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Article

Transcriptomic Analysis Identifies GhSACPD-Mediated Fatty Acid Regulation in the Cotton Boll Abscission

by
Guangling Shui
1,†,
Zewei Chang
1,†,
Peng Han
1,
Qi Zhang
1,
Zhibo Li
1,
Hairong Lin
1,
Xin Wang
2,*,
Yuanlong Wu
1 and
Xinhui Nie
1,*
1
Key Laboratory of Oasis Ecology Agricultural of Xinjiang Bingtuan, Agricultural College, Shihezi University, Shihezi 832003, China
2
Cotton Institute, Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2025, 15(20), 2166; https://doi.org/10.3390/agriculture15202166 (registering DOI)
Submission received: 8 September 2025 / Revised: 8 October 2025 / Accepted: 15 October 2025 / Published: 18 October 2025
(This article belongs to the Section Crop Genetics, Genomics and Breeding)
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Abstract

Boll abscission in cotton (Gossypium spp.) is a key factor that limits yield; however, the molecular mechanisms underlying this process remain poorly understood. In this study, boll abscission characteristics were uncovered in four cotton varieties that exhibited extreme differences in boll abscission rates via tissue sectioning. Transcriptome analysis was performed on the four cotton varieties. Using weighted gene co-expression network analysis (WGCNA) of the transcriptome data, we identified a stearoyl-(acyl-carrier-protein) desaturase (SACPD) as a potential key regulator of boll abscission. We also performed evolutionary analyses on the SACPD gene family across five cotton species and identified 63 members that were classified into four evolutionary clades, with duplication-polyploidization events being a major driver of gene expansion. Tissue-specific expression profiling revealed that Gossypium hirsutum GhSACPD19 is highly expressed in the abscission zone. Our findings suggest a role of GhSACPD19 in regulating boll abscission, likely through metabolism of jasmonate, a well-known positive regulator of abscission. Our work offers new insights into the regulation of organ abscission at cellular and molecular levels and presents a valuable resource for cotton yield improvement.

1. Introduction

Cotton (Gossypium spp.) is a globally important fiber and oilseed crop [1,2], accounting for 22–25% of the annual global fiber demand [3], and contributes substantial economic value to cotton production. In cotton cultivation, boll abscission can lead to yield losses of 60–70%, representing a major limitation to productivity. Boll abscission represents a paradigmatic model of plant organ abscission, which is a tightly regulated physiological process driven by genetic mechanisms that facilitate the separation of organs from the parent plant, including those that are unpollinated, damaged, diseased, nutrient-deficient, or mature [4]. Cellular activation of the abscission zone involves a coordinated cascade of biological events including selective protein degradation, dynamic remodeling of the cell wall, dissolution of pectin in the middle lamella, altered plasma membrane permeability, accumulation of reactive oxygen species (ROS), and programmed cell death (PCD). PCD within the abscission zone (AZ) is a crucial event in plant organ abscission and is characterized by typical cytological features such as loss of cell viability, nuclear morphological changes, DNA fragmentation, and ROS accumulation [5]. Lipid metabolism plays a significant role in regulating abscission processes, as evidenced by studies on olives which demonstrated that sphingolipids (LCBs) regulate spatially restricted PCD upstream of the salicylic acid (SA) and jasmonic acid (JA) signaling pathways. These events coincided with a marked accumulation of polar lipids (including sphingolipids) and enhanced endocytosis during abscission [6]. These findings highlight the essential contribution of spatiotemporal lipid dynamics and membrane remodeling processes to the mediation of organ abscission signaling pathways.
Lipids are essential structural components of biological membranes that play critical roles in plant growth, development, disease resistance, and insect predation [7,8,9,10]. In plastids, stearoyl-ACP desaturase (SACPD/SSI2) catalyzes the conversion of stearic acid (18:0) to oleic acid (18:1), which is crucial for the synthesis of chloroplast membrane lipids [11]. SACPD, particularly the SSI2 isoform, plays a pivotal role in regulating plant growth, stress responses, and senescence by modulating fatty acid levels, which in turn influence JA- and SA-mediated signaling pathways [12,13]. Dysfunction of SSI2 activity impairs the conversion of stearic to oleic acid, resulting in chlorophyll deficiency, compromised photosynthetic efficiency, and necrotic lesion formation [14,15]. Both ssi2 and the fatty acid biosynthesis 2 (fab2) mutants show significant accumulation of stearic acid (18:0), which alters the composition of membrane lipids [16]. Notably, the double mutant of restorer of defective cross-talk 2 (rdc2) and restorer of defective cross-talk 8 (rdc8) suppressed ssi2 mutant loss-of-function defects, restoring wild-type cellular morphology and pathogenesis-related (PR) gene expression patterns [17]. Interestingly, although the rdc8 mutation completely suppressed the spontaneous cell death phenotype of ssi2, the rdc2 mutation exerted only a partial rescue effect on this phenotype, suggesting the involvement of distinct suppressor pathways [18]. In Arabidopsis, the ssi2 mutant exhibits an enhanced hypersensitive response (HR)-like cell death phenotype accompanied by increased SA accumulation and elevated disease resistance [19]. Reduced 18:1 levels stabilize the plastid-localized protein NOA1, thereby promoting nitric oxide (NO) accumulation and activating defense signaling pathways [20]. Under abiotic stress conditions including elevated temperature and drought, remodeling of membrane lipid composition, notably catabolic hydrolysis of phosphatidylcholine to lysophosphatidylcholine (LPC), induces β-galactosidase (GLB1) expression, thereby accelerating cellular senescence [21]. Furthermore, enzymes such as NPC4 and PLDζ2 facilitate the reutilization of phosphorus from senescent membranes, which further accelerates the senescence process [22]. Additionally, sphingomyelin (d18:0) acts as a positive regulator of the abscission process by enhancing ROS production and activating the SA/JA signaling pathways [23]. These findings underscore the critical role of SACPD in lipid signaling, particularly in regulating plant cell senescence [13,24]. Despite these insights, the precise mechanisms by which SACPD genes regulate senescence through lipid signaling pathways remain unclear.
In this study, to investigate the regulatory mechanism of square-boll abscission, we employed weighted gene co-expression network analysis (WGCNA) on transcriptome data from samples exhibiting extreme differences in boll abscission rates to construct a gene co-expression network, successfully identifying the key regulatory gene SACPD as being involved in cotton boll abscission. Through comprehensive genome-wide multidimensional analyses, we elucidated the molecular mechanism by which SACPD regulates cotton boll abscission by modulating lipid metabolism. This discovery provides a novel target for understanding the molecular mechanisms underlying plant organ abscission and establishes a theoretical foundation for the genetic improvement of crop yield traits.

