Genetic Characterization of RNA Recognition Motif (RRM)-Containing Genes in Coconut Palm

Abstract

RNA recognition motif (RRM)-containing proteins are important regulators involved in diverse cellular processes, including splicing, stability, transport, and translation of transcripts. However, their comprehensive characterization remains limited in perennial tropical crops like Cocos nucifera. In this study, we performed a genome-wide analysis of RRM genes in coconut, identifying a total of 326 CnRRM genes. Phylogenetic classification based on complete RRM domain sequences grouped these proteins into eleven clades (I–XI), each exhibiting distinct variations in motif length and domain architecture. Transcriptome profiling revealed diverse expression patterns across coconut tissues, ranging from constitutive to highly tissue-specific. The CnHRLP1 gene, encoding an hnRNP-like multi-RRM protein, was selected for further functional analysis. Subcellular localization showed that the CnHRLP1 protein is predominantly nuclear, and its constitutive overexpression in Arabidopsis led to a severe dwarf phenotype. RNA-seq analysis demonstrated that CnHRLP1 overexpression broadly reshaped the transcriptome. KEGG pathway enrichment highlighted a significant impact on plant hormone signaling, particularly the gibberellin (GA) pathway. CnHRLP1 overexpression induced the coordinated downregulation of key GA biosynthetic genes (KO, KAO1/2, GA20ox, GA3ox) and the upregulation of GA catabolic genes (GA2ox2/6), suggesting its role in modulating GA homeostasis. In conclusion, this study provides a genomic and functional overview of the coconut RRM protein family and establishes that the hnRNP-like protein CnHRLP1 functions as a transcriptional regulator that inhibits vegetative growth, potentially through the suppression of gibberellin biosynthesis.

1. Introduction

RNA processing is a fundamental regulatory layer of gene expression in eukaryotes, enabling organisms to fine-tune developmental programs and environmental responses beyond transcriptional control [1,2]. Among the molecular components governing post-transcriptional regulation, RNA-binding proteins (RBPs) play a central role by controlling pre-mRNA splicing, RNA stability, transport, and translation [3,4]. One of the most abundant and evolutionarily conserved RNA-binding domains found in RBPs is the RNA recognition motif (RRM). RRM-containing proteins are present across all eukaryotic lineages and constitute a significant class of splicing factors and regulatory RBPs [5,6]. Their high degree of conservation underscores their essential biological functions, particularly in regulating transcriptome complexity through alternative splicing [7,8].

The RRM is typically a 90-amino-acid domain defined by conserved RNP1 and RNP2 motifs that facilitate sequence-specific RNA binding. Proteins containing single or tandem RRMs are integral to the spliceosome’s architecture, mediating critical steps such as splice-site recognition and assembly [9,10,11]. In plants, RRM proteins are classified into several major families, including serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and poly(A)-binding proteins (PABPs) [5,12]. Through the dynamic modulation of alternative splicing, these proteins enable plants to generate a vast array of transcript isoforms from a limited gene set, providing the flexibility needed for phase transitions and rapid responses to fluctuating environmental cues [13,14].

RNA recognition motif (RRM)-containing proteins are increasingly recognized as essential regulators of plant growth and development. Through their roles in RNA processing, RRM proteins influence key developmental processes, such as flowering time and organ growth [15,16,17]. Several hnRNP-like RRM proteins have been reported to regulate developmental transitions by modulating the expression of growth-related genes in model plants, highlighting the contribution of post-transcriptional regulation to plant developmental control [18]. Genome-wide studies have further revealed substantial expansion and functional diversification of RRM gene families in several crop species, including Oryza sativa, Zea mays, Brassica napus and Hordeum vulgare [5,19,20,21]. Functional studies indicate that individual RRM proteins participate in the regulation of growth-associated pathways through post-transcriptional control of hormone-related genes, including hormone receptors and biosynthetic enzymes [22,23]. Despite these advancements in annual crops, the regulatory role of the RRM superfamily remains unexplored, mainly in complex perennial tropical species.

