Skip to main content
Download PDF

Genome-wide identification of Ankyrin (ANK) repeat gene families in three Dendrobium species and the expression of ANK genes in D. officinale under gibberellin and abscisic acid treatments

BMC Plant Biology volume 24, Article number: 762 (2024) Cite this article

Abstract

Background

Dendrobium Sw. represents one of the most expansive genera within the Orchidaceae family, renowned for its species’ high medicinal and ornamental value. In higher plants, the ankyrin (ANK) repeat protein family is characterized by a unique ANK repeat domain, integral to a plethora of biological functions and biochemical activities. The ANK gene family plays a pivotal role in various plant physiological processes, including stress responses, hormone signaling, and growth. Hence, investigating the ANK gene family and identifying disease-resistance genes in Dendrobium is of paramount importance.

Results

This research identified 78 ANK genes in Dendrobium officinale Kimura et Migo, 77 in Dendrobium nobile Lindl., and 58 in Dendrobium chrysotoxum Lindl. Subsequently, we conducted comprehensive bioinformatics analyses on these ANK gene families, encompassing gene classification, chromosomal localization, phylogenetic relationships, gene structure and motif characterization, cis-acting regulatory element identification, collinearity assessment, protein-protein interaction network construction, and gene expression profiling. Concurrently, three DoANK genes (DoANK14, DoANK19, and DoANK47) in D. officinale were discerned to indirectly activate the NPR1 transcription factor in the ETI system via SA, thereby modulating the expression of the antibacterial PR gene. Hormonal treatments with GA3 and ABA revealed that 17 and 8 genes were significantly up-regulated, while 4 and 8 genes were significantly down-regulated, respectively. DoANK32 was found to localize to the ArfGAP gene in the endocytosis pathway, impacting vesicle transport and the polar movement of auxin.

Conclusion

Our findings provide a robust framework for the taxonomic classification, evolutionary analysis, and functional prediction of Dendrobium ANK genes. The three highlighted ANK genes (DoANK14, DoANK19, and DoANK47) from D. officinale may prove valuable in disease resistance and stress response research. DoANK32 is implicated in the morphogenesis and development of D. officinale through its role in vesicular transport and auxin polarity, with subcellular localization studies confirming its presence in the nucleus and cell membrane. ANK genes displaying significant expression changes in response to hormonal treatments could play a crucial role in the hormonal response of D. officinale, potentially inhibiting its growth and development through the modulation of plant hormones such as GA3 and ABA.

Peer Review reports

Background

Dendrobium Sw., a prominent genus within the Orchidaceae family, boasts numerous species with significant medicinal and ornamental value [1]. These plants are typically rich in flavonoids, alkaloids, and polysaccharides, contributing to their high therapeutic and aesthetic worth [2]. With the market demand for Dendrobium officinale Kimura et Migo surging, the cultivation industry has experienced rapid growth. Consequently, plant diseases and hormonal imbalances can impact its growth and yield. Therefore, identifying functional genes related to stress and hormone responses at the genomic level is crucial for D. officinale.

Ankyrin (ANK) repeats domain, which is a common conserved protein domain distributed throughout the organism [3], was first found in these two proteins: yeast cell-cycle regulator Swi6/Cdc10 and Drosophila signal protein Notch [4]. The primary structure of ANK repeat sequence contains 33 amino acids repeats of residues, which constructs a specific protein structure. Although the specific functions of ANK repeats were not clear, and they were found in many proteins with different functions in biology. In yeast and animals, ANK protein plays an important role in growth and development, signal transduction and transcriptional regulation [3]. Similarly, ANK protein has been proved to participate in many important physiological pathways in plants.

In higher plants, the first reported ANK protein is the AKR protein in Arabidopsis thaliana (L.) Heynh., which plays an important role in the regulation of the light system and plant growth and development [5]. ANK protein also plays a crucial role in plant response to biotic and abiotic stresses. For example, CaKR1, a protein containing ANK repeats in tomatoes, is crucial for its resistance to Phytophthora infestans [6]. In Arabidopsis, AKR2, the gene encoding ANK domain, may be related to antioxidant metabolism in plant stress and disease resistance response [7]. NPR1 transcription factor, which is involved in plant hormone signal transduction, is a gene regulated and expressed by SA and plays an important role in plant disease resistance [8]. The ANK repeat protein family is involved in many physiological processes such as stress, hormone response, and growth of plants, so it is necessary to study the ANK gene family.

