Comparative Analysis of Alternative Splicing in Moso Bamboo and Its Dwarf Mutant, Phyllostachys edulis ‘Tubaeformis’

Abstract

Internode length is a crucial phenotypic trait of bamboo, significantly impacting its processing and utilization. Phyllostachys edulis ‘Tubaeformis’ (Shengyin Bamboo), a variety of Moso bamboo, exhibits drastically shortened internodes, making it a valuable ornamental bamboo species. We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin bamboo, and compared the differences between the two. Our sequencing data unveiled 139,539 AS events, with retained introns as the most prevalent events. A large number of genes were differentially alternatively spliced (DAS) between Moso bamboo and Shengyin bamboo, and genes related to RNA splicing were most significantly enriched. The high expression of SR isoforms in the 24th internode of Moso bamboo is likely the main factor leading to its greater number of alternative splicing events. Alternative splicing affects the functional domains of partial GRF, E2F, and NAM isoforms, leading to the loss of domains in some isoforms and enabling some isoforms to acquire new functional domains, and this phenomenon is more common in Shengyin bamboo. AS modifies the functional domains of certain GRF isoforms, frequently resulting in domain losses or endowing isoforms with novel domains, and this phenomenon is more common in Shengyin bamboo. We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin Bamboo and compared the differences between the two.

1. Introduction

Moso bamboo (Phyllostachys edulis) is a highly adaptable plant utilized in various industries, including food production, papermaking, and construction. It is considered a critical non-timber forestry resource in East Asia due to its rapid growth rate, one of the fastest among plant species [1,2,3]. The Phyllostachys edulis ‘Tubaeformis’ (Shengyin bamboo) is a mutant variant of Moso bamboo, with shortened and swollen internodes. Typically, it reaches a height of 4–5 m, with locally concave internodes. The lower portion is large, gradually enlarging towards the base, forming a trumpet shape [4]. So far, the regulatory mechanisms played by alternative splicing in the abnormal internode development of Phyllostachys edulis ‘Tubaeformis’ remain unclear.

Alternative splicing (AS) is a pervasive mechanism in complex organisms, not only orchestrating the post-transcriptional regulation of isoform diversity but also impacting co-transcriptional regulation [5]. Co-transcriptional regulation is the shared period between transcriptional regulation and post-transcriptional regulation. In this process, the rapid kinetic nature of RNA polymerase itself and the relatively longer span of reaction time create conditions for the occurrence of numerous associated reactions, such as mRNA splicing, capping, and modification [6,7]. Moreover, important RNA regulatory events, including mRNA splicing, m6A methylation, and A-to-I editing, primarily take place during this segment. By doing so, alternative splicing significantly enhances the diversity of both transcripts and proteins. [6,7]. Four primary AS events exist: alternative 5′ splice site (3′SS), alternative 5′ splice site (5′SS), intron retention (IR), and exon skipping (ES). In the human genome, nearly 95% of genes undergo AS, with exon skipping being the most common type, while in plants, intron retention prevails [8]. Apart from these, alternative transcription start or termination sites (aTSS or aTTS) contribute to post-transcriptional regulation, influencing protein translation efficiency and mRNA stability, distinct from the regulatory role of AS. Pre- and post-transcriptional regulatory mechanisms are instrumental in physiological tuning and adaptive responses, underpinning plant development, growth, and resilience to environmental cues [9,10]. Moreover, research underscores the specificity of AS events to certain species and tissues, highlighting their dynamic adjustment in reaction to environmental signals [11,12].

Currently, research on the internode shortening of Phyllostachys edulis ‘Tubaeformis’ has been conducted. At the anatomical level, the difference in fiber length between Shengyin bamboo and Moso bamboo suggests that the shortening of internodes in Shengyin bamboo is due to the reduction in length of fiber cells [13]. The AP2 and gibberellin-regulated gene GASR7 may be involved in the regulation of internode shortening in Shengyin bamboo [13]. However, it is still unclear whether there are differences in alternative splicing events between Moso bamboo and Shengyin bamboo, and whether alternative splicing is involved in regulating the dwarfing of Shengyin bamboo.

