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Genome-wide identification of the Remorin genes in Adiantum nelumboides and their responses to diverse abiotic stresses

BMC Plant Biology volume 25, Article number: 1397 (2025) Cite this article

Abstract

Adiantum nelumboides is a traditional Chinese medical herb and an endangered fern, and its population is threatened by various abiotic stresses. The plant-specific Remorin (REM) genes play important roles in coping with abiotic stresses; however, the information about REMs in A. nelumboides is scarce. In this study, a total of 34 AnREMs were identified in the latest released genome of A. nelumboides. These AnREMs could be assigned into three groups with 16 genes in Group 1, 3 genes in Group 4 and 15 genes in Group 6. All the REM proteins contained the conserved Remorin_C domain, while only AnREM1.2e harbored the Remorin_N domain. Plenty of cis-elements related to stress and hormone response were identified in the promoters of most AnREM genes. Transcriptomic analysis showed that several AnREMs (such as AnREM1.1a, AnREM1.4b, AnREM1.4a, AnREM4.2b) were significantly induced by re-watering and/or heat stresses, while AnREM1.2b, AnREM1.4b, AnREM6.5a, AnREM6.5c and AnREM1.4e were down-regulated in response to half-waterlogging stress. A co-expression gene network centered around AnREMs was constructed, pathway enrichment analysis showed the potential roles of AnREMs in regulating reactive oxygen species (ROS) accumulation and stabilizing the cell membrane. Taken together, the results from this study indicated that AnREM1.1a, AnREM1.4b, AnREM1.4a and AnREM4.2b could serve as the potential molecular targets for improving the abiotic stress tolerance in A. nelumboides.

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Introduction

Remorins (REMs) are plant-specific proteins that are widely identified in all land plants ranging from mosses to angiosperms [1, 2]. The REM proteins are characterized as a highly conserved C-terminal REM-related domain (Remorin_C, PF03763) and a variable N-terminal REM-related domain (Remorin_N, PF03766) [3]. The Remorin_C domain consists of a coiled-coil domain and a set of hydrophobic amino acid residues [1]. Therefore, the hydrophilic profile of REM proteins confers them the capacity to attach to the plasma membrane (PM) [2]. The oligomeric filamentous structures facilitated by coiled-coil domains of the C-terminal domains in vitro imply the roles of plant REM proteins in the plant cytoskeleton and/or the membrane skeleton [4, 5]. Currently, plenty of studies show that plant REM genes play important roles in plant growth and development, in hormone signaling, and in coping with diverse abiotic and biotic stresses, such as heat stress [6,7,8,9,10,11].

In pepper, CaREM1.4 is involved in regulating resistance to Ralstonia solanacearum by triggering cell death [12]. Its interaction with RPM1-interacting protein 4–12 (CaRIN4-12) hinders its role in the plant immune response [12]. Similarly, heterologous overexpression of tomato SlREM1 in Nicotiana benthamiana stimulates cell death by increasing the accumulation of reactive oxygen species (ROS) in response to the necrotrophic fungus Botrytis cinerea [13]. In citrus, CsREM1.1 positively regulates the fruit’s resistance to Penicillium digitatum [14]. In addition to biotic stresses, the expression of GmREM2.1 in soybean is highly induced by the infection of rhizobia, silencing of GmREM2.1 significantly reduces nodule formation [15], suggesting its role in plant-microbe interactions. Furthermore, a rice REM gene named Grain setting defect1 (GSD1) positively regulates grain setting by regulating the translocation of carbohydrates from leaves to the phloem with the help of the S-acylation and its interaction with actin 1 protein [16, 17]. For abiotic stress, the heterologous overexpression of Populus euphratica PeREM6.5 improves the tolerance to water stress in Arabidopsis transgenic plants by increasing the mRNA levels of genes involved in water transport and ROS scavenging [18]. Overexpression of Setaria italica SiREM6 in Arabidopsis significantly improves the salt tolerance of seedlings [19]. Additionally, overexpression of rapeseed BnaREM1.3–4 C-1 in the Arabidopsis rem1.3 mutant rescues the phenotypes of mutant plants under normal, salt and osmotic stresses, suggesting the roles of BnaREM1.3–4 C-1 in regulating plant growth and development, as well as in coping with abiotic stresses [20]. Growing evidence shows that plant REM genes play important roles in the normal growth of plants and in response to diverse stressful conditions; however, little information is available for REM genes in fern, especially their roles in coping with adverse environmental conditions.

