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. 2023 Feb 10;4(5):100554. doi: 10.1016/j.xplc.2023.100554

Single-cell RNA landscape of the special fiber initiation process in Bombax ceiba

Yuanhao Ding 1,2,6, Wei Gao 4,6, Yuan Qin 5,6, Xinping Li 1,6, Zhennan Zhang 4, Wenjie Lai 1, Yong Yang 1, Kai Guo 3, Ping Li 1, Shihan Zhou 1, Haiyan Hu 1,2,∗
PMCID: PMC10518721  PMID: 36772797

Abstract

As a new source of natural fibers, the Bombax ceiba tree can provide thin, light, extremely soft and warm fiber material for the textile industry. Natural fibers are an ideal model system for studying cell growth and differentiation, but the molecular mechanisms that regulate fiber initiation are not fully understood. In B. ceiba, we found that fiber cells differentiate from the epidermis of the inner ovary wall. Each initiated cell then divides into a cluster of fiber cells that eventually develop into mature fibers, a process very different from the classical fiber initiation process of cotton. We used high-throughput single-cell RNA sequencing (scRNA-seq) to examine the special characteristics of fiber initiation in B. ceiba. A total of 15 567 high-quality cells were identified from the inner wall of the B. ceiba ovary, and 347 potential marker genes for fiber initiation cell types were identified. Two major cell types, initiated fiber cells and epidermal cells, were identified and verified by RNA in situ hybridization. A developmental trajectory analysis was used to reconstruct the process of fiber cell differentiation in B. ceiba. Comparative analysis of scRNA-seq data from B. ceiba and cotton (Gossypium hirsutum) confirmed that the additional cell division process in B. ceiba is a novel species-specific mechanism for fiber cell development. Candidate genes and key regulators that may contribute to fiber cell differentiation and division in B. ceiba were identified. This work reveals gene expression signatures during B. ceiba fiber initiation at a single-cell resolution, providing a new strategy and viewpoint for investigation of natural fiber cell differentiation and development.

Key words: Bombax ceiba, fiber initiation, single-cell RNA-seq

This study documents the novel mechanism of fiber cell initiation in B. ceiba, in which initiated fiber cells undergo an extra division process that results in a series of fiber cell clusters. Single-cell sequencing and comparative analysis with cotton (Gossypium hirsutum) were used to reconstruct the fiber initiation process of B. ceiba, revealing key regulators involved in fiber initiation.

Introduction

Bombax ceiba L., commonly known as the red silk cotton tree, is a tall, deciduous, diploid tree with a height up to 40 m. B. ceiba is widely distributed in tropical and subtropical regions and is an important component of the tropical dry deciduous forest ecosystem (Trapnell et al., 2014). B. ceiba is a multipurpose tree species of tropical forests with medicinal, economic, and ecological benefits (Sharma et al., 2020). The fiber from mature B. ceiba fruits is a new natural fiber source for the textile industry because it is extremely thin, light, and soft (Rijavec, 2008). B. ceiba fibers also have many special features that give them a wide range of uses: they are bright, antibacterial, moth-proof, mildew-proof, difficult to entangle, warm, hygroscopic, impervious to water, and not conductive of heat (Sunmonu and Abdullahi, 1992). The physical properties of B. ceiba fibers are well studied, but the biological processes that underlie their development remain to be clarified.

Both B. ceiba and cotton (Gossypium hirsutum) produce single-cell fibers, and both are species from the Malvaceae family, which suggests that their molecular mechanisms of fiber development are likely to be similar. Cotton fibers are the trichomes of the seed and develop from the outer epidermis of the ovule (Haigler et al., 2012). Previous studies have shown that the molecular mechanisms of fiber development in cotton may be similar to those of trichome development in Arabidopsis (Wang et al., 2013). The R2R3-MYB/bHLH/WD40 (MBW) complex of GL1 (GLABROUS1), TTG1 (TRANSPARENT TESTA GLABRA1), and GL3 (GLABRA 3) or EGL3 (ENHANCER OF GLABRA3) transcription factors (TFs) regulates GL2 (GLABRA 2), a downstream TF that is involved in trichome initiation in Arabidopsis (Ishida et al., 2008). Cotton homologs of these genes, such as GaMYB2 (homolog of AtGL1), GaHOX1 (homolog of AtGL2), GhDEL65 (homolog of AtGL3), and GhTTG1 and GhTTG3 (homologs of AtTTG1), can often rescue the phenotypes of homologous gene mutants in Arabidopsis and function in regulating cotton fiber initiation (Wang et al., 2004; Humphries et al., 2005; Guan et al., 2008; Shangguan et al., 2016). Interestingly, researchers have found that several important TFs have species-specific roles in cotton fiber initiation; these include GhMYB25, GhMYB25-like, PROTODERMAL FACTOR1 (GhPDF1), class-IV HD-ZIP factor (GhHD1), and Gh14-3-3(Machado et al., 2009; Walford et al., 2011, 2012; Deng et al., 2012; Zhou et al., 2015). Various hormone signals have also been shown to participate in cotton fiber initiation (Hu et al., 2016; Zhang et al., 2017; Zeng et al., 2019). Despite these findings, we lack a unified theory for fiber initiation. The natural fibers of B. ceiba are unusual in that they differentiate from the inner ovary wall, but the fiber initiation process in B. ceiba has received little attention.

