Transcriptome analysis provides insights into aril development regulated gene network in lychee, longan, and rambutan

Abstract

Background

Lychee (Litchi chinensis Sonn.), longan (Dimocarpus longan Lour.), and rambutan (Nephelium lappaceum L.) are popular tropical fruits in the family Sapindaceae, known for their succulent arils—specialized seed appendage with significant biological and commercial value. Despite their agricultural relevance, the molecular mechanisms underlying aril development in these species remain poorly understood.

Results

We conducted RNA-sequencing to profile transcriptomes during aril development, complemented by in-situ hybridization to validate the spatial expression of LcLBD1. OrthoFinder identified species-specific and shared differentially expressed genes (DEGs), while functional enrichment analyses (GO, KEGG) and transcriptional network modeling elucidated regulatory pathways. After detailed analyses of transcriptomes, species-specific and shared DEGs were identified across lychee, longan, and rambutan using OrthoFinder. Members of the bHLH and MYB gene families were implicated in early aril development. Species-specific DEGs were primarily enriched in metabolic pathways. From shared DEGs, we identified ten transcription factors (AGL8, AP3, SHP1, WOX13, LBD1, LBD3, OBP1, SPL2, SPL3, and SPL9) and three genes (IAA8, CSLD5, and CYCD3;2) as key regulators. Interestingly, in-situ hybridization localized LcLBD1 expression to funicle and small aril cells, suggesting roles in cell differentiation and division.

Conclusion

We have identified ten transcription factors and three genes affecting aril development in lychee, longan, and rambutan, and validated the expression of LcLBD1 in funicle and aril cells. These results offer a new perspective on the molecular mechanism of aril development and lay the groundwork for future research into the functions and regulatory mechanisms of candidate genes.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06861-8.

Introduction

Lychee, longan, and rambutan—the most well-known members of the Sapindaceae family—produce edible arils: sweet, translucent flesh derived from the funicle [1]. Lychee is grown extensively in China, India, Vietnam, and Thailand, whereas longan is primarily grown in China, Vietnam, and Thailand [2]. Although rambutan is indigenous to Indonesia and Malaysia, it is also widely cultivated throughout Southeast Asia, Hawaii, Central America, Africa, and China [3, 4]. The formation of aril in lychee, longan, and rambutan is similar, as they all originate from funicle. Although aril is present in many species, its significance and value vary depending on the species. Aril serves as a seed propagator and protector in Momordica charantia and Carica papaya, but it also partially inhibits seed germination, which presents breeding challenges. The economic benefits of fleshy arils, like those of lychee, longan, and rambutan, are determined by their size. Therefore, investigating the early stages of aril development and the enlargement of fleshy aril is valuable from both a biological and commercial standpoint. However, due to the long growth period of fruit trees and the lack of model plants having aril, there still exists a significant gap in understanding the molecular and genetic mechanisms that regulate fleshy structure development in lychee, longan, and rambutan.

The fleshy, white aril that envelops the seeds comprises irregularly shaped parenchyma cells that the seed developed to adapt to its surroundings. The development of the aril differs by species, with origins in either the integument or the funicle. It can form partially or wholly, with differences in origin, location, size, shape, structure, and color. Zu et al. revealed that the aril in C. orbiculatus develops from cells in the exosomal region of the outer integument [5]. Huang et al. found compelling microscopic evidence in lychee that the aril originates from the funicle rather than the obturator [6]. There is substantial evidence that aril production in Sapindaceae is independent of seeds, leading to the proposal of the ‘ball skin vs bladder effect’ [7]. The embryo with cotyledon invades the space provided by the pre-formed pericarp for the aril and competes for assimilation [8, 9], confirming the origin of the aril from the funicle. The morphological observations in lychee allow for the division of the early aril development into two stages: in stage I, the pericarp and seed coat are the main focus of growth; however, stage II focuses on the development of the embryo and aril [7]. Only a few studies have investigated the genes that regulate aril development in Sapindaceae. In Ginkgo and Taxus, B- (APETALA3 and PISTILLATA), C- (AGAMOUS), D- (SEEDSTICK and SHATTERPROOF), and E-class (AGL6 and SEPALLATA) flower development genes modulate aril formation [10]. In lychee, two hydroxy methyl glutaryl CoA reductase genes (LcHMG1 and LcHMG2) govern early cell division and late expansion during aril development, respectively [11].