Beyond developmental regulation, RRM-containing proteins have been implicated in plant responses to biotic and abiotic stresses, including drought, salinity, temperature extremes, and pathogen infection. RRM-mediated RNA processing contributes to stress-responsive gene expression and adaptive signaling pathways [24,25]. Emerging evidence further suggests that RNA-binding proteins participate in hormone-regulated developmental processes by influencing the expression of genes involved in hormone biosynthesis and signaling [26]. In particular, post-transcriptional regulation has been linked to gibberellin-related pathways that control plant growth response [27,28]. Such regulatory potential is consistent with the role of RNA-binding proteins as modulators of developmental hormone responses rather than direct regulators of hormone biosynthesis.

Coconut palm (Cocos nucifera) is an economically important crop valued for food production, oil extraction, and construction materials [29]. Despite considerable research aimed at unraveling the molecular mechanisms governing its growth, development, flowering and responses to stress [30,31,32]. The publication of high-quality reference genomes has recently provided a breakthrough in understanding coconut evolution, particularly regarding plant height and fiber content [18]. Transcriptomic studies have increasingly recognized it as a critical process that contributes to the functional diversification of gene products in this economically vital species. [33]. However, the upstream regulators of RNA processing, particularly RRM binding proteins, have not been systematically analyzed in coconut. Given the unique architectural and physiological traits of the coconut, a systematic analysis of its RRM family is critical to uncovering the molecular basis of its growth regulation.

Given the complex architecture, long life cycle, and perennial growth habit of the coconut, a comprehensive analysis of the RRM gene family is essential for understanding post-transcriptional regulation underlying coconut growth and development. In this study, we conducted the first genome-wide identification and characterization of RRM genes in C. nucifera, analyzed their phylogenetic relationships, domain architectures, and expression patterns across tissues, and functionally characterized a representative hnRNP-like protein, CnHRLP1, through subcellular localization, transcriptomic profiling, and transgenic validation in A. thaliana. Our results establish an association between RRM-mediated RNA processing and gibberellin (GA) associated developmental regulation, providing a foundation for future mechanistic studies in coconut.

2. Results

2.1. Identification and Classification of CnRRM Proteins

To comprehensively identify and characterize the RNA-binding proteins (RBPs) in coconut (Cocos nucifera), a systematic bioinformatic analysis was initiated. For the accurate and sensitive genome-wide identification of RRM-containing proteins, a profile Hidden Markov Model (HMM)-based search strategy was employed. A total of 325 RNA-binding motif (RRM) genes were identified (Table S1). To resolve the evolutionary relationships among coconut RRM proteins, a maximum-likelihood phylogenetic tree was constructed using full-length RRM protein sequences (Table S2 and Figure 1A and Figure S1 for detail information). The resulting topology partitioned the CnRRM family into eleven distinct clades (I–XI). While CnRRM members were represented across the entire phylogeny, the numerical distribution per clade was non-uniform. Clades III, IV, V, VII, and VIII constituted the most significant expansions, whereas clades I, IX, X, and XI were notably smaller, suggesting lineage-specific conservation or contraction.

An analysis of RRM motif architecture revealed significant variation in length across the identified clades (Figure 1B). Individual RRM domains spanned a range of approximately 60 to 140 amino acids. Structural divergence was clade-dependent: Clades III and IV exhibited the greatest variability in motif length, with higher median motif lengths. Conversely, clades VI, VII, IX, and X were characterized by more constrained length distributions, maintaining shorter, more uniform RRM domains. Differences in RRM motif length were observed among clades independently of clade size. Conserved motif composition and domain organization were further analyzed for representative proteins from each clade (Figure 1C). Distinct RRM subtype combinations were associated with specific phylogenetic groups. Proteins in clade I predominantly contained U1A-like and CELF-like RRM motifs, whereas clade II included PTBP-, La-, and CIDB-like RRM domains. Clades V–VIII exhibited more complex domain architectures, frequently comprising multiple RRM subtypes, including RBM, FET, ABT1, NIKF-like, and SARFH-related motifs within individual proteins. In contrast, proteins in clades IX–XI generally displayed simpler domain organizations, often dominated by hnRNP-related RRM motifs.

2.2. Domain Organization and Structural Diversity of CnRRM Proteins

A positive correlation was observed between RRM domain multiplicity and overall protein length. Monomeric RRM domain proteins generally represented the shortest polypeptides within the family, whereas those harboring tandem RRMs exhibited a moderate increase in total size. A marked expansion in both median length and distributional breadth was noted in proteins containing three or four RRMs. Members possessing five RRMs were infrequent and were characterized by consistently high molecular weight (Figure 2A).