The complexity of plant cells at the single-cell level has long been an active area of interest in plant biology (Ziegenhain et al., 2017). The application of single-cell RNA sequencing (scRNA-seq) methods has provided new insights into the heterogeneity of gene expression in different cells, the dynamics of cell types during development, and the expression characteristics of rare cell types (Wagner et al., 2016; Hwang et al., 2018). scRNA-seq has been widely used in model and nonmodel plants and yields complete cell-type-specific information for different tissues (Zhang et al., 2019; Liu et al., 2021). scRNA-seq can reveal the gene expression of cells throughout their differentiation (Liu et al., 2020). For example, the developmental trajectories from meristematic mother cells to guard mother cells in the stomatal lineage were analyzed by scRNA-seq in Arabidopsis (Liu et al., 2020). scRNA-seq of rice (Oryza sativa) roots was used to reconstruct the optimal developmental trajectory of epidermal cells and ground tissues, revealing conserved and differentiated root developmental pathways between dicots and monocots (Zhang et al., 2021). As a classical single-cell differentiation phenomenon, fiber initiation is a dynamic process that involves the transformation of epidermal cells into fiber cells. scRNA-seq offers researchers a chance to investigate changes in gene expression during fiber initiation at the single-cell level.

Fiber initiation serves as an ideal model system for studying cell growth and differentiation (Yang and Ye, 2013). B. ceiba fibers are single-cell fibers that develop from the epidermis of the inner ovary wall. The number of fiber initiation cells is one of the important factors that determine fiber yield. In this study, protoplasts isolated from inner ovary wall cells of B. ceiba were used to select a suitable cell population for scRNA-seq to clarify the key developmental processes and expressed genes involved in B. ceiba fiber initiation. The study of B. ceiba fiber initiation provides insights into plant cell fate specification and generates new gene resources for improving fiber yield and quality, especially in cotton.

Results

Identification of fiber initiation and protoplast isolation from B. ceiba

To confirm the timing of fiber initiation in B. ceiba, a large number of flowers and capsules were collected and observed at different days post-anthesis (DPA), and the developmental statuses of the ovaries and fibers were recorded (Figure 1A–1L). The results showed that the buds undergo a long period of vegetative growth before flowering (Figure 1A). At −3 DPA, the flower buds enter a rapid growth stage, and the petals clearly begin to elongate (Figure 1B). At −1 DPA, the flower buds have elongated and swelled to their maximum size (Figure 1C). The flower then blooms at midnight and remains open until the next morning (0 DPA, flowering day), with the petals curling outward (Figure 1D). The flower remains in full bloom for 2–4 days, after which time the petals gradually retract and fade, and the ovary begins to expand. The diameter and length of the fertilized capsule increase dramatically over time (Figure 1D–1L). After capsules have matured, soft fibers are shed from the inner wall and tightly surround the seeds, waiting for the wind to carry them far from the parent plant (Supplemental Figure 1). Our measurements indicated that the length of mature B. ceiba fibers ranges from 21 to 26 mm, and no fuzz fibers were found (Supplemental Figure 2A).

Figure 1.

Figure 1

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Identification and application of scRNA-seq in fibre initiation of B. ceiba.

(A–L) The development of the flower and capsule in B. ceiba: (A–E) development processes of flowers at −10, −3, −1, 0, and 3 days post anthesis (DPA); (F–L) development processes of capsules at 8, 11, 14, 16 and 30 DPA.

(M–O) Scanning electron microscopy (SEM) of the 11 DPA B. ceiba inner wall. Scare bars: 1 mm in (M); 500 μm in (N); 100 μm in (O).

(P–R) Workflow of the isolation of the inner ovary wall of B. ceiba protoplast cells and their loading onto the 10x Genomics platform.

From 8 to 11 DPA, some epidermal cells of the inner ovary wall begin to differentiate into fiber cells (Supplemental Figure 2B). The number of initiated fiber cells continues to increase as the capsules develop. Scanning electron microscopy revealed that some epidermal cells of the inner ovary wall had completed the initiation process, including fiber cell differentiation and division, by 11 DPA (Figure 1M–1O and Supplemental Figure 2B). Therefore, inner ovary walls of 11-DPA capsules were collected for protoplast isolation (Figure 1P–1Q). Samples with approximately 20 000 protoplasts were prepared and immediately mixed with 10x Genomics single-cell reaction reagents and cell barcodes. Processed samples were then used for library construction and sequencing on the 10x Genomics scRNA-seq platform (Figure 1R).

scRNA-seq and cell-type cluster identification

Transcriptome data were obtained from 16 000 valid single cells using the 10x Genomics scRNA-seq platform (Supplemental Figure 3). To ensure the quality of the single-cell data, only cells with gene counts between 300 and 6000 were retained (Supplemental Figure 4A), and cells with more than 20 000 unique molecular identifiers (UMIs) were removed (Supplemental Figure 4B). Finally, 15 567 high-quality cells were retained, and 43 450 genes were identified, with an average of 3167 UMIs and 1896 genes per cell (Supplemental Table 1). The 15 567 single cells were classified into 10 clusters by unsupervised clustering analysis, and the data were visualized by Uniform Manifold Approximation and Projection (UMAP) (Figure 2A). The number of cells varied among clusters, from cluster 0 with 6958 cells (45.49%) to cluster 9 with 37 cells (0.24%) (Figure 2B).

Figure 2.

Figure 2

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Identification and application of scRNA-seq in fibre initiation of B. ceiba.

(A) Uniform manifold approximation and projection (UMAP) visualization of 10 cell clusters. Individual cells are represented by dots; n = 15567; cell clusters are coloured differently.