The current work used lychee, longan, and rambutan transcriptomes to uncover the molecular mechanisms that drive aril development. We acquired the aril tissue through manual dissection, and the global gene expression profiles of aril development in lychee, longan, and rambutan were generated using RNA-seq technology. The transcriptome data from three species provided an improved viewpoint for analyzing molecular-level data and identifying the critical functional genes of aril development. In addition, the function of candidate genes was confirmed by an in-situ hybrid experiment.

Materials and method

Plant materials

The plant materials of lychee (‘feizixiao’, ‘guiwei’, and ‘hushanwanli’) and longan (‘96–1’, ‘68–1’, and ‘DAW’) used in this study were donated by the Fruit Tree Research Institute of Fujian Academy of Agricultural Sciences. ‘feizixiao’, ‘guiwei’, ‘hushanwanli’, ‘96–1’, and ‘68–1’ were the main cultivars in China, while ‘DAW’ is a Thai longan variety. Rambutan materials (‘Baoyan 7’, ‘Baoyan 4’, and ‘Baoyan 2’) were collected in Tropical Crops Institute of Hainan Baoting in China. We selected five distinct developmental stages of aril in lychee, three stages of aril in longan and rambutan (Figure S1 and Table S1). Each sample had three replications. They were divided into two processes: early fruit development (Lcstage1-Lcstage3, Dlstage1-Dlstage2, Nlstage1-Nlstage2) and fruit expansion (Lcstage3-Lcstage4, Dlstage2-Dlstage3, Nlstage2-Nlstage3). Since it was difficult to distinguish between the Lcstage1, Lcstage2, Dlstage1, and Nlstage2 of aril and seeds (Fig. 1), these sample stages were combinations of aril and seeds. The thin aril completely encased the seeds in the Lcstage3, Dlstage2, and Nlstage2 stages. The aril thickened during the Lcstage4, Dlstage3, and Nlstage3 phases.

Fig. 1.

Morphological observation of early development of longan fruit by paraffin section (A) and scanning electron microscopy (B). ar, aril; ob, obturator

RNA extraction, cDNA library construction, and sequencing

Total RNA was extracted from plant materials using TIANGEN’s RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit, following the manufacturer’s protocol. Novogene Co., Ltd. employed the Illumina HiSeq TM 2500 sequencing platform to create the cDNA library construction and sequence the transcriptome.

RNA-seq data processing and fundamental analysis

The raw data were processed by the software Trimmomatic (v0.38), which removed adaptors and low-quality sequences to give clean reads [12]. In addition, RSEM [13] package of Trinity(v2.8.6) [14] was used to analyze the FPKM (fragments per kilobase of exon per million fragments mapped) in different samples, and the genome of lychee [15], rambutan [16] and longan (which is from our lab and unpublished) were used as reference. The running process was “/trinityrnaseq/util/align_and_estimate_abundance.pl–transcripts cds.fa–seqType fq–left R1.fq.gz–right R2.fq.gz–est_method RSEM–aln_method bowtie–trinity_mode–prep_reference–gene_trans_map gene_trans_map.tab–output_dir outfile”. The differentially expressed genes (DEGs) were further analyzed by edgeR (v3.30.0) [17], with the screening criterion of Adjusted P value (FDR) ≤ 0.001 and|log2 (fold change)|≥ 2. The expression trend clustering of DEGs was determined using software R (v4.3.1). Genes with FPKM values below 0.3 were considered to be unexpressed in this study.

Gene functions were annotated using the website eggNOG-mapper (http://eggnog-mapper.embl.de/) [18], and KO and GO numbers were extracted as background files. DEG enrichment was conducted by GO and KEGG using the OmicShare platform (https://www.omicshare.com/tools/) and visualized using R packages ‘clusterProfiler’ [19], ‘AnnotationDbi’, and ‘tidyverse’. DEG protein sequences were retrieved, functional annotation of DEGs was performed using OrthoFinder, BLASTP algorithm, and KOBAS [20], and homologous DEG sequences were screened among lychee, longan, and rambutan.

