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Transcriptome sequencing and gene expression analysis revealed early ovule abortion of Paeonia ludlowii

BMC Genomics volume 24, Article number: 78 (2023) Cite this article

Abstract

Background

Paeonia ludlowii (Stern & G. Taylor D.Y. Hong) belongs to the peony group of the genus Paeonia in the Paeoniaceae family and is now classified as a “critically endangered species” in China. Reproduction is important for this species, and its low fruiting rate has become a critical factor limiting both the expansion of its wild population and its domestic cultivation.

Results

In this study, we investigated possible causes of the low fruiting rate and ovule abortion in Paeonia ludlowii. We clarified the characteristics of ovule abortion and the specific time of abortion in Paeonia ludlowii, and used transcriptome sequencing to investigate the mechanism of abortion of ovules in Paeonia ludlowii.

Conclusions

In this paper, the ovule abortion characteristics of Paeonia ludlowii were systematically studied for the first time and provide a theoretical basis for the optimal breeding and future cultivation of Paeonia ludlowii.

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Introduction

Paeonia ludlowii (Stern & G. Taylor D. Y. Hong) is a wild endemic plant with conservation status II in China (National Forestry and Grassland Administration, 2021). It is a tall plant with multiple yellow flowers and it can develop secondary branches around florescence in spring which was distinguished from other species and cultivars of Paeonia Sect. It has breeding, medicinal, ornamental, and exploitative values [1,2,3,4] (Fig. 1). Wild P.ludlowii is distributed only in the Nyingchi region of Tibet, China [5], while P.ludlowii grown in other countries has been introduced from the Nyingchi region in China [6].

Fig. 1
figure 1

Paeonia ludlowii (Mirui Township, Bayi District, Nyingchi, Tibet); a and b: May 2021; c: October 2021

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In the late twentieth and early twenty-first centuries, P.ludlowii was considered an independent species. Subsequently, its geographical distribution was further defined [5] and introduced in Gansu and Henan Provinces [7, 8]. Significant progress has been made to understand the genetic resources [9], diversity [10, 11], nutritional value and applications of P.ludlowii [3, 12,13,14], including endophytic fungi [15], seed germination, seedling establishment [16, 17], pollen germination, storage characteristics [18], and saline-alkali stress [19]. Its self and heterozygous affinity, but its fruit set rate is very low [7, 20, 21], resulting in a high seed defeat rate. However, no research has assessed seed defeat in P.ludlowii.

Other endangered plants such as dove trees (Davidia involucrata) [22], Caryocar brasiliense [23], and Liriodendron chinense [24] also experienced severe seed abortion. Recent studies have demonstrated that seed abortion is a complex plant behavior influenced by a combination of internal and external conditions [25]. As molecular biology theories and techniques have progressed, research related to seed abortion has gradually shifted from physiological and morphological levels to molecular and genetic levels. Currently, the main research focus is on the dove tree [22, 26], pomegranate (Punica granatum) [27], longan (Dimocarpus longan Lour.) [28], peanut (Arachis hypogaea) [29], hazelnut (Corylus heterophylla) [30, 31], grapes (Vitis vinifera) [32], rice (Oryza sativa) [33], and other species in which significant progress has been made identifying and obtaining some candidate genes or proteins. However, unlike many of the above species, P.ludlowii seeds within the same carpel are not completely aborted. Yet aborted and normal seeds coexist, leading us to speculate that unique regulatory mechanisms exist in these plants.

To reveal the regulatory mechanism of ovule abortion in P.ludlowii, we sequenced the transcriptome using high-throughput sequencing technology based on morphological observations. We constructed the first transcript catalog of P.ludlowii seeds. We also identified and analyzed differentially expressed genes in normal and aborted seeds through multiple pathways based on the transcriptome sequencing results, which provides valuable insights into the molecular regulatory mechanisms of ovule abortion in woody perennials.

Materials and methods

Acquisition of experimental materials

Paeonia ludlowii materials were collected from three individual flowering trees as the methods used by our laboratory before [34]. The materials we obtained were all from the Ex-situ Conservation Centre of Chinese Paeoniceae Wild Species (Luanchuan County, Henan Province, China) (111°21′36.15″E, 33°56′4.99″N, 1 408 m altitude). The plant materials used in our experiments were collected in compliance with relevant institutional, national and international guidelines and legislation. Li Qingdao, former Secretary of the Party Committee and professor level senior engineer of the Sui and Tang Dynasties City Ruins Botanical Garden in Luoyang, Henan Province, China undertook the formal identification of the plant material used in our study. And We have sent the voucher specimen of this material to the Museum of Beijing Forestry University, the herbarium collection number is HNLC052101. Three to five carpels were periodically dissected to observe ovule development beginning when the flowers opened on 2020 21 May. The ovules were also selected from the same flowering tree from 25 May 2020 to 15 June 2020, and the carpels were collected at 9 d after flowering (DAF), 10 DAF, 11 DAF, 12 DAF, 13 DAF, 15 DAF, and 17 DAF. The ovules were removed after rapid peeling and photographic recording (Fig. 2). Ovule samples were named Pl-1 (ovules at 9 DAF), Pl-2 (ovules at 10 DAF), Pl-3 (ovules at 11 DAF), Pl-4 (ovules at 12 DAF), Pl-5N (normal ovules at 13 DAF), Pl-5A (aborted ovules at 13 DAF), Pl-6N (normal ovules at 15 DAF), Pl-6A (aborted ovules at 15 DAF), Pl-7N (normal ovules at 17 DAF), and Pl-7A (aborted ovules at 17 DAF). All samples were quickly frozen in liquid nitrogen and stored at − 80 °C for subsequent experiments.

Fig. 2
figure 2

Test materials: a-d: ovules at 9, 10, 11, 12 DAF; e–g: normal and aborted ovules at 13, 15, 17 DAF. Normal and aborted ovules are indicated by red and blue arrows, respectively

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Ultrastructure observation

Samples were prepared according to the methods used by Donghui Wang (2017) [35]. After properly trimming the embedding samples, they were sectioned using an ultrathin sectioning machine (Leica EM UC7; Wetzlar, Germany), stained with a small droplet staining method 2% UO2 acetate for 1 h, rinsed 2–3 times with ultrapure water, and blotted dry with filter paper. The same small droplet staining method was used for lead citrate staining for 5–10 min, after which it was rinsed 2–3 times with ultrapure water, dried naturally, and observed by TEM (JEOL JEM-1010, Japan).

RNA extraction, cDNA library construction, and transcriptome sequencing

Our laboratory's preliminary experimental results showed structural differences between normal and abortion ovules from 9 DAF [36]. Therefore, we selected the ovule samples from the 9th to 17th DAF for transcriptome sequencing. Total RNA was extracted using an RNA prep Pure Plant Kit (Polysaccharides& Polyphenolics-rich) (Tiangen, Beijing, China). Three biological replicates were prepared for each sample. RNA purity was checked using a Nano Photometer® spectrophotometer (IMPLEN, CA, USA), and OD260/OD280 readings between 1.8 and 2.1. After RNA samples were validated, RNA-seq libraries were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample and sequenced at Novogene Co. Ltd (Beijing, China). Transcriptome sequencing was conducted on the Illumina HiSeqTM2000 high throughput sequencing platform.