This study utilized PacBio Isoform-Sequencing and second-generation sequencing technology to identify genome-wide isoforms in the bamboo shoots of both Moso bamboo and Shengyin bamboo. The analysis then focused on alternative splicing (AS) to explore its potential roles in gene regulation and reveal transcriptomic complexity within the growing culms of Shengyin bamboo. Furthermore, the study delved into the underlying causes of the abnormal growth of Shengyin bamboo, which is regulated by AS, aTSS, and aTTS. These findings offer new insights into the regulatory functions of AS, aTTS, and aTSS in modulating conserved domains crucial for protein function in Moso bamboo and Shengyin bamboo shoot growth.

2. Materials and Methods

2.1. Sample Collection and RNA Extraction

Shoot samples of Phyllostachys edulis ‘Tubaeformis’ (Shengyin bamboo) and the corresponding WT, Moso bamboo, were harvested at the Anji Bamboo exposition park, Zhejiang Province, at 10 a.m. on 8 April 2022. The planting and management conditions for Shengyin bamboo and Moso bamboo are consistent, with no slope, similar forest density, and no surrounding trees to block sunlight or growth. Three bamboo shoots of Moso bamboo and three bamboo shoots of Shengyin bamboo about 120-cm-long with similar growth conditions and free from diseases and pests were selected, respectively, as three biological replicates for subsequent research (Figure S1). The selected bamboo shoots are first stripped of their sheaths. The internode with no roots at both the upper and lower nodes, starting from the base of the bamboo shoot, is defined as the first internode. The sixth, fifteenth, and twenty-fourth internodes were selected by counting from bottom to top were selected for further PacBio Isoform-Sequencing and second-generation sequencing.

RNA extraction was performed on each bamboo stem sample with TRIzol reagent procured from Invitrogen (Carlsbad, CA, USA). The assessment of RNA purity and integrity was carried out dual-prongedly: via the Agilent 2100 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) for integrity verification and the Nano Photometer spectrophotometer from IMPLEN (Westlake Village, CA, USA) for purity confirmation. Only RNA samples that surpassed stringent quality metrics were selected for subsequent library construction.

2.2. Observation Based on Scanning Electron Microscope

Longitudinal sections of the 6th, 15th, and 24th internodes from mature stems of Moso bamboo and Shengyin bamboo were prepared and used for SEM observation. The method is as follows: Following fixation with 2.5% glutaraldehyde, tissues underwent four washes with 0.1 M PBS. A progressive ethanol dehydration series followed, prior to four exchanges with isoamyl acetate for further dehydration. The bamboo samples were then subjected to critical point drying with CO₂ in a Quorum EMS850 system (London, UK), before being gold-coated utilizing an EIKO IB-3 coater (EIKO, Ibaraki, Japan). High-resolution imagery was captured via an EDAX-equipped JSM-6360LV SEM (JEOL, Tokyo, Japan) at an acceleration voltage of 2 kV.

2.3. PacBio Single-Molecule Long-Read Sequencing Sequencing and Analysis

The Pacific Biosciences single-molecule long-read sequencing libraries were constructed based on equally combined total RNA from the 6th, 15th, and 24th internodes of Shengyin bamboo (marked as S6, S15, and S24, respectively), and the 6th, 15th, and 24th internodes of Moso bamboo (marked as M6, M15, and M24, respectively). Following PCR amplification, the resultant cDNA was processed into SMRT Bell templates adhering to PacBio’s Iso-Seq protocol. These libraries were sequenced utilizing the PacBio RS II platform, deploying three separate SMRT cells, to capture comprehensive isoform sequences.

The analysis of Iso-Seq data was conducted via SMRTlink version 4.0, adhering to established protocols. The process began with the generation of reads of inserts (ROIs), which entailed the cleanup of subreads by discarding adapters and artifacts. Classification into full-length and non-full-length reads was executed with ‘pbclassify.py’, utilizing standard parameters [14]. An isoform-level clustering step ensued, yielding high-confidence consensus sequences, each verified by post-correction accuracies exceeding 99% and quality scores meeting or exceeding 30. Alignment of these refined isoforms against the reference genome was accomplished using GMAP, followed by the construction of transcript architectures through the TAPIS pipeline, ensuring detailed annotation of transcript structures [15]. Raw sequencing data can be obtained from the NCBI under accession numbers PRJNA1113140.