The CnRRM family exhibits a clear bias in domain copy number distribution, with mono-RRM architecture being the most prevalent (142 members), followed by dual-RRM configurations (92) (Figure 2B). Higher-order complexity was significantly less frequent, with proteins containing three (44) or four (24) RRMs comprising a smaller fraction of the total proteome. Only two proteins were identified with a pentameric RRM arrangement, representing the most complex structural variants. These variations in domain topology highlight the diverse structural frameworks within the coconut RRM superfamily (Figure 2C). CnU2B contained two U1A-like RRM domains arranged in tandem. CnNUC exhibited a multi-RRM structure comprising NUCL-type domains together with extended non-RRM regions. CnHRLP1 contained multiple hnRNP-R/Q-like RRM domains arranged in proximity. CnRBM19 exhibited a complex architecture, comprising several RBM19-related RRM domains and a region annotated for poly(A) binding. Based on conserved domain composition and phylogenetic placement, these representative genes belong to distinct RRM subfamilies: CnU2B corresponds to a U2 small nuclear ribonucleoprotein–associated RRM protein (U2B/U1A-like), CnNUC belongs to the nucleolin-like RRM subfamily, CnHRLP1 represents an hnRNP-like multi-RRM protein, and CnRBM19 was classified within the RBM19-type RRM subfamily. Gene nomenclature was assigned according to the closest annotated homologs identified through sequence similarity and phylogenetic inference, in combination with conserved domain architecture, with the prefix “Cn” indicating Cocos nucifera origin.

2.3. Expression Patterns of CnRRM Genes Across Coconut Tissues

Transcriptome analysis was used to evaluate the expression patterns of CnRRM genes across various vegetative and reproductive tissues (Figure 3A). The family showed a wide range of transcript abundance, as represented by log₁₀(FPKM + 1) values. The heatmap, organized by the coefficient of variation (CV), revealed a transparent gradient: a group of genes at the top maintained high, stable expression across all tissues (low CV). In contrast, genes at the bottom showed highly variable or tissue-specific patterns (high CV). Notably, distinct expression signatures were observed in the leaf, shoot, and floral tissues, as well as across the developmental stages of the endosperm and mesocarp (Figure 3A).

The expression profiles of five representative RRM groups were characterized: the hnRNP group (hnRNP-R-Q superfamily), implicated in pre-mRNA processing; the PABP group (PABP-1234 superfamily), involved in RNA transport; the SF-CC1 group (SF-CC1 superfamily) and U2AF group (U2AF_lg superfamily), both functioning as splicing factors; and the spliceosome-associated group (e.g., U2AF35B and U1A), which contributes to spliceosome assembly. Among the five groups, the U2AF subclass exhibited the highest median expression levels, whereas the PABP group showed the most incredible spread in transcript abundance (Figure 3B). Analysis of expression stability confirmed these trends; PABP-like genes had the highest average CV values, indicating they are more likely to perform specialized, tissue-dependent roles. In contrast, the hnRNP and U2AF groups showed lower CV values, suggesting more consistent activity across the plant.

To place these expression patterns into a functional context, the expression profile of a single representative gene belonging to the hnRNP group, CnHRLP1, was examined across the same tissue set. CnHRLP1 exhibited a tissue-dependent expression pattern that was consistent with the broader trends observed for hnRNP-like RRM genes. The selection of CnHRLP1 for further functional characterization was guided by its hnRNP-like multi-RRM domain architecture, predicted nuclear localization, phylogenetic placement within an expanded RRM clade, and moderate-to-high expression across multiple tissues, all of which support its suitability for investigating the functional relevance of RRM-mediated RNA regulation in coconut (Figure 3A).

2.4. Subcellular Localization and Phenotypic Effects of CnHRLP1

The 3D structure of CnHRLP1 protein was predicted (Figure S1). To determine the subcellular localization of CnHRLP1 protein, a 35S::CnHRLP1–eGFP fusion construct was transiently expressed in Nicotiana benthamiana leaf epidermal cells together with a nuclear marker (35S::OsGhd7-RFP–RFP). Confocal microscopy revealed that the eGFP fluorescence signal largely overlapped with the RFP signal, indicating predominant nuclear localization of CnHRLP1 (Figure 4A). The functional effects of CnHRLP1 were further examined by constitutive overexpression of the gene in A. thaliana under the control of the CaMV 35S promoter. All independent transgenic lines analyzed (L1, L3, L4, and L5) exhibited a pronounced dwarf phenotype compared with wild-type plants (Figure 4B). After 30 days of growth, wild-type plants reached heights of approximately 300–350 mm, whereas CnHRLP1 overexpression lines remained below 100 mm. (Figure 4C). Quantitative analysis confirmed a substantial reduction in plant height in all overexpression lines relative to wild-type controls. Together, these results demonstrate that CnHRLP1 protein was predominantly localized to the nucleus and that elevated CnHRLP1 expression is associated with potent inhibition of vegetative growth in Arabidopsis.