Transcriptome assembly and functional gene annotation

There is currently no reference genome for P.ludlowii, so we sequenced the non-reference transcriptome. The transcriptome data of P. ludlowii have been uploaded to NCBI (http://www.ncbi.nlm.nih.gov/) (PRJNA818047). Clean data is obtained by removing reads containing aptamers after sequencing data is complete (clean reads), ploy-N, and low-quality reads from the raw data. At the same time, Q20, Q30, GC-content, and sequence duplication levels of the clean data were calculated. All downstream analyses were based on clean data with high quality. We analyzed the variation between each group of three samples and added a PCA plot based on the expression of all single genes in each sample (Additional Fig. 1). We spliced clean reads from sequencing into transcripts using Trinity (v2.4.0) [37] and performed hierarchical clustering with Corset (version 4.6) program transcripts [38]. Each cluster was defined as “Gene”. Finally, the longest transcript in each cluster was as the “unigene” of the gene for subsequent analysis. We used BUSCO (Benchmarking Universal Single-Copy Orthologs) software to evaluate the splicing quality of the data obtained from the splicing and evaluate the splicing quality, also evaluate the accuracy and integrity of the splicing results according to the proportion and integrity of the comparison (Additional Fig. 2). Gene functions were annotated based on the following databases and tools. Nr (NCBI non-redundant protein sequences, diamond v0.8.22); Nt (NCBI non-redundant nucleotide sequences, NCBI blast 2.2.28 +); Pfam (Protein family, HMMER 3.0 package); KOG/COG (Clusters of Orthologous Groups of proteins, diamond (v0.8.22)); Swiss-Prot (A manually annotated and reviewed protein sequence database, diamond (v0.8.22)); KO (KEGG Ortholog database, KAAS (r140224)); GO (Gene Ontology, blast2go (b2g4pipe_v2.5)).

Take the unigenes obtained from Trinity splicing as the reference sequence (Ref), and map the clean reads of each sample to Ref. The comparison process was completed by calling bowtie2 with RSEM software [39], and the used bowtie2 parameter is the default parameter (mismatch 0). The number of reads matched to a gene is called read count. Then, we used FPKM (expected number of Fragments Per Kilobase of translation sequence per Millions base pairs sequenced) to convert the read count to FPKM [40]. The FPKM calculation formula is:

$$\mathrm{FPKM}=\frac{\mathrm{mapped}\;\mathrm{fragments}\;\mathrm{of}\;\mathrm{transcript}}{\mathrm{Total}\;\mathrm{Count}\;\mathrm{of}\;\mathrm{mapped}\;\mathrm{fragments}\;(\mathrm{Millions})\;\times\;\mathrm{Length}\;\mathrm{of}\;\mathrm{transcript}\;(\mathrm{kb})}$$

Differential expression analysis and enrichment analysis

Differential expression analysis was performed using the DESeq R package (1.10.1). The screening conditions were: False Discovery Rate (FDR) adjusted p-value < 0.05 and | log2FC |> 1. We then used GO seq [41] and KOBAS software [42] to perform GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) [43] pathway enrichment analysis of the DEGs (differentially expressed genes). Based on GO and KEGG enrichment analysis of DEGs, structural genes related to plant nutrition, plant hormone signal transduction, programmed cell death, ovule development and abortion were screened from differential genes, and the expression pattern of structural genes was analyzed.

QRT-PCR validation

Liu et al. demonstrated that RPS9 was the most suitable internal reference gene for peony seeds at different developmental stages [44]. Therefore, we used the peony RPS9 gene as an internal reference gene in this paper. The primer sequences used in this paper are shown in Additional file 1. The qRT-PCR reactions were performed on a qTOWER2.2 fluorescent qPCR instrument (jena, Germany), according to the instructions included in the NovoStart® SYBR qPCR SuperMix Kit (Novoprotein). A negative control was set for each gene during the reaction, and three biological replicates were designed for all experiments. Finally, the relative expression of the target gene was calculated according to the 2−ΔΔCT algorithm [45].

Results

Structural differences between normal and aborted ovules in the early stages of embryonic development in Paeonia ludlowii

After ultrastructural observation of ovules with significantly different external morphology 15 DAF after natural pollination, we found significant differences in their ultrastructures. In normal ovules, the free nucleus of the endosperm was intact (Fig. 3a), the nucleolus and chromatin structures could be distinguished, and a large amount of cytoplasm was distributed around the free nucleus (Fig. 3b). In the cells of the inner beads, the nucleolus, nuclear membrane, chromatin structures could be distinguished, and a large amount of secretion was outside the cell wall. In contrast, a large number of mitochondria was distributed along the cell wall (Fig. 3c). In the aborted ovules, the endosperm-free nucleus and cytoplasm were degraded. Only some traces remained, a large number of inner beads near the embryo sac were reduced in cell contents (Fig. 3d), the plasma membrane was wrinkled, the cell wall was thinner and more deeply stained than in normal ovules, almost no secretion was observed outside the cell wall, and chromatin in some of the nuclei tended to be marginalized (Fig. 3e, f). Similar to the characteristics of programmed cell death (PCD) in some plant cells [46,47,48], this suggests that the PCD process may initiated early in P.ludlowii ovules, and premature PCD could be a cause of their early embryo and endosperm abortion.

Fig. 3
figure 3

Ultrastructure of normal ovules and aborted ovules 15 days after natural pollination: CW: cell wall; EX: exudate; FN: free nuclear; Nu: nucleolus; a-c: The endosperm free nucleus of the normal ovule is intact; d-f: The free nuclear endosperm of aborted ovules is completely degraded, and the inner integument cells are significantly different from normal ovules

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Transcriptome sequencing results of Paeonia ludlowii

The data was filtered and screened to obtain high-quality clean reads for subsequent analysis. As a result, 6.0 Gb of clean reads were obtained for each sample: the GC content of reads in the samples ranged from 41.36% ~ to 44.25%, and the percentage of Q30 bases exceeded 92.19% for all samples (Additional file 2).

Splicing the clean reads with Trinity yielded 256,789 transcripts with an average length of 1,238 bp and an N50 value of 1,881. The longest transcript in each gene was considered a representative of that gene, which is called a unigene. In total, 114,350 unigenes were obtained. All unigenes obtained by trinity splicing were compared with seven public databases, and 50,323 unigenes were annotated.

Nr function annotation for unigenes

Based on the analysis of the annotation results of unigenes in Nr, it was found that 3.3% of the sequences of unigenes shared more than 95% identity with sequences of other species, 29.6% had 80%-95% identity of other species, and 46.3% had 60%-80% identity of other species (Fig. 4a). The function of Nr was annotated among the unigenes by diamond software (v0.8.22) with parameters set to e−value = 1e−5, Nr function was annotated among the unigeneswith 7995 unigene sequences that were highly homologous with those of Vitis vinifera, accounting for 19.8% of the total, followed by Camellia sinensis (5.9%), Actinidia chinensis (5.2%), Quercus suber (4.3%), and Rosa chinensis (3.6%) (Fig. 4b).

Fig. 4
figure 4

Nr Database comparison chart: a The similarity distribution map of unigenes compared with other species in the Nr annotation results; b Species distribution map on Nr library comparison

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Screening for differentially expressed genes (DEGs)

Since Paraffin sections revealed that cell degeneration already occurred inside in the embryo sac of aborted ovules at 9 DAF, so DEGs were screened between samples from four adjacent ovule developmental periods at 9 DAF (Pl_1), 10 DAF (Pl_2), 11 DAF (Pl_3), and 12 DAF (Pl_4) (Pl_1 vs. Pl_2, Pl_2 vs. Pl_3, Pl_3 vs. Pl_4) (Figs. 5a-c, Fig. 6a). Removing duplicate genes yielded a total of 8,800 DEGs (Fig. 6b). Of them, the fewest DEGs existed between (Pl_1 vs. Pl_2), with only 1,176, including 643 up-regulated genes and 533 down-regulated genes (Fig. 5a); the most DEGs existed between (Pl_2 vs. Pl_3) 2,821 up-regulated expressions and 2,481 down-regulated expressions (Fig. 5b); the rest of (Pl_3 vs. Pl_4), with 2,489 up-regulated genes and 2,444 down-regulated genes (Fig. 5c).