(B) Number and percentage of cells in each cluster.

(C) Expression patterns of cluster-specific marker genes. The size of the circle represents the percentage of cells with expression (pct.exp.), and the colour represents the average expression after scaling (avg.exp.scale).

(D) RNA in situ hybridization of HD1, TUA9, CPC and CUT1 in 11 DPA capsules with the sense probe as a negative control. Red arrows indicate fibre cells, and black arrows indicate non-fibre epidermic cell. Scale bar, 50 μm.

We sought to identify two specific cell types associated with B. ceiba fiber initiation: initiated fiber cells and inner ovary wall epidermal cells. Marker genes specifically expressed in initiated fiber cells of cotton were used to identify the initiated fiber cell cluster in B. ceiba; these included MYBMIXTA-Like 4 (MML4) (Tian et al., 2020), MYB25 (Machado et al., 2009), HD-ZIP homeobox 3 (HOX3) (Shan et al., 2014), DEL65 (Shangguan et al., 2016), HD1 (Walford et al., 2012), and PDF1 (Deng et al., 2012). The homologous B. ceiba genes were specifically expressed in cluster 6 (Figure 2C). Likewise, marker genes involved in differentiation of stomatal guard cells and inhibition of fiber initiation, such as CPC (Liu et al., 2015), HIC (Gray et al., 2000), MYB61 (Liang et al., 2005), FLP (Lai et al., 2005), and CUT1 (Millar et al., 1999), were found in cluster 3 (Figure 2C and Supplemental Table 2). RNA in situ hybridization with four representative genes (HD1, TUA9, CPC, and CUT1) from cell clusters 3 and 6 provided additional support for the annotation of these clusters as epidermal and fiber cells (Figure 2D). Thus, cells from cluster 6 were initiated fiber cells, and cells from cluster 3 were epidermal cells of the inner ovary wall.

Identification of marker genes involved in B. ceiba fiber initiation

To identify the specific marker genes involved in B. ceiba fiber initiation, 347 specifically expressed genes (SEGs) were identified in fiber initiation cell cluster 6 (FICC6) (Supplemental Table 3). The expression levels of the top 54 most highly expressed genes are depicted as a heatmap (Figure 3A); these genes were specifically expressed in FICC6. Six highly expressed representative genes from FICC6 (SCL28, WPRB, AGP1, PIP2-8, RAD50, and AUR1) are displayed in UMAP map clusters (Figure 3B). We found that these genes were most highly expressed and concentrated in FICC6. RNA in situ hybridization with PIP2 and SCL28 confirmed their gene expression patterns in FICC6 (Figure 3C). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that the 347 SEGs from FICC6 were enriched mainly in RNA modification, ribonucleoprotein complex biogenesis, and the ribosome pathway (Figure 3D and 3E).

Figure 3.

Figure 3

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Identification of a new marker gene for fibre initiation clusters in B. ceiba.

(A) Heatmap showing the top specifically expressed genes (SEGs) with the highest expression levels (log2fold change) in the fibre initiation cluster.

(B) Expression patterns of six new marker genes on the UMAP map.

(C) RNA in situ hybridization of PIP2 and SCL28 in 11 DPA capsules with the anti-sense probes, and sense probes were used as negative controls. Red arrows indicate staining fibre cells, and black arrows indicate unstaining fibre cells from negative controls. Scale bar, 50 μm.

(D) GO enrichment analysis of all SEGs in the fibre initiation cluster.

(E) KEGG enrichment analysis of all SEGs in the fibre initiation cluster.

Differentiation trajectory of initiated fiber cells from the epidermis

The ability of scRNA-seq to capture cells in different developmental states makes it possible to study successive differentiation trajectories during tissue or cell development. To further explore the developmental processes of initiated fiber cells, pseudotime trajectory analysis was performed using cell clusters 3 (inner wall epidermis) and 6 (initiated fiber cells) in Figure 2A. For fibers that developed from the epidermis, the epidermal cell cluster was considered the origin of the developmental trajectory, and the bifurcation point marked cells with changes in differentiation state (Figure 4A). The pseudotime trajectory showed that cells eventually differentiated into two cell types: differentiated epidermal cells (branch 1) and initiated fiber cells (branch 2) (Figure 4A and 4B and Supplemental Figure 5). To further classify the cell types of branches 1 and 2, expression levels of the epidermal marker gene HIC and the fiber initiation marker gene PDF1 are shown in the branches. HIC was found at the end of branch 1 (Figure 4C) and is mainly involved in differentiation of mature stomatal guard cells, whereas PDF1 was mainly found at the end of branch 2 (Figure 4D) and is related to fiber initiation. Expression patterns of six more genes from branches 1 and 2 further supported the cell-type classifications of these branches (Supplemental Figure 6). Cells from epidermal and initiated fibers were continuously distributed along branch 2 (Figure 4B), demonstrating that pseudotime analysis essentially revealed the development of fiber initiation. The top 10 SEGs involved in cell differentiation from epidermal cells to other tissues or fiber cells are shown in Figure 4E. These genes may play a role in the differentiation of stomata and fiber initiation cells from the inner epidermis of the ovary.

Figure 4.

Figure 4

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Differentiation trajectory of epidermis and fibre initiation.

(A) Pseudotime trajectory of fibre initiation and epidermal cell development. Each dot indicates a single cell.

(B) Pseudotime trajectory of single-cell transcriptomics data coloured according to cluster. Each dot indicates a single cell.