Advancements in third-generation sequencing technologies, such as PacBio and nanopore, have facilitated the sequencing and publication of the genomes of D. officinale, Dendrobium nobile Lindl., and Dendrobium chrysotoxum Lindl. [9,10,11]. In our previous work, we assembled the first chromosome-level genome of D. officinale by combining PacBio long-reads, Illumina short-reads and Hi-C sequencing [9]. The genome size of D. officinale is 1.21 Gb, with a very high heterozygosity of 1.27% and a repeat content of 64.39% [9]. This progress enables researchers to investigate the evolution of ANK gene families in three Dendrobium species and identify functional genes.

In this study, we identified 78, 77, and 58 ANK genes in D. officinale, D. nobile, and D. chrysotoxum, respectively. We conducted extensive bioinformatics analyses on the ANK gene families, which included gene classification, chromosomal localization, phylogenetic relationships, gene structure and motif composition, cis-acting elements, collinearity, protein-protein interaction networks, and gene expression profiles. Moreover, we identified three DoANKs (DoANK14, DoANK19, and DoANK47) in D. officinale with potential research value for disease resistance and stress response. DoANK32, associated with the ArfGAP gene in the endocytosis pathway, influences vesicle transport and auxin polar transport, thereby affecting organ formation and development in D. officinale. DoANK genes exhibiting significant expression changes under hormonal treatment may play a crucial role in the hormonal response of D. officinale, potentially inhibiting growth and development by modulating plant hormones such as GA3 and ABA.

Results

Genome-wide identification of ANK domain containing proteins in DendrobiumSw

A total of 78, 77, and 58 members of the ANK gene family were identified in three Dendrobium species: D. officinale, D. nobile, and D. chrysotoxum, respectively (Tables S3), using the HMM profile (PF00023) and BLASTP method (S1). Compared to D. chrysotoxum, D. officinale and D. nobile have a greater number of candidate members in the ANK gene family. All ANK proteins contain conserved ANK domains and are encoded by genes named DoANK01-DoANK78, DnANK01-DnANK77, and DcANK01-DcANK58, respectively (Table S2, S3). The physical and chemical properties of ANK proteins in the three Dendrobium species indicate that the amino acid lengths of DoANK, DnANK, and DcANK proteins range from 93 to 1964, 76 to 1621, and 133 to 1696 amino acids, respectively (Table S4). The molecular weights of DoANK, DnANK, and DcANK proteins range from 9.76 to 217.34, 8.49 to 178.86, and 14.12 to 187.15 kDa, respectively (Table S4). Their isoelectric points range from 4.03 to 9.86, 3.93 to 10.18, and 3.95 to 10.14, respectively (Table S4).

Conserved domains distribution of ANK genes in DendrobiumSw

Based on domain composition, all ANK genes in D. officinale are categorized into 10 subfamilies. Of these, 33 members contain only the ANK domain, while the remaining 45 members contain additional typical domains (Tables 1, S4). Nine DoANK genes containing transmembrane domains are classified as the ANK-TM subfamily (Table 1). Eleven members, designated as ANK-RF, possess ring finger domains (Table 1). Eight genes feature a zinc finger domain, belonging to the ANK-ZnF subfamily, and two members contain a tyrosine kinase catalytic domain, classified as the ANK-PK subfamily (Table 1).

Table 1 Quantitative characteristics of ANK subfamilies in different species
Full size table

In D. nobile, 20 of the 77 DnANK genes contain only the ANK domain, while the remaining 57 members have additional domains (Table 1). The ANK-TM subfamily includes 16 DnANK genes, the ANK-RF subfamily comprises 10 members, the ANK-ZnF subfamily includes 6 genes with zinc finger domains, and the ANK-PK subfamily contains 3 genes with tyrosine kinase catalytic domains (Table 1).