Adiantum nelumboides is an endemic and endangered fern that is narrowly distributes along the Chongqing Three Gorges Reservoir area ranging from the east of Wanshou to the west of Shizhu [21]. A. nelumboides has been used as a traditional Chinese medicinal herb for thousands of years, and it plays roles in clearing heat, detoxifying the body, and promoting diuresis [21]. Besides being a medicinal herb, it also has the potential to be cultivated as an ornamental ferns [21]. A. nelumboides prefers high soil water content, acidic soil with low pH, and also steep slopes and cliff walls, the steeper slopes prevent it from competing with other plants and from human excavation [21]. However, A. nelumboides is threatened by the low genetic differentiation, human activities, low natural renewal ability, and also the climate changes in its habitat [21, 22]. Studies show that environmental conditions (such as temperature and precipitation) play essential roles in its future distribution [21, 22]. However, little information is available regarding A. nelumboides in response to environmental stresses.

To address this issue, a recent study integrated both transcriptomic and metabolomic approaches to uncover the essential roles played by phytohormone signaling and ROS scavenging of A. nelumboides in adapting to drought and half waterlogging stresses [23]. However, the potential roles of AnREM genes in coping with abiotic stresses remain to be explored. In this study, taking advantage of the latest released genome information of A. nelumboides [24], the genome-wide identification of AnREM members was conducted. The gene and protein structures of AnREMs, as well as the cis-elements on the promoters of AnREMs, were analyzed. Furthermore, the transcriptional changes of AnREMs in response to heat, drought and half-waterlogging stresses were systematically evaluated. Finally, the co-expression network centered around AnREMs was constructed to unravel the potential roles of AnREMs in comping with diverse abiotic stresses. The results obtained will offer new insights into the roles of AnREMs in plant adaptation to abiotic stresses and provide the genetic targets for enhancing environmental stress tolerance.

Methods and materials

Plant material and heat stress treatment

The plants of A. nelumboides X. C. Zhang were propagated and cultured at the Yangtze River Institute of Rare Plants (Hubei Province, China) according to the previous publication [23]. Healthy and uniform daughter ramet plants with 3–4 leaves were selected for heat stress treatment. Before heat stress treatment, the plants were cultured in a growth chamber at 25℃ (day)/20℃ (night) under the photoperiod of 16 h for one week. Afterwards, the plants were exposed to a condition of 35℃ (day)/28℃ (night) for heat stress with the same photoperiod. The leaves of A. nelumboides were harvested at 0, 6, 12, 24 and 48 h after heat stress, harvested samples were immediately immersed in liquid nitrogen, and stored at −80℃ for subsequent analysis. All the identified AnREM protein sequences are present in Table S1.

Identification of REMs in A. nelumboides

The identification of REM gene members was conducted as described in the previously published papers [25, 26]. Briefly, the protein sequences of Arabidopsis REM genes were acquired from the TAIR database. Subsequently, a local blastp was conducted using AtREM proteins as queries against the genome of A. nelumboides [24] with a cut-off lower than 10E-5 using Blast Several Sequences to a Big Database tool in TBtools-Ⅱ software [27]. Then, the hidden Markov model (HMM) of Remorin_C (PF03763) was retrieved from the Pfam database, and used as the queries against the A. nelumboides protein sequences [24] with HMMER3.0 software [28]. The results obtained from both blastp and HMMER analyses were combined, and the protein sequences of candidate AnREMs were submitted to the hmmscan database (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan) to verify the presence of the conserved Remorin_C domain. Only the AnREM proteins that contained the Remorin_C domain were considered for further analysis.