(C–D) Expression of marker genes by cell type on a pseudotemporal trajectory. Colours indicate gene expression level in individual cells.

(E) Pseudotime expression trajectories of branch-dependent top 10 SEGs. Branch 1 indicates the differentiation directions to epidermic cell, and initiated fibre cell of Branch 2. State 1-3 represent three differentiated situations of single cells.

(F) Trends in gene expression with branching in cells based on different differentiation fates. The GO term enriched significantly in each cluster is shown on the right.

(G) A transcription factor (TF) regulatory network constructed by all identified TFs from initiated fibre cell clusters and epidemic cell clusters. Red blocks represent TFs reported associate to fibre initiation; yellow blocks show TFs related to fibre development or elongation; purple blocks mean TFs related to cell division. Line thickness indicates the strength of association between TFs.

The expression levels of all SEGs of inner wall epidermal cells (branch 1) and fiber initiation cells (branch 2) are shown in a heatmap (Figure 4F), and the SEGs were classified into five modules (M1–5) according to their expression profiles (Supplemental Table 4). Genes from M1 and M2 were extremely highly expressed in cells from the ends of branches 1 and 2 (Figure 4E), suggesting that these genes functioned in the processes of stomatal cell differentiation and fiber initiation, respectively. Genes from M4 were highly expressed in cells of the prebranch, indicating that they may help to maintain the undifferentiated state of primitive epidermal cells or to prepare for the differentiation stage. Genes from M3 and M5 were more highly expressed in cells of the transition state, implying that they probably functioned in the differentiation of fiber cells or stomatal cells. GO analysis of genes from M1–5 showed that genes in M2 were mainly enriched in structural molecule activity and ribosome-related processes (Figure 4F) and possibly functioned in differentiated fiber cell growth. On the other hand, genes from M1 and M5 were enriched in phenylpropanoid metabolic processes and ubiquitin-like protein ligase binding processes, respectively, and genes from M3 and M4 were mainly involved in response to stimuli (Figure 4F). GO analysis thus showed that genes from different modules were enriched in specific biological processes, implying that strict temporal and spatial regulation of gene expression determines the direction of epidermal cell differentiation in B. ceiba.

Fiber initiation is controlled by many TFs. To identify TFs that potentially regulate fiber initiation in B. ceiba, we identified those that were specifically expressed in the inner wall epidermal cell cluster and the initiated fiber cell cluster. In total, 528 TFs were identified, and members of the MYB (43 genes) and bHLH families (54 genes) were the most highly specifically expressed TFs in the initiated fiber cell cluster (Supplemental Figure 7 and Supplemental Table 5). All identified TFs were used to construct a regulatory network showing their relationships (Figure 4G). Within this network were some important TFs reported to participate in fiber initiation, such as HD1 (Walford et al., 2012), BZR1 (Zhou et al., 2015), MYB2 (Wang et al., 2004), TRY (Liu et al., 2015), and HOX1 (Huang et al., 2021) (Figure 4G). Several TFs strongly related to cell division were also found, such as WOX3 (Shimizu et al., 2009), DOF3.4 (Skirycz et al., 2008), SCL28 (Goldy et al., 2021), and MYB88 (Yang et al., 2014) (Figure 4G), as were fiber development- or elongation-related TFs such as SLR1 (Ueguchi-Tanaka et al., 2008), MYB106 (Jakoby et al., 2008), ETC1 (Kirik et al., 2004), PDF2 (Qin et al., 2022), and GEBPL (Shaikhali et al., 2015) (Figure 4G). Specifically expressed TFs in the fiber initiation cluster were then subjected to GO and KEGG enrichment analysis. The cell cycle process was the most enriched GO biological process (Supplemental Figure 8A), and enrichment of the ribosome, spliceosome, and starch and sucrose metabolism pathways was revealed by KEGG pathway analysis (Supplemental Figure 8B).

Similarities and differences in fiber initiation between cotton and B. ceiba

Cotton fibers originate from the outer epidermal cells of the ovule, whereas B. ceiba fibers differentiate from the epidermis of the inner ovary wall. To explore the evolutionary conservation and differences in fiber cell initiation between cotton and B. ceiba, we performed an integrated analysis with single-cell datasets of initiated fiber cells from G. hirsutum (TM-1) (Qin et al., 2022) and B. ceiba. A total of 518 SEGs related to cotton fiber initiation were identified in the single-cell dataset from epidermal cells of cotton ovules (Supplemental Table 7). These 518 SEGs were aligned to the B. ceiba genome, revealing 277 orthologous genes (Supplemental Table 8). Of the 347 SEGs identified in FICC6 of B. ceiba, only 74 were shared with cotton (Figure 5A). By contrast, 203 and 273 SEGs were specifically expressed in cotton and B. ceiba FICC6, respectively (Supplemental Tables 8 and 9), and were then used for GO enrichment analysis. Most SEGs from cotton and B. ceiba were enriched in the same biological processes, but only SEGs from B. ceiba were enriched in mitotic cytokinesis and cell division (Figure 5B and 5C). In contrast to cotton fibers, initiated fiber cells of B. ceiba undergo continuous mitosis, producing many initiated cell clusters on the epidermis of the inner ovary wall, which then elongate into mature fibers (Supplemental Figure 2B). SEGs related to cell division in B. ceiba included CYCB2-1 (Lee et al., 2003), CYCB1-2 (Romeiro Motta et al., 2022), CDKB 2-2 (Andersen et al., 2008), MYB88 (Yang et al., 2014), and SCL28 (Goldy et al., 2021), all of which participate in the mitotic cycle of plant cells (Figure 5D). SCL28 was located in the center of the TF network and is related to the known important regulator HD1 (Figure 4G). Thus, we speculate that SCL28 may have an important role in cell cluster formation during fiber initiation in B. ceiba.