2.5. Transcriptomic Impact of CnHRLP1 Overexpression

To investigate the regulatory role of CnHRLP1, we conducted RNA sequencing (RNA-seq) analysis on both CnHRLP1-overexpressing (OE, Col-0 background) lines and wild-type (Col-0) controls. Successful transformation was confirmed by transcript abundance levels, with OE-HRLP1 lines exhibiting nearly 1000 FPKM, whereas expression in WT remained negligible (Figure 5A). The RT-qPCR analysis further confirmed a significantly higher expression level of the exogenous CnHRLP1 gene in the OE-HRLP1 transgenic plants compared to the Col-0 controls, consistent with the transcriptomic findings. Principal Component Analysis (PCA) revealed a clear separation between the WT and OE-HRLP1 transcriptomes, with PC1 accounting for 98% of the total variance, indicating that CnHRLP1 overexpression significantly reshaped the global gene expression profile (Figure 5B).

A comparative analysis of global transcript levels revealed a slight upward shift in the median log₂ transferred FPKM values for the OE lines compared to WT (Figure 5C). Differential expression analysis identified many Differentially Expressed Genes (DEGs) (Figure 5D). Specifically, thousands of genes were significantly up-regulated or down-regulated in response to CnHRLP1 overexpression. The DEGs were further categorized by their Fold Change (FC) and expression range (Figure 5E). Most down-regulated genes showed a (FPKM ≥ 10) between −1 and −2, while a substantial number of up-regulated genes exhibited high transcript abundance (FPKM > 10). These findings suggest that CnHRLP1 is associated with widespread and indirect remodeling of transcript abundance, consistent with an RNA-mediated regulatory mechanism, and influences a broad array of downstream targets involved in coconut biological processes.

2.6. Functional Enrichment and Functional Regulation of the GA Pathway

To elucidate the biological impact of CnHRLP1 overexpression, differentially expressed genes (DEGs) were subjected to KEGG pathway enrichment analysis (Figure 6A). The results revealed that DEGs were predominantly involved in plant hormone signal transduction, plant-pathogen interactions, and starch and sucrose metabolism. Notably, a significant number of up-regulated genes were enriched in pathways related to phenylpropanoid biosynthesis and various secondary metabolite pathways, suggesting that CnHRLP1 overexpression is associated with transcriptional reprogramming of secondary metabolism and stress-related pathways (Figure 5A).

Given the enrichment in hormone signaling, we specifically examined the Gibberellin (GA) biosynthetic pathway (Figure 6B). Overexpression of CnHRLP1 led to a coordinated downregulation of several key rate-limiting enzymes in GA biosynthesis. Transcript levels for ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO1/2) were significantly reduced in the OE lines. Furthermore, genes encoding active GAs, including multiple isoforms of GA20ox and GA3ox, showed decreased expression (Figure 6B). In contrast, genes involved in GA inactivation, such as GA2ox2 and GA2ox6, showed marked up-regulation in the OE-HRLP1 lines. This simultaneous suppression of biosynthetic genes and activation of catabolic genes suggests that CnHRLP1 overexpression is associated with altered transcript abundance of genes involved in GA biosynthesis and inactivation, consistent with an indirect effect on GA-related gene networks.

3. Discussion

RRM proteins are essential eukaryotic RNA-binding proteins (RBPs) that serve as central nodes in post-transcriptional networks, governing pre-mRNA splicing, mRNA stability, and translational control [3,34,35]. In plants, the expansion of RRM families is intrinsically linked to the regulatory complexity required for environmental adaptation and developmental flexibility [36,37]. This study presents a comprehensive genome-wide characterization of the RRM family in Cocos nucifera, integrating phylogenetic resolution with the functional analysis of the representative member, CnHRLP1.