Construction of phylogenetic tree, analyses of gene and protein structures and three‑dimensional structure prediction

A neighbour-joining (NJ) phylogenetic tree of REM genes from A. nelumboides, Arabidopsis, and rice was constructed using MEGA11 software [29], subsequently the phylogenetic tree was visualized in the iTOL website [30]. The conserved domains of AnREM proteins were predicted using the MEME website [31]. The Gene Structure View (Advanced) tool in TBtools-Ⅱ software [27] was used to visualize the gene and protein structures with the phylogenetic tree (Newick file), the gff3 file, the xml file from MEME website and the results from hmmscan as described above.

The three‑dimensional structure prediction of AnREM proteins was carried out with the AlphaFold3 [32], and the results were visualized with the Pymol software (https://pymol.org/).

Bioinformation analysis of AnREMs

The physicochemical properties (including protein length, molecular weight, theoretical isoelectric point (pI) and grand average of hydropathicity) were calculated using Protein Paramter Calc tool in TBtools-Ⅱ software [27]. The subcellular localizations of AnREM proteins were predicted on Plant-mPLoc website [33]. The locus information of each AnREM gene was retrieved using the GXF Gene Position & Info Extract tool in TBtools-Ⅱ software [27].

Cis-element analysis of AnREM genes in A. nelumboides

The 3000 bp sequences upstream of each AnREM gene were retrieved using gff file and genome sequences with an in-house script. Subsequently, the 3000 bp sequences of promoters were submitted to the PlantCARE database [34] to predict the possible cis-elements.

RNA extraction and RNA-seq analysis

The harvested samples were milled into fine powder in a mortar with liquid nitrogen. Subsequently, the total RNAs of foliar samples were extracted by the CTAB method according to the previous publication [35]. After a quality check, the total RNAs of the samples were sent to Novogene Corporation (Tianjin, China) for library construction and RNA-seq analysis. The RNA-seq was conducted on the DNBSEQ-T7 platform (MGI Tech Co., Ltd., Shenzhen, China) in 150 bp paired-end mode.

Transcriptome analysis

To analyze the gene expression of AnREMs under drought and waterlogging stresses, the transcriptomes of A. nelumboides under control condition (CK), 30% (w/w) pot soil water content for 5 days (SW05), rewatering to normal field water capacity for 5 days (SWF05), half-waterlogging treatment for 5 (DW5) and 10 (DW10) days were retrieved from NCBI database under the accession of PRJNA898650. The information about the treatments was detailed in the previous publication [23]. The iSeq software was used to download the transcriptomes from the database. The transcriptomes from the online resource and this study were analyzed according to the following procedures. First, the low-quality reads were filtered by fastp [36], then the clean sequences were mapped onto the genome of A. nelumboides [24] using bowtie2 software [37]. The gene count quantifications among different samples were accomplished in featureCounts [38]. The differentially expressed genes were calculated using the DEseq2 package [39] in R software. The genes with absolute values of fold changes above 2, and the false discovery ratio (FDR) lower than 0.05 were defined as significantly differentially expressed genes. The heatmap representing the expression levels of AnREMs under different comparisons was drawn in R software with the pheatmap package [40].

Co-expression analysis of AnREMs and pathway enrichment analysis

The WGCNA package [41] in R software was used to construct the co-expression network, subsequently the AnREMs-centered co-expression network was extracted from the whole network output from the WGCNA analysis using R software. The co-expression network of AnREMs was visualized in Cytoscape software [42] as suggest by the previous publication [43].

For GO and KEGG enrichment analysis, the all-protein sequences of A. nelumboides were annotated using the EggNOG database [44]. Subsequently, the GO and KEGG enrichment was analyzed in R software with the clusterProfiler package [45]. The bubble figure representing the enrichment results was drawn in R software with the ggplot2 package as suggested by the previous publication [46].