Figure 5.

Figure 5

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Evolutionary conservation and divergence of fibre initiation cell types in cotton and B. ceiba.

(A) Venn diagram showing the number of homologous and SEGs in B. ceiba and cotton fibre initiation cell types.

(B–C) GO analysis of genes specifically expressed in B. ceiba and cotton.

(D) Specific expression of B. ceiba genes in cell cycle regulation. Four stages of cell mitotic cycle are Gap1 (G1), Synthesis (S), Gap2 (G2) and Mitotic (M). The complexes surround mitotic cycle are regulators for each stage. The specific genes found in B. ceiba initiated fibre cell clusters are marked in red.

(E) Gene family expansion and contraction in B. ceiba and G. hirsutum. Red blocks showed extremely more members found in G. hirsutum of DRP1A gene family. Gray and brown blocks represent gene numbers of gene family. Red, green and black font gene names represent genes reported function in fibre initiation, speculative function in fibre initiation and related to cell division process, respectively.

(F) Heatmap showing the expression level of representative genes at single-cell level in fibre and epidermic cell clusters in B. ceiba and G. hirsutum. Meaning of red, green and black font gene names were the same as above description.

(G) RNA in situ hybridization of two cell division related genes, CYCB1-2 and CYCB2- 1 in 11 DPA capsules with the sense (negative control) and antisense probes. Red arrows indicate staining fibre cells, and black arrows indicate unstaining fibre cells from negative controls. Scale bar, 50 μm.

To further analyze differences in fiber initiation between B. ceiba and cotton at the genome level, we identified gene family expansions and contractions of key fiber initiation regulators in B. ceiba and G. hirsutum and analyzed their expression differences at the single-cell level (Figure 5E and 5F). Most of these gene families were expanded in G. hirsutum compared with B. ceiba, especially DRP1A (red bar), which has 271 members in G. hirsutum, 10 times more than in B. ceiba (Figure 5E). Heatmap analysis showed that cell division–related genes (black font) were highly expressed in the B. ceiba fiber cell cluster compared with cotton, which is consistent with differences in fiber initiation between the two species; i.e., initiated fiber cells of B. ceiba undergo additional cell divisions that do not occur in cotton (Figure 5F). Some genes with previously reported functions in fiber initiation (red font) were highly expressed in the fiber cell cluster (Supplemental Table 10). We performed RNA in situ hybridization with CYCB1-2 (Romeiro Motta et al., 2022) and CYCB2-1 (Lee et al., 2003) to validate the expression patterns of cell division–related genes in the B. ceiba ovary and found that these genes were highly expressed in initiated fiber cells (Figure 5G).

Initiation process of fiber cells in B. ceiba

A summary of the regulatory factors associated with fiber initiation in B. ceiba is shown in Figure 6. Like cotton fiber development, B. ceiba fiber development comprises three stages: fiber initiation, elongation, and maturation. In cotton, fiber cells initiate from seed epidermal cells and enter the elongation stage directly. By contrast, B. ceiba fiber cells undergo an extra cell division process after differentiation from the epidermal cells of the inner ovary wall, resulting in a series of fiber cell clusters (Figure 1O and Supplemental Figure 2B) that subsequently elongate into long fiber cells. SEGs from B. ceiba, including PDF1 (Deng et al., 2012), HD1 (Walford et al., 2012), HOX1 (Huang et al., 2021), DEL65 (Shangguan et al., 2016), MYB2 (Wang et al., 2004), BZR1 (Zhou et al., 2015), and CPC/TRY (Liu et al., 2015), are known to function in fiber initiation in cotton. In this study, we suspected that GEBPL (Shaikhali et al., 2015), HOX3 (Shan et al., 2014), MYB106 (Jakoby et al., 2008), and SLR1 (Ueguchi-Tanaka et al., 2008) might be involved in fiber initiation in B. ceiba. The primary initiated fiber cells of B. ceiba continue to divide into multicellular groups, and cell cycle regulators such as CDKB2-2 (Andersen et al., 2008), CYCB1-2 (Romeiro Motta et al., 2022), CYCB2-1 (Lee et al., 2003), SCL28 (Goldy et al., 2021), and MYB88 are considered to function in fiber initiation of B. ceiba. Enriched microtubule-associated proteins such as MAP65-1 (Smertenko et al., 2006), DRP1A (Mravec et al., 2011), TPX2 (Bird and Hyman, 2008), and KIN12E (Herrmann et al., 2021) may also participate in fiber cell mitosis during this process. Once fiber initiation is complete, fiber cells transition to the elongation stage.

Figure 6.

Figure 6

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Schematic diagram of gene regulation related to cell differentiation and development in B. ceiba-initiating fibres.

Early developmental process of B. ceiba are divided into three stages including protrusion, cell division and elongation; protrusion and cell division stages are together composed the fibre initiation process. Regulators and network upon ‘Protrusion’ block are reported proteins involved in fibre initiation process, proteins with red markers were proteins found in B. ceiba. Regulators under ‘Protrusion’ block are suspected proteins that potential related to the fibre initiation of B. ceiba according to the network analysis in Figure 4G that strong connected with the reported regulators. Reported regulators related to ‘Cell division’ were divided into cell cycle and microtubule-associated related types and marked with green blocks.