2. Materials and Methods

2.1. Investigation of Plant Materials and Squares-Bolls Abscission Rate

To study the regulatory mechanism of squares-bolls abscission, two abscission-resistant Gossypium hirsutum materials, Xinluzao 32 (L32) and Xinluzhong 50 (L50), and two abscission-sensitive Gossypium hirsutum materials, Xinluzhong 18 (H18) and Xinluzao 35 (H35), were selected as the experimental materials. Field trials were conducted from 2018 to 2019 at two ecological regions: Shihezi (SHZ, N 44°18′, E 86°13′) and Korla (KEL, N 41°38′, E 86°06′) in Xinjiang. The squares-bolls abscission rate (AR1) was measured at the initial abscission stage (10 d after flowering) under four environmental conditions (2018-SHZ, 2018-KEL, 2019-SHZ, and 2019-KEL). The abscission rate was calculated using the following formula:
Number of abscised squares and bolls/(number of abscised squares and bolls + number of retained squares and bolls) × 100
To investigate the abscission zone of the cotton pedicels, four cultivars (L32, L50, H35, and H18) were grown in a greenhouse at Shihezi University. During the experimental period, greenhouse conditions were maintained as follows: day/night temperature of 28 ± 2 °C, photoperiod of 16 h light/8 h dark, and relative humidity of 30–40%. Following Kućko’s sampling method [25], pedicel tissues exhibiting senescence and yellowing symptoms within 10 days after flowering were defined as naturally abscised abscission zones (AZ). In contrast, green, non-abscised pedicels collected 10 days after flowering were classified as non-abscised abscission zones (NAZ) (Figure S1). Using sterilized surgical blades, pedicel segments (approximately 5 mm in length, including 2.5 mm above and 2.5 mm below the junction between the pedicel and fruiting branch) were excised, immediately frozen in liquid nitrogen, and stored at −80 °C for transcriptome analysis.

2.2. Cellular Structure Observation of Boll Pedicel Abscission Zone

The cellular architecture of the pedicel abscission zones was comprehensively analyzed using scanning electron microscopy (SEM) and paraffin sectioning. For SEM observation, the samples were fixed in 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.2) with vacuum infiltration at 4 °C overnight, followed by triple rinsing with ddH2O (20 min each), freeze-drying for 8 h, gold sputtering, and examination using a SU8010 field-emission SEM. For paraffin sectioning, AZ and NAZ samples collected at 1, 4, 7, and 10 days post-anthesis were processed for histological analysis by FAA fixation, ethanol dehydration series (50%, 70%, 85%, 95%, and 100%; 30 min per concentration), xylene clearing, and paraffin embedding. Serial 5 μm sections were prepared using a rotary microtome, stained with toluidine blue, and imaged under a Leica T13000 Ergo stereomicroscope (Leica, Wetzlar, Germany).

2.3. RNA-seq Profiling and Computational Analysis of Boll Pedicel Abscission Zone Transcriptomes

Total RNA was extracted from the AZ and NAZ samples L32, L50, H35 and H18, using the EASYspin Plus Complex Plant RNA Kit (Cat. No. RN53) (Aidlab, Beijing, China). Sequencing was performed on an Illumina NovaSeq 6000 system (Illumina, San Diego, USA) to generate 150 bp paired-end reads. Quality control and preprocessing of the raw paired-end sequencing data were performed using fastp (v0.23.2). The process involved removing low-quality bases and adapter sequences, filtering out reads containing excessive ambiguous bases (N), and generating clean data. To enhance processing efficiency, eight threads (-w 8) were used, and paired-end filtering was applied separately to the raw sequencing files of R1 and R2, resulting in the corresponding clean reads files. Clean reads were aligned to the Gossypium hirsutum cultivar Xinluzao 31 (HC04) genome [26] using HISAT2 (v2.2.0) with default parameters, employing the options --rna-strandness RF and --dta to enable downstream transcript assembly. Gene expression was quantified as fragments per kilobase of transcript per million mapped reads (FPKM) using StringTie (v2.1.5) with parameters -e and -B to estimate expression levels for known transcripts and generate Ballgown input files. For differential expression analysis, read counts per gene were generated using featureCounts (v2.0.1) with options -p -B -C -s 2 for paired-end, strand-specific data, and statistically analyzed using DESeq2 (v1.46.0) with default settings and adjusted p-values calculated by the Benjamini–Hochberg method. The false discovery rate (FDR) was calculated using the Benjamini–Hochberg procedure. Significantly differentially expressed genes (DEGs) were defined as those with an adjusted p < 0.01 and |log2 (fold change)| > 2. A principal component analysis (PCA) was conducted using the DESeq2 plotPCA function. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using clusterProfiler (v4.10.0) [27]. A gene co-expression network was constructed using the WGCNA in R v1.6.1 [28,29]. Hub genes were identified by calculating intramodular connectivity (KME) values, followed by betweenness centrality (BC) analysis using the CytoNCA plugin in Cytoscape 3.9.1 [30].

