Single-cell RNA sequencing reveals a high-resolution cell atlas of petals in Prunus mume at different flowering development stages

Abstract Prunus mume (mei), a traditional ornamental plant in China, is renowned for its fragrant flowers, primarily emitted by its petals. However, the cell types of mei petals and where floral volatile synthesis occurs are rarely reported. The study used single-cell RNA sequencing to characterize the gene expression landscape in petals of P. mume ‘Fenhong Zhusha’ at budding stage (BS) and full-blooming stage (FS). Six major cell types of petals were identified: epidermal cells (ECs), parenchyma cells (PCs), xylem parenchyma cells, phloem parenchyma cells, xylem vessels and fibers, and sieve elements and companion cells complex. Cell-specific marker genes in each cell type were provided. Floral volatiles from mei petals were measured at four flowering development stages, and their emissions increased from BS to FS, and decreased at the withering stage. Fifty-eight differentially expressed genes (DEGs) in benzenoid/phenylpropanoid pathway were screened using bulk RNA-seq data. Twenty-eight DEGs expression increased from BS to FS, indicating that they might play roles in floral volatile synthesis in P. mume, among which PmBAHD3 would participate in benzyl acetate synthesis. ScRNA-seq data showed that 27 DEGs mentioned above were expressed variously in different cell types. In situ hybridization confirmed that PmPAL2, PmCAD1, PmBAHD3,5, and PmEGS1 involved in floral volatile synthesis in mei petals are mainly expressed in EC, PC, and most vascular tissues, consistent with scRNA-seq data. The result indicates that benzyl acetate and eugenol, the characteristic volatiles in mei, are mostly synthesized in these cell types. The first petal single-cell atlas was constructed, offering new insights into the molecular mechanism of floral volatile synthesis.


Introduction
Floral fragrance is an important trait of ornamental plants that are used in perfume production and plays a crucial role in attracting pollinators and adapting to biotic and abiotic stresses.Floral volatiles are a mixture of secondary metabolites in plants, which are divided into three major classes: terpenoids, benzenoids/phenylpropanoids (BPs), and fatty acid derivatives [1].These volatiles are mainly synthesized in petals [2].Some genes involved in f loral volatile synthesis exhibit specific or high expression in epidermal cells (ECs) of petals, such as linalool synthase gene (CbLIS) in Clarkia breweri, benzoic acid methyltransferase gene (AmBAMT) in Antirrhinum majus, and orcinol O-methyltransferase gene (RhOOMT) in rose petals [3][4][5].Several structural genes are involved in the biosynthesis pathway of a given f loral volatile [6].However, whether these genes are expressed in a single cell remains to be proven.
Prunus mume (mei), a traditional ornamental f lower in China, has a long cultivation history.It belongs to the Prunus genus of Rosaceae family and originates in the Yangtze River basin in southern China, which usually blooms in early spring.Furthermore, mei produces a characteristic f loral fragrance compared to other Prunus species, such as peach and apricot [7].Thus, it is important to explore the potential molecular mechanism of f loral volatile synthesis in mei to develop aromatic cultivars in the Prunus genus.Some studies have shown that benzaldehyde, benzyl alcohol, benzyl acetate, and eugenol are characteristic aroma components of mei [8].These volatiles are synthesized through the BP pathway [9].A series of genes involved in f loral scent biosynthesis in mei have been studied using bulk RNAseq: phenylalanine ammonia-lyase gene (PmPAL), 4-coumarate-CoA ligase gene (Pm4CL), cinnamate-CoA ligase gene (PmCNL), cinnamate 4-hydroxylase gene (PmC4H), cinnamoyl-CoA reductase gene (PmCCR), cinnamyl alcohol dehydrogenase gene (PmCAD), benzyl alcohol acetyltransferase gene (PmBEAT), and eugenol synthase gene (PmEGS) [10][11][12].However, such studies have mostly selected the whole f lower as the experimental material.Thus, it might be difficult to explain the expression pattern of key genes in different cell populations.Assuming that scent-producing cells can be separated from the rest of the cells in the entire petal, the priority of key genes in f loral volatile synthesis will be better explored [13].
Single-cell RNA sequencing (scRNA-seq) is a new technology that analyses transcriptomes of thousands of single cells simultaneously, identifies unknown cell types, and explores the potential roles of different cell types in plant organs to highlight functional specialization in many plants, such as Oryza sativa roots, poplar stem, and strawberry leaves [14][15][16][17].ScRNA-seq has also been applied to investigate the cell types of petals and f loral fragrance synthesis at the cellular level.For example, cell types in Nicotiana attenuata corolla were identified using scRNA-seq, and the molecular mechanism of the cell type-specific metabolism of benzyl acetone biosynthesis was revealed [13].However, there are no relevant studies on non-model plant petals at the singlecell level.Analysis of a single-cell transcriptome in mei petals will enable us to identify the major cell types, focus on the expression dynamics of differentially expressed genes (DEGs) among different cell clusters, and discover cellular interaction and diversity at the precise time of volatile synthesis.It is also a reference for further understanding the molecular mechanism underlying the cell type specificity of BP biosynthesis.
In this study, P. mume 'Fenhong Zhusha' (FHZS) petals at budding stage (BS) and full-blooming stage (FS) were selected as the main materials to construct a single-cell gene expression map of woody plant petals using scRNA-seq.Floral volatiles and bulk RNA-seq data of variety 'FHZS' petals at four f lowering development stages [BS, initial f lowering stage (IFS), FS, and withering stage (WS)] were measured.Through combining scRNA-seq and bulk RNA-seq transcriptome data, candidate genes related to f loral volatile synthesis in mei petals were screened, and their expression patterns in different cell types were analysed at BS and FS.The main expression sites of genes involved in f loral volatile biosynthesis in mei petals were verified at the cellular level via in situ hybridization, enhancing the systemic understanding of the mechanism of f loral volatile synthesis.