2.4. RNA-Sequencing Library Preparation, Sequencing, and Analysis

RNA extraction was performed with the Trizol kit (Invitrogen, Carlsbad, CA, USA) following standard procedures. RNA integrity was assessed with an Agilent 2100 Bioanalyzer and visually confirmed via RNase-free agarose gel. Eukaryotic mRNA was selectively captured using Oligo(dT) beads (Invitrogen, Carlsbad, CA, USA), after which it was fragmented and reverse-transcribed into cDNA with a NEBNext Ultra RNA Library Prep Kit (NEB #7530, New England Biolabs, Ipswich, MA, USA). Double-stranded cDNA was then subjected to adapter ligation, end-repair, and PCR amplification, with product purification done via AMPure XP Beads (1.0X). Sequencing was executed on an Illumina Novaseq6000 platform by Gene Denovo Biotech (Guangzhou, China). To generate clean reads, adapter sequences and low-quality reads were removed using fastp (version 18.0) [16]. The filtered reads were subsequently aligned to the reference genome using HISAT2 2.4 [17], with standard settings. Expression quantification was achieved by calculating FPKM values, providing a standardized measure of transcript abundance.

2.5. Functional Annotation of Genes and Isoforms

Gene and isoform annotations were derived through BLASTX alignment against major databases—gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-Prot, and Pfam protein domain—with a stringent E-value cutoff of 1 × 10⁻⁵. Specifically, the KEGG annotations were based on the Oryza Sativa (rice) genome. For functional enrichment insights, topGO v2.1 software was employed to conduct GO term analysis.

2.6. Alternative Splicing Events

Four primary alternative splicing (AS) event types—namely, intron retention, exon skipping, alternative 5′ splice site, and alternative 3′ splice site—were identified using AStalavista v4.0.1 [18,19]. Transcript isoforms were translated based on the longest open reading frame (ORF) employing TBtools [20]. Additionally, the conserved domain of each isoform was identified through Pfam protein domain analysis [21]. PSI (percent spliced in) and DAS (differential alternative splicing events) among various samples were quantified using rMATS [22]. The criteria for filtering differential alternative splicing are FDR ≤ 0.05 and ∣IncLevelDiff∣ ≥ 0.2.

3. Results

3.1. Observation of Mature Culm Internodes

Statistical analysis of internode lengths in different parts of Shengyin bamboo and Moso bamboo shoots reveals that internode length in Shengyin bamboo is shortest at the base and gradually increases with height (Figure S2). In contrast, the internode length of Moso bamboo initially increases and then decreases, with the peak occurring at the 15th to 20th internodes from the base upwards. The length of the 15th internode in Moso bamboo shows the greatest difference compared to Shengyin bamboo, reaching a multiplier of 3.94 times. Based on preliminary observation results, we selected the 6th, 15th, and 24th internodes for subsequent scanning electron microscope observation and transcriptome sequencing work.

Comparative analysis of cell lengths in the internodes of mature culms of Shengyin bamboo and Moso bamboo revealed that the cell lengths of the 6th, 15th, and 24th internodes in Moso bamboo were significantly longer than those in Shengyin bamboo (Figure 1), with differences reaching around threefold. This indicates that the main cause of dwarfing in Shengyin bamboo is the inhibition of elongation growth in internode cells.

3.2. Overview of Isoform Sequencing (Iso-Seq)

To improve the current annotations and to identify the AS events in Moso bamboo and Shengyin bamboo, we sequenced the transcriptome of the 6th, 15th, and 24th internodes of Moso bamboo and Shengyin bamboo using the single-molecule long-read sequencing platform, which provides long reads that are often up to transcript length. A total of 1,023,811 FLNC (error-corrected, full-length, and non-chimeric) reads were acquired (Table S1). After correcting the SGS data (Table S2), high-quality consensus transcripts and FLNC reads were re-mapped to the Moso bamboo genome, identifying 124,105 unique isoforms. These comprised 50,927 isoforms matching existing genome annotations, 66,412 isoforms linked to known gene sets and 6757 novel isoforms from 3892 putative new gene sites, unaccounted for in prior annotations (Figure 2A).