For D. chrysotoxum, out of 58 DcANK genes, 16 contain only ANK domains, while the remaining 32 have additional domains (Table 1). Seven DcANK genes, identified as the ANK-TM subfamily, contain transmembrane domains, and nine members, designated as ANK-RF, possess ring finger domains (Table 1). Additionally, five genes belong to the ANK-ZnF subfamily, and one gene, containing a tyrosine kinase catalytic domain, is classified as the ANK-PK subfamily (Table 1).

Chromosomal locations of the ANK genes in DendrobiumSw

Among the 213 ANK genes identified, 5 were located on scaffolds, while 208 (97.65%) were distributed across 19 chromosomes of Dendrobium with varying densities (Table S3). In a single chromosome, the ANK genes were distributed in all regions: at the telomere end, near the centromere, etc., it is scattered or clustered separately. Gene clusters were found on chromosomes DOchr3, DOchr17, DOchr19, DNchr18, and DNchr19 (Fig. 1). In D. officinale, DOchr3 contained the most DoANK genes, with eleven, whereas DOchr5, DOchr6, DOchr9, and DOchr12 each had only two (Fig. 1A). In D. nobile, DNchr4 and DNchr18 each harbored ten DnANK genes, the highest number, while DNchr7, DNchr10, and DNchr11 each contained only two (Fig. 1B). For D. chrysotoxum, DCchr17 and DCchr19 each had seven DcANK genes, the most among its chromosomes, whereas DCchr6, DCchr8, DCchr9, and DCchr15 each had only one (Fig. 1C).

Fig. 1
figure 1

Chromosomal locations of ANK proteins in the D. officinale (A), D. nobile (B), and D. chrysotoxum (C). The number of each chromosome is displayed above it. Scale represents chromosome length in Mbp

Full size image

Phylogenetic analysis of ANK Protein in DendrobiumSw

To thoroughly understand the phylogenetic relationships and evolutionary history of ANK gene, and infer its function according to homologous genes, ClustalX2.1 was used to compare the protein sequences of Dendrobium and 105 ANK protein sequences of Arabidopsis (S1). A rootless phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replications (Fig. 2). The phylogenetic analysis revealed that all ANK protein amino acid sequences were divided into six major groups (Fig. 2). Consistent with the subfamily classification, members of the same subfamily generally clustered together. For instance, all members of the ANK-ZnF subfamily, except DoANK76, were grouped together (Fig. 2). Similarly, all members of the ANK-BTB subfamily were in the same group (Fig. 2). However, members of the ANK-M subfamily were distributed across all branches of the phylogenetic tree (Fig. 2). Generally, most members within the phylogenetic tree shared similar domain compositions, though domain-based classification did not entirely align with phylogenetic classification.

Fig. 2
figure 2

Phylogenetic relationships of ANK genes in D. officinale (green), D. nobile (blue), D. chrysotoxum (purple) and A. thaliana (yellow). The maximum likelihood phylogenetic tree were constructed with 1000 bootstrapping runs using RAxML. ANK subfamilies are represented by small ICONS of different colors

Full size image

Gene duplication events and syntenic analysis of DoANKs, DnANKs, and DcANKs

The phylogenetic tree was constructed using the amino acid sequences of DoANKs, DnANKs, and DcANKs (Fig. 3). In general, members with similar domains were more closely related in the phylogenetic tree, although their associations were not always exact. The ANK genes in D. officinale were divided into three branches (Fig. 3A), a pattern also observed in D. nobile (Fig. 3B) and D. chrysotoxum (Fig. 3C). In order to analyze the motif composition of DoANKs, DnANKs, and DcANKs, submitted this amino acid sequence to the MEME website, and the number of predicted motifs was set to 15, with a length between 15 and 50 amino acids (Fig. 3). Among the 15 motifs, Motif 2 was widely found in almost all ANKs (Fig. 3). The location and number of exons and introns could be used as one of the evidences of phylogenetic grouping, and play a very important role in the evolution of the whole gene family. The location and number of exons and introns of ANK genes in Dendrobium were shown in the Fig. 3. The number of exons of ANK genes in Dendrobium was significantly higher than that of introns. Introns of genes play a key role in plant phylogenetic analysis. Newly evolved plant species tend to had fewer introns than their ancestors. The structure of many ANK genes was relatively simple, including only one CDS, such as DoANK01, DoANK05, DnANK36, DnANK40, DcANK13, and DcANK17, etc. (Fig. 3).

