensor to direct auxin flow toward the structure (Friml et al., 2003;Weijers et al., 2005).In tomatoes (Solanum lycopersicum), SlPIN1 heavily influence leaf development and formation of leaf clefts (Koenig et al., 2009).SlPIN4 is highly expressed in tomato flowers and fruit, particularly in the placenta during fruit development (Mounet et al., 2012;Pattison and Catala, 2012).The "short" PIN is primarily localized in the endoplasmic reticulum, where they regulate intracellular auxin homeostasis.In Arabidopsis, auxin transport is primarily overseen by PIN5 and PIN8.PIN5 is predominantly expressed in hypocotyls, cotyledon vascular tissue, and guard cells, while PIN8 exhibits specific expression in anthers, male gametophytes, and sporophytes (Mravec et al., 2009;Barbez et al., 2012;Ding et al., 2012).Moreover, PIN6 displays dual localization and can be observed on both the cell membrane and endoplasmic reticulum (Bender et al., 2013;Cazzonelli et al., 2013;Simon et al., 2016).
Abscission represents a crucial developmental stage during which organs are separated from plant bodies in a region known as the abscission zone (AZ), which exhibits a high sensitivity towards shedding signals (Tucker and Yang, 2012;Wilmowicz et al., 2018;Shi et al., 2023).The timing of organ detachment is governed by plant hormones such as ethylene, auxin, and abscisic acid (ABA) signaling the activation of various Metabolic processes (Dal Cin et al., 2007;Sun, 2009;Meir et al., 2010;Parra-Lobato and Gomez-Jimenez, 2011;Tadeo et al., 2012a).Early abscission is prevented by auxin synthesized in distal organs and transported through PIN proteins, which inhibit the responses to ethylene signaling (Zhu et al., 2011;Kühn et al., 2016).Once a reduction in auxin is detected in the AZ, cells become responsive to these signals and trigger abscission (Meir et al., 2006).During rose (Rosa hybrida) petal abscission, there is a reduction in the amount of auxin and RhSUC2 from the phloem of AZ to the petals.This auxin influences the shedding process by binding to response factor RhARF7, which then regulates the RhSUC2 promoter (Liang et al., 2020).Furthermore, previous research investigating tomato pedicel abscission has characterized the regulatory roles of SlPIN1 and SlPIN4, which facilitate the basal transportation of auxin to the AZ and promote pedicel abscission (Shi et al., 2017;Liang et al., 2020).
The tree peony (Paeonia suffruticosa Andr.) is a woody flowering plant that has long been cultivated for its vibrant colors, elegant posture, and expansive floral patterns.However, the species' ornamental and economic value is hindered by a short flowering period, with plants often blooming for less than one week under natural conditions.Although a growing body of research supports an association between auxin and abscission, few studies have explored the role of PIN-mediated auxin transport proteins in this process.In this study, we comprehensively investigate the PIN gene family by using genome sequence data from tree peony to analyze gene structures, chromosomal localization, phylogenetic relationships, motif, transmembrane structures, and cis-elements.Additionally, we analyzed gene expression profiles in various tissues to speculate on their potential functions while striving to identify members involved in initiating AZ formation.Our findings lay a foundation for further functional PsPIN investigations and explore the potential connections between PINs, auxin signaling pathways, and plant organ abscission.

Plant materials and treatments
A total of 30 tree peonies (cv.'Xueyingtaohua') were planted and grown for four years in a greenhouse at Qingdao Agricultural University (Qingdao, China) (22 ± 3 °C day, 15 ± 3 °C night, natural 10 h light/14 h dark photoperiod).Five slightly opening flowers in similar bud states were selected and removed from each test plant.Samples were delivered to the laboratory within one hour of harvest and stems were pruned to 15 cm, immersed in distilled water, and placed in an incubator (20 °C, 14 h light/10 h dark) for 36 hours.Twenty cut flowers were randomly selected from each group and treated with either a solution containing 100 mM, 200 mM IAA (Indole-3-Acetic Acid, IAA) and 100 mM TIBA (2,3,5triiodobenzoic acid, TIBA).The remaining samples were still placed in distilled water as the control group.10 cut flowers were randomly selected from each treatment group and control group for shedding rate statistics.Following treatment, organs of interest were individually removed from the flowers.Double-sided blades were used to excise the ovary by cutting along its boundary with the receptacle.Whole stamens were removed and kept intact.The abscission zone was represented by the 2 mm segment located at the junction between the petals and the receptacle.The proximal and distal portions of this section were represented by the upper 2 mm (including petal tissue) and lower 1mm (including the ring-shaped receptacle), respectively.Three cut flowers were selected for each sampling stage for in situ hybridization, fluorescence quantification, and endogenous IAA determination experiments, respectively.