Results

Identification of AnREMs in A. nelumboides

A total of thirty-four AnREMs were identified in the genome of A. nelumboides, the protein length of which ranged from 127 to 965 aa (Table 1). The estimated molecular weights of AnREM proteins varied from 14.56 to 105.96 kDa (Table 1). The subcellular localizations of AnREM proteins were predicted to be in the cell membrane and/or nucleus (Table 1). The theoretical pIs of AnREM1.4b, AnREM1.4c, AnREM1.4f, AnREM6.1a, AnREM6.1b, AnREM6.1c, AnREM6.1e and AnREM6.1f were lower than 7.00, suggesting they were acidic proteins, and the remaining AnREM proteins were alkaline proteins with estimated theoretical pIs above 7.00 (Table 1). All the values of the grand average of hydropathicity were below 0 (Table 1), suggesting that they were hydrophilic proteins.

Table 1 The REM family members in A. nelumboides
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Gene and protein structures of AnREMs in A. nelumboides and phylogenetic tree

The gene structure analysis of AnREMs showed that most AnREMs had 5’-untranslated region (UTR) and/or 3’-UTR, expect for AnREM1.1b, AnREM1.4e, and AnREM1.2c (Fig. 1A). A large number of introns were found in the coding sequence (CDS) of all the AnREMs, for instance, AnREM1.1b and AnREM1.4e had 3 and 4 introns, respectively (Fig. 1A). Furthermore, long introns (defined as longer than 5 kb) were detected in the CDS of AnREM1.2c, AnREM1.2d, AnREM6.1b, and AnREM6.1d (Fig. 1A). An ultra-long intron (longer than 25 kb) before the last exon was detected for AnREM6.1b and AnREM6.1d (Fig. 1A). A total of seven conserved protein motifs have been detected among all the AnREM proteins (Table 2; Fig. 1B), the motifs 1 and 2 were conserved in all the AnREM proteins (Fig. 1B). All the AnREM proteins from Group 1 contained motif 1, 2, 3 and 7, except that motif 7 was absent in AnREM 1.4a and AnREM1.4b (Fig. 1B). All the AnREM proteins harbored the conserved Remorin_C domain, and only AnREM1.2e contained the Remorin_N domain (Fig. 1C). Additionally, the Remorin_C domains of AnREM6.1c and AnREM6.1e proteins were truncated, whereas the AnREM6.5b represented the shortest protein with an entire Remorin_C domain (Fig. 1C). From the three-dimensional structure prediction, it can be concluded that all AnREM proteins contained a conserved Remorin_C domain, whose structure was relatively stable (Fig. 2). Additionally, the N-terminal regions of AnREMs exhibited structural diversity (Fig. 2), which may allow these proteins to perform a variety of functions.

Fig. 1
figure 1

The gene structures (A), the protein conserved motifs (B) and the known HMM motifs (C) of AnREMs in A. nelumboides

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Table 2 The conserved protein domains of AnREM proteins
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Fig. 2
figure 2

Three‑dimensional structure prediction for AnREM proteins. The Remorin_N domain and the Remorin_C domain were represented in yellow and red, respectively

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A NJ phylogenetic tree of REMs from A. nelumboides, Arabidopsis, and rice was constructed, which could be divided into six subgroups, i.e., Group 0.2, Group 1 & 3, Group 4, Group 5, and Group 6 (Fig. 3). This was consistent with the previous studies [47, 48]. All the identified AnREMs in A. nelumboides belonged to Groups 1, 4, and 6 (Table 1; Fig. 3), suggesting that REM members from these groups were more ancient in the evolutionary history.