Among the genes detected by Iso-Seq, 8952 genes exhibited two isoforms, while only 733 genes displayed more than 11 isoforms. As the number of isoforms per gene rose, both the count of newly discovered genes and that of annotated genes declined. Furthermore, annotated genes in the Moso bamboo genome database tended to possess more isoforms than newly identified genes, with over half of the novel genes featuring just a single isoform (Figure 2B). Based on principal component analysis of homologous isoform expression levels, it was found that M24 and S8, which represented the upper part of Moso bamboo and the lower part of Shengyin bamboo, respectively, showed significant differences compared to the other four bamboo shoot samples (Figure S3).

3.3. AS Increased Transcriptome Complexity in Moso Bamboo and Shengyin Bamboo Growing Culms

In total, 13,9539 AS events were identified without transcriptome assembly, avoiding potential artificial results (see Supplementary Table S3). Each AS gene produced 7.08 AS events on average. We further classified these AS events into five distinct types: 63,484 retained introns (RI), 10,034 skipping exon (SE), 25,349 alternative 5′ splice-site (A5), 15,524 alternative 3′ splice-site (A3), and 25,148 other types (Figure 3A). For each bamboo shoot sample, M22 exhibits the highest number of alternative splicing events, whereas M6 records the least occurrence of alternative splicing events. But the difference in the number of alternative splicing events among different bamboo shoot samples is not significant (Figure 3B).

Isoform complexity arises from alternative transcription start and end sites, as well as alternative splicing (AS), all of which are crucial in gene expression regulation. To unravel interplay among these events, a Venn diagram analysis illustrated that 26,844 genes experienced both alternative start and stop transcription processes, with limited genes experiencing these individually (Figure 3C). Most genes showing AS involvement also exhibited aTSS/aTTS activity. Differential expression analysis across three internode stages in Shengyin and Moso bamboo revealed 8016 DEGs (FDR ≤ 0.05 and ≥2-fold change), of which 5500 (or 68.6%) were associated with isoform-generating events. Specifically, 3476 of these DEGs involved AS, while 4807 implicated both aTSS and aTTS, highlighting their substantial impact on gene expression variation.

The likelihood of genes undergoing alternative splicing increases with the number of exons and the overall mRNA length in bamboo shoots (Figure 4A,B). In a counterpoint, our data revealed a telling inverse relationship between the frequency of alternative splicing and both the length of exons and the GC content (Figure 4C,D). These observations collectively attest to the significant influence that specific gene characteristics exert on the occurrence of alternative splicing, highlighting the intricate interplay between gene structure and transcriptome diversity.

3.4. Analysis of Differences in Alternative Splicing Events between Moso Bamboo and Shengyin Bamboo

To explore the differences in alternative splicing during the growth and development of Moso bamboo and Shengyin bamboo shoots, we used rMATs to compare the differences in alternative splicing events among the upper (24th internode), middle (15th internode), and lower (6th internode) internodes of Moso bamboo and Shengyin bamboo shoots, separately. The results showed that a large number of genes were differentially alternatively spliced (DAS) between Moso bamboo and Shengyin bamboo (Figure S4 and Table S4).

The comparison with the highest number of DASs is M6 vs. S6, followed by M15 vs. S15. This suggests that there are less alternative splicing differences between the young tissues of Moso bamboo and Shengyin bamboo shoots (Figure 5A). However, the vast majority of genes undergoing differential alternative splicing do not exhibit differential expression. Therefore, it is speculated that during the growth and development of Moso bamboo and Shengyin bamboo shoots, gene expression regulation and alternative splicing regulation are two independent regulatory systems, but both play important roles in the abnormal internode growth of Shengyin bamboo shoots. The analysis of differentially expressed genes in the comparisons of the three groups revealed that there were more common differentially expressed genes between the middle (M15 vs. S15) and the base of the bamboo shoots (M6 vs. S6), while at the top, there were more unique differentially expressed genes (M24 vs. S24) (Figure 5B). The results of the analysis of differential alternative splicing events were similar to those of the differential expression gene analysis (Figure 5C).