Species homologous and specific genes analysis

OrthoFinder was used to identify homologs and unique genes in lychee, longan, and rambutan based on shared DEGs between cultivators. The homologs and species-specific genes were further analyzed and annotation by KEGG enrichment, GO enrichment and transcription factor analysis.

Construction of transcriptional regulatory network (TRN)

Transcription factors (TFs) were predicted by Plant TFDB [21] and PlantPAN4.0 [22]. The input file consisted of the homologous gene expression of the three species. GENIE3 in R examined the relationship between transcription factors and gene expression [23]. In addition, the 2 kb region before the start codon of homologous DEGs among lychee, longan, and rambutan were retrieved as the promoter region. The Analysis of Motif Enrichment (AME) tool (https://meme-suite.org/meme/tools/ame) was used to enrich the cis-regulatory elements (CREs) in DEG promoter regions [24], while the regulatory network between TFs and downstream genes was visualized by Cytoscape v2.3 [25].

Real-time quantitative PCR (RT-qPCR)

ACTIN2 was used as an internal control in the RT-qPCR dye method quantification kit (Takara) used to measure the expression of the genes. Reaction mixtures (10 µl) included 1 µl cDNA, 5 µl of 2 × SYBR Green Master Mix, 0.4 µl each of forward and reverse primers (10 mmol l⁻¹), and 3.2 µl ddH₂O. RT-qPCR was carried out using the following protocol: 95 °C for 5 min, followed by 42 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. The 2^−ΔΔCt method was applied to determine the relative expression level. The primers used in RT-qPCR are presented in Table S2.

In situ hybridization (ISH) histochemistry assay

Fresh tissues were immediately dissected and fixed in PFA solution mixed with diethyl pyrocarbonate (DEPC) water for 24 to 48 h. The samples were placed in an H₂O₂-HAc mixture (1:1, v/v), vacuumed for 30 min, treated for 3 h, and then moved to a covered glass bottle containing 70% EtOH-DAG mixture (1:1, v/v) for two days at 50 °C [26]. The tissues were then gradient dehydrated (4 °C), cleared (room temperature), and impregnated with wax (40 °C ~ 58 °C). The next step was embedding samples into paraffin blocks. The RNA probe preparation and hybridization process adhered to the guidelines provided by Min-Feng Wu et al. [27]. The LcLBD1 forward primer sequence was 5’-GTGTTGAGAAGTGCGTTTT-3’ and reverse primer was 5’-AATAGACTGCGTTGCTGAC-3’. The paraffin section process of longan was referred to the paraffin section process of in situ hybridization.

Subcellular localization assay

The cDNA sequence of LcLBD1 was connected to pmACT2-FGFP to construct the re combinant plasmid 35S::LcLBD1-GFP. The constructed fusion vector was injected into N. benthamiana leaves through agroinfiltration. The infected N. benthamiana was placed in darkness for one day and cultured under light for two days. N. benthamiana leaves were imaged using a confocal laser scanning microscope (Zeiss LSM880). Before that, the leaves were soaked in 0.1 µg/ml DAPI solution for 30 min.

Result

Morphology and structure of aril during its early development in longan

Observations on the 5th, 10th, and 15th days revealed a protruding tissue at the base of the seed, present exclusively on one side of the seed (Fig. 1A). The protruding structure appeared to be an obturator. The obturator vanished on the 20th and 25th days, although remnants of it could still be observed. The 30th day marked the reappearance of the projecting protrusion, the aril. The aril continued to protrude from the 30th to the 45th day, and the surface shapes of the primordium aril and obturator were similar. The 60th day saw the emergence of the aril, which grew upward from the base of the seed to enclose it. The obturator and aril have an ambiguous relationship; it is unclear if the obturator eventually disappears during development or transforms into the aril.