Single-cell transcriptome sequencing of P. mume petals
ScRNA-seq on the petals of variety 'FHZS' was performed using the 10X Genomics Chromium platform (Fig. 1A).Free protoplasts from petals at BS and FS were obtained by enzyme digestion.After cell viability testing, 20 000 cells of each sample were used to construct two libraries sequenced by the PE150 mode of the Illumina sequencing platform.In total, 7983 and 8965 high-quality cells were successfully captured from petals at BS and FS, respectively.Correspondingly, 478 182 852 and 449 158 706 reads were obtained, with 97.2% and 97.8% valid barcodes, respectively, and 91.0% and 92.1% of reads were confidently mapped to P. mume var.tortuosa genome.A total of 19 072 and 18 296 genes were identified through scRNA-seq of petals at BS and FS, with 3358 and 2646 median genes per cell.The median unique molecular identifier (UMI)  S1, see online supplementary material).Cells from two f lowering development stages were visualized on a uniform manifold approximation and projection (UMAP) plot (Fig. 1B).At the same time, bulk RNA-seq of petals was performed at four f lowering development stages.The datasets are shown in Tables S2 and S3 (see online supplementary material).Pearson correlation analysis was used to compare gene expression between scRNA-seq and bulk RNAseq profiles.Results showed strong correlations (R = 0.736 and 0.721 for petals at BS and FS, respectively; P < 2.2e-16; Fig. 1C).