Fig. 5
figure 5

DEG volcano plot analysis of Paeonia ludlowii: a Pl_1 vs. Pl_2; b Pl_2 vs. Pl_3; c Pl_3 vs. Pl_4; d Pl_5A vs. Pl_5N; e Pl_6A vs. Pl_6N; f Pl_7A vs. Pl_7N. Red indicates upregulation, and green represents downregulation

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Fig. 6
figure 6

DEGs of Paeonia ludlowii: a The number of DEGs; b Venn diagram of DEGs during the adjacent period of early abortion; c Venn diagram of DEGs between early aborted ovules (A) and normal ovules (N); d Venn diagrams of all DEGs

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The external morphology of the ovules of P.ludlowii begins to show small differences in the volume of normal and aborted ovules at 12 DAF, a clear distinction between normal and aborted ovules was explored at 13 DAF. To understand the mechanism of aborted ovule formation in P.ludlowii, the transcriptome data of aborted ovules (A) and normal ovules (N) of P.ludlowii were analyzed for differences between sample groups (Pl_5A vs. Pl_5N, Pl_6A vs. Pl_6N, Pl_7A vs. Pl_7N) (Figs. 5d-f, Fig. 6a). After the comparison we removed the genes in the overlapping part of the Venn diagram, so that the duplicates only appear once, and then 20,522 DEGs were finally obtained between A and N (Fig. 6c). Among them, 4,488 DEGs, 2,689 up-regulated genes, and 1,799 down-regulated genes were obtained between A and N at Pl_5 (Fig. 5d); 7,359 up-regulated genes and 5,624 down-regulated genes were obtained between A and N at Pl_6 (Fig. 5e); 12,122 DEGs, containing 5,613 up-regulated genes and 6,509 down-regulated genes, were screened at Pl_7 (Fig. 5f).

Notably, 6,619 of the 8,800 DEGs in the adjacent period duplicated DEGs between A and N. To focus the analysis on genes that are truly differentially expressed, 22,703 DEGs were available for subsequent analysis after removing duplicates (Fig. 6d).

GO function annotation for differentially expressed genes

A total of 10,288 DEGs were annotated into 133 secondary terms of 3 major categories: biological process (BP), cellular component (CC), and molecular function (MF). Secondary classification and enrichment analysis of the 22,703 DEGs were performed together (Fig. 7, Additional file 3). The BP processes with the highest enrichment and high DEGs include the metabolic, single-organism, and biosynthetic processes. DEGs are primarily associated with the cell, cell parts, and intracellular elements relating to CC. The DEGs involved in MF, on the other hand, were mainly associated with catalytic activity, followed by oxidoreductase activity and nucleic acid binding transcription factor activity. These results suggest that metabolic and cellular differences during development could contribute to ovule abortion.

Fig. 7
figure 7

GO enrichment diagrams of DEGs (Top 48): Unigenes were annotated in three categories: biological processes, cellular components, and molecular functions

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KEGG function annotation for differentially expressed genes

To identify the major biometabolic pathways and signal transduction pathways involved in DEGs during ovule abortion in P.ludlowii, the identified DEGs were enriched for KEGG metabolic pathway functions [43]. A total of 4258 DEGs were annotated into 118 pathways belonging to five categories (Additional file 4). Of them, many DEGs were enriched to pathways related to genetic information processing, such as ribosomes. For biosynthetic pathways, DEGs are primarily annotated to phenylalanine biosynthesis and flavonoid biosynthesis (Fig. 8). These results indicate that normal and aborted ovules differ significantly in tissue development and nutrient accumulation during development. Additionally, plant hormone signal transduction, plant-pathogen interaction endocytosis, and phagosome pathways are also enriched in many DEGs, which are thought to play a key role in regulating plant seed abortion (Additional file 4).

Fig. 8
figure 8

KEGG Enrichment and functional classification of DEGs in Paeonia ludlowii: a DEGs involved in KEGG pathways; b KEGG enrichment analysis. The Y-axis indicates the pathway name, the X-axis shows the richness factor, the size of the points represents the number of DEGs in the pathway, and the color of the points corresponds to different p-value ranges

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Expression of DEGs associated with carbohydrate metabolism during ovule abortion

Glycolysis/gluconeogenesis, citrate cycle (TCA cycle), and pentose phosphate pathway (PPP) are the three major pathways of plant energy metabolism [49]. Analysis of DEGs revealed that many DEGs encode the three pathways in normal ovules and aborted ovules (Fig. 9, Additional file 5). The process includes genes encoding key glycolytic pathway enzymes: hexokinase genes PlHK and PlHK3, pyruvate kinase genes PlPK, PlKPYA, PlPKP2, and PlPKP4 (Fig. 9a). The pyruvate dehydrogenase E1 gene PlPDHB, the pyruvate dehydrogenase E2 gene PlDLAT, and the 6-pyruvate dehydrogenase genes PlPFKA and PlALDH7B4, are responsible for the entry of pyruvate into the TCA cycle (Fig. 9b). Genes encoding key enzymes involved in the TCA cycle include pyruvate dehydrogenase citrate (Pro-S), cleavage enzyme gene PlACLY, aconitase genes PlACO and PlACO1, and malate dehydrogenase genes PlMDH1 and PlMDH2 (Fig. 9c). The key enzyme genes encoding the pentose phosphate pathway include the 6-phosphogluconate dehydrogenase gene PlPGD and the transketolase gene PlTKLA (Fig. 9d). Analysis of DEGs involved in the three major pathways demonstrates that the transcript levels of some unigenes continued to increase with ovule development and then decreased significantly from Pl_4. In the subsequent developmental period, the unigenes were mostly up-regulated in normal ovules and down-regulated in aborted ovules. This indicates that plant energy metabolism levels decreased during ovule abortion. Meanwhile, we identified unigenes involved in the three major pathways, including two glycolysis/gluconeogenesis unigenes PlCTPI (Cluster-20879.33466), PlHK1(Cluster-20879.18261) and one pentose phosphate pathway (PPP) unigene, PlTKTA (Cluster-20879.70023), which were also up-regulated in aborted ovules during Pl_5-Pl_7. The low expression of these genes in aborted ovules of P. ludlowii indicates that the carbohydrate synthesis pathway of aborted ovules is affected, and we hypothesize that the nutrient and energy supply of aborted ovules would be severely impaired for this reason, confirming the low viability of aborted ovules [34].

Fig. 9
figure 9

Expression patterns of DEGs associated with carbohydrate metabolism of Paeonia ludlowii. a Expression pattern of DEGs in glycolysis pathway; b Expression pattern of DEGs in pyruvate dehydrogenase; c Expression pattern of DEGs in citrate cycle; d Expression pattern of DEGs in pentose phosphate pathway. The unigene ID is on the left of each step. The Heatmap was constructed based on log10 (FPKM)

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Expression of DEGs associated with phytohormone signaling during ovule abortion

During phytohormone signaling in aborted ovules of P.ludlowii, there are changes in the expression of genes related to multiple phytohormone signaling pathways. We focused on the differential expression of genes related to seven phytohormones: abscisic acid (ABA), gibberellin (GA), cytokinin (CTK), auxin (IAA), ethylene (ETH), brassinosteroids (BR), and jasmonic acid (JA) (Fig. 10). These include the ABF transcription factor family associated with the ABA signaling pathway, the GA receptor GID1 gene, the CTK receptor AHK gene, the CTK response factor ARR protein, the CTK regulatory protein AHP, the IAA early response gene family SAUR (small auxin up RNA), the IAA input vector AUX/IAA gene, the IAA response gene GH3, the JA receptor JAZ protein, JAR genes involved in JA biosynthesis, and JA signal response factor MYC2. Homologs of the BR response transcription factor BES1 were up-regulated in normal ovules during the Pl_1-Pl_4 and Pl_5-Pl_7 periods and down-regulated in aborted ovules. In addition, the EIN3 and ERF1 genes related to ETH signal response factor, the ETH receptor ERS gene, BR receptor kinase BRI1, and the BR signal kinase gene BSK were down-regulated in normal ovules and up-regulated in aborted ovules during the ovule development period. Many genes homologous to phytohormones were involved, indicating that the phytohormone key-related genes in vivo play an essential role in ovule abortion in P.ludlowii.