Fig. 3
figure 3

Phylogenetic relationships, conserved motifs and exon-intron structures of ANK genes in D. officinale (A), D. nobile (B), and D. chrysotoxum (C). The conserved motifs of proteins were identified using MEME and visualized by TBtools. Different colors represent 15 different motifs. Green and yellow boxes are respectively indicating CDS and UTR. The black lines indicate introns. The scale bar in the bottom represents the gene length in kb

Full size image

Gene duplication events and syntenic analysis of DoANKs, DnANKs, and DcANKs

BLASTN and MCScanX were employed to analyze the synchrony of DoANKs, DnANKs, and DcANKs, and to study replication events among ANK genes. Eight, nine, and four pairs of homologous genes were identified in DoANKs (Fig. 4A), DnANKs (Fig. 4B), and DcANKs (Fig. 4C), respectively. In D. officinale, DoANKs homologous genes were located on chromosomes DOchr1, DOchr2, DOchr3, DOchr10, and DOchr19 (Fig. 4A); in D. nobile, DnANKs homologous genes were located on chromosomes DNchr1, DNchr5, DNchr9, DNchr11, DNchr13, DNchr14, DNchr15, DNchr16, and DNchr18 (Fig. 4B); however, in D. chrysotoxum, DcANKs homologous genes were located on chromosomes DCchr10, DCchr12, DCchr14, DCchr16, DCchr17, and DCchr19 (Fig. 4C). The eight pairs of homologous genes in DoANKs were DoANK02-36, DoANK05-34, DoANK07-08, DoANK11-12, DoANK16-18, and DoANK69-70-72 (Fig. 4A). The nine pairs of homologous genes in DnANKs were DnANK06-36-48, DnANK24-25, DnANK40-66, DnANK49-59, and DnANK57-58-74 (Fig. 4B). The four pairs of homologous genes in DcANKs were DcANK23-34, DcANK31-39, DcANK29-53, and DcANK46-53 (Fig. 4C).

Fig. 4
figure 4

The gene duplications of ANK genes from three Dendrobium. Blue, yellow, green lines highlight the syntenic gene pairs in D. officinale (A), D. nobile (B), and D. chrysotoxum (C), respectively. ANK subfamilies are represented by small ICONS of different colors

Full size image

Calculating the Ka/Ks value of duplicate gene pairs provides insights into the driving force of gene evolution. The Ka/Ks ratio reflects the rate of gene evolution and the type of selection pressure. When Ka/Ks = 0, the selection pressure was neutral. When Ka/Ks < 1, it was a negative selection; when Ka/Ks > 1, it was a positive selection. The Ka/Ks ratios of 21 pairs of Dendrobium genes ranged from 0.001 to 1.409 (Table S5). Among them, the Ka/Ks ratio of 19 pairs was < 1 (Table S5), indicating negative selection in the evolutionary process [12]. These data suggest that the ANK gene family in Dendrobium has undergone purifying selection, which removes deleterious alleles, thereby contributing to the long-term stability of these genes during evolution.

In order to study the expansion and contraction of ANK gene family members in the evolutionary process, the collinearity between ANK genes in three Dendrobium species and Arabidopsis thaliana was studied by collinearity analysis. There were 17 homologous gene pairs between the ANK genes of D. officinale and Arabidopsis (Fig. 5C), 48 pairs with D. chrysotoxum (Fig. 5A), and 66 pairs with the ANK genes of D. nobile (Fig. 5B). Collectively, the ANK genes of the three Dendrobium species and Arabidopsis exhibited conserved collinearity across chromosome regions, despite some deviations in duplicate gene pairs.