The identification of 325 CnRRM genes in Cocos nucifera reflects a significant evolutionary expansion compared to model species such as Arabidopsis and rice [8,38]. This expansion suggests that the coconut genome has retained a high degree of structural plasticity to manage its complex woody anatomy and specialized fruit development. Our data demonstrates a significant positive correlation between RRM domain stoichiometry and total polypeptide length (Figure 2A), supporting the “modular assembly” of RBP evolution. The multiplication of RRM domains allows for higher specificity and affinity for complex RNA targets, enabling more intricate post-transcriptional control [39,40]. The prevalence of mono-RRM architecture likely represents a conserved basal splicing function. At the same time, the complex, multi-RRM configurations (e.g., the five-RRM CnRBM19) may have evolved to facilitate specialized long-distance RNA transport or sequestration in the palm’s extensive vascular system [41].

The classification of CnRRM proteins into eleven phylogenetic clades reflects evolutionary diversification patterns reported for RRM families in A. thaliana, O. sativa, and other plant species, where distinct clades correspond to functional subclasses such as hnRNPs, splicing factors, and poly(A)-binding proteins [39,42]. Notably, the number of CnRRM members differed markedly among clades (Figure 1A), indicating that subfamily expansion has not occurred uniformly during coconut genome evolution. This non-uniform distribution can be discussed in relation to structural differences among clades: expanded clades are associated with broader variation in RRM motif length (Figure 1B) and more complex domain architectures with diverse RRM subtype combinations (Figure 1C). These features may increase RNA-binding versatility and favor retention following duplication events. In contrast, clades with fewer members predominantly comprise proteins with simpler RRM configurations, which may be subject to stronger functional constraints. Variation in RRM motif length and subtype composition among clades is consistent with structural and biochemical studies demonstrating that differences in RRM architecture influence RNA-binding specificity and interaction dynamics [7,34,41]. The presence of multi-RRM proteins in several clades suggests functional specialization, as proteins harboring multiple RRMs are frequently involved in higher-order ribonucleoprotein complexes that coordinate diverse aspects of RNA metabolism [43]. The coexistence of single- and multi-RRM proteins within the CnRRM family highlights functional stratification. Multi-RRM proteins, particularly those containing hnRNP- or RBM-related domains, are commonly associated with broader regulatory capacity at the RNA level, reflecting their ability to engage multiple RNA targets or protein partners [43,44]. Such structural complexity consists of roles in integrating multiple RNA processing events to achieve coordinated gene regulation.

Transcriptome profiling across six coconut tissue categories, including multiple developmental stages of mesocarp and endosperm (7–12 months after flowering), revealed that CnRRM genes are not uniformly expressed but instead exhibit distinct expression patterns and levels of expression stability across tissues [45]. The high transcript abundance and low coefficient of variation (CV) observed in the U2AF group suggest a constitutive role in fundamental splicing processes across all tissues [36]. The relatively stable expression of hnRNP-type CnRRMs aligns with their conserved roles in basal nuclear RNA metabolism. In contrast, greater variability among PABP-like genes likely reflects context-dependent functions in mRNA stability and translation [8,46]. CnHRLP1 emerged as a particularly notable member of the CnRRM family. Its predominant nuclear localization is consistent with previous studies demonstrating that hnRNP-like RNA-binding proteins primarily function in the nucleus to regulate RNA processing and transcript fate [16,47]. Constitutive overexpression of CnHRLP1 in A. thaliana resulted in severe growth inhibition, indicating that tight regulation of CnHRLP1 expression is required for normal plant development. Similar dwarf or growth-restricted phenotypes have been reported in plants with altered expression of RNA-binding proteins, underscoring the sensitivity of developmental programs to post-transcriptional regulatory imbalance [37,48]. These findings indicate that precise regulation of CnHRLP1 expression is essential for normal vegetative development. Transcriptomic analysis further demonstrated that CnHRLP1 overexpression induces extensive, yet selective, changes in gene expression. The clear separation between wild-type and overexpression lines, together with comparable global transcript abundance distributions, indicates that CnHRLP1 does not globally disrupt transcription but instead modulates specific gene networks. Such selective transcriptomic reprogramming is a characteristic feature of RNA-binding proteins, which typically influence gene expression indirectly through RNA processing, stability, or translational regulation rather than direct transcriptional control [7,49].