The ovule of longan is an anatropous ovule that has both inner and outer integuments. The term ‘obturator’ refers to a protuberant that is close to the micropyle and encircled by tissue that resembles dotted lines (Fig. 1B). The obturator appeared between the 0th and 25th days following anthesis, with significant variance in the overall fruit size. There was no evidence of an aril primordium, and seeds, fruits, and nucellus developed rapidly. The obturator vanished on the 30th, and aril primordium appeared symmetrically. The aril primordium exhibited an upward growth trend on the 35th day.

Transcriptomes data analysis of aril developmental stages in the three species

RNA-seq was utilized to quantify the samples featuring three stages of aril development: aril inception (Lcstage1-Lcstage2, Dlstage1, and Nlstage1), aril development (Lcstage3-Lcstage4, Dlstage2-Dlstage3, and Nlstage2-Nlstage3), and aril maturity (Lcstage5) stage where Lc, Dl, and Nl stand for lychee, longan, and rambutan, respectively (Figure S1). Pearson correlation coefficients were used to evaluate biological repeatability and consistency between samples. The biological repeatability of lychee samples was high, and there was a substantial correlation between the three cultivars at all stages (except for Lcstage1) (Figure S2A). Furthermore, the samples from Lcstage3 to Lcstage5 clustered together, indicating a significant degree of similarity between them (Figure S2A). The biological repeatability between various samples of longan was higher, with a stronger correlation between different varieties and stages. However, there was a reduced correlation between longan ‘DAW’ and other varieties at Dlstage1 (Figure S2A), possibly due to notable regional variations. Using Pearson correlation analysis, it was preliminarily established that there was a significant degree of biological repeatability, and the similarity of different varieties at different stages was high. PCA analysis showed that the samples had good repeatability, and these transcriptome data could be used for subsequent analysis (Figure S2B).

The number of genes with FPKM ≥ 0.3 in rambutan and longan was similar across different periods, but the difference was more significant in lychee (Figure S2C). There was more balance in the level of gene expression amongst the varieties. The FPKM value allows for their classification into four categories: 0.3 ≤ FPKM < 1, 1 ≤ FPKM < 10, 10 ≤ FPKM < 100, and FPKM ≥ 100. Among these, over 70% of genes at each stage, irrespective of species and varieties, had an FPKM range between 1 and 100, with only a few genes exhibiting high or low expression (Figure S2C). According to these findings, most genes had moderate expression levels aligned with the gene transcription level rule.

Differentially expressed genes in the transcriptome of lychee, longan, and rambutan arils

For the three cultivars of lychee, longan, and rambutan, the number of common differentially expressed genes (DEGs) was 3126, 2219, and 2418, respectively (Figure S3). Given that aril development is a dynamic regulatory process, the expression of shared genes from the three cultivars of lychee, longan, and rambutan was retrieved. Based on the trend in gene expression, these genes were categorized into four groups: cluster 1 (down-regulated profile), cluster 2 (first down-regulated, then up-regulated profile), and cluster 3 (first up-regulated, then down-regulated profile), and cluster 4 (up-regulated profile) (Figure S4A-C). GO enrichment and transcription factor enrichment were employed to understand the biological processes of various clusters (Figure S4A-C).

Cluster 1, 2, 3, and 4 mainly enriched in plant development-related GO terms, and different Clusters had different significantly GO terms. In addition, transcription factors enrichment showed that different transcription factors families played various roles in aril development stages, such as, bHLH and MYB in cluster 1, ERF and WRKY in cluster 2, bHLH in cluster 3, and ERF in cluster 4. Most genes were initially down- or up-regulated and then down-regulated throughout aril development. The genes of cluster 2 and 4 is up-regulate during thin aril to flesh aril. These genes might regulate aril enlargement. Therefore, we calculated the percentage of genes in cluster 2 and 4. Longan and rambutan had approximately 25% of their genes grouped into clusters 2 and 4, whereas lychee had 54.6%. Thus, approximately 50% of the genes might be involved in cell division or differentiation during the early stages of aril development.