Identification of cell types in P. mume petals
After filtering out low-quality cells, an unsupervised clustering analysis of 16 948 cells was conducted.Eleven clusters were formed (Fig. 2A).Because there were no cell-specific marker genes and a high-revolution atlas in petals reported, clusters were assigned based on orthologs of single-cell markers in other plants.Cluster 0 was ECs, which contained the marker genes GPAT6 and LTPG1 that are known to be involved in the export of epidermal components and cutin biosynthesis in petal formation [18,19].Clusters 1 and 2 were parenchyma cells (PCs) for high expression of CAP10A and CAB40 (orthologs of FvLHCB2.1), which are marker genes for PC [17,20].Clusters 3 and 5 were xylem parenchyma cells (XPCs) and expressed the marker genes NPF2.11 and ABR1 [21,22].Cluster 4 was defined as phloem parenchyma cells (PPCs) because of the expression of bZIP9 that was specifically expressed in PPCs [23].It was speculated that cluster 6 might be likely PPCs due to the enrichment of the same genes with cluster 4. Cluster 8 was xylem vessels and fibers (XVFs) with the marker gene WAT1, which determines the secondary cell wall thickness of wood fibers and is associated with developing XVFs in Arabidopsis thaliana [16,24].PmWAT1, expressed predominantly in xylem vessels, was observed using in situ hybridization (Fig. S1, see online supplementary material).Cluster 10 was the sieve element companion cell complex (SECC) in phloem and contained the marker genes PP2-A1 and PP2-A4 [25,26].Cell types of clusters 6, 7, and 9 could not be determined using the current marker genes and were named unknown cells (Fig. 2B).
These cell-specific DEGs were enriched in various biological processes in Gene Ontology terms (Fig. S2a, see online supplementary material).DEGs in cluster 0 were rich in lipid metabolic process and fatty acid biosynthetic process, which were related to ECs.DEGs in clusters 1 and 2 were enriched in ribosome biogenesis and organonitrogen compound metabolic process.Clusters 3 to 5, 8, and 10 were thought to be vascular tissues, and DEGs in these clusters were mainly involved in signal molecule conduction and transport.DEGs in clusters 6, 7, and 9 were unavailable for further analysis.Based on the Kyoto Encyclopedia of Genes and Genomes annotation, the enriched pathways in ECs (cluster 0) were mostly involved in metabolic pathways, fatty acid metabolism, steroid biosynthesis, amino sugar and nucleotide sugar metabolism, and so on.DEGs in PCs (clusters 1 and 2) were primarily related to photosynthesis-antenna proteins of petals.DEGs in vascular tissues (clusters 3 to 5, 8, and 10) were mainly enriched in ribosome, secondary metabolic biosynthesis, endocytosis, and plant hormone signal transduction pathways (Fig. S2b, see online supplementary material).

Discovery of cell-specific marker genes in P. mume petals
After identifying cell clusters, the number of relevant upregulated DEGs ranged from 447 in cluster 10 to 1834 in cluster 0 (Fig. S3, see online supplementary material).The top five cluster-specific genes in each cluster were collected (Fig. 3).MALD3, MLP423, FDH, EXL3, and CER2 were highly enriched in ECs (cluster 0).MALD3 and FDH (the orthologous of NaKCS10), were the epidermal-specific marker genes in A. thaliana and N. attenuata, respectively [13,19].AtMALD3 was found to be responsible for trafficking cutin and wax to the plant surface for assembly and deposition of the cuticle, and NaKCS10 was involved in cuticular wax and cutin biosynthesis.PaEXL3 in Prunus armeniaca might be associated with lipid metabolism to protect plants against cold stress [27].UGT94E5, JGB, EIX2, CAB21, and AMT1-2 were highly expressed in PCs (clusters 1 and 2).CAB (chlorophyll a/b binding protein of LHCII type) proteins are related to photosynthesis, providing energy for plant development [28].PmAMT1-2 might also be related to f loral volatile synthesis because PmAMT1-2 expression was positively correlated with benzyl acetate and benzaldehyde production in P. mume [10].The expression of MLP328, GAF1, AOMI, PUB21, and WRKY18 showed high specificity in XPCs (clusters 3 and 5).ABCC14, INVA, RAV2, AVT6A, and PLAT1 were expressed more in PPCs (cluster 4).VIT1, UGT74G1, PME2.1, SAUR32, and CHIT5A were mainly expressed in cluster 8 and considered as the marker genes in XVFs.PmSAUR32 was highly expressed in P. mume 'WuYuyu' stems, which was involved in hormone and sugar metabolism [29].MYR2, CLE25, GRXS1, RL6, and PHO1-H1 were mostly expressed in cluster 10, which were thought to be the marker genes in SECC (Table S4, see online supplementary material).AtPHO1-H1 was expressed in the vascular cylinder of roots and shoots in A. thaliana under phosphate-deficient conditions to support plant growth and survival [30].These marker genes would contribute to recognizing the petal cell types of mei.