Fig. 10
figure 10

Expression patterns of DEGs associated with phytohormones signaling of Paeonia ludlowii. a DEG expression pattern of genes related to ABA; b Expression pattern of DEGs in GA pathway; c Expression pattern of DEGs in CTK pathway; d Expression pattern of DEGs in IAA pathway. e Expression pattern of DEGs in ETH pathway. f Expression pattern of DEGs in JA pathway. g Expression pattern of DEGs in BR pathway. The unigene ID is on the left of each step, and the sample name is on the bottom. The Heatmap was constructed based on log10 (FPKM)

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Several phytohormone-related genes were under-expressed to undetected levels during aborted ovules, relevant information is displayed in Additional file 6. The expression profiles of genes encoding differentially expressed proteins related to the synthesis, response, and transport of different phytohormones in P.ludlowii showed a diverse distribution after the initiation of abortion.

Differential analysis of apoptosis and programmed cell death (PCD)-related genes in ovules

Among the DEGs of P.ludlowii ovules, several genes are involved in the regulation of apoptosis., These include genes encoding the F-box protein family and the molecular chaperone HSP70/HSc70. Additionally, a small number of genes related to TGA, NAC, cysteine-rich receptor-like protein kinase, BAG, and aspartic proteinase are related to programmed cell death in P.ludlowii. The expression level of TGA genes in aborted ovules is higher than that in normal ovules during Pl_5-Pl_7. On the contrary, the NAC gene was highly expressed in normal ovules during the Pl_5-Pl_7 period. Aspartic proteinase genes are highly expressed in aborted ovules during Pl_6-Pl_7. Finally, we focused on HSP and F-box-related genes; their expression patterns in normal ovules and aborted ovules were diverse during the developmental process of P.ludlowii. Therefore, we speculated that HSP and F-box-related genes played various roles in programmed ovule death in P.ludlowii (Fig. 11, Additional file 7).

Fig. 11
figure 11

DEGs involved in PCD of Paeonia ludlowii: a DEGs expression pattern of TGA genes; b DEGs expression pattern of NAC genes; c DEGs expression pattern of BAG genes; d DEGs expression pattern of F-box genes; e DEGs expression pattern of HSP70 genes; f DEGs expression pattern of aspartic proteinase genes; g DEGs expression pattern of cysteine-rich receptor-like protein kinase genes; The left side of each step in the expression pattern diagram is the gene ID, and the bottom is the sample name. Heatmap was constructed based on log10 (FPKM)

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Analysis of DEGs related to ovule development and abortion

Key regulators of Arabidopsis ovule development include AG, INO, SPL, ANT, and BELI [50,51,52]. A total of 19 DEGs homologous to AG and ANT were obtained from the P.ludlowii ovule samples (Fig. 12). After the ovules of P.ludlowii were aborted (Pl_5-Pl_7 period), one ANT homolog PlANT1 (Cluster-20879.38482) and three AG homologs, PIAGL11-like (Cluster-20879.35874), PlAGL62 (Cluster-2543.0), and PlAGL62 (Cluster-3694.0) were consistently up-regulated in normal ovules during the Pl_5-Pl_7 period, down-regulated or not expressed in aborted ovules, and significantly more highly expressed in normal ovules than in aborted ones (Fig. 12, Additional file 8).

Fig. 12
figure 12

Expression of DEGs involved in the development ovule of Arabidopsis in Paeonia ludlowii. a Expression of ANT and AIL relevant DEGs involved in the development ovule of Arabidopsis in Paeonia ludlowii; b Expression of MADS, AG and AGL relevant DEGs involved in the development ovule of Arabidopsis in Paeonia ludlowii. The unigenes shown in red are the key genes mentioned in the article

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The Arabidopsis gene sequence database (https://www.Arabidopsis.org/) was searched for the keyword “ovule abortion” and produced nine genes related to ovule abortion. When these genes were knocked out, the ovule abortion rate of Arabidopsis mutants was significantly higher than that of wild plants [53] (Table 1). Using the above nine genes as a reference, combined with the expression of DEGs, GO, and KEGG enrichment results, 12 homologs were mined, including two ERS homologs PlERS1 (Cluster-20879.39834), one OVA6 homolog PlOVA6 (Cluster-20879.20949), one OVA7 homolog PlOVA7 (Cluster-20879.36837). The expression of PlOVA8 (Cluster-20879.38120) was more down-regulated in aborted ovules after the apparent defeat (Pl_5-Pl_7) and up-regulated in normal ovules. Additionally, the expression level in aborted ovules was significantly lower than in normal ovules (Fig. 13, Additional file 9).

Table 1 Genes associated with ovule abortion in Arabidopsis
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Fig. 13
figure 13

Expression of DEGs involved in ovule abortion of Paeonia ludlowii. a Expression of OVA8 relevant DEGs involved in ovule abortion of Paeonia ludlowii; b Expression of OVA6, OVA7, OVA1, OVA9 and ERS relevant DEGs involved in ovule abortion of Paeonia ludlowii. The unigenes shown in red are the key genes mentioned in the article

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Additionally, a number of homologs were obtained in DEGs associated with ovule abortion in plants other than Arabidopsis (Fig. 14). These include a CesA homolog PlCesA8 (Cluster-20879.67422), an HD-zip transcription factor family homolog PlHB14 (Cluster-20879.48787), a JMT homolog PlJMT (Cluster-20879.15575), an SMT2 homolog PlSMT2 (Cluster-20879.38371), and an ETH signaling pathway transcription factor EIL1 (Ethylene-insenstives3-like) homolog PlEIL1 (Cluster-20879.37106), all of which were consistently up-regulated in aborted ovules at Pl_5-Pl_7. Its expression was significantly higher than that of normal ovules at the same time. Two HD-zip transcription factor family homologs, PlHB12L (Cluster-20879.18846) and PlHB7 (Cluster-20879.18847) were also up-regulated in aborted ovules at Pl_5-Pl_7. At the same time, they were not expressed in normal ovules of the same period or were expressed at undetectable levels (Fig. 14). We selected 15 key genes for future research into abortion of P.ludlowii: ANT homolog PlANT1 (Cluster-20879.38482) and three AG homologs, PIAGL11-like (Cluster-20879.35874), PlAGL62 (Cluster-2543.0), and PlAGL62 (Cluster-3694.0), ERS homolog gene (Cluster-20879.39834), OVA6 homolog PlOVA6 (Cluster-20879.20949), OVA7 homolog PlOVA7 (Cluster-20879.36837), OVA8 homolog PlOVA8 (Cluster-20879.38120), CesA homolog PlCesA8 (Cluster-20879.67422), the 3HD-zip transcription factor family homologs PlHB14 (Cluster-20879.48787), PlHB12L (Cluster-20879.18846), and PlHB7L (Cluster-20879.18847), JMT homolog PlJMT (Cluster-20879.15575), SMT2 homolog PlSMT2 (Cluster-20879.38371), PlEIL1 (Cluster-20879.37106). However, further investigation is required to establish whether the expression of these genes is related to ovule abortion in P.ludlowii.

Fig. 14
figure 14

Expression of DEGs involved in aborted ovules of Paeonia ludlowii. a Expression of CESA relevant DEGs involved in involved in aborted ovules of Paeonia ludlowii; b Expression of EIL relevant DEGs involved in involved in aborted ovules of Paeonia ludlowii; c Expression of LAC relevant DEGs involved in involved in aborted ovules of Paeonia ludlowii; d Expression of WRKY relevant DEGs involved in involved in aborted ovules of Paeonia ludlowii; e Expression of CMT, SMT, LAMT, MPBQ, IMT, DRM, METTLI and AMT relevant DEGs involved in involved in aborted ovules of Paeonia ludlowii; f Expression of HBI, HB, HAT, GL, HDGIIL, HAD and ATMLIL relevant DEGs involved in involved in aborted ovules of Paeonia ludlowii. The unigenes shown in red are the key genes mentioned in the article

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QRT-PCR validation of DEGs

To verify the credibility of the RNA-seq results and assess the cause of ovule abortion in P.ludlowii, 16 differential genes were selected for qRT-PCR analysis. The results demonstrated that the expression patterns of 16 of these genes in the qRT-PCR analysis were similar to those in the transcriptome sequencing (Fig. 15). This indicates that transcriptome data and qRT-PCR analysis data were accurate and reliable and that the genes screened in this study that play a key role in ovule abortion in P.ludlowii were reliable.