Identification of species-specific DEGs in lychee, longan, and rambutan

To further investigate the differences and similarities of aril development in lychee, longan, and rambutan, OrthoFinder was used to align the protein sequences of DEGs shared across cultivars. Lychee, longan, and rambutan had 3126, 2219, and 2418 common DEGs across cultivars (Figure S3). In contrast, 810, 329, and 369 DEGs in lychee, longan, and rambutan were species-specific (Figure S5). Figure S5 shows that 1030 DEGs of lychee, 994 DEGs of longan, and 1049 DEGs of rambutan were homologous. Lychee and rambutan have more homologous genes during aril development than longan.

We retrieved the protein sequences of species-specific DEGs to identify transcription factors in lychee, longan, and rambutan. Lychee features bZIP (3), E2F/DP (1), LSD (1), MYB (1), and MYB-related (1) transcription factors, six of which were up-regulated from Lcstage2 to Lcstage4, while LITCHI024386.m1, an E2F/DP family member was exclusively expressed at Lcstage2 (Fig. 2A). In longan, B3 (1), BBR-BPC (1), bHLH (1), bZIP (1), ERF (1), GRAS (1), HB-other (1), M-type_MADS (1), SBP (1), and Trihelix (1) were detected. Seven genes were down-regulated during aril development, while three genes showed expression only at Lcstage3. We identified multiple transcription factor types in rambutan, including bZIP (3), C2H2 (1), GRAS (2), NAC (1), and Nin-like (1). Seven genes were expressed at Nlstage1 and Nlstage2, whereas Nl09g05900, a bZIP family member, showed high expression at Nlstage3.

Fig. 2.

Functional annotation of specific DEGs in lychee, longan, and rambutan. A The expression profiles of specific transcription factors. B KEGG enrichment of specific DEGs in lychee

Across the three species, KEGG enrichment analysis revealed that genes specific to their respective species were enriched in various metabolic pathways (Fig. 2B and Figure S6A-B). Species-specific genes showed enrichment of linoleic acid metabolism, glycerophospholipid metabolism, and carbon metabolism in lychee (Fig. 2B); glutathione metabolism, carotenoid biosynthesis, starch, and sucrose metabolism in longan (Figure S6A); and photosynthesis, glycosaminoglycan degradation, and porphyrin metabolism in rambutan (Figure S6B).

Transcription factors might influence aril development by regulating plant development-related DEGs

GO enrichment analysis revealed that 1030 DEGs in lychee, 994 DEGs in longan, and 1049 DEGs in rambutan were primarily involved in nucleic acid binding transcription factor activity, cell periphery, cell communication, post-embryonic development, hormone-mediated signaling pathway, plant organ development, plant-type cell wall, and fruit development (Fig. 3 and Table S3). The DEGs of the aril development-associated pathway were retrieved, followed by visualization of their expression. IAA8 and BG1 were selectively expressed in the early stages (Lcstage2, Dlstage1, and Nlstage1) of the hormone-mediated signaling pathway. SPL family genes (SPL2, SPL3, and SPL9) exhibited similar expression trends and were highly expressed in the early stages of plant organ development. In addition, genes related to the cell cycle (such as CSLD5, CYCD3;2, and HIK) and cell differentiation (such as AP3, AGL8, and SHP1) were up-regulated in early aril development (Figure S7A-C).

Fig. 3.

The homologous DEGs of GO enrichment analysis in three species. The green color represented the number of genes. The red boxes indicated the GO terms we were interested in

Lychee, longan, and rambutan shared 111, 111, and 147 differentially expressed common transcription factors, respectively. MYB was the second most abundant transcription factor among them, with bHLH coming in first (Fig. 4A). In addition, there was an enrichment of transcription factors involved in plant development and morphogenesis, including Dof, LBD, MIKC-MADS, SBP, and WOX. Among these transcription factors, the bHLH transcription factors were expressed in the early stages (Lcstage2, Dlstage1, and Nlstage1). The erratic expression of MYB genes and their distinct expression trends during aril development suggest that these genes have different functions in regulating the early or expansion stages of aril development. Early aril development was characterized by unique expression of aril development-related transcription factors, including OBP1, LBD1, LBD38, AG, SHP1, AGL8, AP3, and WOX13 (Fig. 4B-D).