Dynamic changes in floral volatiles and potential genes related to their synthesis in P. mume petals
The f loral volatiles of variety 'FHZS' petals were analysed at four f lowering development stages using gas chromatographymass spectrometry (GC-MS).Benzyl alcohol, benzyl acetate, eugenol, cinnamaldehyde, and benzaldehyde, which originated from BP biosynthesis, were detected.Their contents during release increased from BS to FS and decreased at WS, reaching the peak at FS.The amounts of benzaldehyde and cinnamaldehyde were low at the four stages (Fig. 4A).
The f loral fragrance profiles in mei were dominated by benzyl acetate and eugenol.It has been reported that PmBAHD5 (orthologs of PmBEAT36) catalyzed benzyl alcohol to benzyl acetate and PmBAHD3 (the homologous gene of PmCFAT1) was involved in eugenol synthesis [7,31].Because of the substrate diversity of acyltransferases, this study delved deeper into the functional roles of PmBAHD3 by heterologous expression in Escherichia coli.The purified recombinant protein was obtained (Fig. S4, see online supplementary material), and enzymatic activity was performed in vitro with benzyl alcohol as a substrate.Benzyl acetate was detected in the reaction production using GC-MS (Fig. 4C).PmBAHD3 would function on benzyl acetate synthesis, suggesting that benzyl acetate formation in P. mume might be the result of a combination of multiple alcohol acyltransferases.

Exploration of cell types related to floral fragrance synthesis in P. mume petals
We analysed the cells percentage of 'FHZS' petals at different f lowering development stages.From BS to FS, the proportions of ECs decreased sharply, whereas that of PCs increased obviously.The percentages of vascular tissue cells changed slightly between BS and FS (Fig. S5a, see online supplementary material).DEG expression in each cell type of 'FHZS' petals at BS and FS was also obtained, which provided data to further analyse the expression of genes involved in f loral fragrance synthesis in different cell types.In general, there were 2169 upregulated and 3132 downregulated genes at FS compared with those at BS (Fig. S5b, see online supplementary material).
To further understand the cell types related to f loral fragrance synthesis, this study explored the expression patterns of the 28 candidate genes mentioned above in scRNA-seq data: 2 PmPALs, 1 PmC4H, 1 PmCNL, 3 Pm4CLs, 5 PmCCRs, 4 PmCADs, 10 PmBAHDs, and 2 PmEGSs.Among them, 27 genes were detected, except for Pm4CL4 (Table S6, see online supplementary material).The expression of 10 genes (Pm4CL3, PmCCR4, PmCAD4, PmBAHD8,10,12,14,17,24, and PmEGS2) was low in each cell type at BS and FS.The expression of 13 genes, including PmPAL1,2, PmC4H, PmCNL, PmCCR1,2,3,5, PmCAD1,3, PmBAHD2,5, and PmEGS1 was higher at FS than at BS in all cells we obtained.Although these genes are expressed in almost every cell type, their expression varies among different cell types.For instance, the expression levels of PmPAL1,2, PmC4H, PmCNL, and PmCCR5 in ECs and PCs were higher than those in other cell types at FS.The expression levels of PmCCR1 and PmCCR2 were slightly higher in PCs at BS and FS, whereas that of PmCCR3 was higher in SECC.The expression of PmCAD1 and PmCAD3 was higher in XPCs and XVFs.PmBAHD2 was expressed mainly in ECs and PPCs, which increased markedly from BS to FS.Despite its significant increase from BS to FS, PmBAHD5 expression in PCs and XPCs was higher than that in other cell types.PmEGS1 expression was slightly higher in PCs at FS. Besides, the expression levels of PmCAD2 and PmBAHD3,6 were lower in all cells at FS than in those at BS. PmCAD2 expression was slightly higher in ECs, XPCs, PPCs, and XVFs at FS. PmBAHD3 was also expressed in each cell type at FS, except for SECC, and exhibited higher expression in XPCs, PPCs and XVFs at FS.However, PmBAHD3 expression increased significantly in ECs from BS to FS.There were no significant differences in Pm4CL1 expression at these two f lowering development stages, but its expression in ECs significantly increased from BS to FS (Fig. 5A).
Furthermore, the main expression sites of several genes, including PmPAL2, PmCAD1, PmEGS1, PmBAHD3, and PmBAHD5, were confirmed by in situ hybridization.As shown in Fig. S6 (see online supplementary material), PmPAL2 expression in all cell types at FS was higher than that in cells at BS.And PmPAL2 was primarily expressed in PCs at BS, whereas its expression in ECs and PCs was higher at FS, suggesting that scRNA-seq data were reliable.The cell types where the other four genes were expressed at FS were detected.PmCAD1 and PmEGS1 were expressed in each cell type of petals.Although PmBAHD3 and PmBAHD5 were detected in most cells, PmBAHD3 expression in PPCs and XVFs was higher than that in others and PmBAHD5 was highly expressed in XPCs, consistent with the scRNA-seq data.These results suggested that f loral volatiles might be synthesized in ECs, PCs, and most vascular tissue cells of P. mume petals (Fig. 5B).These results also indicated the reliability of cell cluster classification in this study (Fig. 2).