Fig. 5
figure 5

Collinearity analysis of ANK genes between D. officinale and three other plants, including D. chrysotoxum (A), D. nobile (B), and A. thaliana (C). Grey lines indicate the collinear blocks. Syntenic genes of ANK gene family is exhibited with red lines

Full size image

Promoter analysis of DoANKs, DnANKs, and DcANKs

Cis-acting regulatory elements could bind to certain transcription factors to control gene transcription. In order to further analyze the potential functions of DoANKs, DnANKs, and DcANKs and their responses to various signal factors, cis-acting elements were identified and classified in the 1500 bp promoter region (Table S6). Different cis-acting elements had significant differences in the function and regulation of corresponding genes (Table S6). Therefore, the identified cis-acting elements were mainly divided into four categories: hormone responses, light responses, stress responses, and plant growth (Fig. 6; Table S6). There were nine cis-acting elements associated with hormone response: ABRE, TCA-element, ERE, GARE-motif, P-box, TATC-box, TGACG-motif, TGA-element, and CGTCA-motif (Fig. 6; Table S6). Seven cis-acting elements related to light response: G-box, GATA-motif, i-box, TCT-motif, AE-box, GT1-motif, and Box 4 (Fig. 6; Table S6). Seven stress response elements predicted, including TC-rich repeats, MBS, Myc, Myb, box S, W box, LTR, and WUN-motif (Fig. 6; Table S6). Two cis-elements related to plant growth were detected, mainly including GCN4-motif and CAT-box (Fig. 6; Table S6). In DoANKs, most cis-acting elements were related to hormone response categories, light response categories, and stress response categories (Fig. 6A). The proportions of the three categories of DnANKs and DcANKs were similar to DoANKs (Fig. 6). These results suggest that ANK proteins in Dendrobium may respond to various hormones, light, stress, and other factors, necessitating further experiments to confirm their functions.

Fig. 6
figure 6

Information of cis-acting elements in ANK genes of D. officinale (A), D. nobile (B), and D. chrysotoxum (C). The gradient red colors in the grid indicate the number of different cis-elements. The proportions of the four different types of cis-acting elements in ANK genes are shown in the pie chart. Different colors in the pie chart represent cis-acting elements for each category

Full size image

Analysis of protein-protein interaction network of ANK protein in D. officinale

In order to further explore the function of ANK protein, PPI network was analyzed and STRING online website was used to detect the interaction between ANK protein and related proteins. 98 proteins were finally identified and linked (Table S7). Finally, 98 proteins were identified and connected, with a total of 560 connections (Fig. 7). ABI5 transcription factor interacting with ANK protein was the key transcription factor of ABA signal transduction, which inhibits seed germination and plant growth and development (Table S7). The results indicate that ANK proteins in D. officinale play diverse and crucial roles in plant growth and development.

Fig. 7
figure 7

Protein-protein interaction (PPI) networks of ANK proteins in D. officinale. The gradient circle size indicates the different degrees of importance

Full size image

Data analysis of RNA-Seq

To investigate the molecular response mechanism and gene expression of D. officinale under plant hormone stress, Illumina NovaSeq was used to sequence the transcriptome of two hormones (GA3 and ABA) stress, and 9 qualified RNA libraries were constructed, each treating 3 repeats. The raw sequencing data were filtered to produce 365,283,218 clean reads and 49.1 Gb clean bases. The Q20 value for each sample was above 98%, with an error rate below 1%. The Q30 value exceeded 95%, with an error rate below 0.1%, and the GC content ranged from 45.45 to 45.84% Table S9). Therefore, the RNA-seq data were reliable for subsequent analysis.

Differential expression analysis in GA3 and ABA treatment

To identify the differentially expressed genes (DEGs) under GA3 and ABA treatments, the screening thresholds of DEGs were padj < 0.05 and |log2FoldChange| > 1. As compared with the control, a total of 3341 DEGs (2055 up-regulated and 1286 down-regulated) and 1028 DEGs (465 up-regulated and 563 down-regulated) were detected in GA3 and ABA treatment, respectively (Figure S4).