Discussion

Traditional transcriptome sequencing techniques measure the average expression levels of genes in a population of cells, precluding accurate assessment of gene expression in individual cells or cell types and thus obscuring the heterogeneity of expression among different cells (Tang et al., 2011). Single-celled natural fibers, including those of cotton and cottonwood, are the best material for studying single-cell differentiation and development. scRNA-seq is an excellent method for investigating the molecular mechanisms that underlie fiber cell initiation and development at the single-cell level. The natural fibers of B. ceiba have unique properties and specific developmental processes not found in cotton fibers, indicating the presence of a species-specific regulatory network and offering potential genetic resources for research on fiber development. Using scRNA-seq to study the initiation of cotton fiber cells can provide important reference data and new genetic resources for fiber initiation in cotton.

Characteristics of natural fiber differentiation

Based on scRNA-seq combined with GO and KEGG enrichment analyses, 347 SEGs were identified in B. ceiba FICC6 and found to be strongly enriched in ribosome-related pathways (Figure 3D and 3E). Previous studies have shown that the nucleus is significantly enlarged in epidermal cells that differentiate into fiber-initiating cells (de Moura et al., 2020), indicating that ribosomal RNA (rRNA) biosynthesis is necessary for fiber cell differentiation (Bahadori, 2019). In a recent cotton scRNA-seq study (Qin et al., 2022), the GO terms “regulation of ethylene-activated signalling pathway,” “sucrose alpha-glucosidase activity,” and “cuticle hydrocarbon biosynthetic process” were enriched in a gene module analysis of cotton fiber initiation, and these terms were previously reported to be enriched during cotton fiber elongation (Shi et al., 2006). Our pseudotime analysis showed that the epidermis differentiated into two main cell types: initiated fiber cells and differentiated epidermal cells (Figure 4A and 4B). A large number of guard cells are distributed on the epidermis of the inner ovary wall surface of B. ceiba (Supplemental Figure 2B). In cotton, ovule epidermal cells can also develop into fibers and stomata (James Mc, 1975). Here, the marker gene HIC (Gray et al., 2000), which has been reported to function in stomatal development, was highly expressed in differentiated epidermal cell clusters (Figure 4C, Branch 1). Thus, natural fiber initiation involves at least two developmental trajectories: initiated fiber cells and stomatal cells.

Genes associated with the differentiation of B. ceiba fiber cells

The initiation of fiber development is a complex process whose molecular regulatory mechanisms remain to be fully clarified. Some important genes such as HD1 and MYB25 have been shown to play essential roles in cotton fiber initiation, but very little is known about the analogous process in B. ceiba. In cotton, fiber initiation is regulated by many TFs such as R2R3-MYB (GhMML4 and GhMYB25) (Machado et al., 2009; Tian et al., 2020), bHLH (GhDEL65) (Shangguan et al., 2016), and HD-ZIP IV (GbPDF1 and GhHD1) (Deng et al., 2012; Chen et al., 2017). Here, their orthologs were specifically expressed in FICC6 of B. ceiba (Figure 2C), indicating that the fiber initiation mechanisms of cotton and B. ceiba share some similarities. Orthologs of other cotton TFs such as TRY (Ishida et al., 2008), BZR1 (Zhou et al., 2015), MYB2 (Wang et al., 2004), and HOX1 (Huang et al., 2021), which are involved in regulating cotton fiber initiation, were also specifically expressed in FICC6 of B. ceiba (Figure 4G). In Arabidopsis, the TRY protein has been shown to move to neighboring cells and compete with GL1 for GL3/EGL3 binding, resulting in inhibition of trichome development (Ishida et al., 2008). In this study, TRY was expressed in FICC6 of B. ceiba (Figure 4G), indicating that it also functions in B. ceiba’s fiber initiation process. Some other TFs whose genes were specifically expressed in B. ceiba, such as SLR1 (Ueguchi-Tanaka et al., 2008), MYB106 (Jakoby et al., 2008), HOX3 (Shan et al., 2014), and GEBPL (Shaikhali et al., 2015), were found to interact with these reported genes in the regulatory network (Figure 4G). In cotton, GhSLR1 is a blocker of the phytohormone gibberellic acid; it interferes with stabilization of the GhHOX3–GhHD1 complex, inhibits transcription of downstream target genes, and ultimately suppresses fiber elongation (Shan et al., 2014). GEBPL encodes a GLABROUS1 enhancer-binding protein that promotes trichome generation (Curaba et al., 2003). GL1 is an important regulator of the MYB-bHLH-WD40 (MBW) ternary complex in Arabidopsis, controlling the production of trichomes (Larkin et al., 1994). Thus, we suspect that GEBPL, MYB106, HOX3, and SLR1 play important roles in fiber initiation of B. ceiba.