Fig. 15
figure 15

QRT-PCR validation of DEGs

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Discussion

Nutrients affect early seed abortion in Paeonia ludlowii

In P.lutea, a large number of DEGs between normal and aborted seeds were distributed in “metabolic process”, “binding” and “catalytic activity” [54]. Similar to the research results of P.lutea, during the seed development and abortion of P.ludlowii, a large number of DEGs were enriched in the pathways related to nutrient metabolism, including “biosynthetic process”. At the same time, a large number of DEGs were enriched in “phenylpropane biosynthesis” and other metabolic pathways. The expression of a large number of DEGs encoding TCA cycle and PPP pathway in Pl_1 ~ Pl_4 was down-regulated, and then in aborted seeds of Pl_ 5 ~ Pl_7 was down. The results showed that the abortion of P.ludlowii was related to the decomposition and accumulation of nutrients and energy metabolism. Previous studies found that the abortion of P.ludlowii was related to the catabolism and accumulation of nutrients [34]. In this paper, the low expression of genes related to the energy metabolism pathway in aborted seeds confirmed the low vitality of aborted seeds. It is speculated that the seeds accumulated a certain amount of energy before the abortion. Due to the gradual development in the later stage, the energy supply of the aborted seeds was insufficient, and the endosperm-free nucleus and cytoplasm degenerated, but some energy metabolism processes still existed.

Phytohormones affect early seed abortion in Paeonia ludlowii

Phytohormones have an important regulatory role in ovule development [52]. Our results indicate that the expression profiles of genes encoding differentially expressed proteins of different phytohormones in ovules showed a diverse distribution, suggesting that the mechanisms of phytohormone regulation are complex. The ABA, BR, and ETH-related genes were heavily up-regulated in aborted ovules during Pl_5 to Pl_7 (Fig. 10). ABA levels are closely related to plant growth and development, and increases in ABA levels in aborted seeds of maize, date, and chrysanthemum have been described [55]. Also, the increase in ABA levels in lychee embryos alters the balance ratio of endogenous hormones and could affect embryo development, eventually leading to embryo abortion [56]. Brassinolide is a sterol plant hormone that participates in plant growth and various physiological reactions. It has been reported that BR can regulate plant growth and development by interacting with the ABA hormone. In this study, the contents of ABA and BR hormones increased in aborted ovules during the later stages of ovules development. Therefore, we speculate that ABA and BR interaction plays a vital role in regulating plant ovule abortion. Transcriptome analysis demonstrated that the EIN3 and ERF1 genes, which are related to ETH signal response factors and the ETH receptor ERS gene, were up-regulated in aborted ovules. Wanghui revealed the molecular mechanism of ethylene involved in regulating ovule abortion in seedless pears [57], demonstrating that ethylene-insensitive 3-like 1 (EIL1) is involved in regulating ovular abortion and senescence in seedless pears by directly affecting the transcription of CYSTEINE PROTEINASE 1(Cysp1), a cell senescence-related gene. Therefore, excessive accumulation of ethylene in the ovules of P.ludlowii could lead to premature senescence and death of the ovules. We believe that phytohormones play an essential role in the development of P.ludlowii ovules, and that regulating ovule development in P.ludlowii by external phytohormone application should be considered.

Key regulatory genes for ovule development and abortion regulate early ovule abortion in Paeonia ludlowii

ANT is an AP2 subfamily transcription factor that encodes an ANT gene closely related to ovule primordium formation and ovule development and can regulate seed size [58,59,60]. Arabidopsis plants overexpressing ANT increased ovule size but did not change the ovule number [61]; the Arabidopsis MEE45 (MATER NAL EFFECT EMBRYO ARREST45) transcription factor regulates seed size by inducing ANT expression [62]. The PlANT1 (Cluster-20879.38482) gene was up-regulated in normal seeds after the ovules of P.ludlowii were aborted, and their expression was significantly higher than that of aborted ovules. This suggests that the ANT homolog PlANT1 could play a key role in regulating ovule abortion in P.ludlowii.

AG genes regulate the abortion and development of floral organs, fruit and seed development [63]. In this chapter, three AG homologs, PlAGL11-like (Cluster-20879.35874), PlAGL62 (Cluster-2543.0), and PlAGL62 (Cluster-3694.0), were up-regulated in normal ovules relative to aborted ovules. Studies have demonstrated that the AGL11 gene has an essential regulatory role in tomato ovule development and seed formation [64]. We found that the differential expression of PlAGL11-like between normal and aborted ovules indicates that PlAGL11-like plays a vital role in regulating seed abortion in P.ludlowii. During ovule development, AGL62 is a major regulator of endosperm cellularization and is expressed only in the endosperm, where it inhibits endosperm cellularization at the syncytial stage and promotes nuclear proliferation [65]. In wild Arabidopsis seeds, AGL62 is highly expressed at the endosperm cytosolic stage. It suddenly decreases before cellularization, while the deletion of AGL62 leads to early endosperm cellularization, reduced nuclei, and lethal seed defects [66]. The high expression of two PlAGL62 in normal ovules of P.ludlowii suggests that it could play the same regulatory role in regulating endosperm development in P.ludlowii, and that the low expression of PlAGL62 in aborted ovules prevents the free nuclei of the endosperm from continuing to proliferate, leading to ovule abortion.

The ERS, OVA6, OVA7, and OVA8 genes are important regulators of ovule development and abortion in Arabidopsis. In Arabidopsis, 26% of ovules of plants with knockout of the ERS and OVA8 genes, respectively, were aborted compared with 6% of seeds of the wild type; 56% of ovules of plants with knockout of the OVA6 gene were aborted compared with 5% of seeds of the wild type; and the ovule abortion rate of OVA7 knockout plants was also much higher than that of wild-type plants [53]. This suggests that these genes play a positive regulatory role in ovule development, and their deletion will lead to abnormal ovule development and abortion. In this chapter, the ERS homolog PlERS1 (Cluster-20879.39834), OVA6 homolog PlOVA6 (Cluster-20879.20949), OVA7 homolog PlOVA7 (Cluster-20879.36837), and OVA8 homolog PlOVA8 (Cluster-20879.38120) were up-regulated in normal ovules during Pl_5-Pl_7. Their expression level was significantly higher than that of aborted ovules, and was similar to the expression pattern of these genes in Arabidopsis, presumably with a similar regulatory pattern on ovule development of P.ludlowii.

Key regulatory genes of ovule abortion regulate early ovule abortion in Paeonia ludlowii

Several CesA genes in dove trees are significantly up-regulated in aborted ovules compared to normal ovules, and the fibrillin content in aborted ovules is significantly higher than in normal ovules [67]. Similar to dove trees, the expression of the CesA homolog PlCesA8 (Cluster-20879.67422) was significantly higher in aborted ovules than in normal ovules during the Pl_5-Pl_7 in P.ludlowii. This suggests that PlCesA8 plays a vital role in ovule abortion in P.ludlowii, and that the gene could regulate cellulose synthesis, leading to early ovule formation and ovule abortion.

Many genes in the HD-Zip family are related to embryonic development [68], including VvHDZ05, VvHDZ09, VvHDZ13, VvHDZ17, VvHDZ23, VvHDZ24, VvHDZ27, and VvHDZ28 in the HD-Zip I subfamily and VvHDZ01 in the HD-Zip II subfamily. VvHDZ01 in II was associated with grape embryo abortion [69]. In this chapter, the HD-zip transcription factor family homologs PlHB14 (Cluster-20879.48787), PlHB12L (Cluster-20879.18846), and PlHB7 (Cluster-20879.18847) were significantly highly expressed in aborted ovules of P.ludlowii and displayed low or no expression in normal seeds. This suggests that these three genes play an essential role in ovule abortion of P.ludlowii, and that their high expression in aborted ovules could affect the normal development of ovules and lead to ovule abortion.