Fig. 4.

Transcription factors analysis in lychee, longan, and rambutan. A Transcription factors enrichment of DEGs homologous in lychee, longan, and rambutan. B-D The expression profiles transcription factors in lychee, longan, and rambutan

The transcriptional regulatory network of DEGs showed that hub genes from the gene families MIKC_MADS (AGL8, AP3, and SHP1), WOX (WOX13), LBD (LBD1 and LBD38), Dof (OBP1), and SBP (SPL2, SPL3, and SPL9) were linked with other genes (belonging to ‘post-embryonic development’, ‘cell division’, ‘plant-type cell wall’, ‘cell communication’, ‘hormone-mediated signaling pathway’, ‘plant organ development’, ‘fruit development’, and ‘meristem development’) to regulate aril development (Fig. 5). We selected six genes from species-shared DEGs to verify the expression trend by real-time quantitative polymerase chain reaction (RT-qPCR), and the genes showed downward trends during aril development and specific expression in Dlstage1 in longan. The results were consistent with those from RNA-seq data, confirming the validity of our analysis (Figure S8).

Fig. 5.

Transcriptional regulatory network of lychee (A), longan (B) and rambutan (C) species-shared genes. The different colour was used to displayed different GO terms. The transcription factors are displayed by triangles, and circles showed genes

LBD1 could be involved in cell differentiation and division during aril development

We selected ten TFs (AGL8, AP3, SHP1, WOX13, LBD1, LBD3, OBP1, SPL2, SPL38, and SPL9) and three genes (IAA8, CSLD5, and CYCD3;2) based on function annotation, expression trends, and transcriptional regulatory network from homologs in three species. Of these genes, LBD1 in lychee, longan, and rambutan was similar to CpLBD1 in Carica papaya, whose seed was also encased in aril (Fig. 6A). This result suggested a potential role for LcLBD1, DlLBD1, and NlLBD1 in aril development. A 300 bp RNA probe of the gene was used for in-situ hybridization in lychee to demonstrate the function of LBD1 better. LcLBD1 was expressed in the funicle, placenta, early aril cell across Lcstage3 (thin aril), and the edge placenta across Lcstage4 (fleshy aril) (Fig. 6B). In addition, the LBD1 transcription factor translocated in the nucleus, which is consistent with the properties of transcription factors (Fig. 6C).

Fig. 6.

LBD transcription factors analysis. A Phylogenetic tree of LBD1 during plants with aril and without aril. The star represented the species with aril. BLcLBD1 of in situ hybridization histochemistry assay in lychee. LcLBD1 was located in funicle, placentation, and small aril cell. C Subcellular localization of LcLBD1 protein. F, funicle; ar, aril

Discussion

The Sapindaceae family includes lychee, longan, and rambutan, each with a distinct flesh structure. The fruit ripening process includes morphogenesis [28–31] and bioactive substances [32–39], and these are the primary focus of most studies. Studies on the molecular mechanisms regulating aril development, including differentiation, division, and expansion of aril, are limited. RNA-seq was used in our investigation to uncover the genes critical for aril development and to predict the molecular models that regulate aril development.

We compared the RNA-seq data of aril in lychee, longan, and rambutan and analyzed the species-specific and homologous genes. The species-specific genes of lychee, longan, and rambutan were enriched for different metabolic pathways. These metabolic pathways may significantly influence fruit development and flavor accumulation in lychee, longan, and rambutan. LITCHI024386.m1 (E2F/DP) was only expressed at Lcstage2. It has been reported that E2F/DP gene family might be involved in cell proliferation [40–42]. Hence, LITCHI024386.m1 (E2F/DP) might regulate the cell clycle and cell division in lychee. In longan, Dil.10g002130.1.t1 (ERF), Dil.08g005650.1.t1 (M-type_MADS), and Dil.01g003180.1.t1 (Trihelix) were specifically expressed at Dlstage3. These genes may influence the size and ripening of longan. Additionally, 8 specific transcription factors were identified in rambutan. These transcription factor families are mainly involved in plant stress and development [43–46].