Next, we categorized the GO function of these DASs (Table S5), and the GO terms that related to RNA splicing, including RNA splicing via transesterification reactions (GO0000375), RNA splicing (GO:0008380), RNA processing (GO:0006396), etc., which were most significantly enriched in all three comparison groups (Figure S5). These results indicate that genes regulating differential alternative splicing between Moso bamboo and Shengyin bamboo are themselves undergoing alternative splicing.

To investigate the impact of AS, aTSS, and aTTS products, isoforms, on the growth and development of two types of bamboo shoots, we conducted an analysis of the expression levels of isoforms from the SR (serine/arginine-rich protein splicing factor), GRF (growth regulating factor), E2F, and NAM (no apical meristem) gene families. In the Moso bamboo genome, a total of 59 potential SR family members were identified, generating a total of 262 isoforms. Six direct homologs of SR30 were identified, generating 37 isoforms through the combined action of AS, aTSS, and aTTS. Among them, the majority were highly expressed in M24, with most containing intact RRM1 and RRM2 domains, while a minority of isoforms had incomplete RRM2 domains (Figure 6A). Two isoforms, PB.23545.8 and PB.23545.7, lacked both RRM1 and RRM2 domains and exhibited high expression in S8. Eight genes were identified as direct homologs of SR45a, generating a total of 47 isoforms. Similar to SR30, the majority of these isoforms were highly expressed in M24 and S24, with the highest expression observed in S24. Additionally, four isoforms lacked any structural domains, with the exception of PB.5333.21, which exhibited high expression in S8, while the others were highly expressed in S24. The high expression of SR30 and SR45 in M24 also led to a greater occurrence of alternative splicing events in M24.

The GRF family is extensively involved in plant growth and development, correlating with cell division and growth [23,24]. Expression analysis reveals that in M24, the highest expression is observed in the majority of the most abundant homologous isoforms, followed by S24 (Figure 7A). The analysis of the number of differentially expressed GRF isoforms between the 6th, 15th, and 24th internode samples of Moso bamboo and Shengyin bamboo indicates that the greatest difference in GRF expression occurs between M6 and S6, with 19 isoforms showing differential expression, all of which are highly expressed in M6. In contrast, there are 7 and 10 differentially expressed isoforms between M15 and S15 and M24 and S24, respectively, with some isoforms showing higher expression in Moso bamboo and others showing higher expression in Shengyin bamboo. Most GRF members contain intact WRC and QLQ domains, while a few members acquire new domains such as the t-SNARE complex subunit, the synta domain, and the Syntaxin N-terminal domain through alternative splicing. The majority of them are highly expressed in S8 and S24. Additionally, there is an isoform, PB.19613.1, which lacks any structural domains and is highly expressed in S24.

In the E2F/DP family, an expression analysis revealed that the majority of isoforms are highly expressed in either M24 or S24 (Figure 7B). There are two isoforms, PB.3056.1 and PB.3056.2, that have lost all complete conserved domains and are both highly expressed in S24, while there are two isoforms that have acquired the secretory_peroxidase domain and are both highly expressed in M24. The greatest difference in expression among isoforms is between M6 and S6, with a total of 11 isoforms showing differential expression, all of which are highly expressed in M6.

In the NAM family, most NAM members tend to express more highly in the basal region in both Moso bamboo and Shengyin bamboo (Figure 8). Among the isoforms showing differential expression in M15 vs. S15 and M24 vs. S24, most exhibit higher expression levels in moso bamboo. One isoform lacks the NAM domain and shows the highest expression abundance in M6. There are six homologous isoforms with incomplete NAM domains, among which four are highly expressed in samples related to sacred bamboo.

4. Discussion

As an important post-transcriptional regulatory mechanism, alternative splicing (AS) is widely involved in various biological processes in plants [1,25,26] such as enhancing transcriptome adaptation to stresses and aiding plants in coping with pathogen defense [27,28]. Our study also revealed regulatory differences in alternative splicing events between different genotypic mutants of the same species. Analysis of differential alternative splicing and gene expression in Moso bamboo and Shengyin bamboo shoots revealed that most differentially alternatively spliced genes do not exhibit differential expression.