Sequence retrieval and phylogenetic analysis
The known PIN protein sequences of Arabidopsis thaliana [TAIR -Home Page (arabidopsis.org)]and Oryza sativa [Rice Genome Annotation Project (uga.edu)]weredownloaded to blast tree peony genomewide (Yuan et al., 2022).The HMMER software was utilized with default parameters to conduct a search for candidate PIN protein sequences according to transmembrane domain (PF03547.22),followed by further identification of the sequences through a homology search using the BLAST default parameters.The candidate tree peony PIN genes were validated using the SMART web tool [SMART: Main page (embl-heidelberg. de)], and manually curated to remove truncated and redundant proteins.The identified tree peony PIN proteins were designated based on their phylogenetic relationships with Arabidopsis homologs.The subcellular localization information of the PsPIN protein sequences were analyzed by the Softberry service platform ProtComp 9.0 (SoftBerry -ProtComp (Plant) HELP) (Yu et al., 2008a).The MapInspect software (http://mapinspect.software.informer.com/)(Wu et al., 2019) was used to analyze physical position and chromosomal localization of PsPIN genes.The MEME web server [Introduction -MEME Suite (meme-suite.org)] (Bailey et al., 2015) was employed to identify intricate motifs of PsPIN proteins, utilizing the default parameter settings except for a maximum motif count of 20.A total of 112 PIN amino acid sequences from 11 species (Arabidopsis thaliana, Paeonia ostii, Solanum lycopersicum, Solanum tuberosum, Gossypium raimondii, Zea may, Oryza sativa, Cystopteris fragilis, Picea abies, Populus trichocarpa, and Glycine max) were retrieved from the NCBI database.Multiple sequence alignment was performed using ClustalW program in MEGA-X software (Kumar et al., 2018), and the phylogenetic tree was constructed using maximum likelihood (ML) with 1000 bootstraps.

Gene structure, cis-acting elements and phosphorylation sites analysis
The PsPIN gene structure (exon-intron) was defined using the online software Gene Structure Display Server (GSDS) [Gene Structure Display Server 2.0 (gao-lab.org)](Hu et al., 2015).DNAMAN software (https://www.lynnon.com/pc/framepc.html)and TMHMM Serverv.2.0 (http://www.cbs.dtu.dk/services/TMHMM/) were applied to perform protein family sequence alignment and transmembrane structure analysis.The cis-regulatory elements in the promoter region (2,000 bp upstream of the starting codon) of the PsPINs were searched by the online program of PlantCARE [PlantCARE, a database of plant promoters and their cis-acting regulatory elements (ugent.be)](Lescot et al., 2002).Gene structure, cis-acting element numbers and responsive functions were visualized using TBtools (Chen et al., 2020).NetPhos 3.1 web server (NetPhos 3.1 -DTU Health Tech -Bioinformatic Services) was used to analyze the phosphorylation sites of the PsPIN proteins.

Real-time quantitative RT-PCR
Total RNA was extracted from 100 mg of each sample using a Steady Pure Plant RNA Extraction Kit (TaKaRa, Beijing, China) according to the manufacturer's instructions.The first strand of cDNA was synthesized from 1 µg of total RNA using a HiScript III RT SuperMix (Vazyme, Nanjing, China) for qPCR reverse transcriptase at a final volume of 50 µL.qRT-PCR was performed using a SYBR ® Premix Ex Taq ™ II Kit (TaKaRa, Dalian, China).All reactions were performed in triplicate.The relative expression level was calculated by the 2 -DDCt method (Livak and Schmittgen, 2001).The primers used for qRT-PCR are listed Supplementary Table S8.

IAA quantifcation by ELISA
IAA standards, antigens, and antibodies were obtained from Qingdao Agricultural University.All samples were homogenized and extracted in cold 80% methanol (4°C) containing 1mmol/L butylated hydroxytoluene for 4 h.Samples were then centrifuged at 4000rpm for 15 min (4°C).Extracts were passed through a C18 Sep-Pak cartridge (Waters, USA), dried using N 2 , and then dissolved in a mixture comprising 2 mL PBS (pH 7.5), 0.1% Tween 20 (v/v), and 0.1% gelatine (w/v).Next, 96-well microfiltration plates were coated with an IAA antigen in NaHCO 3 buffer (50 mmol/L, pH 9.6) and incubated overnight at 4°C.Plates were then washed four times with a mixture of PBS (pH 7.5) and 0.1% Tween 20 (v/v).Subsequently, all wells were filled with 50 mL of IAA antibodies along with a 50 mL aliquot of either a sample extract or IAA standard.Plates were then incubated at 37°C for 30 min.After an additional wash with the PBS and Tween 20 mixture, wells were supplemented with IgG horseradish peroxidase, incubated for 30min at 37°C, and washed a final time.Finally, orthophenylenediamine was supplied to act as a substrate before a final 30 min incubation at 37°C.Absorbance was detected at 490 nm.