2.4. Identification and Characterization of SACPD Gene Family Members

Genomic data for four cotton species: Gossypium arboreum (A2) ‘SXY1’ genome HAU_v1 (Ga), G. herbaceum (A1) ‘ZhongCao1’ genome HAU_v1 (Ghe), G. raimondii (D5) ‘Grai D502’ genome HAU_v1 (Gr), and G. barbadense ‘3-79’ genome HAU_v2_a1 (Gb) were obtained from the CottonGen database, whereas G. hirsutum cv. Xinluzao 31 genome was sourced from Li et al. [26]. Candidate SACPD genes were initially identified through BLASTp analysis against Arabidopsis thaliana SACPD protein sequences as queries, with a word size of 3, the BLOSUM62 matrix, and a stringent E-value cutoff of 1 × 10−10. Putative genes were further validated by conserved domain prediction using NCBI-CDD to exclude pseudogenes. Subsequent characterization included analysis of physicochemical properties, subcellular localization prediction with DeepLoc-2.0, chromosomal distribution mapping, and exon–intron structure determination using TBtools-II v2.357 [31]. Conserved protein motifs were identified using MEME suite analysis with parameters set to identify 10 distinct motifs.

2.5. Phylogeny and Synteny Analysis Reveal Evolutionary Patterns of the SACPD Gene Family

The evolutionary relationships of the SACPD gene family were investigated using comprehensive phylogenetic and comparative genomic analyses. A comprehensive evolutionary analysis was performed on 70 SACPD protein sequences from Ga (9), Gb (19), Ghe (7), Gr (9), Gh (19), and Arabidopsis thaliana (7). The phylogenetic tree was constructed using the Maximum Likelihood method in MEGA-12. Multiple sequence alignment was performed with the MUSCLE algorithm, and the JTT+G model was selected as the best-fit model using the built-in model selection function, with default parameters. The robustness of the phylogeny was evaluated with 1000 bootstrap replicates. The resulting tree was visualized and annotated using iTOL [32]. Genomic synteny analysis performed using TBtools revealed segmental duplications and homologous gene pairs, while OrthoVenn2 facilitated the identification of orthologous clusters among the cotton species. For the calculation of nonsynonymous-to-synonymous substitution ratios (Ka/Ks), full-length amino acid sequences of SADPC gene pairs in Gossypium hirsutum were selected, and the Ka/Ks ratios were determined using TBtools with the default parameters.

2.6. Expression Pattern Analysis of SACPD Genes Across Cotton Tissues

The expression patterns of the SACPD gene family across different tissues were systematically analyzed using published transcriptome data [26,33], with data normalization performed using the ‘rank invariant’ algorithm. To validate the expression profiles of key genes, pedicel abscission zones from the cotton cultivar L32 were used as the experimental material. Total RNA was extracted using RNAiso Plus reagent (Takara, Beijing, China), followed by cDNA synthesis using M-MLV reverse transcriptase (Takara). Quantitative real-time PCR (qRT-PCR) was performed using TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China), with the UBQ7 gene serving as an internal reference control. All experiments included at least three independent biological replicates, and the primer sequences used are listed in Supplementary Table S1.

3. Results

3.1. Identification of the Bolls Abscission Phenotype and Anatomical Characterization

To reveal the mechanism of squares-bolls abscission, boll abscission rates (AR1) were evaluated across four environments using two abscission-resistant materials (L32 and L50) and two abscission-sensitive materials (H18 and H35). Statistical analysis revealed that the resistant materials exhibited an average AR1 of 10.19%, which was significantly lower (p < 0.01) than that of the sensitive materials (average AR1 of 35.46%) (Table 1). To reveal the cytological differences between the materials, scanning electron microscopy (SEM) analysis of the pedicel abscission zone fracture surfaces was performed. It was demonstrated that cellular structural differences in the pedicle detachment zone served as decisive factors for organ abscission. The abscission-sensitive materials displayed pronounced tissue heterogeneity, with distinct separation phenomena observable at the xylem–phloem interface, loosely arranged epidermal cell structures at the fracture surfaces, and compromised intercellular connection integrity (Figure 1A). These ultrastructural features reflected the contrasting abscission propensities of the two materials.
To investigate the key developmental stages of pedicel abscission zone (AZ) cell differentiation in the materials, two abscission-resistant (L32 and L50) and two abscission-sensitive (H18 and H35) cotton germplasms were cultivated under greenhouse conditions. Paraffin sections of the pedicels at 1, 4, 7, and 10 days post-anthesis (DPA) were analyzed (Figure 1B). The abscission-sensitive line H35 exhibited 27 layers of abscission zone cells, with 27 small and densely arranged abscission layer cells present in the pedicel abscission zone at 1-day post-anthesis (1 DPA). However, as boll development progressed, the number of abscission cell layers initially decreased, followed by an increasing trend, with ultimately 92% of the cells (25 layers) differentiating into abscission layer cells. In contrast, the abscission-resistant line, L32, initially contained only 20 layers of abscission cells. During boll development, the number of abscission cell layers increased continuously, reaching 25 layers by 10 DPA. Histological examination revealed that the abscission zone in cotton pedicels initiates formation 1 DPA and completes differentiation by 10 DPA, achieving a final count of 25 cell layers in the abscission layer (Figure S2). These findings identified 10 DPA as the critical developmental stage for abscission zone formation, warranting further transcriptome analysis to elucidate the underlying molecular mechanisms.

3.2. Comparative Transcriptome Analysis of AZ Tissues Between Abscission-Sensitive and Abscission-Resistant Cotton Lines

To elucidate the molecular basis underlying pedicel abscission zone differentiation, we conducted transcriptome profiling of the NAZ and AZ tissues at 10 DPA in abscission-resistant (L32 and L50) and abscission-sensitive (H18 and H35) cotton lines. A total of 24 libraries were constructed with clean reads aligned to the Gossypium hirsutum TM-1 reference genome. Each sample yielded a minimum of 34 million clean reads, with alignment rates ranging from 89.50% to 94.03% and Q30 scores exceeding 97.58% (Table S2). Differential gene expression analysis was performed using stringent thresholds (|log2(Fold Change)| ≥ 2 and FDR ≤ 0.01). Transcriptome profiling of abscission zone (AZ) versus non-abscission zone (NAZ) tissues at 10 DPA revealed substantial differential gene expression across the four cotton genotypes, with abscission-sensitive lines H18 and H35 showing more extensive transcriptional changes than the resistant lines L32 and L50. The analysis identified 6325 DEGs in H18 (47.35% upregulated), 7328 in H35 (51.14% upregulated), 5498 in L32 (53.26% upregulated), and 4843 in L50 (45.86% upregulated), totaling 9964 unique DEGs, including 2844 conserved across all genotypes (Figure 2). Notably, the abscission-sensitive genotypes displayed an excess of 3312 DEGs compared to the resistant genotypes, with line H35 demonstrating the most significant transcriptomic alterations. The identification of 2844 conserved DEGs provides key targets for understanding the molecular mechanisms underlying cotton boll abscission, and the scale of changes (9964 DEGs) underscores the complexity of this developmental process.