JMT is a critical enzyme in the biosynthesis of MeJA, a jasmonic acid derivative. It is responsible for the methylation of JA into MeJA, which plays a vital role in plant seed development [70]. For example, AtJMT overexpression leads to increased MeJA accumulation and mediates stress signals leading to increased ABA levels, which affects the differentiation of rice spikes and ultimately leads to lower grain yields [71]. AtJMT overexpression leads to reduced seed numbers in Arabidopsis [72], while PlJMT (Cluster-20879.15575) was consistently up-regulated in aborted ovules of P.ludlowii and was highly expressed than in normal ovules. In Perilla frutescens, PfJMT gene expression was down-regulated as seeds developed [70]. The high expression of PlJMT in aborted ovules of P.ludlowii could affect ovule development and lead to abortion.

S-adenosylmethionine (SAM) is involved in various important physiological processes in plants, such as transaminopropyl, transmethylation, and transsulfuration, and is a critical methyl donor [56]. S-adenosylmethionine synthase genes (SAMs) are abundantly expressed in litchi aborted embryos, leading to embryo abortion in litchi [56]. SAM-related methyltransferase genes such as LcCMT1, LcCMT2, and LcDRM2 have also been associated with 'Guiwei' litchi embryo abortion [73]. In P.ludlowii, SMT is a SAM-dependent methyltransferase, and the expression level of SMT2 homolog PlSMT2 (Cluster-20879.38371) was sharply increased in aborted ovules, suggesting it plays an essential role in ovule abortion in P.ludlowii.

EIL1 is a positive regulator of the ETH signaling pathway and primarily regulates the expression of most downstream ETH signaling genes [74]. PbEIL1 is significantly up-regulated in seedless pears when seeds start to septate, and the ETH signaling pathway is activated, leading to excessive accumulation of ETH in the seeds and premature seed senescence and death. Its high expression in pears was associated with seed senescence in a transgenic experiment in tomatoes [75]. PlEIL1 (Cluster-20879.37106) was consistently up-regulated in aborted ovules of P.ludlowii at the Pl_5-Pl_7 stage, and its expression was significantly higher than that of normal ovules. The expression pattern was similar to that of PbEIL1, indicating that PlEIL1 plays a similar regulatory role in P.ludlowii as PbEIL1 in seedless pears. The high expression of PlEIL1 in aborted ovules may led to ovule abortion. All of these genes could be related to the defeat of P.ludlowii. However, the aborted functions of these genes must be further investigated. These findings will provide genetic resources for improving ovule abortion and expanding P.ludlowii production.

Conclusion

In this paper, we investigated the characteristics and internal regulatory mechanisms of ovule abortion in P.ludlowii through physiological observations and transcriptome sequencing analysis. Transcriptome sequencing was performed on ovules at key stages during the early abortion in P.ludlowii, and the expression changes of key genes at different stages of ovule development were compared, including carbohydrate metabolism, phytohormone signal transduction, PCD, ovule development, and ovule abortion. Additionally, 15 key genes were screened for future research. We found that certain nutrients and phytohormones are essential for regulating ovule abortion, and that the exogenous application of nutrients and hormones could be an effective method of alleviating ovule abortion in P.ludlowii. This paper presents the first analysis of the mechanism of ovule abortion in P.ludlowii, which has significant implications for the conservation, propagation, and introduction of this species.

Availability of data and materials

The datasets analyzed during the current study are available in the NCBI repository, the accession number to datasets: PRJNA818047.

References

  1. Li JJ, Zhao XF, Zhao XQ. Tree peony of China (in Chinese). Beijing, China: Encyclopedia of China Publishing House; 2011. p. 17–23.

    Google Scholar 

  2. Yuan T, Chen TQ, Tang Y. Observation on secondary development characteristics of Branches of Paeonia ludlowii (in Chinese). Acta Horticulturae Sinica. 2021;48(01):117–26.

  3. Li J, Wang ZH.: Nutrients, fatty acid composition and antioxidant activity of the flowers and seed oils in wild populations of Paeonia ludlowii. Emirates J Food Agricult. 2019:206–213.

  4. Hong DY, Pan KY. Notes on taxonomy of Paeonia sect. Moutan DC. (Paeoniaceae). Acta Phytotaxonomica Sinica. 2005;42(2):169–77.

  5. Hong DY, Zhou SL, He XJ, Yuan JH, Zhang YL, Cheng FY, Zeng XL, Wang Y, Zhang XX. Current status of wild tree peony species with special reference to conservation (in Chinese). Biodiversity Science. 2017;25(7):781–93.

    Article  Google Scholar 

  6. Cheng FY, Li JJ, Yu L. Exportation of Chinese tree peonies (Mudan) and their developments in other countries II: wild species (in Chinese). J Northwest Normal Univ (Natural Science). 1998;03:3–5.

    Google Scholar 

  7. Li JJ, He LX, Chen DZ, Chen FH, Cheng FY. Preliminary report of introduction experiment of Paeonia ludlowii in Tibet (in Chinese). Plant Introduction and Acclimatization. 1995;10:105–10.

    Google Scholar 

  8. NI SW. Introduction and Ex-situ conservation of Paeonia delavayi, Paeonia lutea, Paeonia ludlowii (in Chinese). Beijing, China: Beijing Forestry University; 2009.

  9. Dong L, Hong M, Li ZH, Liu XX, Zhang YL. Karyotypic Studies of five Paeonia ludlowii populations from China. Caryologia. 2011;64(4):370–6.

    Google Scholar 

  10. Wang SQ, Li HM, Wang L, Cheng J, Tang L. On the meiosis in Paeonia ludlowii (Stern & Taylor) D. Y. Hong, an endangered species of SE Tibet, PR China. Wulfenia, 2012. 19: 97–106.

  11. Zhang JM, Lopez-Pujol J, Gong X, Wang HF, Vilatersana R, Zhou SL. Population genetic dynamics of Himalayan-Hengduan tree peonies, Paeonia subsect. Delavayanae Molecular Phylogenetics and Evolution. 2018;125:62–77.

    Article  PubMed  Google Scholar 

  12. Lu YZ, Zhang CQ, Yu BX, Zhang EH, Quan H, Yin X, Cai H, Yuan F, Li LQ, Xu YJ. The seed oil of Paeonia ludlowii ameliorates Abeta25-35-induced Alzheimer’s disease in rats. Food Sci Nutr. 2021;9(5):2402–13.

  13. Yu SY, Du SB, Yuan JH, Hu YH. Fatty acid profile in the seeds and seed tissues of Paeonia L. species as new oil plant resources. Sci Rep. 2016;6(1):26944.

  14. Zhang CQ, Xu YJ, Lu YZ, Li LQ, Lan XZ, Zhong ZC. Study on the fatty acids, aromatic compounds and shelf life of Paeonia ludlowii Kernel Oil. J Oleo Sci. 2020;69(9):1001–9.

    Article  CAS  PubMed  Google Scholar 

  15. Lu YZ, Zhang EH, Hong MS, Yin X, Cai H, Yuan L, Yuan F, Li LQ, Zhao KT, Lan XZ. Analysis of endophytic and rhizosphere bacterial diversity and function in the endangered plant Paeonia ludlowii. Arch Microbiol. 2020;202(7):1717–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Qiu YY, Zhang L, Ni SW, Yuan T. Preliminary study on characteristics relates to germination and rootinh techology of Paeonia ludlowii seeds (in Chinese). J Gansu Agric Univ. 2016;51(06):58–63.

    Google Scholar 

  17. Ma H, Li ZH, Zhang YL, Wang Y, Liu XX, Wan YM. Release of seed dormancy of Paeonia ludlowii (in Chinese). Scientia Silvae Sinicae. 2012;48(09):62–7.

    Google Scholar 

  18. Jia WQ, Wang YL, Guo YZ, Wang Z, Qi Q, Yan SN, Liu HC, He SL. Characterization of Pollen Germination and Storage of Paeonia ludlowii (in Chinese). Scientia Silvae Sinicae. 2021;57(02):82–92.