In situ hybridization
The AZ samples were collected at 0, 24, 48, and 72 hours after flowering for this assay.The in vitro transcription vector pGEM-T was constructed using a PsPIN4-specific fragment amplified.with the Antisense probe sequence is listed in Supplementary Table S9.Needle labeling was performed following the protocols provided by the DIG RNA Labeling Kit (SP6/T7) (Roche, USA) and DIG Nucleic Acid Detection Kit (Roche, USA).The probe concentrations used was 211.7 ng/ul for PIN.The protocol for in situ hybridization was modified based on Abcam's guidelines (In situ hybridization (ISH) protocol | Abcam).Detailed steps for paraffin sectioning referred to Shi (Shi et al., 2017), finally, the sections were stained overnight with NBT/BCIP (Roche, Germany) at 37°C.Subsequently, the samples were examined under a Nikon Eclipse ci optical microscope (Nikon, Japan).

Generation of transgenic plants and floral organs abscission assay
The overexpression vector 35S:PsPIN4 was generated by cloning the CDS of PsPIN4 into the vector pCAMBIA1300.The primers used for PsPIN4 CDS amplification are listed in Supplementary Table S8.Transgenic Arabidopsis plants were obtained according to the floral dip transformation method (Clough and Bent, 1998) and were then used for phenotype observation and further assays.The pBS was quantified as the force in gram equivalents required for pulling a petal from flower (Butenko et al., 2003), as determined by using a digital force gauge (Model: HF-2, Lunjie Electromechanical Instrument Co., Ltd., Shanghai).A total of 20 petals per position were measured.

Histochemical GUS assays
A promoter sequence of 2,000 bp upstream of the starting codon was cloned and fused into pCAMBIA1381 vector with a GUS reporter gene.The primers used for PsPIN4 promoter amplification are listed in Supplementary Table S8.Arabidopsis homozygous T 3 generation plants were utilized for GUS staining analysis.Samples at different stages (1, 2, 3, and 4 weeks) as well as floral organs of different positions (P1-P7) were selected to observe the expression of ProPsPIN4 throughout the whole plant using GUS staining.The samples were performed in the staining solution (50 mM phosphate buffer (pH7.2),0.1% (v/v) Triton ™ X-100, 0.5 mM K 4 Fe (CN) 6H 2 O, 0.5 mM K 3 Fe (CN) 6 , and 0.5 mM X-Gluc) at 37°C in the dark.Then 95% ethanol was applied to decolorize the tissues and examined under the ZEISSV11 stereoscope (Nikon Japan).

Western blot analyses
For immunoblot analyses, total proteins were extracted from 0.1 g of four transgenic lines (Arabidopsis thaliana at 1,2,3, and 4 weeks of age and floral organs at 2,4, and 6 positions) and NT plants in 3 ml of extraction buffer [50 mM Tris-HCl (pH 8), 10 mM MgCl 2 , 1 mM EDTA 0.5 M (pH 8), 5% glycerol, 1 mM DTT, 0.1% Triton X-100 and 1 mM (PMSF)].The homogenate was placed on ice for 5 min, and then centrifuged at 13,000 × g for 15 min.The proteins concentrations of the resulting supernatant were determined by the Bradford method (Bradford, 1976).Total proteins samples were then electrophoretically separated on 12% (w/v) SDS-PAGE gels and blotted by the semi-dry method (iBlotTM Gel Transfer Stack) onto a nitrocellulose membrane (Invitrogen).GUS protein detection was carried by the same protocol described by Ben Saad et al. (Ben-Saad et al., 2015).

Subcellular localization of PsPIN4
The PsPIN4 ORF without stop codon was amplified with the primers PsPIN4-GFPF and PsPIN4-GFPR (Supplementary Table S8).The PsPIN4::GFP fusion expression vector was constructed and used to transform Agrobacterium (Agrobacterium tumefaciens) GV3101.Agrobacterium was cultured in LB liquid medium (with kanamycin and rifampicin) until an OD 600 of 0.6-0.8 was obtained.Further, the cells were centrifuged at 5,000 rpm for 10 minutes.The cell pellet was diluted to obtain an OD 600 of 0.6-0.8 with a solution of 10 mM MES, 200 mM acetylsyringone, and 10 mM MgCl 2 (pH 5.6), and injected into tobacco leaves.The infected plants were cultured in darkness for 24 hours, and then in normal conditions (25°C, 16 hours light/8 hours dark) for 24 hours.The nuclear labeled protein GHD7::CFP and the membrane labeled protein PIP2;1::RFP were interchanged for accurate localization, and the fluorescence was observed using a laser confocal microscope.

Statistical analysis
Statistical analysis was performed by GraphPad Prism 7 (GraphPad Software Inc.San Diego, CA.USA) and t-tests with P < 0.05 ( * ) and P < 0.01 ( * * ) was used to indicate statistically significant level.All results were presented as the means ± standard deviation (SD) and at least three replicates were set up.