Analysis of KEGG and GO gene function annotation of DEGs

To investigate the functional significance of DEGs under GA3 and ABA stress, we conducted GO enrichment analysis. All DEGs can be classified into three categories: cellular component (CC), molecular function (MF), and biological processe (BP). The top 20 enriched terms for all three categories were selected for plotting. Under GA3 treatment, DEGs were primarily enriched in molecular functions such as binding with carbohydrates, iron ions, ADP, molecular oxygen and heme, oxidoreductase activity, monooxygenase activity, etc. Additionally, extracellular regions and defense responses were also included (Fig. 8A, Table S10). Under ABA treatment, in terms of molecular function, DEGs were mainly enriched in carbohydrate binding, heme binding, transmembrane transporter activity, acyltransferase activity, etc. In addition, extracellular domain and transcriptional regulation were also included (Fig. 8A, Table S10). It is speculated that hormone treatment may stimulate the expression of genes associated with binding to certain molecules.

Fig. 8
figure 8

The GO and KEGG enrichment analysis. (A) The GO enrichment analysis with GA3 and ABA treatment. Red, orange, and green represent cellular_component, molecular_function, and biological_process, respectively. (B) The KEGG enrichment analysis with GA3 and ABA treatment. Red, brown, green, blue and purple represent Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, Organismal Systems, respectively

Full size image

Through KEGG enrichment analysis, annotated pathways were classified into five categories: Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, and Organismal Systems (Fig. 8B, Table S11). The results showed significant enrichment of pathways such as Environmental adaptation, Lipid metabolism, Global and overview maps, Carbohydrate metabolism, Biosynthesis of other secondary metabolites, Amino acid metabolism, Translation, Folding, sorting and degradation, Signal transduction, and Transport and catabolism (Fig. 8B, Table S11). The enrichment of these pathways may indicate that hormonal stress promotes their activation.

Analysis of KEGG and GO gene function annotation of DoANKs

After GA3 treatment, GO enrichment analysis revealed 110, 473, and 396 DEGs in the three categories of CC, MF, and BP, respectively (Fig. 9A). After ABA treatment, GO enrichment analysis showed that there were 41, 234, 177 DEGs in the three categories of CC, MF, and BP, respectively (Fig. 9A). Under ABA treatment, only DoANK72 gene was enriched into the category of cell composition, while under GA3 treatment, in addition to DoANK72 gene, there were seven DoANKs (DoANK02, DoANK17, DoANK20, DoANK30, DoANK31, DoANK32, and DoANK70) enriched into the category of CC (Fig. 9A). In the MF category, only one gene, DoANK01, was enriched with GA3 treatment (Fig. 9A). In the BP category, two DoANKs (DoANK14 and DoANK19) were enriched in this category under GA3 treatment, and two DoANKs (DoANK19 and DoANK47) were enriched in this category under ABA treatment (Fig. 9A).

Fig. 9
figure 9

Functional annotation analysis of GO (A) and KEGG (B) genes of DoANKs treated with GA3 and ABA, and subcellular localization analysis of DoANK32 (C)

Full size image

In order to explore the function of ANK genes in D. officinale, KEGG databases were used to analyze the functional annotation of 78 DoANKs. According to KEGG annotation results, two genes were annotated into the plant hormone signaling pathway (K14508) in response to NPR1 protein under GA3 (DoANK14 and DoANK19) and ABA treatment (DoANK19 and DoANK47), respectively (Fig. 9B). NPR1 (nonexpressor of pathogenes related genes 1) is a key gene regulating plant disease resistance, which can also regulate SAR (systemic acquired resistance), and ISR (induced systemic resistance). NPR1 regulates PR gene expression through interaction with TGA transcription factors and it binds to WRKY transcription factor and plays a key role in regulating and balancing SA transmission pathway (Fig. 9B). Through KEGG enrichment, DoANK32 gene was annotated to ArfGAP gene in endocytosis pathway (K12489) (Fig. 9B). The protein encoded by the ArfGAP gene can interact with the small G protein ARF on the cell membrane, thereby regulating lipid transport, membrane fusion and endocytosis of the cell membrane (Fig. 9B).

Subcellular localization of DoANK32

The subcellular localization of DoANK32 was detected by transient expression system in tobacco leaves. DoANK32-GFP was injected into tobacco leaves and transient expression of DoANK32 was observed, showing localization in the cell membrane and nucleus (Fig. 9C).