    CAS  Google Scholar 

  19. Ge QS, Gao Y, Wang H, Fang JP. Photoresponse process and model comparison of Paeonia ludlowii under saline-alkali stress. Meteorological and Environmental Research. 2019;10(04):84–7.

    Google Scholar 

  20. Tang Y, Yuan T, Chen TQ. Flowering characteristics and breeding system of Paeonia ludlowii (in Chinese). Acta Bot Boreal-Occident Sin. 2021;41(5):0782–94.

    Google Scholar 

  21. Yang XL, Wang QJ, Lan XZ, Li CY. Numeric dynamics of the endangered plant population of Paeonia ludlowiii (in Chinese). Acta Ecol Sin. 2007;27(3):1242–7.

    Google Scholar 

  22. Li M, Dong XJ, Peng JQ, Xu W, Ren R, Liu J, Cao FX, Liu ZM. De novo transcriptome sequencing and gene expression analysis reveal potential mechanisms of seed abortion in dove tree (Davidia involucrata Baill.). BMC Plant Biol. 2016;16:82.

  23. Collevatti RG, Estolano R, Garcia SF, Hay JD. Seed abortion in the bat pollinated Neotropical tree species, Caryocar brasiliense (Caryocaraceae). Botany. 2009;87(11):1110–5.

  24. Huang SQ, Guo YH, Wu Y, Liu Q, Zhang F. Flower numerical variation and seed set in natural populations of Liriodendron chinense (in Chinese). Acta Botanica Sinica. 1998;40(01):27–32.

    Google Scholar 

  25. Meyer KM, Soldaat LL, Auge H, Thulke H. Adaptive and selective seed abortion reveals complex conditional decision making in plants. Am Nat. 2014;183(3):376–83.

  26. Ren R, Huang FZ, Gao RX, Dong XJ, Peng JQ, Cao FX, Li M. Selection and validation of suitable reference genes for RT-qPCR analysis in dove tree (Davidia involucrata Baill.). Trees. 2019;33(3):837–49.

  27. Chen LN, Zhang J, Li HX, Niu J, Xue H, Liu BB, Wang Q, Luo X, Zhang FH, Zhao DG. Transcriptomic analysis reveals candidate genes for female sterility in pomegranate flowers. Front Plant Sci. 2017;8:1403.

    PubMed  PubMed Central  Google Scholar 

  28. Liu H, Liu YZ, Zheng SQ, Jiang JM, Wang P, Chen W. Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion. Planta. 2010;231(4):847–60.

  29. Chen H, Yang Q, Chen K, Zhao SS, Zhang C, Pan RL, Cai TC, Deng Y, Wang XJ, Chen YT. Integrated microRNA and transcriptome profiling reveals a miRNA-mediated regulatory network of embryo abortion under calcium deficiency in peanut (Arachis hypogaea L.). BMC Genomics. 2019;20(1):392.

  30. Liu JF, Xing JY, Fang J, Ai PF, Cheng YQ. New insight into ovary abortion during ovary development of hazelnut through a combined proteomic and transcriptomic analysis. Sci Hortic. 2018;234:36–48.

    Article  CAS  Google Scholar 

  31. Cheng YQ, Liu JF, Zhang HD, Wang J, Zhao YX, Geng WT. Transcriptome analysis and gene expression profiling of abortive and developing ovules during fruit development in hazelnut. PLoS ONE. 2015;10(4): e122072.

    Article  Google Scholar 

  32. Nwafor CC, Gribaudo I, Schneider A, Wehrens R, Grando MS, Costantini L. Transcriptome analysis during berry development provides insights into co-regulated and altered gene expression between a seeded wine grape variety and its seedless somatic variant. BMC Genomics. 2014;15(1):1030.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Wu LM, Zhu XW, Cui XY, Liu ZJ, Liu YT, Xu PZ, Chen XQ, Wu XJ, Zhang HY. Analysis of genes regulating seed abortion in heteroploid crosses of rice (in Chinese). Chin Sci Bull. 2015;60(17):1592–601.

    Google Scholar 

  34. Chen TQ, Yuan T, Qiao HY, Xu K. Effects of secondary branches on seed setting rate and physiological mechanism of ovule abortion in Paeonia ludlowii (in Chinese). Journal of Northwest A&F University (Natural Science Edition). 2022;09:2–15.

  35. Wang DH. Experimental techniques commonly used in plant developmental biology (in Chinese). Peking University Press; 2017. p. 124.

  36. Chen T, Xie M, Jiang Y, Yuan T. Abortion occurs during double fertilization and ovule development in Paeonia ludlowii. J Plant Res. 2022;135:295–310.

  37. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Nadia MD, Alicia O. Corset: enabling differential gene expression analysis for de novo assembled transcriptomes. GeneBiology. 2014;15(7):410.

    Google Scholar 

  39. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12(1):323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):174–511.

    Article  Google Scholar 

  41. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11(2):R14.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Wu J, Mao X, Cai T, Luo J, Wei L.: KOBAS server: a web-based platform for automated annotation and pathway identification. Nucleic Acids Res, 2006. 34(Web Server issue).

  43. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.

  44. Liu HF, Gao LX, Hu YH. Reference genes discovery and selection for quantitative real- time pcr in tree peony seed and petal tissue of different development stages (in Chinese). J Agricultur Biotechnol. 2015;23(12):1639–48.

    CAS  Google Scholar 

  45. Livak KJ, Schmittgen TD.: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.), 2001. 25(4):402–408.

  46. Sun XB, Dai XM, Wang YJ, Han LB. Advances in programmed cell death in plants (in Chinese). Biotechnol Bull. 2010;11:1–6.

    Google Scholar 

  47. Ni XL.: Aerenchyma formation and their relationship to programmed cell death in three plants (in Chinese). Xi’an, Shaanxi, China:Northwest University, 2014.

  48. Huang WJ, Sun XC, Li B, Wang N, Yue ZG, Zhang YQ, Zhao ZY, Tang ZS. Programmed cell death induced by drought stress in sprout tumble of Pinellia ternata (in Chinese). China J Chin Materia Med. 2019;44(10):2020–5.

  49. Catav SS, Elgin ES, Dag C, Stark JL, Kucukakyuz K. NMR-based metabolomics reveals that plant-derived smoke stimulates root growth via affecting carbohydrate and energy metabolism in maize. Metabolomics. 2018;14(11):143.

    Article  PubMed  Google Scholar 

  50. Gasser C, Hauser B, Broadhvest J. Genetic analysis of ovule development. Annu Rev Plant Physiol Plant Mol Biol. 1998;49:1.

    Article  CAS  PubMed  Google Scholar 

  51. Cucinotta M, Colombo L, Roig-Villanova I. Ovule development, a new model for lateral organ formation. Front Plant Sci. 2014;5:117.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Barro-Trastoy D, Dolores Gomez M, Tornero P, Perez-Amador MA. On the way to ovules: the hormonal regulation of ovule development. Crit Rev Plant Sci. 2020;39(5):431–56.

    Article  CAS  Google Scholar 

  53. Berg M, Rogers R, Muralla R, Meinke D. Requirement of aminoacyl-tRNA synthetases for gametogenesis and embryo development in Arabidopsis. Plant J. 2005;44(5):866–78.

    Article  CAS  PubMed  Google Scholar 

  54. Zhang S, Li Y, Li Y, Zhao F, Zeng X. Insights on seed abortion (endosperm and embryo development failure) from the transcriptome analysis of the wild type plant species Paeonia lutea. Bioinformation. 2020;16(8):638–51.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Zhang F, Wang Z, Dong W, Sun C, Wang H, Song A, He L, Fang W, Chen F, Teng N. Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding. Sci Rep. 2014;4(1):1–10.

    Google Scholar 

  56. Zhang YS, Xiang X, Fu JR, Huang SZ. Full-length amplification and sequence analysis of S-adenosylmethionine synthase gene from aborted litchi embryos (in Chinese). Acta Horticulturae Sinica. 2004;31(02):160–4.