Identification of PIN proteins in tree peony
In this study, a total of 8 PsPIN genes were identified in tree peony and were renamed PsPIN1, PsPIN2a-b, PsPIN4, PsPIN5, PsPIN6a-b and PsPIN8 (Table 1) according to Arabidopsis homologs.Information regarding these genes and their corresponding proteins including gene ID, location, number of exons, protein length (aa), molecular weight (MW), theoretical isoelectric point (pI), and subcellular location are showed in Table 1 and Supplementary Table S1.Among the 8 PsPIN proteins, the lengths and molecular weights varied greatly, ranging from 90 aa (PsPIN6b) to 654 aa (PsPIN4) and from 10192.01 Da to 70955.96Da, respectively.Our prediction of subcellular localization revealed that PsPIN proteins are primarily distributed on the cell membrane.In addition, the presence of shorter protein sequences may be attributed to the genome annotation process.
Through comparisons with the tree peony genome, our results indicate that the newly identified PsPIN genes are distributed across three chromosomes (Figure 1), the majority of which were located on chromosomes I and V (each containing three PsPIN members).In view of this, we searched the tree peony genome for segmental and tandem duplicate gene pairs and found no repeat events among PsPIN family members.

Multiple sequence alignment, phylogenetic analysis and motif assessment of PsPINs
To elucidate the evolutionary relationship within the PsPIN gene family, we used the MEGA-X software to construct a phylogenetic tree from the inferred full-length amino acid sequences of 112 PIN protein sequences collected from 11 species.Based on the classification methods of the AtPINs, the tree revealed six distinct branches (AtPIN1, AtPIN2, AtPIN5, AtPIN6, AtPIN8, and AtPIN3/4/7), The 8 newly identified PsPIN proteins were categorized into six groups: PsPIN1, PsPIN2a-b, PsPIN4, PsPIN5, PsPIN6a-b and PsPIN8.The evolutionary analysis revealed a robust correlation between peony and poplar PIN.We employed the MEME (Multiple Em for Motif Elicitation) to unveil the motif compositions of the 112 PIN proteins and identified 20 conserved motifs through structural analysis (Figure 2).In the "long" PIN group, PsPIN1 lacked only the 17th motif, whereas PsPIN2a lacked motifs 9, 12, 17, 18 and 20.Potentially as a result of incomplete genome annotation, only one or two motifs were identified in PsPIN2b, PsPIN6a, and PsPIN6b.Conversely, "short" PINs such as PsPIN5 and PsPIN8 were found to contain shorter central hydrophilic rings and lack the typical 10, 15, 11, 9, 18, 17, 12 and 14 motifs.S2. (B) Conserved PsPIN protein motifs in tree peony.Motifs are differentiated with colored boxes and numbers (1-20).Gray lines represent the non-conserved sequences.Sequence information for all motifs is provided in Supplementary Table S3.Sun et al. 10.3389/fpls.2024.1380417Frontiers in Plant Science frontiersin.org

Structure and transmembrane domains analysis of the PsPINs
The structural characteristics of the gene family were investigated by integrating the PIN genes from Arabidopsis, rice, and tree peony.The results illustrated great variability, with the quantity of introns among the PsPINs ranging from 3 to 6 (Figure 3A).Specifically, PsPIN5 and PsPIN8 each contained four introns, while PsPIN1, PsPIN2a, and PsPIN4 contained five introns, and PsPIN2b and PsPIN6a contained six introns.Due to potentially incomplete genomic annotation, only three introns were detected in PsPIN6b.It is important to highlight the absence of UTR regions associated with PsPIN2 and PsPIN6, which also displayed shorter exon segments.Additionally, PsPIN2b possessed notably longer gene spans.
The transmembrane motif of the PsPIN protein was presented for further structural analysis (Figure 3B).We identified a distinct central The structure and transmembrane domain analysis of PsPINs.Notably, the tyrosine (Y) in PsPIN8 was replaced with histidine (H).Amino acid fragments are shown in Supplementary Table S4.
The online software NetPhos 3.1 Server was used to predict post-translational protein phosphorylation.Phosphorylation sites were abundant among the 8 PsPIN proteins, with serine (Ser) representing the most frequently modified site, followed by threonine (Thr) and tyrosine (Tyr) (Figure 5; Table 2).The concentration of prediction sites in the center of the hydrophilic ring resulted in significantly fewer sites accounting for PsPIN5 and PsPIN8.