Fig. 3
figure 3

The neighbour-joining phylogenetic tree represents the REM proteins from A. nelumboides (An), A. thaliana (At) and rice (Os)

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Cis-element analysis of AnREM promoters

To uncover the possible transcriptional regulations of AnREMs, the cis-elements in the promoters of AnREMs were analyzed (Fig. 4). Plenty of cis-elements were detected in the promoter of each AnREM gene, and these identified cis-elements could be generally categorized into three groups, i.e., light response, plant growth and development, as well as stress and hormone response (Fig. 4). In the category of light response, G-box was widely identified in the promoters of AnREM1.1b, AnREM1.2a, AnREM1.2f, AnREM1.4e, AnREM1.4f, AnREM4.2a, AnREM4.2b, AnREM4.2c, AnREM6.1e, AnREM6.2b, AnREM6.3a, AnREM6.3c, AnREM6.3d and AnREM6.5b (Fig. 4A). For the stress and hormone response category, the abscisic acid response elements (ABREs) were widely identified in most AnREMs promoters, three AnREMs (e.g., AnREM1.2a, AnREM6.3d and AnREM6.5b) contained more than 10 ABREs in their promoter regions (Fig. 4A). Additionally, the CAAT-box was detected in large numbers in the promoters of all AnREMs (Fig. 4A). The cis-elements belonging to plant growth and development occupied the smallest proportions of all detected cis-elements for each AnREM gene, which ranged from 10% to 12% (Fig. 4B). On the other hand, approximately 22%−73% and 24%−68% of detected cis-elements were assigned into light response and stress and hormone response, respectively (Fig. 4B), suggesting that most AnREMs were transcriptionally regulated by environmental clues.

Fig. 4
figure 4

The numbers (A) and proportions (B) of cis-elements identified in the promoters of each AnREM gene. The numbers and red color in the cells indicate the number of certain identified cis-elements for each gene

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Expression analysis of AnREMs under drought and half-waterlogging stresses

The transcriptional changes of AnREMs in response to drought and rewatering, as well as half waterlogging stress were analyzed (Fig. 5) using the published online resources [23]. For the drought and rewatering treatment, AnREM1.2d and AnREM1.4d were significantly down-regulated after 5 days of drought stress, whereas the mRNA levels of AnREM1.1b, AnREM1.2h, AnREM6.3d and AnREM6.5c were significantly decreased under 5 days drought treatment (Fig. 5A). However, no significantly differentially expressed genes were identified in the comparison of SWF05 vs. CK (Fig. 5A). Compared to drought for 5 days, the expression levels of AnREM1.4b, AnREM6.1f, AnREM6.2b, AnREM6.3d, AnREM6.1a, AnREM6.5a and AnREM6.5c were significantly induced after 5 days of rewatering (Fig. 5A). Nevertheless, the mRNA levels of AnREM6.1b, AnREM6.1c and AnREM6.1e were significantly down-regulated when comparing SWF05 with SW05 (Fig. 5A).

Fig. 5
figure 5

The heatmaps represent the expression levels of AnREM genes in response to drought and re-watering treatments (A), and to half-waterlogging stress (B). The bule to red colors indicate the relative expression levels ranging from low to high. The stars in the cells indicate significant expression under the given comparisons. CK, control; SW05, 30% (w/w) pot soil water content for 5 days; SWF05, rewatering to normal field water capacity for 5 days; DW5, half-waterlogging treatment for 5 days; DW10, half-waterlogging treatment for 5 days

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The expression levels of AnREM1.2b and AnREM1.4b were significantly down-regulated by half-waterlogging stress after both 5 and 10 days (Fig. 5B). Additionally, compared with the control condition, AnREM6.5a was down-regulated by half waterlogging stress for 5 days, while AnREM6.5c and AnREM1.4e were both significantly down-regulated by half waterlogging stress for 10 days (Fig. 5B).

Transcriptional changes of AnREMs in response to heat stress

The mRNA levels of AnREM6.2b, AnREM6.3b and AnREM6.5b were significantly down-regulated, while those of the AnREM4.2b and AnREM1.1a were significantly induced after 6 h of heat exposure (Fig. 6). Compared with 0 h, the 12 h heat stress led to an increase in the mRNA levels of AnREM6.3a, AnREM1.4a, and AnREM4.2b, and to a decrease in the transcriptional expression of AnREM6.1d and AnREM6.1e (Fig. 6). In the comparisons of 24 h vs. 0 h and 48 h vs. 0 h, the mRNA levels of most AnREMs showed similar expression patterns, for instance, the mRNA levels of AnREM1.2f, AnREM6.3b, AnREM6.1e, AnREM6.2a and AnREM6.3a were significantly decreased, whereas the mRNA of AnREM1.4a genes was significantly highly accumulated (Fig. 6). Additionally, several other AnREMs (e.g., AnREM1.2 h, AnREM6.3d, AnREM6.1a, AnREM1.2b and AnREM1.4b) were only down-regulated after 48 h of heat stress (Fig. 6).