While lychee, longan, and rambutan have different flavors, their morphological development is consistent. We also detected homologous DEGs in lychee, longan, and rambutan. Thirteen key DEGs were identified among the development-related pathway genes and transcription factors, comprising three function genes (IAA8, CSLD5, and CYCD3;2) and ten transcription factors (AGL8, AP3, SHP1, WOX13, LBD1, LBD3, OBP1, SPL2, SPL3, and SPL9). In Ginkgo and Taxus, the ABCDE model genes of floral development regulated the onset of fleshy structures [10]. The D-class genes, STK and SHP1, play a vital role in aril development [16]. The LBD gene family participates in the formation of plant lateral organs and secondary growth [47, 48]. SPL can also participate in many biological processes, such as flowering, stress response, and fruit development [49–51]. MADS-box, WOX, LBD, and SPL might be responsible for mediating aril development.

In-situ hybridization revealed the expression site of LcLBD1. LcLBD1 is primarily expressed in the funicle, placenta, and small aril cells at Lcstage2, but only expressed in the placenta at Lcstage3 (Fig. 6B). These results suggest that LcLBD1 could play a vital role in early stages of aril development by regulating aril cell differentiation and division or funicle development. Prior research has demonstrated that AtLBD1, AtLBD3, AtLBD4, and AtLBD11 could accelerate cell growth and division, enhancing the secondary growth in Arabidopsis [52]. A similar phenomenon involving LBD1 has been reported in poplar stems [53]. The developing part of the aril is the funicle containing phloem, xylem, and vascular cambium, among which only the vascular cambium has the differentiation function, suggesting that vascular cambium may play a significant role in aril formation. Our preliminary investigation focused on the expression of LcLBD1 in the funicle; however, additional validation is necessary to determine whether it also shows expression in the vascular cambium.

Despite the RNA-seq analysis-based screening of these aril development genes, the molecular process remains unverified due to the long growing period of lychee, longan, and rambutan. Fruit trees cannot quickly produce genetically stable positive seedlings, making it impossible to see the phenotype associated with aril development in a short time. Thus, it is necessary to validate the candidate genes of aril development in multiple ways, including creating a transient silencing system and selecting plants with short growth cycles and stable genetic transformation systems as model plants for studying the development of arils.

Conclusion

Lychee, longan, and rambutan develop differently from other fruits because they originate from a funicle, the molecular mechanism of which is still unclear. This study analyzed species-specific and homologous DEGs by RNA-seq in lychee, longan, and rambutan to investigate variations and similarities during aril development.

Lychee, longan, and rambutan differ primarily in their metabolic processes, which may influence their flavor and oxidation rate. In addition, analysis of homologous DEGs by GO enrichment and TRN revealed three genes (IAA8, CSLD5, and CYCD3;2) and ten transcription factors (AGL8, AP3, SHP1, WOX13, LBD1, LBD3, OBP1, SPL2, SPL3, and SPL9) as hub genes in the gene regulatory network of aril development. Furthermore, we experimentally discovered that LcLBD1 was expressed in the funicle placenta and small aril cells, indicating a potential role for LBD1 in cell differentiation and division. In light of the above results, we propose that aril developed from the cambium of funicle in these fruits.

Authors’ contributions

R.M. conceived this project; W.S. and W.Z. designed experiments; D.F. and W.Z. collected samples of longan, lychee and rambutan. W.S., D.F., M.Y., Y.Y., W.Y., and X.Y. performed most of the experiments and analysis. W.S. wrote the manuscript. R.M. and W.Z. revised the manuscript. All authors read and approved the final manuscript.

Funding

The research was supported by the startup fund from Fujian Agriculture and Forestry University, the Project of Sanya Yazhou Bay Science and Technology City, Grant No:SCKJ-JYRC-2023–21, and the startup fund from Hainan University.

Data availability

The RNA-seq data of Baoyan 7 used the NCBI GenBank under accession number PRJNA728838 [39], while the other raw data have been deposited in the Genome Sequence Archive [54] in National Genomics Data Center [48, 55], China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA025136) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/.

Declarations

Ethics approval and consent to participate

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Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.