  57. Wang H, Zhang S, Qu Y, Gao R, Xiao Y, Wang Z, Zhai R, Yang C, Xu L. Jasmonic acid and ethylene participate in the gibberellin-Induced ovule programmed cell death process in seedless pear '1913' (Pyrus hybrid). Int J Mol Sci. 2021;22(18):9844.

  58. Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQ, Gerentes D, Perez P, Smyth DR. AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell. 1996;8(2):155.

  59. Klucher K M, Chow H, Reiser L, Fischer R L. The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2. Plant Cell. 1996;8(2):137.

  60. Galbiati F, Sinha Roy D, Simonini S, Cucinotta M, Ceccato L, Cuesta C. An integrative model of the control of ovule primordia formation. Plant J. 2013;76(3):446–55.

    Article  CAS  PubMed  Google Scholar 

  61. Barro-Trastoy D, Gomez MD, Blanco-Touriñán N, Tornero P, Perez-Amador MA. Gibberellins regulate ovule number through a DELLA–CUC2 complex in Arabidopsis. Plant J. 2022;110:45.

  62. Li YJ, Yu Y, Liu X, Zhang XS, Su YH. The Arabidopsis MATERNAL EFFECT EMBRYO ARREST45 protein modulates maternal auxin biosynthesis and controls seed size by inducing AINTEGUMENTA. Plant Cell. 2021;33(6):1907–26.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Zhang C, Wei L, Wang W, Qi W, Cao Z, Li H, Bao M, He Y. Identification, characterization and functional analysis of AGAMOUS subfamily genes associated with floral organs and seed development in Marigold (Tagetes erecta). BMC Plant Biology. 2020;20(1):439–55.

  64. Busi MV, Bustamante C, D’Angelo C, Hidalgo-Cuevas M, Boggio SB, Valle EM, Zabaleta E. MADS-box genes expressed during tomato seed and fruit development. Plant Mol Biol. 2003;52(4):801–15.

  65. Kang I, Steffen JG, Portereiko MF, Lloyd A, Drews GN. The AGL62 MADS domain protein regulates cellularization during endosperm development in Arabidopsis. Plant Cell. 2008;20(3):635–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Hehenberger E, Kradolfer D, Köhler C. Endosperm cellularization defines an important developmental transition for embryo development. Development. 2012;139(11):2031–9.

    Article  CAS  PubMed  Google Scholar 

  67. Xiong YL, Cao FX, Liu ZM, Dong XJ, Li M. Cloning and expression analysis of CesA genes involved in seed abortion in dove tree (Davidia involucrata) (in Chinese). Plant Physiology Journal. 2016;52(10):1481–90.

    Google Scholar 

  68. Ariel FD, Manavella PA, Dezar CA, Chan RL. The true story of the HD-Zip family. Trends Plant Sci. 2007;12(9):419–26.

    Article  CAS  PubMed  Google Scholar 

  69. Li Z, Zhang C, Guo Y, Niu W, Wang Y, Xu Y.: Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes (Vitis vinifera L.). BMC Genomics, 2017. 18(1):1.

  70. Bai H, Lu G, Lu J X, Guan L, Tang X, Zhang T. Cloning and expression analysis of Perilla jasmonate hydroxylmethyltransferase gene PfJMT (in Chinese). Scientia Agricultura Sinica. 2019;52(09):1657–66.

  71. Kim EH, Kim YS, Park S, Koo YJ, Choi YD, Chung Y, Lee I, Kim J. Methyl Jasmonate reduces grain yield by mediating stress signals to alter spikelet development in rice. Plant Physiol. 2009;149(4):1751–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Cipollini D. Constitutive expression of methyl jasmonate-inducible responses delays reproduction and constrains fitness responses to nutrients in Arabidopsis thaliana. Evol Ecol. 2010;24(1):59–68.

  73. Xie D, Ma X, Rahman MZ, Yang M, Huang X, Li J, Wang H. Thermo-sensitive sterility and self-sterility underlie the partial seed abortion phenotype of Litchi chinensis. Sci Hortic. 2019;247:156–64.

  74. Li ZH, Peng JY, Wen X, Guo HW. Ethylene-insensitive3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing mir164 transcription in Arabidopsis. Plant Cell. 2013;25(9):3311–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Wang H, Zhang H, Liang F, Cong L, Song L, Li X. PbEIL1 acts upstream of PbCysp1 to regulate ovule senescence in seedless pear. Horticulture Res. 2021;8(1):59.

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to Ex-situ Conservation Centre of Chinese Paeoniceae Wild Species (Luanchuan County, Henan Province, China) for kindly providing the experiment materials. And We are grateful for the technical and theoretical guidance provided by Secretary Li of the Luoyang Sui Tang City Heritage Botanical Garden.

Funding

This work is supported by National Forestry and Grassland Administration Industry Standard Project “Peony Complex” (2018-LY-054) and Characteristic Development Guidance Funds for Beijing Forestry University (2019XKJS0324).

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Author notes

  1. Ting-qiao Chen and Yue Sun contributed equally to this work

Authors and Affiliations

  1. Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing Laboratory of Urban and Rural Ecological Environment, Engineering Research Center of Landscape Environment of Ministry of Education, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, School of Landscape Architecture, National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing, 100083, China

    Ting-qiao Chen, Yue Sun & Tao Yuan

  2. School of Geography and Environmental Science/School of Karst Science, Guizhou Normal University, Guiyang, 550001, China

    Ting-qiao Chen

Authors
  1. Ting-qiao Chen
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  2. Yue Sun
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Contributions

TQC and YS conceived and designed the experiments. TQC and YS performed the experiments, analyzed the data, provide the qPCR data and wrote the paper. TQC, YS and TY revised and approved. All authors read and approved the manuscript.

Corresponding author

Correspondence to Tao Yuan.

Ethics declarations

Ethics approval and consent to participate

Article 5 of the Regulations of the People's Republic of China on the Protection of wild plants points out that the State encourages and supports scientific research on wild plants and on-site protection and ex-situ protection of wild plants. There are three wild peony introduction and domestication bases in China, including Lanzhou Yuzhong, Gansu Forestry Science and Technology Extension Station, and the Ex-situ Conservation Centre of Chinese Paeoniceae Wild Species built by our research group in Luanchuan County, Henan Province, China, which collected all wild tree peony species and part of the herbaceous peony group. We have obtained the support of the local Forestry Bureau of Luanchuan County and signed the project application for the cultivation of new tree peony varieties. The project number is 2014hhlm012. The plant materials used in our experiments were collected in compliance with relevant institutional, national and international guidelines and legislation.

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The authors declare no competing or financial interests.

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Supplementary Information

Additional file 1.

qRT-PCR primers information.

Additional file 2.

Summary of Sequencing Data Quality.

Additional file 3.

  GO enrichment analysis of DEGs.

Additional file 4.

KEGG enrichment analysis of DEGs.

Additional file 5.

DEGs involved in carbohydrate metabolism.

Additional file 6.

DEGs involved in phytohormones signaling of Paeonia ludlowii.

Additional file 7.

DEGs involved in PCD of Paeonia ludlowii.

Additional file 8.

DEGs involved in the development ovule of Arabidopsis in Paeonia ludlowii.

Additional file 9.

DEGs involved in ovule abortion of Paeonia ludlowii.

Additional file 10.

DEGs involved in aborted ovules of Paeonia ludlowii.

Additional file 11. Fig. 1.

Principal component analysis of this experimental samples.

Additional file 12. Fig. 2.

Results of BUSCO evaluation of spliced transcripts (different colours represent different types of spliced transcripts).

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Chen, Tq., Sun, Y. & Yuan, T. Transcriptome sequencing and gene expression analysis revealed early ovule abortion of Paeonia ludlowii. BMC Genomics 24, 78 (2023). https://doi.org/10.1186/s12864-023-09171-1

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Keywords

BMC Genomics

ISSN: 1471-2164

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