Similarities and differences in fiber initiation of cotton and B. ceiba

There are two major similarities between B. ceiba fibers and cotton fibers: both are single cells generated from epidermal cells, and both undergo a similar developmental process that includes initiation, elongation, and dehydration. Many key regulators of cotton fiber initiation were also expressed during fiber initiation in B. ceiba, including PDF1, TRY, BZR1, HD1, HOX1, and HOX3, indicating that fiber initiation in the two species involves similar regulatory mechanisms. However, there are also two main differences in fiber initiation between cotton and B. ceiba. First, fibers of B. ceiba originate from epidermal cells of the inner ovary wall (Figure 1M–1O and Supplemental Figure 2B), and second, the initiated fiber cells of B. ceiba undergo cell division and form clusters of single fiber cells that eventually develop into mature fibers (Figure 1M–1O and Supplemental Figure 2B). Cotton fibers are individual cells initiated from outer epidermal cells of the ovule that elongate and dehydrate to become mature fibers (Stewart, 1975). Here, gene expression profiling revealed that SEGs in FICC6 from B. ceiba were enriched in GO terms related to mitosis and cell division (Figure 5B), and TF network analysis indicated that the cell cycle TF SCL28 might be an important regulator of cell cluster generation (Figure 4G). SCL28 belongs to the GRAS family (Goldy et al., 2021); it accumulates during the G2/M phase of the cell cycle, facilitates the progression of cell division through G2/M, and regulates the selection of cytokinesis planes (Goldy et al., 2021). Other SEGs related to the cell cycle or mitotic cytokinesis, such as CYCB2-1, CYCB1-2, CDKB2-2, and MAP65-1, were uniquely expressed in B. ceiba FICC6 (Figure 6). CYCBs have been reported to regulate the G2/M transition and induce mitotic cycles in endoreduplicating Arabidopsis cells (Schnittger et al., 2002), and the cyclin-dependent kinase CDKB2-2 is essential for normal cell cycle progression and meristem integrity (Andersen et al., 2008). Cell division depends on the control of microtubule dynamics and organization (Banerjee et al., 2020). MAP65-1 has been shown to have a cell cycle–dependent microtubule binding pattern (Smertenko et al., 2006), and it was specifically expressed in FICC6 of B. ceiba. In conclusion, fiber initiation in cotton and B. ceiba involves similar regulatory mechanisms, but B. ceiba fiber initiation also involves cell division and corresponding regulators, which may be applicable for improvement of fiber yield in cotton.

Methods

Observation of B. ceiba fiber initiation

Plant materials were collected from B. ceiba trees (>10 years of age) planted on the campus of Hainan University in Haikou, China (20°3′23.07″N, 110°19′29.388″ E). To confirm the timing of fiber initiation in B. ceiba, capsules were photographed from 0 DPA to 11 DPA using an SU3500 scanning electron microscope (Hitachi, Japan) in order to observe the numbers of initial cells on the inner ovary walls. Mature fibers of B. ceiba form fiber agglomerates that facilitate seed dispersal by the wind. Mature capsules of B. ceiba were collected from five different plants, and more than 200 fiber agglomerates were randomly selected from each plant for fiber length measurements.

Protoplast isolation from the inner ovary wall of B. ceiba

All 11-DPA capsules of B. ceiba were observed by scanning electron microscopy to ensure that fiber cells had been initiated. Selected capsules were dissected, ovules were removed, and the innermost ovary wall layers were obtained and cut into 1- to 2-mm strips. Samples were then placed in a tube containing 20 ml of enzyme solution (2% [w/v] Cellulase R10; 1% [w/v] Macerozyme R10; 1% hemicellulase; 20 mM MES, pH 5.7; 0.5 M mannitol; 20 mM KCl; 10 mM CaCl2; 0.1% BSA [w/v]). After digestion at 26°C with shaking at 25 rpm for 4 h, an equal volume of PBS solution (without calcium and magnesium) was added to the tube, and the tube was gently shaken to release the protoplasts. Protoplasts were then filtered through a 40-μm nylon mesh, centrifuged at 300 g for 5 min at 4°C, and washed three to four times with PBS solution. A small amount of single-cell suspension was taken, and an equal volume of 4% Trypan blue was added for viability determination. Samples were considered acceptable if the percentage of viable cells was greater than 85% of the total cell number. The cells were counted with a Countess II Automated Cell Counter, and the live cell concentrations were adjusted to 1500 to 2000 cells/μl.

scRNA-seq library construction

scRNA-seq libraries were sequenced on the Illumina sequencing platform by Gene Denovo Biotechnology (Guangzhou, China). Protoplasts were loaded onto a 10x Genomics Chromium Controller to generate single-cell gel beads in emulsion (GEMs) in which lysis and barcoded reverse transcription of mRNA were performed. The products of all GEMs were mixed, and a standard sequencing library was constructed. The cDNA was amplified by PCR using the sequencing primers R1 and R2 and ligated to Illumina sequencing adapters for the P5 and P7 flow-cell binding sites. High-throughput sequencing of the constructed libraries was performed on the Illumina sequencing platform to generate 150-bp paired-end reads.

scRNA-seq data processing and analysis

The scRNA-seq data were aligned to the B. ceiba reference genome (Sequence Read Archive PRJNA429932) (Gao et al., 2018). Raw scRNA-seq data were aligned, filtered, and normalized using Cell Ranger 3.1.0 software (10x Genomics), resulting in a gene expression matrix for each cell. The Seurat (v3.1.0) R toolkit was used to explore QC indicators, filter cells, visualize gene and molecular counts, plot their relationships, and exclude cells with clearly abnormal numbers of gene tests as potential multiples (Butler et al., 2018). Data normalization was performed using the global-scaling normalization method “LogNormalize” (Risso et al., 2014). To overcome the extensive technical noise for any single gene in scRNA-seq data, we used the UMAP method to place cells with similar neighborhoods in high-dimensional space into lower dimensional space for data visualization (Becht et al., 2018).