Notably, genes involved in gibberellin (GA) biosynthesis and metabolism were strongly affected in CnHRLP1 overexpression plants. Gibberellins are key regulators of stem elongation and vegetative growth, and reductions in bioactive GA levels are well known to result in dwarf phenotypes [50,51]. The coordinated repression of GA biosynthetic and activating genes, together with increased expression of GA-inactivating GA2ox genes, provides a credible molecular explanation for the growth inhibition observed in OE-CnHRLP1 plants. Although CnHRLP1 is unlikely to regulate GA metabolic enzymes, the observed expression patterns directly suggest that RNA-mediated regulatory processes may indirectly influence GA-associated gene networks, consistent with broader roles of RNA-binding proteins in coordinating hormone-dependent developmental responses [52,53]. Overall, these findings indicate that CnHRLP1 functions as a nuclear RNA-binding protein that restricts vegetative growth by selectively modulating gene expression networks, with distinct effects on gibberellin-associated pathways. Further evaluation of CnHRLP1 overexpression lines across additional developmental stages, stress or nutrient-related conditions, and under exogenous gibberellin treatment, including germination and early seedling growth responses should be explored in future studies.

4. Materials and Methods

4.1. Plant Materials

The coconut leaf, mesocarp, embryo, and endosperm tissues were sampled from a 10-year-old coconut tree in Hainan, China, on 10 May 2024. These samples were subsequently used for cDNA synthesis and full-length coding sequence amplification of CnHRLP1. Total RNA was extracted from a composite sample of the collected samples according to the protocol described by Xiao et al. (2012) [54]. The complementary DNAs (cDNAs) were prepared using the ABScript Neo RT MasterMk kit (RK20433, ABclonal, Nanjing, China). Gene transformation experiments utilized Arabidopsis ecotype Columbia-0 (Col-0) wild-type plants, cultivated in a controlled environment chamber under a 16 h light/8 h dark photoperiod, a temperature of 21 °C, and 65% relative humidity. Col-0 seeds were surface-sterilized with sodium hypochlorite, rinsed with deionized water, and subsequently plated onto solidified ½ Murashige and Skoog (MS) medium supplemented with 1% (w/v) sucrose and 0.6% (w/v) phytoagar (Duchefa Biochemie, Haarlem, The Netherlands). To ensure uniform germination, plates were stratified at 4 °C in the dark for 24 h before transferring to the growth chamber. For plant transformation procedures, Col-0 seedlings were directly sown into soil-filled pots and maintained in a growth chamber at 22 °C, under a 16 h light/8 h dark photoperiod, with a light intensity of approximately 125 μmol m⁻² s⁻¹.

4.2. RRM Gene Identification and Characterization

The gene models of the coconut palm were used from the reference for the high-quality coconut genome analysis [55]. Specifically, the curated RRM domain profile (PF00076) was retrieved from the PFAM database (http://pfam.xfam.org/, accessed on 20 September 2023). HMMER (v3.4) was used to identify the conserved domain of deduced peptides from transcripts and screen for genes with RNA-binding domains. The homologous genes of the identified RRM genes in A. thaliana were identified via BLAST (v2.14.1) analysis using the corresponding Arabidopsis gene. The paralogous gene copy was determined via the BEST-hits criteria for multiple-copy genes. A phylogenetic tree of the RRM genes was constructed by CLUSTALW and visualized via the iTOL website (https://itol.embl.de/, accessed on 20 June 2025).

CnHRLP1 protein sequence was used to generate a PDB file and a protein model of CnHRLP1. The primary sequence was analyzed in intensive modeling mode, which uses hidden Markov models to identify structural templates. The resulting PDB file was processed and visualized to highlight secondary structure elements and critical side-chain orientations.

4.3. Characterization of Expression Pattern of RRM Genes

The coconut palm transcriptome dataset, encompassing six tissue types—leaf, stem, female flower, mesocarp, and endosperm—was obtained from our previous study and has been deposited in the China National Center for Bioinformation under accession number CRA004778 (https://ngdc.cncb.ac.cn/gsa, accessed on 20 September 2023). The raw data from RNA-seq were processed with FastQC and trimmomatic (parameters were set as: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 HEADCROP:8 MINLEN:36 HEADCROP:15). The clean reads were mapped to the reference genome using hisat2 [56] and were assembled by the StringTie software (v2.2.2) with default parameters [57]. The TPM values derived from the combination analysis of the two kinds of software were used to represent gene expression levels. The R package Dseq2 (v1.50.2) was used to analyze the differentially expressed genes.