3.3. Lipid Metabolism Is a Key Metabolic Pathway in Cotton Boll Abscission as Revealed by GO and KEGG Analysis

To elucidate the functional roles of DEGs associated with cotton boll abscission, we performed GO and KEGG enrichment analyses on two DEG sets: 2844 co-differentially expressed genes and 9964 abscission-related DEGs. GO analysis revealed that at the molecular function level, both sets were significantly enriched in oxidoreductase activity (especially diphenol-related redox reactions), copper ion binding, DNA-binding transcription factor activity, and glucosyltransferase functions. Genes in the ‘biological process’ category were involved in transcriptional regulation (DNA-templated transcription and RNA biosynthesis), RNA processing, cell wall organization and biogenesis, and polysaccharide metabolism. Analysis of GO terms in the cellular component category indicated a strong enrichment in cell envelope structures such as cell wall, plasma membrane, extracellular matrix, and cell periphery (Figure 3A). These findings suggest a coordinated role for these genes in maintaining the redox balance, regulating gene expression, and mediating cell wall remodeling during abscission.
KEGG pathway analysis further revealed distinct metabolic signatures between the two gene sets. Both DEG sets were enriched in secondary metabolite biosynthesis and environmental stress response pathways, suggesting that these conserved processes were activated during abscission-related stress. In the larger 9964 DEG set, pathways related to α-linolenic acid and glycerolipid metabolism were significantly enriched, indicating involvement in membrane lipid remodeling, phospholipase activity, and jasmonate biosynthesis (Figure 3B). Conversely, the 2844 co-DEG set showed higher enrichment in carbohydrate metabolism pathways, such as starch and sucrose metabolism, reflecting a possible emphasis on energy supply (Figure S3). This divergence implies functional specialization, with abscission-related genes contributing to both cell envelope adaptation and metabolic energy coordination. Collectively, these results highlight that while multiple metabolic pathways, including carbohydrate metabolism, contribute to boll abscission, lipid metabolism plays a particularly critical regulatory role in this process.

3.4. The SACPD Gene as the Key Hub Gene Identified by WGCNA-Based Construction of Gene Co-Expression Networks

Seven distinct co-expression modules were identified from 9964 DEGs using WGCNA with dynamic hybrid cutting (Table S3). The module sizes ranged from 47 genes (MEred) to 5887 genes (MEturquoise). An optimal scale-free topology was achieved using a soft-threshold power of 12 for network construction (R2 stabilization, mean connectivity <500; Figure S4). Using the abscission rates of the four cotton materials in the module-trait correlation analysis, we found that the MEyellow module was significantly associated with H35-N (p < 0.01) and the MEbrown module was significantly associated with H35-T (p < 0.01), suggesting their potential functional importance in abscission regulation (Figure 4B). To systematically explore the intramodular signaling pathways and elucidate the molecular regulation of cotton boll abscission, we conducted comprehensive GO and KEGG pathway analyses of 230 genes from the MEyellow and MEbrown modules. GO enrichment analysis indicated significant enrichment (p < 0.01) in biological processes such as antioxidant/oxidative stress response, lipid metabolism regulation, macromolecule catabolism, and stress response mechanisms (Figure 4C). KEGG pathway analysis demonstrated prominent enrichment in phenylpropanoid biosynthesis, terpenoid metabolism, and plant hormone signal transduction pathways (Figure 4D). These results strongly imply that redox homeostasis (particularly the oxidative stress response) and phenylpropanoid biosynthesis pathways play pivotal roles in regulating cotton boll abscission and provide novel insights into the molecular basis of this agronomic trait.
To identify key genes associated with cotton boll abscission, we integrated Module Membership (MM) and Gene Significance (GS) analyses of 230 candidate genes from the MEyellow and MEbrown modules. Stringent thresholds (MM > 0.8 and GS > 0.25) revealed 21 hub genes. The co-expression network constructed and visualized in Cytoscape revealed that HC04_D10013030, which encodes a stearoyl-(acyl-carrier-protein) desaturase (SACPD) family protein containing a conserved SACPD domain, was the primary hub gene (MM = 0.83, GS = 0.28) (Figure 4E). Previous studies have shown that SACPD family proteins are involved in fatty acid metabolism and plant immunity and are closely associated with oxylipin biosynthesis and stress responses [34]. The linoleic and linolenic acids catalyzed by SACPD serve as precursors for jasmonic acid (JA) biosynthesis, which subsequently induces the expression of cell wall-degrading enzymes such as cellulases, directly promoting cell separation in the abscission zone [35]. Accordingly, we hypothesize that SACPD regulates cotton boll abscission by modulating the JA signaling pathway through lipid metabolism.