Flowering plants possess a significant number of SR proteins, with Arabidopsis having 18 members [29], Brachypodium [17,30], rice [24,31], and grape (Vitis vinifera) [18,32]. In Arabidopsis, several sequence-conserved splicing factors and regulators have been functionally characterized, playing crucial roles in regulating various aspects of development and stress responses [33,34]. Due to the polyploidy and various duplication events in the Moso bamboo genome [15], a large number of SR family members have been identified. we conducted a blast similarity search using the [19] SR genes identified in Arabidopsis, revealing 59 SR genes in moso bamboo—more than in the model plants mentioned. This increase is likely attributed to its tetraploid origin around 7–12 million years ago. Notably, both plant and animal genes encoding SR proteins frequently undergo AS themselves. In Arabidopsis, only two SR genes (RSZ22 and SCL28) generate a single pre-mRNA, while the rest collectively yield over 90 transcripts, significantly enhancing the complexity of the SR gene family transcriptome [35]. In our study, 59 potential SR family members generated a total of 262 transcripts, thus dramatically increasing the complexity of the SR gene family transcriptome. Taking Arabidopsis SR30 as an example, it encodes a serine-arginine rich RNA binding protein that plays a role in the regulation of splicing, including its own splicing [36]. It exists as three alternatively spliced forms, which exhibit differential expression patterns. However, in Moso bamboo, there are six orthologous genes which generate [37] isoforms during Moso bamboo and Shengyin bamboo shoot growth. Similar phenomena were observed in SR45a, a crucial component of the spliceosome, plays a significant role in the post-transcriptional regulation of salinity tolerance as well as other stress responses in Arabidopsis and Maize [37,38]. Eight genes were identified as direct homologs of SR45a, generating a total of 47 isoforms in the Moso bamboo genome. These expanded SR family members play crucial roles in the pre-mRNA splicing process of Moso bamboo and Shengyin gene, and increased the richness of bamboo genome transcripts.

Expression analysis reveals that the majority of SR30 and SR45a isoforms are highly expressed in M24 and S24, representing the upper sections of Moso bamboo shoots and Shengyin bamboo shoots. This region is characterized by vigorous growth and active cell division of intercalary meristematic cells [34,39]. Studies have indicated that periods of rapid plant growth and development may lead to an increased incidence of alternative splicing events [40]. Consequently, a notable number of alternative splicing events occur in both M24 and S24. The regulation of self-splicing by SR also results in the loss of conservative functional domains in certain splicing isoforms, while another member acquires a new structural domain. As a result of these functional domain alterations, these isoforms lose their original RNA splicing functions, with the majority being highly expressed in S24. Hence, this could potentially contribute to the higher occurrence of alternative splicing events in M24 compared to S24.

An anatomical analysis based on scanning electron microscope indicates that the length of the internode cells in Moso bamboo is significantly longer than that of Shengyin bamboo, with the difference being around threefold. Considering that the average height of Moso bamboo after maturation is about three times that of Shengyin bamboo, we infer that the key factor causing the dwarfing of Shengyin bamboo is the hindered elongation growth of the internode cells. This is different from the dwarfing mechanism of some previously reported bamboo plant mutants, where the dwarfing of Pseudosasa japonica var. tsutsumiana is caused by a combination of inhibited cell division and cell growth caused by abnormal brassinosteroid synthesis [41,42].

Domains can be considered as discrete functional and structural units of a protein. Loss or acquisition of domains is also associated with changes in protein function [43]. The GRF transcription factor is a plant-specific protein that plays a broad and important role in the growth and development of various plant organs by regulating the expression of genes related to cell proliferation and cell size [44]. In rice, Overexpression of OsGRF3 and OsGRF10 in rice results in thinner stems, increased stem height, and reduced tillering [45,46]. Therefore, we retrieved isoforms of the GRF gene family generated by alternative splicing to assess whether their conserved domains are affected by alternative splicing. During the growth and development of Moso bamboo and Shengyin bamboo shoots, many GRF isoforms lost the WRC or QLQ domains due to alternative splicing. Additionally, we compared the expression of alternative splicing—isoforms in bamboo shoot tissues of Moso bamboo and Shengyin bamboo. A total of five isoforms exhibit loss of conserved domains due to alternative splicing or replacement of conserved domains, with four of them being highly expressed in the shoot internode tissues of Shengyin bamboo, indicating that alternative splicing has a certain degree of impact on the internode growth of Shengyin bamboo.