Discussion
P. mume is famous for its unique fragrance, which mainly comes from its petals [11].Petals are generally composed of ECs, PCs, and vascular tissues [32].Previous studies showed that PCs or ECs of petals is involved in volatile synthesis [33,34].Indeed, studies on A. majus showed that petal adaxial epidermal cell is the main site to produce f loral fragrance [4].Subepidermal parenchyma cells of ray f lorets of Centaurea cyanus exhibit many features of scent-producing cells, confirming their involvement in the production of volatile aromatic compounds in petals [35].However, the specific cell types or layers in mei petals where f loral volatiles generally occur remain unclear.ScRNA-seq has made it possible to annotate cell types of petals, which is a crucial step for subsequent analysis.A previous study discovered the major cell types in N. attenuata corolla by scRNA-seq, including ECs, PCs, phloem, xylem, and chlorenchyma cells, and benzyl acetone was mainly synthesized in ECs [13].In this study, ECs, PCs, and vascular bundles were observed in 'FHZS' petals (Fig. S7, see online supplementary material), and six major cell types were identified using scRNA-seq (Fig. 2A).The cell-specific marker genes previously identified were validated in P. mume, including PmWAT1 (Fig. S1, see online supplementary material), and novel marker genes of P. mume petals were provided.For instance, PmMALD3 and PmFDH were specifically expressed in ECs, PmCAB21 and PmAMT1-2 were highly expressed in PCs, and PmSAUR32 was mostly expressed in vascular tissues (Fig. 3).These novel marker genes could be used to identify the cell types of woody plant petals.
The emission of f loral scent compounds exhibits temporal rhythms [6].In 'FHZS' petals, the emission levels of unique volatile compounds originating from the BP pathway, such as benzyl alcohol, benzyl acetate, and eugenol, increased sharply from BS to FS and decreased at WS (Fig. 4A), consistent with previous studies [8,36].A series of structural genes are involved in the biosynthesis of benzyl acetate and eugenol, which are important characteristic f loral scent compounds in mei [10].PAL is the first and key enzyme in BP pathway that catalyzes the conversion of trans-cinnamic acid from Phe [6].Then, trans-cinnamic acid generates eugenol and benzyl acetate via the C 6 -C 3 and C 6 -C 1 pathways, respectively, with the involvement of enzyme genes such as C4H, CNL, and 4CL [6].The expression levels of PmPAL2, PmC4H, PmCNL, and Pm4CL1 were consistent with the emission levels of benzyl acetate and eugenol from BS to WS, suggesting that they may participate in their synthesis.PmCCR1,2,3 and PmCAD1 expression continued to increase from BS to WS and showed a strong correlation with the emission level of eugenol.PmCCR1,2,3 might provide coniferyl aldehyde, which was converted into coniferyl alcohols by PmCAD1 for eugenol formation.PmBAHD3 (ortholog of PmCFAT1) played a role in eugenol synthesis and might catalyze coniferyl alcohol to coniferyl acetate [31] that was the substrate to produce eugenol catalyzed by a specific enzyme EGS [37].Elevated transcript levels of PmBAHD3 and PmEGS1 from BS to WS also showed a strong correlation with the emission level of eugenol.Due to its higher expression, PmEGS1 might be responsible for eugenol synthesis in mei.By integrating the transcriptome database with the emission levels of volatile compounds, a potential biosynthetic pathway for eugenol in mei was summarized.The expression of PmBAHD5 and PmBAHD3 played important roles in the synthesis of benzyl acetate from benzyl alcohol, displaying an overall uptrend from BS to WS (Fig. 4B).Interestingly, PmBAHD3 played a role in the synthesis of both benzyl acetate and eugenol because of the sub-strate diversity of acyltransferases.Whether other