3.5. Genome-Wide Identification and Characterization of Stearoyl-Acyl Carrier Protein Desaturase Genes in Cotton Species

SACPD, also known as salicylic acid insensitive (SSI), plays a crucial role in plant fatty acid metabolism and immunity. To investigate the involvement of SACPD in cotton boll abscission, we performed genome-wide identification of SACPD genes across five cotton species using BLASTP and HMMER analyses. A total of 63 unique SACPD protein sequences were identified, and domain verification was performed using the SMART, CDD, and PFAM databases. Genomic distribution analysis revealed the presence of nine SACPD genes in G. arboreum, seven in G. herbaceum, nine in G. raimondii, and 19 each in the tetraploid species G. hirsutum and G. barbadense, indicating gene expansion in the latter species. During this process, GhSACPD13, annotated as a pseudogene with an incomplete coding region, was excluded from further analyses.
Comparative analysis of the physicochemical properties of SACPD proteins in diploid and tetraploid cotton (Table S4) revealed notable structural differences. Diploid SACPD proteins displayed conserved lengths (387–397 amino acids), whereas tetraploid proteins exhibited greater variability in length (344–397 amino acids). The molecular weights of diploid proteins ranged from 43.68 to 45.77 kDa, while those of tetraploid proteins ranged from 39.46 to 45.88 kDa. Additionally, isoelectric points differed between diploid and tetraploid SACPD proteins: diploid proteins had isoelectric point values in the weakly acidic to neutral range (5.28–7.68), while tetraploid proteins spanned a broader range (5.09–8.13), reflecting greater functional versatility. These structural and biochemical differences underscore the functional diversification and adaptive specialization of lipid metabolism pathways following the polyploidization of cotton.
A comprehensive genome-wide analysis of the subcellular localization of the SACPD gene family members revealed distinct compartmentalization patterns, each with significant functional implications. The prediction results showed that the majority of SACPD proteins (61 of 63, 96.82%) were localized in the chloroplasts, consistent with their well-established role in fatty acid biosynthesis within the plastids. Notably, one exceptional member, GbSACPD11 from sea island cotton (G. barbadense), was predicted to localize in the cytoplasm. GbSACPD11 exhibited remarkable structural divergence, displaying a nearly two-fold increase in protein length compared with canonical SACPD proteins. This non-canonical subcellular targeting, coupled with its unique structural features, suggests that GbSACPD11 may have evolved specialized roles in cytoplasmic lipid modification or transport. These findings provide compelling evidence of functional diversification within the SACPD gene family during cotton polyploidization, which may contribute to the enhanced metabolic plasticity observed in tetraploid cotton species.
To explore the evolutionary relationships between SACPD proteins across cotton species, a phylogenetic tree was constructed using MEGA-12, incorporating 63 SACPD sequences from five cotton species (G. arboreum, G. raimondii, G. herbaceum, G. barbadense, and G. hirsutum). For comparative analysis, seven Arabidopsis SACPD proteins (AtSACPD1 to AtSACPD7) were included as reference sequences to establish evolutionary context. Phylogenetic analysis classified the SACPD gene family into four distinct subgroups (Groups 1–4) with clear evolutionary relationships with Arabidopsis homologs (Figure 5). Groups 1 and 3, containing three and two Arabidopsis SACPD members, respectively, represented the conserved core subgroups. Notably, Group 2 comprised 14 cotton SACPD genes, showing the highest homology to Arabidopsis AtSACPD1, whereas Group 4, the largest clade containing 49 members, exhibited the strongest evolutionary conservation with AtSACPD2. This phylogenetic architecture underscores both the conserved orthologous relationships and substantial gene family expansion in cotton, particularly in the AtSACPD2-related subgroup.

3.6. Structure and Motif Analysis of SACPD Family Genes

Using the MEME suite for the systematic motif characterization of SACPD genes in five cotton species (G. arboreum, G. herbaceum, G. raimondii, G. hirsutum, and G. barbadense), we identified 10 conserved motifs with distinct evolutionary trajectories (Figure S5). Diploid cotton displayed targeted motif losses, with G. arboreum missing motif 8 in specific members (GaSACPD1/2), G. herbaceum lacking motif 8 (GhSACPD1/5), and G. raimondii lacking motif 9 (GrSACPD1/5) (Figure S6). The tetraploid species exhibited more complex patterns (Figure 6); G. hirsutum had six members (GhSACPD1/3/6/12/13/16) deficient in motif 9, whereas G. barbadense showed particularly intricate variations, including extensive motif 10 loss, motif 8 duplications (GbSACPD7/19), and a unique double-motif deletion (GbSACPD16 missing both motifs 3/10). These progressive motif alterations from diploids to tetraploids reveal an evolutionary trend toward increased structural complexity, which may facilitate functional diversification in cotton polyploidization.

3.7. Characterization of Chromosome Distribution and Gene Duplication in the SACPD Gene Family

Chromosomal distribution and gene duplication analyses revealed distinct evolutionary patterns in SACPD genes across cotton species (Figures S7 and S8). In diploid cottons (G. arboreum, G. herbaceum, and G. raimondii), SACPD genes show conserved chromosomal localization, with 7–9 members distributed across five chromosomes (carrying either one or three genes per chromosome) in each species. Notably, chromosomes 2, 5, and 7 consistently harbored SACPD genes in all three diploids, demonstrating remarkable evolutionary conservation. The allotetraploids (G. hirsutum and G. barbadense) exhibited double chromosome numbers (10 homologous chromosomes: A02, A05, A07, A10, A12, D02, D03, D05, D07, and D10) with strict subgenome asymmetry mirroring their diploid progenitor patterns (the A-genome from G. arboreum and the D-genome from G. raimondii). Interestingly, the absence of SACPD genes on the A03/D12 chromosomes and the singleton distributions on the D03/A12 chromosomes were perfectly conserved between tetraploids and their ancestral diploids, providing compelling evidence of subgenome stability following polyploidization. These results demonstrate deep evolutionary conservation and precise genomic inheritance during cotton speciation.

3.8. Genomic Collinearity and Homologous Evolution Analysis of the SACPD Gene Family in Cotton

To elucidate the expansion mechanism of the SACPD gene family, a collinearity analysis was performed on five cotton species using MCScanX (v1.0) and Circos (v0.69-9) software. In diploid cotton, only G. raimondii exhibited a single collinear gene pair (Figure S9), whereas the tetraploid cotton species showed nine collinear gene pairs in Gossypium barbadense, and seven pairs in Gossypium hirsutum (Figure 7A,B). Notably, each collinear gene pair was located on different chromosomes, indicating that both segmental duplication and polyploidization events were the major driving forces underlying the expansion of the SACPD gene family in tetraploid cotton. Further comparative analysis revealed that 14 homologous SACPD gene pairs were shared between G. arboreum, G. hirsutum, and G. barbadense (Figure 7C,D). In contrast, G. raimondii displayed fewer homologous pairs: 12 with G. hirsutum and 10 with G. barbadense. An intriguing observation was the duplication of SACPD genes located on chromosome 10 of G. raimondii, which appeared on chromosomes A02 and D02 in G. hirsutum but not in G. barbadense. Moreover, the finding that all Ka/Ks ratios in the Gossypium hirsutum SADPC gene family are below one (Table S5) provides evidence that this gene family has been subject to ongoing purifying selection and considerable functional constraints, which have acted to preserve the stability of its core biological functions. These findings highlight the divergent evolutionary trajectories between tetraploid cottons, with gene duplication in G. hirsutum suggesting a key adaptive or domestication-related trait that enables environmental adaptation.