Fig. 6
figure 6

The heatmap represent the gene expression profiles of AnREM genes in the leaves of A. nelumboides exposed to heat stress condition for 0, 6, 12, 24 and 48 h. The bule to red colors indicate the relative expression levels ranging from low to high. The stars in the cells indicate significant expression under the given comparisons

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Co-expression of AnREMs under stress conditions

To unravel the possible biological functions played by AnREMs under stress conditions, a WGCNA-based co-expression network centered around AnREMs was constructed (Fig. 7A). In the co-expression network, a total of 2807 genes were connected by 6747 co-expression relationships with 13 AnREM members (Fig. 7A). AnREM1.2h, AnREM1.4b, AnREM1.4c, AnREM6.1a, AnREM6.1b and AnREM6.5a were tightly co-expressed with 2502 other genes (Fig. 7A), suggesting that they might be involved in biological pathways. Additionally, AnREM4.2b and AnREM4.2c, as well as AnREM6.5c and AnREM1.4e shared some co-expressed genes (Fig. 7A). However, the networks centered by AnREM6.1d, AnREM6.3a and AnREM6.2b were separated from other AnREM-centered networks without any overlapping co-expressed genes (Fig. 7A). The GO and KEGG enrichment analysis revealed that AnREMs potentially played roles in responding insects, cellular response to ethylene stimulus, hydrogen peroxide biosynthetic process, plant-type cell wall organization or biogenesis, plant-type cell wall cellulose metabolism, plant-type cell wall biogenesis, as well as amino sugar and nucleotide sugar metabolism (Fig. 7B).

Fig. 7
figure 7

The co-expression gene network of AnREMs under abiotic stresses (A), and the GO and KEGG enrichment analyses of the genes identified in the co-expression network (B). The blue and grey nodes represent AnREMs and their co-expressed genes, respectively. The edges between the nodes represent the correlations between the genes

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Discussion

In recent years, the REM gene family has been widely identified at the whole-genome level in many plant species, such as Arabidopsis thaliana [2], Brassica napus [20], Cajanus cajan [9], Saccharum spontaneum [10], S. italica [7], Citrus sinensis [14], and Populus trichocarpa [49]. However, the information of REM gene family in fern was quite scarce. In the current study, a total of 34 AnREM gene members were identified by genome-wide approaches, the number of AnREM genes in A. nelumboides was significantly greater than that of P. trichocarpa (22), C. sinensis (10), and S. italica (21) [7, 14, 49]. The large number of AnREM gene members in A. nelumboides might be attributed to its tetraploid genome [24]. Similarly, the allotetraploid B. napus contains 47 BnaREM genes [20]. The plant REM gene family could be divided into six groups (e.g., Group 1–6) and an additional Group 0.2 [2, 20]. Notably, REM members from Group 2 are specific for legumes and poplar [2, 49], which were also absent in the genome of A. nelumboides. The MtREM5.1 of Medicago truncatula was the major expressed REM gene in seeds [2], which suggesting the roles of REM5 members in seed development. However, the REM5 members were absent in the genome of A. nelumboides, which suggested that REM5 members might be specific for spermatophyte, further studies are need to verify this point.

The identified AnREMs were assigned into three groups with 16 genes in Group 1, 3 genes in Group 4 and 15 genes in Group 6, suggesting that REM members from Groups 1, 4 and 6 were more ancient than those from other groups. The AnREM members from Group 1 and 3 were quite close in the phylogenetic tree, which were similar to the previous studies in tomato and wheat [47, 48]. Indeed, a previous systematically phylogenetic analysis also showed that members from Group 1 and Group 3 were in the same clade [2]. The Group 0.2 in this study contained OsREM0.2, AtREM6.6, AtREM6.7 and AtREM6.6, which were consist with the results obtained from tomato and wheat [47, 48].