members of the alcohol acyltransferase gene family in this study, such as PmBAHD2 and PmBAHD17 also participated in benzyl acetate and coniferyl acetate biosynthesis still needs to be further explored.In addition, the release of f loral aroma volatiles might be inf luenced by cellular structure of the petals from BS to FS.More cell-cell spaces were formed in mei petals at FS than those at BS due to a significant increase in proportion of PCs exhibiting a lax arrangement (Fig. S5a, see online supplementary material).This structure might make the emission of fragrant compounds easier in mei petals.
Genes involved in the pathway of a given f loral organic volatile compound might be expressed in the same scent-producing cells.This study explored the expression sites of these relevant genes at the cellular level using single-cell expression profiles in mei petals.The process by which PmPAL catalyzes the conversion of phenylalanine to trans-cinnamic acid may occur in all petal cell types because of PmPAL2 nonspecific expression.Trans-cinnamic acid was used as a substrate to produce eugenol through several enzymatic reactions catalyzed by enzymes encoded by PmC4H, Pm4CL1, PmCCR1,2,3, and PmCAD1, PmBAHD3, and PmEGS1 genes, exhibiting similar expression patterns as PmPAL2, indicating that these successive catalytic actions might occur in all cell types in petals, except for SECC, suggesting that the entire set of genes involved in eugenol production might play synergistic roles in a single cell of every cell type in petals and do not exhibit cell type specificity.Among these genes, PmCAD1 and PmBAHD3 showed high expression in XVFs than in others at FS, probably because eugenol formation shares the same initial biosynthetic steps with the lignin biosynthetic pathway [6].PmBAHD3 and PmBAHD5 converted benzyl alcohol to benzyl acetate in P. mume [7], and their encoded genes were also expressed in all cell types, except for SECC, indicating that benzyl acetate might be synthesized in ECs, PCs, and most vascular bundle cells.Interestingly, PmBAHD5 expression was higher in xylem cells than in other cell types.It was speculated that benzyl acetate synthesized in xylem cells would be transported from petals to stamens for release.This could explain why stamens are the main site for the release and storage of benzyl acetate in addition to petals [38], although PmBAHD5 expression is much higher in petals than in stamens [7], indicating that the expression site of genes in BP pathway might be related to the release of volatile compounds.Moreover, it was speculated that the expression of genes involved in the major Phederived f loral volatile biosynthesis mainly occurs at different sites in plants.NaPAL4 and Na4CL1 from N. attenuata in benzyl acetone (C 6 -C 4 ) biosynthesis derived from Phe are highly expressed in ECs and PCs but not in vascular tissues [13], which are not the same as those in mei petals.
In conclusion, we constructed the first single-cell gene expression atlas of woody plant petals using scRNA-seq and established the marker genes for each cell type in mei petals.Moreover, our results indicated that the f loral volatile synthesis of mei may occur in ECs, PCs, and most vascular tissue cells in petals.These findings can provide references for further exploring cell types in woody plant petals and understanding the potential molecular mechanism of f loral volatile synthesis.However, this study still faced some challenges.For example, it lacked well-established laser microdissection procedures to isolate different cell types in petals, limiting the study at the single-cell level.It was also difficult to obtain the precise contents of various compounds in different cell types.This is critical for a comprehensive understanding of f loral volatile biosynthesis.