Gene ontology and expression patterns analysis of PsPIN genes
Gene ontology analysis was performed to elucidate the functional classifications of PsPIN genes and explore their involvement in various biological processes (Figure 6A).The studied genes were most highly represented in the "Biological Process" ontology by the following GO terms: "regulation of biological quality," "homeostatic process," "regulation of auxin-mediated signaling pathway," and "obsolete auxin homeostasis".Similarly, genes were prevalent in the "cell periphery" and "obsolete intracellular part" terms of the "Cellular Component" ontology.Notably, the only observed cellular function among the 8 genes was related to auxin transmembrane transport activity.This analysis indicates that PsPIN genes are likely involved in various regulatory pathways in plants, including cell morphogenesis, gravitropism, embryo development, and root system development.Furthermore, this analysis revealed new functional categories, such as response to hypoxia, plasmodesma regulation, monosaccharide response, and gametophyte development.Taken together, these findings provide valuable insights for future investigations into PsPIN functional divergence.
To better understand the function of PIN genes in tree peony, we next assessed the expression of PsPIN genes in nine distinct tissues (roots, stems, leaves, petals, receptacles, stamens, ovaries, bracts, and sepals) using qRT-PCR analysis (Figure 6B).The result revealed a minimal expression of PsPIN2 and PsPIN6 in any tissue and an exclusive expression of PsPIN8 in the ovary tissue.In contrast, the expression of PsPIN1, PsPIN4, and PsPIN5 varied across different tissues.Notably, PsPIN4 exhibited a significantly high expression in the petal tissue.

Expression patterns of PsPIN genes during petal abscission
Auxin's influence on the rate of petal shedding in cut peony flowers was examined through the use of IAA and TIBA (Figure 7A).The average abscission time following IAA treatment (153.6 h) was 29.30% longer when compared with the control (118.8 h).Conversely, TIBA treatment was found to significantly accelerated the abscission process, resulting in an average abscission time of only 94.8 hours (Figure 7B).
The quantification of internal plant IAA following exogenous IAA and TIBA treatments was used to reflect auxin's influence.Following IAA treatment, there with a significant increase in the distribution of the hormone within the distal part, proximal part, and AZ.After 72 h, IAA levels within the distal portion and the entire AZ were elevated compared to the control, suggesting that shedding had not been initiated.TIBA treatment, however, decreased the quantities of IAA in the three tissues, especially in the distal portion and AZ.Following 48 h, auxin was only detected in very low quantities in both, reaching stabilization in the middle to late flowering period (Figure 7D).
AZ initiation is dependents on auxin level changes both internally and in surrounding tissues.However, the crucial functions performed by PIN transporters in this process, particularly in flower organ shedding, have seldom been studied.To screen potential PsPINs involved in petal abscission initiation, We analyzed the expression patterns of PsPIN1, PsPIN4, and PsPIN5 in various regions (distal part, proximal part, and AZ) during flowering stages (Figure 7E).The expression of PsPIN1 was consistently low across all tissues.In the distal part, PsPIN4 expression initially increase rapidly at 12 h, followed by a gradual decline.A similar pattern was observed in the proximal part and AZ, with an initial upward trend before 36 h followed by subsequent decline.Notable, the expression of PsPIN4 in the distal part was lower than that in the proximal part after 36 h.The expression of PsPIN5 exhibited a generally declining trend across all three tissues, with a significant increase observed at 60 h in the distal part.These findings highlight PsPIN4 as a candidate gene involved in the regulating of petal abscission.
The application of in situ hybridization enabled the visualization of PsPIN4 expression patterns during petal shedding.The results confirmed the findings of qRT-PCR analysis.Initially, the were only minimal hybridization signals were detected within the AZ, but by 24 h, signals were widespread throughout the entire region (Figure 7C).Nevertheless, strong signals persisted on the upper surface of proximal parts, as well as within the vascular bundles and their periphery.By 72 h of flowering, the signals marginally localized within the vascular bundles of the receptacle, reflecting a decline in auxin transport efficiency mediated by  S5.
PsPIN4.To further investigate the underlying relationship between PsPIN and auxin, exogenous IAA was applied 36 hours after flowering and PsPIN4 expression was analyzed after an additional 48 h (Figure 7F).The localized changes in IAA concentration provided feedback on the expression of PsPIN4, actively regulating the auxin distribution among tissues.A significant increase in PsPIN4 expression was observed in the distal part, AZ, and proximal part.Due to continuous IAA supply, PsPIN4 expression was consistently elevated and did not initiate the abscission layer at 72 h.Furthermore, higher expression levels were more evident with a treatment concentration of 200 mM compared to 100 mM.Overall, our study suggests that PsPIN4mediated auxin transport may be involved in petal abscission.

Ectopic expression of PsPIN4 promoter and subcellular localization of PsPIN4
The ectopic expression of the PsPIN4 promoter was employed to further elucidate the functional role of PsPIN4.The GUS signal in transgenic Arabidopsis was recorded at 1, 2, 3, 4, and 7 weeks respectively.Western blot showed that the GUS fusion protein driven by the PsPIN4 promoter was effectively expressed in the experimental group (Figure 8C).The GUS signal was predominantly distributed in the roots, leaves, petals, sepals and receptacle (Figure 8A).In newly formed leaves, the signal was primarily localized to the main vein.As the leaves matured, apart from the main vein, the signal exhibited a punctate distribution throughout the entire area.However, this punctate distribution was less pronounced in aged leaves.Before three weeks of age, the signal spread throughout the entire root and then weakened at its base while becoming concentrated in lateral and apical regions.Our focus of observation encompassed seven positions from top to bottom of the inflorescence for analyzing the signal within flower organs (Figure 8B).The signal at the apex of the stigma remained relatively stable during the early and middle stages, but exhibited a significant enhancement at positions 6 and 7.The signal within the petals initially increased and subsequently decreased, with peak intensity observed at position 3 and position 4.This signal was primarily localized along the floral veins.Additionally, there was a gradual signal increase at the junction between petals and receptors, which was particularly evident during early pod development.
The subcellular localization analysis revealed that PsPIN4::GFP exhibited co-localization in both the nucleus and plasma membrane, with a slightly stronger fluorescence signal observed in the nucleus compared to the plasma membrane (Figure 8D).Prediction of PsPIN proteins phosphorylation sites.The serine, threonine and tyrosine are indicated in red, green and blue, respectively.Phosphoric acid sites and kinases are listed in Supplementary Table S6.