The E2F/DP transcription factors in higher plants are categorized into E2F, DP, and DEL (DP-E2F-like) groups based on conserved domain, and played important roles in plant growth and development, such as leaf growth [47] and root growth [48]. Two isoforms lacking any complete conserved domain are highly expressed in Shengyin bamboo shoots, similar to what has been observed in GRF. This suggests that certain non-functional isoforms generated by alternative splicing may impact the growth and development of Shengyin bamboo. The comparison of GRF isoforms expression level between Moso bamboo and Shengyin bamboo revealed that the greatest difference was observed in the comparison between M6 and S6, with 19 differentially expressed isoforms, all of which were highly expressed in Moso bamboo. Similar phenomenon were observed in E2F/DP, with 11 isoforms differentially expressed between M6 vs. S6, all of which were highly expressed in Moso bamboo. In the same bamboo shoot, internodes at the base primarily undergo cell elongation, while those at the top are dominated by cell division [39,49]. The higher expression levels of GRF and E2F in M6 than that in S6 may significantly contribute to internode cell growth and development in Moso bamboo, potentially highlighting a comparable difference between Shengyin bamboo and Moso bamboo.

NAM transcription factors, a subgroup of NAC family, are known to play crucial roles in various aspects of plant development, such as developing the shoot apical meristem in petunia embryos, and determining the positions of meristems and primordia [50]. In Arabidopsis, two VND (vascular related NAC-domain) genes, including VND6 and VND7, are involved in xylem differentiation and cell fiber differentiation [51]. Overexpression of PeNAC122 poplar lines exhibited thickened xylem, and accumulated lignin content in stems, and also upregulates the expression of secondary cell wall biosynthetic genes [52]. Expression analysis indicated that most NAM isoforms preferably expressed in Moso bamboo shoot. Combining the aforementioned studies, their much higher accumulation level in Moso bamboo than in Shengyin bamboo might be essential for lignin accumulation and lignification in Moso bamboo.

Alternative splicing events not only lead to the loss of conserved domains in some genes, thereby compromising their function, but also result in some genes acquiring new domains that were originally not part of their gene family. This phenomenon was observed in the E2F, GRF, and NAC families in this study. A typical example is found in two isoforms from E2F/DP, PH02Gene03595.t1 and PH02Gene10996.t1, both of which have acquired a new conserved domain, secretory_peroxidase. Co-linearity analysis revealed that these two genes are located in a synteny blocks, suggesting that the acquisition of the isoforms with the secretory peroxidase domain occurred prior to the genome duplication event in bamboo [53]. The secretory_peroxidase domain belong to class III of the plant heme-dependent peroxidase superfamily. Class III peroxidases are found in the extracellular space or in the vacuole in plants where they have been implicated in hydrogen peroxide detoxification, auxin catabolism and lignin biosynthesis, and stress response [54]. Therefore, these two highly expressed isoforms in bamboo M24 may have acquired some new functions related to growth and development due to the acquisition of this domain. Consequently, the abundant expression of various GRF, E2F/DP and NAC isoforms in Moso bamboo plays a crucial role in promoting the elongation growth of bamboo internodes, thereby contributing to the rapid development of the Moso bamboo shoot.

5. Conclusions

This study explores the previously understudied post-transcriptional mechanisms underlying internode shortening in Shengyin bamboo. Comparative analysis between mature Shengyin and Moso bamboo reveals notable differences in internode cell lengths. Using PacBio sequencing, 139,539 alternative splicing (AS) events were identified, with intron retention being prominent. A large amount of differentially alternatively spliced genes were found between the two bamboos, particularly enriched in RNA splicing-related gene ontology terms. The lower part of the shoots exhibited the highest number of differential AS events. Interestingly, most genes undergoing AS did not show differential expression. Within the GRF gene family, isoforms exhibiting domain loss or domain alteration are predominantly expressed at higher levels in Shengyin bamboo. These findings provide valuable insights into Shengyin bamboo growth regulation and serve as a resource for further research.