The REM proteins from Group 1 are considered the canonical ones with both Remorin_N and Remorin_C domains [2]. For instance, all of four PtREM1 members contained both Remorin_N and Remorin_C domains [49]. However, among the 16 AnREM1s, only AnREM1.2e contained the Remorin_N domain, suggesting that the Remorin_N domain might have been acquired during the course of evolution. The REM6 proteins represent the longest REM proteins among the others [2]. All of the identified AnREM proteins contained the conserved Remorin_C domains, suggesting that they had the capacity to oligomerize into filamentous structures for attaching to the PM. The variable N-terminal regions of AnREM proteins might contribute to the functional diversity [50]. Additionally, several AnREM genes (AnREM6.1b and AnREM6.1d) contained an ultra-long intron before the last exon, reflecting the genome complexity of ferns.

Since plenty of cis-elements belonging to stress and hormone response were detected in the promoter regions of AnREM genes, it was highly possible that the transcriptional regulation of AnREMs was affected by diverse stresses. To address this issue, the transcriptional changes of AnREM genes in response to drought, waterlogging and heat stresses were analyzed. In line with the finding that poplar PeREM6.5 is involved in improving the drought tolerance of the transgenic plants [18], the rewatering treatment significantly induced the mRNA levels of several AnREM genes, including AnREM1.4b, AnREM6.1f, AnREM6.2b, AnREM6.3d, AnREM6.1a, AnREM6.5a and AnREM6.5c, when compared to drought treatment for 5 days. Interestingly, no significantly differentially expressed AnREM genes were detected under the comparison of SWF05 vs. CK, suggesting that AnREMs could rapidly adjust their mRNA levels to changes in soil water content. Little information was available regarding to the transcriptional changes of REMs in response to waterlogging stress. In this study, several AnREM6 genes (e.g., AnREM6.1a, AnREM6.1f, AnREM6.2b, AnREM6.3d, AnREM6.5a and AnREM6.5c) were significantly down-regulated after waterlogging stress for 5 days and/or 10 days, suggesting these AnREM genes might be important for coping with waterlogging stress. For the heat stress, two AnREM1 genes (AnREM1.1a and AnREM1.4a) and AnREM4.2b were significantly induced by heat stress, suggesting these three genes might contribute to the heat tolerance of A. nelumboides. In line with this, the CcREM1.3 gene in C. cajan is also induced by heat stress, and is thought to be a potential target for breeding of heat-tolerant C. cajan [9]. Taken together, the obtained results indicated that AnREM genes play important roles in coping with diverse abiotic stresses, and several AnREM genes (such as AnREM1.1a, AnREM1.4b, AnREM1.4a and AnREM4.2b) could serve as molecular targets for improving tolerances to abiotic stresses.

The genes that participate in the same biological pathways tend to share similar expression levels, therefore, co-expression network analysis has become a powerful approach to uncover the potential roles of given genes [51, 52]. Through the WGCNA analysis, a comprehensive co-expression network centered around AnREM genes was constructed to unravel the possible roles of AnREMs in coping with abiotic stresses. The network revealed that the possible functions of AnREMs in coping with abiotic stresses by regulating ROS accumulation, cell-cell signaling, and the cytoskeleton. Indeed, the previous studies have also shown the close links between the cytoskeleton and REM-induced membrane tubulation in facilitating the stabilization of membrane conformations [53]. Abiotic stresses, such as drought and heat stresses, often trigger the disruption of the cell membrane, which subsequently leads to the accumulation of ROS in plants [54, 55]. Therefore, the REM genes may strengthen the stabilization of cell membrane, and reduce the ROS accumulation. Taken together, the co-expression gene network analysis of AnREM genes imply the roles of AnREMs in the stabilization of cell membrane and the regulation of ROS accumulation.