4.4. Over-Expression Vector Construction

The full-length coding sequence of CnHRLP1 was amplified using primers containing homology arms. The primer sequences were listed in Table S3. We used the Uniclone One Step Seamless Cloning Kit (Genes and Biotech Company, China) to construct the overexpression vector for CnHRLP1, named pc1300-35S-CnHRLP1-eGFP. The pc1300-35S-eGFP plasmid was linearized by digestion with Sal I and Kpn I. Homologous recombination was then employed to ligate the amplified CnHRLP1 coding sequence into the linearized vector. The final binary vector pc1300-35S-CnHRLP1-eGFP was transferred into Agrobacterium tumefaciens strain GV3101 following Agrobacterium-mediated transformation.

4.5. Transient Expression of CnHRLP1 in Tobacco Epidermal Cells for Subcellular Localization

Agrobacterium cultures harboring the pc1300-35S-CnHRLP1-eGFP or the 35S::OsGhd7:RFP (used as a positive control for nuclear localization) constructs were harvested by centrifugation and resuspended in infiltration medium. The empty vectors, pc1300-35S-eGFP and pc1300-35S-RFP, served as negative controls. The resuspended bacterial solution (OD600 = 1.0) was infiltrated into the abaxial side of fully expanded leaves from 4-week-old tobacco plants using a syringe without a needle. GFP signals were observed under a confocal microscope (LMS980, SESIS, Baden-Württemberg, Germany) at 48–72 h post-infiltration.

4.6. Plant Transformation and Transgenic Plants Phenotype Investigation

The overexpression vector for CnHRLP1, designated as pc1300-35S-CnHRLP1-eGFP, was used to transform Arabidopsis thaliana. Transgenic plants were generated through the floral dip method and selected on ½ Murashige and Skoog (MS) plates containing 50 µg/mL hygromycin. Plant height was measured 35 days following the transfer of seedlings from culture plates to soil pots. For phenotypic analysis, ten independent transgenic plants from separate lines were grown under long-day conditions (16 h light/8 h dark) at 22 °C. All transgenic Arabidopsis lines overexpressing CnHRLP1 were generated in the non-transgenic background ecotype Col-0, which was also used as the wild-type (WT) control throughout the phenotypic and transcriptomic analyses.

4.7. Transcriptome Datasets and RT-qPCR for OE-CnHRLP1 Transgenic Plants

To analyze the transcriptional profiles of wild-type and CnHRLP1-overexpressing transgenic Arabidopsis, paired-end RNA-seq reads were aligned to the Arabidopsis thaliana reference genome (TAIR10) using hisat2 [27]. Transcript assembly and abundance estimation were performed with StringTie [28]. Differential expression analysis between the transgenic and wild-type groups was conducted using the DESeq2 package in R, with genes showing an adjusted p-value (FDR) < 0.05 and |log₂FoldChange| > 1 considered significantly differentially expressed. Subsequently, clusterProfiler was employed to perform KEGG pathway enrichment analysis on the set of differentially expressed genes, with terms at FDR < 0.05 deemed significantly enriched.

For the transgenic plants included in the RNA-seq analysis, total RNA was extracted following the protocol described above. Three independent transgenic lines exhibiting a distinct early-flowering phenotype were selected. For each sample, first-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (R312, Vazyme, Nanjing, China) according to the manufacturer’s instructions. Quantitative real-time PCR was then carried out with the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China), following the provided protocol. The reactions were performed on an ABI 7900HT system in 384-well clear optical plates (Applied Biosystems, Foster City, CA, USA) under the following cycling conditions: an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 15 s, and 68 °C for 20 s. The specific primers used in RT-qPCR are listed in Table S3.

5. Conclusions

This study provides a comprehensive characterization of the CnRRM gene family in coconut, revealing substantial phylogenetic, structural, and expression diversity. Functional analyses identified that CnHRLP1 encodes a nuclear-localized RNA-binding protein whose overexpression severely restricts plant growth. Transcriptome profiling indicated that CnHRLP1 selectively remodels gene expression, with prominent effects on gibberellin-associated pathways. To our knowledge, this work provides functional evidence linking CnHRLP1 to transcriptome reprogramming and growth regulation.