Ectopic expression of PsPIN4 in Arabidopsis delays floral organ abscission
To further investigate the functions of PsPIN4, we examined its impact on floral organ shedding in Arabidopsis.In wild-type Col Arabidopsis, the floral organs began to fully drop at position 8. Conversely, transgenic Arabidopsis lines overexpressing PsPIN4 (35S:PsPIN4-3, 35S:PsPIN4-7, and 35S:PsPIN4-8) exhibited a significant delay in petal abscission time, with floral organs fully dropping at position 12 or 13 (Figure 9A).BLADE-ON-PETIOLE (BOP) was used as a gene marker for AZ formation (McKim et al., 2008).The expression of AtBOP1 and AtBOP2 were significantly reduced at position 4 and 5 in 35S:PsPIN4 compared to Col (Figure 9B).Furthermore, the delay in floral organ abscission was quantified by measuring petal break strength (pBS) (Figure 9C).The transgenic lines required more force than Col to tear petals from flowers at positions 5 and 6.Additionally, flowers with "justvisible" white petals were selected and cultured on IAA plates to record the abscission rate (Figure 9D).Initially, the abscission process was found to be significantly delayed in Col plants grown on IAA plates compared to MS plates.However, abscission rates for transgenic lines grown on MS plates and Col grown on IAA plates were similar.Additionally, the 35S:PsPIN4 cultures on the IAA plate demonstrated prolonged organ abscission when compared to all other groups, resulting in only a 47.93% abscission rate at 120 h.While further examining the abscission process of Atpin4, we observed premature abscission of flower organs, with dropping occurring at positions 7 or 8 (Figure 9E).Interestingly, the introduction of PsPIN4 under the control of the CaMV35S promoter into the Atpin4 background seemed to rescue this phenotype, as floral organs completely detached at positions 11 or 12 with a dropout rate similar to that of 35S:PsPIN4 (Figure 9A).The pBS measurement and abscission rate of the IAA-treated plate further confirmed these results (Figure 9F).Moreover, the abscission rates of the Atpin4 and Col groups on the IAA plate were similar at 120 h, while the 35S:PsPIN4/Atpin4 groups displayed significantly lower abscission rates than the other groups (Figure 9G).The pBS at positions 5 and 6 also indicated that the repair line could effectively delay petal shedding (Figures 9E, F).Collectively, this data strongly supports the role of PsPIN4 in delaying floral organ abscission in Arabidopsis.