Plant growth conditions and petal protoplast isolation
'FHZS' petals were grown on the campus of Northwest A&F University.Flowers at BS and FS were harvested.Twenty petals were cut into strips before immersion in a 10 mL enzyme solution [2% cellulase R10, 2% macerozyme R10, 0.4 M mannitol, 20 mM KCl, 20 mM MES (pH 5.7), 10 mM CaCl 2 , 0.1% bovine serum albumin, and 0.5 mM dithiothreitol].Protoplasts were released after shaking at 60 rpm for 2 h at 25 • C in the dark at room temperature.The enzyme containing protoplasts was diluted with the same amount of W5 solution [2 mM MES (pH 5.7), 154 mM NaCl, 125 mM CaCl 2 , and 5 mM KCl], and the reaction was terminated.The protoplast solution was filtered by wetting 40 μm nylon with W5 solution and centrifuged at 100 × g at 4 • C for 6 min.The protoplasts were gently washed twice with W5 solution to obtain pure protoplasts.Cell density and viability were determined with 0.4% trypan blue staining, and protoplasts with a viability of ≥90% and a concentration of ≥1000/μL were immediately processed with the 10X Genomics Single Cell Protocol.

scRNA-seq library construction, sequencing, and raw data quality control
The library was constructed using the Chromium controller and Chromium Next GEM Single Cell 3'eagent Kits version 3.1.The cell suspension was loaded on a 10X Genomics GemCode single-cell instrument to generate single-cell GEMs (gel beads in emulsion).The scRNA-seq library was prepared using Chromium singlecell 30 gel beads and library kits.The library was pair-ended sequenced on the Illumina sequencing platform.In the scRNAseq dataset, Read1 contained a 16 bp GemCode barcode and 10 bp UMI, and Read2 was a cDNA sequence fragment.
Using 10X Genomics Cell Ranger (version 3.1.0),reads with lowquality barcodes and UMI were filtered out, FASTQ sequencing reads converted from raw data were mapped to the P. mume var.tortuosa genome, and a digital gene expression matrix was generated.The gene-cell matrix was loaded into the Seurat package (version 3.1.1)for further quality control, and cells with UMIs ≥8000 or mitochondrial gene percentage ≥10% and expressed <500 or >4000 genes were filtered out.Bimodal cells were removed by DoubletFinder (version 2.0.3).After removing low-quality cells, expression was further homogenized and Harmony was used to merge data to generate the original data for further analysis.R was used to calculate the Pearson correlation coefficient between bulk RNA-seq and scRNA-seq datasets.

Statistics for differential gene expression analysis
Seurat was used to perform differential gene expression analysis of genes in different cell types of 'FHZS' petals at BS and FS.The screening standards were as follows: |log 2 FC| ≥0.36, the proportion of cells expressing target genes in each group ≥0.1, and an adjusted P value (P adj ) ≤0.05.The genes were tested for significance using MAST, and P values were adjusted by the Benjamini-Hochberg method.

Bulk RNA-seq library construction and sequencing
Total RNA was extracted from petals using Trizol reagent according to the manufacturer's instructions.Each sample contained three replicates.High-quality RNA samples with RIN number >7.0 were screened out by Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit (Agilent, Santa Clara, CA, USA) for RNA-seq library construction.Sequencing was conducted by an Illumina Novaseq™ 6000.Clean reads were aligned to the mei reference genome using HISAT2 [39], and expression of all transcripts was estimated by StringTie and ballgown [40].
Collection and determination of floral volatiles in P. mume 'FHZS' petals at four f lowering development stages were collected.After being weighed, petals from different f lowering development stages were separately placed into 25 mL injection vials and then held for 10 min before using extraction fiber to absorb the volatiles for 30 min at 30 • C. GC-MS was used to analyse the emission amounts of f loral volatiles.Helium was the carrier gas in the split mode with a split ratio of 20 and a column f low rate of 27.0 mL/min.The temperature of the GC oven started at 40 • C, was maintained for 2 min, increased to 250 • C (at a speed of 5 • C/min), and held for 6 min.