3.9. Expression Profiling of the SACPD Gene Family in Cotton

To further investigate the role of SACPD family proteins in cotton boll abscission, the expression patterns of 38 SACPD genes across six tissues (root, stem, leaf, petal, anther, and 10-day fiber) in tetraploid cotton were analyzed using the cotton genome database (Figure 8A). The results revealed ubiquitous expression coupled with tissue-specific regulation of the SACPD gene family. Specifically, GbSACPD17 was identified as a leaf-specific gene; GhSACPD6 and GbSACPD16 were specifically expressed in anthers; GhSACPD7 and GhSACPD14 showed fiber-specific expression; and GbSACPD3 and GbSACPD4 were specifically expressed in roots. Notably, SACPD genes were predominantly expressed in the fiber tissues of upland cotton but were significantly enriched in the roots of sea island cotton. Interspecific expression divergence implies that SACPD genes undergo functional differentiation to regulate growth and development in distinct cotton tissues. Moreover, comparative analysis of SACPD expression in cotton pedicel abscission zones revealed differential regulation between abscission-sensitive and -resistant genotypes, which demonstrated that GhSACPD4, -2, -9, -11, -15, and -17 were exclusively expressed in developing pedicel tissues but were undetectable during abscission zone formation. GhSACPD16 is not expressed at either stage. Interestingly, GhSACPD19 showed genotype-specific activation restricted to abscission zone development in abscission-sensitive genotypes (Figure 8B). Comparative real-time PCR analysis showed that GhSACPD19 was significantly upregulated in abscission-sensitive materials compared to their resistant counterparts (Figure 8C and Figure S10). These findings establish the SACPD family, especially GhSACPD19, as key regulators of cotton pedicel abscission, and identify novel targets for dissecting organ abscission mechanisms.

4. Discussion

The abscission zone in plants exhibits high structural diversity and is typically composed of 2–50 layers of tightly arranged small cells, with cell wall degradation usually confined to a separation layer 1–5 cells wide [36,37]. In this study, dynamic cytological observations demonstrated that the sensitive material H35 rapidly differentiated to form 27 layers of abscission zone cells within one day of flowering, which stabilized at 25 layers (accounting for 92% of the initial cell count) after developmental adjustment. This indicated that the abscission zone cells in shedding susceptible cotton exhibited distinct tissue dissociation. The study systematically elucidated the bud and boll shedding regulatory mechanisms at the cellular and molecular levels. Moreover, the rapid response pattern in the abscission-sensitive cotton differed markedly from the gradual developmental mode observed in the abscission-resistant material, L32. Transcriptome sequencing analysis was performed in a critical developmental window of 10 days after flowering, which revealed 9964 DEGs. Functional annotation analysis revealed that key pathways, including α-linolenic acid metabolism, brassinosteroid biosynthesis, and glycerolipid metabolism, participated in shedding regulation by modulating cell membrane stability. While our analysis at this pivotal stage for cotton boll abscission has provided valuable insights, it is likely that additional regulatory events occur earlier or later. Future studies incorporating multiple developmental time points could therefore refine our understanding of the dynamic gene networks and identify stage-specific regulators. Notably, the GhSACPD19 gene (HC04_D10013030) was significantly correlated with cotton boll abscission, indicating that the temporally regulated differentiation of abscission zone cells, coordinated with specific metabolic pathways, constitutes the core molecular and cytological basis for this process. Beyond GhSACPD19, our transcriptome analysis also revealed several other hub genes that may contribute to abscission regulation. For example, HC04_D08019720 may regulate reactive oxygen species and cell wall remodeling, HC04_A01001860 could participate in metabolic or signaling processes, and HC04_A06005780 may function in cell recognition and signal transduction. These genes represent promising candidates for further functional studies to dissect the molecular network controlling cotton pedicel abscission.
In the present study, we conducted a genome-wide analysis of the SACPD gene family across five cotton species, including diploids (G. arboreum [Ga], G. herbaceum [Ghe], G. raimondii [Gr]) and tetraploids (G. barbadense [Gb] and G. hirsutum [Gh]). In total, 63 SACPD genes were identified, with counts of 9 (Ga), 9 (Gr), 7 (Ghe), 19 (Gb), and 19 (Gh) (Figure 5 and Table S4). Notably, tetraploid species harbored approximately twice as many SACPD genes as diploid species, which is consistent with the known polyploidization events that drive gene copy expansion [38,39]. Sequence analysis revealed that all SACPD proteins contained a single conserved PL00179 domain, suggesting a common evolutionary origin. Subcellular localization predictions indicated that most SACPD proteins were Chloroplast-localized (Table S4), which is consistent with their roles in lipid biosynthesis and membrane stability maintenance [40]. Similar structural and functional characteristics have been observed in other species, such as peanut [41] and barley [42]. Furthermore, emerging evidence has demonstrated that SACPD proteins participate in plant defense responses and enhance stress resistance in crops including rice [43] and cotton [44]. These findings suggest that SACPD genes have acquired functional divergence during plant evolution, enabling the coordinated regulation of multiple stress-response pathways and participation in diverse biological processes.
Reconstructing the evolutionary history of gene families provides a theoretical foundation for elucidating their molecular functions and regulatory networks. In the present study, phylogenetic analysis classified cotton and Arabidopsis SACPDs into four distinct evolutionary clades (Figure 5). Notably, cotton SACPDs exhibited high sequence similarity (>80%) with Arabidopsis AtSACPD1 and AtSACPD2, suggesting conserved biological functions in these plant species. Comparative genomic synteny analysis further revealed 10–14 highly conserved homologous gene pairs between diploid (A and D genomes) and tetraploid cotton (At and Dt subgenomes). These homologous genes were distributed across different chromosomes, and their distribution patterns indicated that whole-genome duplication events served as the primary evolutionary mechanism driving SACPD family expansion. This finding is consistent with the genomic doubling characteristics of cotton allopolyploidization [26,45]. Our results not only clarify the expansion history of the SACPD gene family in cotton but also provide novel evolutionary insights into its functional diversification. Previous studies have shown that SACPDs participate in both plant immunity and vegetative growth [46]. Loss of function of SACPD genes leads to the upregulation of salicylic acid (SA), while suppressing jasmonic acid (JA)-mediated defense responses [47]. While most functional studies on SACPDs have focused on their roles in lipid biosynthesis and lipid-derived plant defense mechanisms, reports on the involvement of SACPDs in organ abscission remain scarce. Integrative analysis of tissue-specific expression profiles and differential expression patterns collectively suggested that the SACPD gene family may participate in cotton boll abscission by regulating the developmental process of the pedicel abscission zone. This study not only deepens our understanding of the biological functions of the SACPD gene family but also identifies potential novel targets for high-yield cotton breeding.