Discussion
The number of PIN genes varies widely among plants.We identified 8 PIN genes in tree peony, as many as Arabidopsis, but lower than several other plant species, such as rice, cotton, maize, and soybean, which have 13, 11, 12, and 23 PIN genes, respectively.The genome size of tree peony is about 12.28 Gb (Yuan et al., 2022), while that of Arabidopsis, rice, maize, and soybean is 125 Mb, 430 Mb, 2300 Mb, and1.025Gb, respectively (Arabidopsis Genome, 2000;Burr, 2002;Schnable et al., 2009;Schmutz et al., 2010;Sabot et al., 2011).In this case, it appears that there is no direct correlation between the number of PIN genes and genome size in these plants.The analysis of 20 candidate motifs revealed that the PIN family can be classified into three distinct categories.The "long" PIN class predominantly consists of 20 motifs, whereas the "short" PIN class lacks motifs in the central hydrophilic ring region.Although the motifs found in PsPIN members exhibit variation, they still adhere to this classification scheme.The absence of multiple motifs in PsPIN2b, PsPIN6a and PsPIN6b could be attributed to genome annotation.The structural Gene ontology (GO) and expression patterns of PsPIN genes.(A) GO categories are indicated by green, red, and blue boxes respectively.Annotation details are listed in Supplementary Table S7.(B) Tissues expression of PsPIN genes in the root, stem, leaf, petal, receptacle, stamen, ovary, bract and sepal as determined through qRT-PCR.Error bars are obtained from three measurements.
composition of the central hydrophilic region of PIN proteins essential for classification within this protein family.The modification of this region can result in subfunctionalization through selective deletion or repetition (Zhou and Luo, 2018).Multiple sequence alignment analyses revealed that PsPIN1, PsPIN2a and PsPIN4 share sequences with an identity exceeding 60% in both the central hydrophilic region and each structural domain.The hydrophilic regions of typical PIN proteins have the potential to facilitate specific protein-protein interactions in eukaryotes (Krecek et al., 2009).Conserved PsPIN family proteins may contribute to protein localization overlap and functional synergy, redundancy, or antagonism (Rakusova et al., 2015).For instance, AtPIN3, AtPIN4 and AtPIN7 exhibit compensatory functions in regulating phototropism growth and embryonic development in Arabidopsis (Friml et al., 2003;Paponov et al., 2005), thus, highly conserved PsPIN proteins may also demonstrate similar characteristics.PIN protein, play an important role in intricate regulatory networks within plants auxin signaling as crucial transporter (Friml, 2003).The auxin response of the PIN gene is influenced by both tissue type and developmental stage, which may explain the diverse auxin elements observed in PsPINs promoters.Regulation of PIN by certain hormones also contributes to its interaction with auxin or other hormones.Auxin has been reported to facilitate the degradation of DELLA proteins, thereby enhancing cellular response to gibberellin (GA) (Fu and Harberd, 2003).Conversely, GA promotes localization of the PIN protein on the cell membrane while inhibiting auxin transport (Willige et al., 2011;Loefke et al., 2013).Additionally, treatment with Jasmonic acid (JA) downregulates expression levels of AtPIN7 leading to a deceleration in auxin transport (Jang et al., 2019).The abundance of hormoneresponsive elements in the PsPIN promoters suggests that this protein family interacts with a variety of other hormones.Specific phosphorylation modifications play a crucial role in determining the polar orientation and transport function of PIN proteins (Huang et al., 2010).PsPIN proteins have multiple modification sites primarily within the region spanning 200-300 amino acids.However, further research is needed to describe the regulatory pathway governing PIN phosphorylation during specific growth and development stages.Conserved loci are typically found within TPRXS motifs present in long hydrophilic rings (Zourelidou et al., 2014;Dory et al., 2018).In Arabidopsis, AtPIN8 protein lacking phosphorylation sites within the short ring domain exhibits independent transport activity from kinases (Mounet et al., 2012).The "short" PIN type of PsPIN5 and PsPIN8 correlates to a significant reduction in serine and threonine modification sites, thereby providing further support for this perspective.
The expression of PIN genes is regulated by auxin through the Aux/IAA signal transduction system, and their auxin response varies depending on tissue type (Heisler et al., 2005;Gallavotti et al., 2008;Bayer et al., 2009).In Arabidopsis, auxin treatment induces the expression of AtPIN1, AtPIN3, and AtPIN7 in roots but not in the hypocotyl (Vieten et al., 2005).During shedding, the PsPIN4 gene exhibited a heightened sensitivity to IAA at both the proximal and distal parts.Following the absorption and accumulation of exogenous IAA in the receptacle, local concentration changes were perceived by the tissues, resulting in an upregulation of PsPIN4 expression.Consequently, auxin was transported to the petals through junctions to facilitate flowering, inducing an upregulation of PsPIN4 expression at the distal part.Additionally, PsPIN4 expression depends on IAA concentration as a means to cope with changes in plant auxin levels and actively regulate internal homeostasis.However, regulation of PIN by auxin is complex as prolonged exposure or high concentrations can promote PIN entry into cells resulting in degradation (Sieberer et al., 2000;Vieten et al., 2005).Therefore, further investigation is required to uncover the relationship between PsPIN4 and auxin distribution during the dissociation stage of the organ shedding process.The IAA concentrations of both the and distal parts undergo dynamic changes during organ shedding (Taylor and Whitelaw, 2001).The level of the distal part is consistently decreased to facilitate detachment (Kucko et al., 2020).This study revealed that during the middle and late stages of flowering, PsPIN4 expression at the distal part continued to decline as a response to auxin transport efficiency in preparation for abscission.Furthermore, it appears that the rate of auxin transport in the AZ may have a more direct impact on the abscission process than simply relying on concentration differences between both sides (Liang et al., 2020).During early flowering stages, a significant accumulation of PsPIN4 occurred within and around the AZ to facilitate IAA transport for meeting flowering.However, its distribution within AZ significantly diminished later on, indicating a weakened capacity for IAA transport.The distribution pattern of PsPIN4 observed during late receptacle stages may be attributed to either the partial auxin supply required for protective layer formation post-abscission or the response of the fractured layer to external exposure.Previous studies have demonstrated significant differences in the expression of various PINs (Habets and Offringa, 2014).The ectopic expression of the promoter suggests that PsPIN4 might not only be involved in flowering regulation, but may also assist in the development of roots, leaves, and fruits Furthermore, our analysis of GUS signal distribution characteristics suggest the gene may be active across multiple tissues during flowering.The expression levels observed in petals during early stages followed by continuous decline aligned with expression trends observed in tree peony.Notably, the junction between the receptacle and petal in Arabidopsis, corresponded to the fruit pod growth site where sustained high expression signals were crucial for adequate auxin supply to facilitate fruit pod development.This potentially explains inconsistencies observed in PsPIN4 expression during the late flowering stage of tree peony.
The application of exogenous IAA was found to significantly delay petal abscission in both peony and Arabidopsis plants.The overexpression of the PsPIN4 gene efficiently facilitated IAA transport in the IAA plate, thus sustaining flower presence.However, limitation in the transporter PIN or insufficient IAA content resulted in similar shedding rates following 120 h of 35S:PsPIN4 growth on the MS plate and Col growth on the IAA plate.The premature abscission phenotype observed in mutants suggests the involvement of AtPIN4 in flower organ abscission.The weakening displayed in IAA plates indicate a potential participation by functionally redundant members, requiring further investigation.Our analysis also that 35S:PsPIN4/AtPIN4 could effectively compensate for the mutant's early shedding phenotype.Overall, these findings indicate that PIN4mediated auxin transport plays a crucial role in Arabidopsis flower organ abscission.In summary, our study revealed a total of 8 PsPIN genes were identified from the tree peony genome, which could be classified into "long" and "short" PIN categories depending on their structure.These genes were randomly distributed across three tree peony chromosomes.With the exception of PsPIN2b, PsPIN6a, and PsPIN6b (due to incomplete genome annotation), PsPIN protein sequences exhibited a high degree of conservation, which was characterized by the presence of 4-5 transmembrane domains at both the N-terminal and C-terminal regions.PsPINs promoters were found to contain cis-regulatory elements associated with growth, development, phytohormone responsiveness, and environmental stress.Furthermore, we examined the differential expression patterns of PsPIN genes in various tissues.The presence of diverse phosphorylation sites in the hydrophilic region suggests complex post-translational modification mechanisms, which will require further study.Perhaps most importantly, we discovered that PsPIN4 is highly sensitive to auxin in abscission zones and acts as a floral organ abscission repressor.These findings provide valuable insights for future PsPIN gene investigations and contributes to our understanding of the intricate relationship between PIN-mediated auxin transport and abscission.