Analysis of SEGs

To analyze patterns of transcriptional regulation in individual cell subsets, likelihood-ratio tests were performed to identify SEGs in individual cells and compare them with all other cells. SEGs were identified using the following parameters: p value ≤ 0.01, log2FC ≥ 0.36, and percentage of cells in which the gene was detected in a specific cluster >25%. GO enrichment analyses were performed based on gene expression levels to identify the main functions of the clusters. To analyze marker genes of the initiation cluster, we selected the top genes according to the results of the SEG analysis and used TBtools to draw a gene expression heatmap (Chen et al., 2020).

Pseudotime trajectory analysis

The Monocle package (version 3.0) was used to construct cell differentiation trajectories for selected clusters (Trapnell et al., 2014). The trajectory was visualized as a tree-like structure with tips and branches. Gene expression profiles in Monocle were used to identify genes that were specifically expressed between groups of cells and to assess the statistical significance of those findings (Qiu et al., 2017). Pseudotime analysis of fiber development was performed, and genes with similar expression trends were grouped. The branch point was selected to identify genes that contributed to branching of the developmental trajectory. The differential gene test function was used to identify pseudotime-dependent and branch-dependent genes. Genes that were significantly branch dependent were visualized using the plot-genes-branched-heatmap function.

RNA in situ hybridization

RNA in situ hybridization experiments were carried out as described previously (Zhang et al., 2017). In brief, gene-specific probes were prepared as described in the manual of the DIG Northern Starter Kit (Roche); primers used in this study are listed in Supplemental Table 11. Samples were prepared from 11-DPA capsules of B. ceiba and embedded in paraffin for later use; 10-μm paraffin sections were obtained, de-paraffinized, rehydrated, and incubated overnight with the RNA probe in darkness. After a series of washes with SSC (saline–sodium citrate) and NTE (0.5 M NaCl, 10 mM Tris [pH 8.0], 1 mM EDTA) buffers, the non-specifically bound probe was removed. Alkaline phosphatase–conjugated anti-digoxigenin (anti-Dig-AP, Roche) and nitro-blue tetrazolium/5-bromo-4-chloro-3-inodyl-phosphate (NBT/BCIP) color substrate solution (Roche) were used for color development. Ovary sections of B. ceiba were photographed using a CX33 microscope (Olympus, Tokyo, Japan) in bright-field mode.

Comparative analysis of fiber initiation-related gene clusters generated by scRNA-seq in G. hirsutum and B. ceiba

scRNA-seq data from the outermost layer of cotton ovules were recently published by the Group of Cotton Genetic Improvement, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei, China2 (Qin et al., 2022). To investigate the similarities and differences in fiber development between B. ceiba and cotton, orthologous genes were first identified by blasting the 518 SEGs in the cotton FICC against B. ceiba protein sequences with an e-value threshold of 1e−5. To analyze gene family expansion and contraction, protein sequences were obtained from the G. hirsutum TM-1 genome (Wang et al., 2019) and the B. ceiba genome (Gao et al., 2018); gene family members were identified using blast tools under the screening condition of p value <10−5.

Funding

This work was supported by the National Natural Science Foundation of China (31960437).

Author contributions

H.Y.H. and Y.H.D. conceptualized and designed the experiments. X.P.L. performed most of the experiments under the supervision of H.Y.H. and guidance of Y.H.D. W.G., Y.Q., and Z.N.Z. completed the RNA in situ hybridization experiments, and Y.H.D. captured the figures. W.J.L., K.G., and S.H.Z. helped to collect the samples. Y.Y. and P.L. provided help with bioinformatics analysis. Y.H.D. and X.P.L. wrote the manuscript with the support of all other authors. K.G., Y.Y., and H.Y.H. critically revised the manuscript. W.G. and Y.Q. added the RNA in situ hybridization experiment.

Acknowledgments

The support for Kai Guo and Yong Yang in writing the manuscript is greatly appreciated. We thank Wenjie Lai and Sihan Zhou for support with sample collection and protoplast isolation. Thanks to Kai Guo for help with sample observation by scanning electron microscopy and Yong Yang and Ping Li for support with bioinformatics analysis. We also thank Wei Gao, Yuan Qin, and Zhennan Zhang for help with the RNA in situ hybridization experiments. We show our appreciation to Guangzhou Gene Denovo Biotechnology Co., Ltd. for support with single-cell sequencing. No conflict of interest is declared.

Published: February 10, 2023

Footnotes

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.

Supplemental information

Document S1. Supplemental Figures 1–8
mmc1.pdf (1.2MB, pdf)
Data S1. Supplemental Tables 1–11
mmc2.xlsx (8.5MB, xlsx)
Document S2. Article plus supplemental information
mmc3.pdf (5.9MB, pdf)

Data availability

The raw sequencing data are available at the National Center for Biotechnology Information Sequence Read Archive under BioProject accession number PRJNA850395 and the China National Genomics Data Center (https://ngdc.cncb.ac.cn) under BioProject accession number PRJCA014500.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Figures 1–8
mmc1.pdf (1.2MB, pdf)
Data S1. Supplemental Tables 1–11
mmc2.xlsx (8.5MB, xlsx)
Document S2. Article plus supplemental information
mmc3.pdf (5.9MB, pdf)

Data Availability Statement

The raw sequencing data are available at the National Center for Biotechnology Information Sequence Read Archive under BioProject accession number PRJNA850395 and the China National Genomics Data Center (https://ngdc.cncb.ac.cn) under BioProject accession number PRJCA014500.

Articles from Plant Communications are provided here courtesy of Elsevier

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