5. Conclusions

Comparative transcriptome analysis of abscission zone (AZ) tissues between abscission-sensitive and abscission-resistant cotton lines revealed that GhSACPD19 acts as a key hub in regulating cotton boll abscission. Furthermore, genomic analysis revealed the presence of 63 distinct SACPD proteins in five cotton species (G. arboreum, G. herbaceum, G. raimondii, G. barbadense, and G. hirsutum). We also conducted comprehensive analyses of the gene expansion patterns, evolutionary relationships, and homologous gene clusters within the SACPD gene family. Expression profiling revealed distinct expression patterns of the GhSACPD genes during abscission zone formation. Collectively, these findings significantly advance our understanding of the SACPD gene family in cotton and underscore its potential to enhance fiber yield. These results provide mechanistic insights into the role of SACPD in cotton boll abscission and offer valuable genetic targets for future improvements in cotton boll abscission.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15202166/s1, Figure S1: Sampling scheme of the cotton pedicel abscission zone; Figure S2: Statistical analysis of abscission cell layers; Figure S3: Significant GO terms and KEGG pathway enriched in 2844 DEGs; Figure S4: Integrated Map of Transcriptome Network Construction and Characteristic Evaluation; Figure S5: Conserved motif architecture of cotton gene family; Figure S6: Gene structure and conserved motif analysis of SACPD family members in diploid cotton species; Figure S7: Chromosomal localization of SACPD gene family in diploid cotton; Figure S8: Chromosomal distribution characteristics of SACPD gene family in tetraploid cotton; Figure S9: Analysis of genomic synteny among diploid cotton species; Figure S10: Melting curves for quantitative real-time PCR (qPCR) analysis; Table S1: The primers for qRT-PCR; Table S2: Sample information and summary of sequencing reads; Table S3: Sizes of the co-expression modules identified by WGCNA; Table S4: Predicted Physicochemical Characteristics of SACPD Proteins Identified in Five Gossypium Species; Table S5: Analysis of Ka, Ks, and Ka/Ks of the SACPD Gene Family in G. hirsutum.

Author Contributions

Conceptualization, X.N. and Y.W.; formal analysis, Z.C. and P.H.; data curation, G.S. and Q.Z.; investigation, Z.L., H.L. and X.W.; data curation, Z.C.; writing—original draft, G.S. and Z.C.; writing—review and editing X.N., Y.W. and X.W. All authors have read and agreed to the published version of the manuscript.