FIGURE 1
FIGURE 1Distribution of PsPIN genes in tree peony.Scale bar indicates length of chromosome.
(A) Intron-exon structure of PsPINs.Green boxes represent exons, blue boxes represent UTR, and spaces between the boxes represent introns.The scale bar indicate exon and intron length.(B) The transmembrane and modular loop structure of PsPINs.Prediction of trans-membrane helices within two highly conversed hydrophobic domains located at N-and C-termini are indicated by colored rectangles.Sun et al.  10.3389/fpls.2024.1380417Frontiers in Plant Science frontiersin.orghydrophilic ring (HL), which separates the two hydrophobic domains (HD) located at the N-terminus and C-terminus.The variances in length recorded in the central hydrophilic region contribute greatly to PsPIN diversity.PsPIN2a, PsPIN4, and PsPIN5 possessed five transmembrane helices (Helix1-Helix5) at the N-terminus, whereas PsPIN1 and PsPIN8 lacked the third transmembrane helix.Specifically, only PsPIN2a contained five transmembrane helices at the C-terminus, while PsPIN1 and PsPIN4 had 4 helices, and PsPIN8 had only 3 helices.The eighth helix of PsPIN2b, PsPIN6a, and PsPIN6b differed significantly from other members.The conserved NPXXY structure, which is crucial membrane and receptor proteins interaction in endocytosis, was found near the hydrophobic and hydrophilic regions of the C-terminus.

FIGURE 4
FIGURE 4 Predicted cis-elements in PsPINs promoters.Cis-regulatory elements in PsPINs promoter regions (2,000 bp upstream of the starting codon) are distinguished by shapes and colors.Displayed numbers of varying colors indicate the quantity of each component.Annotations of cis-elements are listed in Supplementary TableS5.
FIGURE 8 Ectopic expression of PsPIN4 promoter and subcellular localization of PsPIN4 (A) GUS expression driven by PsPIN4 promoter in Arabidopsis.a-e: Arabidopsis seedlings at age of 1, 2, 3, 4, and 7 weeks, respectively.Numbers in the d1 represent leaf developmental stages.(B) GUS expression is driven by PsPIN4 promoters in flower organs at different flowering stages.P1~P7: Position of flowers in the inflorescence from top to bottom.(C) GUS protein levels assessed by western blot using GUS antibody in plants treated as in (A) Actin was detected as a loading control.P2, P4, and P6: Corresponding to the different positions of flower organs in panel (B).(D) Subcellular localization of PsPIN4 by fluorescent microscopy with a stimulating wave length of 488 nm, Bar=25 mm.

TABLE 1
The PIN family genes in tree peony.

TABLE 2
The predicted phosphorylation sites of PsPIN proteins.