In situ hybridization
'FHZS' petals at BS and FS were collected and immediately put into an FAA solution (50% ethanol, 5% acetic acid, and 3.7% formaldehyde) for fixation for >12 h.The antisense probe of genes was generated with gDNA as a template according to the DIG High Prime Lab/Detection K (Roche).The primers are shown in Table S7 (see online supplementary material).In situ hybridization was performed as described previously [41].Images were observed using a LeicaMZ10Fmicroscope (Leica Co., Baden-Württemberg, Germany).

Heterologous expression of PmBAHD3 protein in E. coli and in vitro enzymatic assay
To express the PmBAHD3 protein in E. coli, the full-length sequence of the PmBAHD3 gene was amplified with gene-specific primers and seamlessly cloned into the pCold-TF vector.The recombinant plasmid pCold-TF-PmBAHD3 and the empty pCold-TF vector were separately transformed into E. coli ArcticExpress (DE3).After induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 12 • C for 20 h, cells were harvested by centrifugation, resuspended in phosphate-buffered saline (pH 7.2), and broken down by sonication.The recombinant pCold-TF-PmBAHD3 protein and pCold-TF protein were purified using a nickel-Sepharose column (MedChemExpress, USA).The purified products were analysed and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used for subsequent enzyme activity analysis.
Enzyme activity was determined in a 200 μL reaction mixture by mixing 50 μg purified PmBAHD3 protein with 1 mM benzyl alcohol, 250 μM acetyl-CoA, and 10 mM MgCl 2 in 50 mM Tris-HCl (pH 8.0).The mixture was incubated at 30 • C for 1 h, followed by the addition of 100 μL ethyl acetate to the reaction under vortexing and centrifugation.The supernatant was transferred to a sample vial for GC-MS analysis.

Figure 1 .
Figure 1. Isolation and analysis of single-cell transcriptomes from mei petals.A Petal protoplast extraction at BS and FS.B UMAP visualization of samples at BS and FS.C Correlation analysis of scRNA-seq profiling and bulk RNA-seq at BS (a) and FS (b), respectively.

Figure 2 .
Figure 2. Cell cluster identification of single-cell transcriptomes from mei petals.A Visualization of the 11 cell clusters.B Violin plots show the homologous gene expression distribution of the reported marker genes in different cell types in mei.

Figure 3 .
Figure 3. Expression heatmap of cell-specific marker genes in 11 clusters of petals.The expression value of each gene was z-score normalized.EC, epidermal cell; PC, parenchyma cell; PPC, phloem parenchyma cell; SECC, sieve element companion cell complex; UN, unknown cell types; XPC, xylem parenchyma cell; XVF, xylem vessel and fiber.

Figure 4 .
Figure 4. Correlation analysis of f loral volatile compounds emission and the related regulated genes in biosynthesis.A Dynamic changes in the main f loral volatiles released at four f lowering development stages of mei.B Heat map of transcriptome expression of 58 structural genes related to f loral volatile biosynthesis from BS to WS. FPKM values were log10 transformed.BS, budding stage; FS, full-blooming stage; IFS, initial f lowering stage; WS, withering stage.C In vitro analysis of the enzyme activity of PmBAHD3.

Figure 5 .
Figure 5. Cell-specific distribution of genes related to f loral fragrance biosynthesis.A Heat map of the expression of 27 genes related to f loral volatile synthesis in a single-cell transcriptome.Expression values were log10 transformed.BS, budding stage; FS, full-blooming stage.B Localization of the expression of four genes involved in benzyl acetate and eugenol synthesis in 'FHZS' petals by in situ hybridization.Bar = 25 μm.EC, epidermal cell; PC, parenchyma cell; VB, vascular bundle.