Funding

The development fund for Xinjiang Talents XL (XL202403-06), the Tianshan Talents Program for the Top Young Innovative Talents (2023TSYCCX0117), the National Natural Science Foundation of China (No. 32260510) and the Science and Technology Planning Project of Tumusuk city-Open bidding for selecting the best candidates (KY2024JBGS09).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Morphological and cellular characterization of the cotton boll pedicel abscission zone. (A) Scanning electron micrographs showing the fracture surface of cotton boll pedicel between abscission-sensitive (H18, H35) and abscission-resistant (L32, L50) cotton varieties. overview (scale bar = 500 μm) and detail (scale bar = 100 μm). The blue arrows indicate rough and smooth regions within the abscission zone, highlighting structural differences related to abscission susceptibility; (B) cellular structure of the abscission layer in cotton boll pedicel. Paraffin-embedded sections were prepared from samples collected at 1, 4, 7, and 10 days post-anthesis (DPA). The experiment was independently repeated three times (n = 3). Scale bar = 200 μm.
Figure 1. Morphological and cellular characterization of the cotton boll pedicel abscission zone. (A) Scanning electron micrographs showing the fracture surface of cotton boll pedicel between abscission-sensitive (H18, H35) and abscission-resistant (L32, L50) cotton varieties. overview (scale bar = 500 μm) and detail (scale bar = 100 μm). The blue arrows indicate rough and smooth regions within the abscission zone, highlighting structural differences related to abscission susceptibility; (B) cellular structure of the abscission layer in cotton boll pedicel. Paraffin-embedded sections were prepared from samples collected at 1, 4, 7, and 10 days post-anthesis (DPA). The experiment was independently repeated three times (n = 3). Scale bar = 200 μm.
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Figure 2. Analysis of differentially expressed genes in the abscission zone. (A) Distribution of DEGs in extreme materials; (B) number of DEGs in extreme materials. N, non-abscission zone; T, abscission zone.
Figure 2. Analysis of differentially expressed genes in the abscission zone. (A) Distribution of DEGs in extreme materials; (B) number of DEGs in extreme materials. N, non-abscission zone; T, abscission zone.
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Figure 3. Significant GO terms and KEGG pathway enriched in 9964 DEGs. (A) Significant GO terms enriched in 9964 DEG; (B) significant KEGG pathways enriched in 9964 DEGs.
Figure 3. Significant GO terms and KEGG pathway enriched in 9964 DEGs. (A) Significant GO terms enriched in 9964 DEG; (B) significant KEGG pathways enriched in 9964 DEGs.
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Figure 4. Integrative analysis of cotton boll abscission-related gene co-expression networks. (A) Gene co-expression clustering dendrogram with module colors, (B) weighted gene co-expression network analysis (WGCNA), (C) GO enrichment analysis of MEyellow and MEbrown module genes, (D) KEGG pathway enrichment analysis of MEyellow and MEbrown module genes, and (E) co-expression network of module genes.
Figure 4. Integrative analysis of cotton boll abscission-related gene co-expression networks. (A) Gene co-expression clustering dendrogram with module colors, (B) weighted gene co-expression network analysis (WGCNA), (C) GO enrichment analysis of MEyellow and MEbrown module genes, (D) KEGG pathway enrichment analysis of MEyellow and MEbrown module genes, and (E) co-expression network of module genes.
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Figure 5. Phylogenetic analysis of the SACPD gene family in cotton.
Figure 5. Phylogenetic analysis of the SACPD gene family in cotton.
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Figure 6. Structural characteristics and conserved motif distribution of SACPD gene family in tetraploid cotton. (A) G. hirsutum; (B) G. barbadense.
Figure 6. Structural characteristics and conserved motif distribution of SACPD gene family in tetraploid cotton. (A) G. hirsutum; (B) G. barbadense.
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Figure 7. Evolutionary conservation of SACPD gene family synteny and homologs in Gossypium diploids and allotetraploids. (A) Syntenic analysis of the G. hirsutum genome; (B) syntenic analysis of the G. barbadense genome; (C) conservation of homologous genes between G. hirsutum and its diploid progenitors; (D) conservation of homologous genes between G. barbadense and its diploid progenitors.The positions of the genes are marked by red dots.
Figure 7. Evolutionary conservation of SACPD gene family synteny and homologs in Gossypium diploids and allotetraploids. (A) Syntenic analysis of the G. hirsutum genome; (B) syntenic analysis of the G. barbadense genome; (C) conservation of homologous genes between G. hirsutum and its diploid progenitors; (D) conservation of homologous genes between G. barbadense and its diploid progenitors.The positions of the genes are marked by red dots.
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Figure 8. Tissue-specific expression patterns of the SACPD gene family in cotton. (A) Expression profiling of the SACPD gene family across various tissues (from (left) to (right): root, stem, leaf, petal, anther, stigma, and 10-day fiber tissues); (B) expression analysis during abscission zone development in extreme materials (NAZ: non-differentiated abscission zone; AZ: differentiated abscission zone); and (C) relative transcript levels of GhSACPD19 in the abscission zone was analyzed by real-time PCR in four cotton varieties that exhibited extreme differences in boll abscission rates. The UBQ7 gene was used as an internal reference transcript. Results are presented as mean ± SD from three independent experiments (n = 3). One-way ANOVA was used to determine statistical significance (*** p < 0.001).
Figure 8. Tissue-specific expression patterns of the SACPD gene family in cotton. (A) Expression profiling of the SACPD gene family across various tissues (from (left) to (right): root, stem, leaf, petal, anther, stigma, and 10-day fiber tissues); (B) expression analysis during abscission zone development in extreme materials (NAZ: non-differentiated abscission zone; AZ: differentiated abscission zone); and (C) relative transcript levels of GhSACPD19 in the abscission zone was analyzed by real-time PCR in four cotton varieties that exhibited extreme differences in boll abscission rates. The UBQ7 gene was used as an internal reference transcript. Results are presented as mean ± SD from three independent experiments (n = 3). One-way ANOVA was used to determine statistical significance (*** p < 0.001).
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Table 1. The abscission rate of selected cotton materials.
Table 1. The abscission rate of selected cotton materials.
Accession IDDefoliant Sensitivity2018-KEL-AR12018-SHZ-AR12019-KEL-AR12019-SHZ-AR1Mean
L32R8.91 2.06 22.24 3.57 9.20
L50R2.15 1.60 37.60 3.40 11.19
H35S12.72 36.58 53.82 20.77 30.97
H18S25.74 38.72 51.81 43.51 39.95
Note: R, Boll abscission-resistant materials; S, Boll abscission-sensitive materials.
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MDPI and ACS Style

Shui, G.; Chang, Z.; Han, P.; Zhang, Q.; Li, Z.; Lin, H.; Wang, X.; Wu, Y.; Nie, X. Transcriptomic Analysis Identifies GhSACPD-Mediated Fatty Acid Regulation in the Cotton Boll Abscission. Agriculture 2025, 15, 2166. https://doi.org/10.3390/agriculture15202166

AMA Style

Shui G, Chang Z, Han P, Zhang Q, Li Z, Lin H, Wang X, Wu Y, Nie X. Transcriptomic Analysis Identifies GhSACPD-Mediated Fatty Acid Regulation in the Cotton Boll Abscission. Agriculture. 2025; 15(20):2166. https://doi.org/10.3390/agriculture15202166

Chicago/Turabian Style

Shui, Guangling, Zewei Chang, Peng Han, Qi Zhang, Zhibo Li, Hairong Lin, Xin Wang, Yuanlong Wu, and Xinhui Nie. 2025. "Transcriptomic Analysis Identifies GhSACPD-Mediated Fatty Acid Regulation in the Cotton Boll Abscission" Agriculture 15, no. 20: 2166. https://doi.org/10.3390/agriculture15202166

APA Style

Shui, G., Chang, Z., Han, P., Zhang, Q., Li, Z., Lin, H., Wang, X., Wu, Y., & Nie, X. (2025). Transcriptomic Analysis Identifies GhSACPD-Mediated Fatty Acid Regulation in the Cotton Boll Abscission. Agriculture, 15(20), 2166. https://doi.org/10.3390/agriculture15202166

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