Conclusion

In summary, a total of 34 AnREMs belonging to three groups were identified in the genome of A. nelumboides. All the AnREM proteins contained the Remorin_C domain, and only AnREM1.2e harbored the Remorin_N domain. Plenty of cis-elements involved in stress and hormone response were detected in the promoters of AnREM genes, suggesting that they might be regulated by diverse abiotic stresses. Several AnREMs (such as AnREM1.1a, AnREM1.4b, AnREM1.4a and AnREM4.2b) were significantly induced by re-watering and/or heat stresses. These identified AnREM genes could server as molecular targets for improving the stress tolerance of A. nelumboides in the context of global climate change, also provide genetic resources for other plants to improve their performance under abiotic stresses. However, the lack of the efficient genetic transformation technology for A. nelumboides will hinder the taking advantage of these AnREM in genetic improvement, and also the functional verification of these key AnREM genes in A. nelumboides.

Data availability

Data is provided within the manuscript or supplementary information files.

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Funding

This work was supported by the Ecological Environmental Protection Fund of China Three Gorges Corporation (NBZZ202300130), the Hubei Provincial Central Government-Guided Local Science and Technology Development Special Project (2024BS019), the National Natural Science Foundation of China (U2240222) and the discovery and identification of functional genes for the formation of important traits of ecological adaptability of three excellent plants, such as Adiantum nelumboides, Plantago fengdouensis, and Myricaria laxiflora (GCZX-2024-03-044)..

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  1. Linbao Li and Jiayi Gu have contributed equally to this work.

Authors and Affiliations

  1. Yangtze River Biodiversity Research Centre, China Three Gorges Corporation, Wuhan, 443133, China

    Linbao Li, Qianyan Liang, Jinhua Wu, Yang Su, Haofei Zhu, Bicheng Dun, Ganju Xiang, Zhiqiang Xiao, Guangxin Zhang & Di Wu

  2. Hubei Key Laboratory of Rare Resource Plants in Three Gorges Reservoir Area, Yichang, 443100, China

    Linbao Li, Qianyan Liang, Jinhua Wu, Yang Su, Haofei Zhu, Bicheng Dun, Ganju Xiang, Zhiqiang Xiao, Guangxin Zhang & Di Wu

  3. National Engineering Research Center of Eco-Environment Protection for Yangtze River Economic Belt, Three Gorges Corporation, Wuhan, 100083, China

    Linbao Li, Qianyan Liang, Jinhua Wu, Yang Su, Haofei Zhu, Bicheng Dun, Ganju Xiang, Zhiqiang Xiao, Guangxin Zhang & Di Wu

  4. College of Horticulture and Forestry Science, Hubei Engineering Technology Research Center for Forestry Information, Huazhong Agricultural University, Wuhan, 430070, China

    Jiayi Gu

  5. School of Tropical Agriculture and Forestry (School of Agricultural and Rural Affairs, School of Rural Revitalization), Hainan University, Haikou, China, 570228

    Jiayi Gu

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  1. Linbao Li
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  9. Zhiqiang Xiao
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  10. Guangxin Zhang
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Contributions

D.W. was responsible for conceptualization, funding acquisition, project administration, supervision, writing the original draft, and writing review & editing; L.L. and J.G. undertook data curation, formal analysis, investigation, resource provision, validation, visualization, and also participated in writing the original draft as well as writing review & editing; Q.L., J.W., Y.S., H.Z., B.D., G.X., Z.X., and G.Z. were involved in data curation, investigation, and visualization - related work. All authors reviewed the manuscript.

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Correspondence to Di Wu.

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Li, L., Gu, J., Liang, Q. et al. Genome-wide identification of the Remorin genes in Adiantum nelumboides and their responses to diverse abiotic stresses. BMC Plant Biol 25, 1397 (2025). https://doi.org/10.1186/s12870-025-07444-3

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BMC Plant Biology

ISSN: 1471-2229

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