Expression analysis of DoANKs

To further study the function of DoANKs, we analyzed the specific expression of these genes in different tissues. The expression of these genes varies greatly in different tissues. Most genes were hardly expressed in roots but highly expressed in stems, flowers, and leaves (Fig. 10A). Eight genes, including DoANK03, DoANK05, DoANK33, DoANK44, DoANK54, DoANK58, DoANK59, and DoANK75, were highly expressed in various tissues, suggesting that these genes play important roles in plant response to stress (Fig. 10A).

Fig. 10
figure 10

Expression analysis of DoANKs in different tissues, growth stage, GA3 and ABA treatment. (A) Expression profiles of ANK genes of D. officinale in different tissues and growth stage including root, stem, leaf, flower, Young seedling and the adult plants. Z-score transformed FPKM values. (B) Relative expression levels of ANKs under GA3 treatment. (C) Relative expression levels of ANKs under ABA treatment

Full size image

The results of cis-element analysis showed that most DoANKs in D. officinale contained cis-acting elements in response to hormones. Meanwhile, PPI network analysis showed that ABI5 transcription factor interacts with ANK protein as a key transcription factor in ABA signal transduction, which can inhibit seed germination and plant growth and development. Therefore, we used qRT-PCR to analyze the expression pattern and potential function of DoANKs treated with GA3 and ABA. GA3 treatment results showed that, 17 genes (DoANK01, DoANK14, DoANK19, DoANK26, DoANK29, DoANK32, DoANK34, DoANK39, DoANK41, DoANK45, DoANK48, DoANK58, DoANK59, DoANK64, DoANK69, DoANK70, and DoANK72) were significantly up-regulated, 4 genes (DoANK31, DoANK46, DoANK60, and DoANK63) were significantly down regulated, and the remaining genes had no significant changes (Fig. 10B). ABA treatment results showed that, 8 genes (DoANK01, DoANK10, DoANK29, DoANK39, DoANK42, DoANK63, DoANK64, and DoANK65) were significantly up-regulated, 8 genes (DoANK19, DoANK31, DoANK41, DoANK45, DoANK47, DoANK54, DoANK68, and DoANK75) were significantly down regulated, and the remaining genes had no significant changes (Fig. 10C). DoANK genes with significantly different expression levels under hormonal treatment may play an important role in the hormonal response of D. officinale, inhibiting the growth and development of D. officinale by influencing hormones such as GA3 and ABA in plants.

WGCNA of D. officinale genes in GA 3 and ABA treatment

To explore the function of DoANKs, WGCNA analysis was performed using transcriptome data. The results show that the turquoise module has the most positive correlation with GA3 treatment, and the yellow module has the most negative correlation with GA3 treatment. Brown modules were most positively correlated with ABA treatment, and purple modules were most negatively correlated with ABA treatment (Figure S5). DoANK19 belonged to the turquoise module, which was most positively correlated with GA3 treatment and negatively correlated with ABA treatment (Figure S5, Table S12). The results of WGCNA analysis were consistent with the results of DoANK19 gene expression, which was up-regulated under GA3 treatment and down-regulated under ABA treatment (Figs. 10, S5). In the results of qRT-PCR analysis, the expression levels of 17 DoANKs were significantly up-regulated under GA3 treatment. Of these, 15 DoANKs (DoANK01, DoANK14, DoANK19, DoANK26, DoANK32, DoANK34, DoANK41, DoANK45, DoANK48, DoANK58, DoANK59, DoANK64, DoANK69, DoANK70, and DoANK72) belong to the turquoise module and the other two DoANKs (DoANK29 and DoANK39) belong to the blue module (Figure S5, Table S12). The expression levels of 4 DoANKs were significantly down-regulated under GA3 treatment, among which 3 DoANKs (DoANK31, DoANK46, and DoANK60) belonged to turquoise module and the other DoANKs (DoANK63) belonged to yellow module (Figure S5, Table S12). Under ABA treatment, the expression levels of 8 DoANKs were significantly up-regulated, including 2 DoANKs (DoANK10, and DoANK42) belonging to the brown module, 2 DoANKs (DoANK01, and DoANK64) belonging to the turquoise module, 2 DoANKs (DoANK29, and DoANK39) belonging to the blue module, DoANK63 belonging to the yellow module, and DoANK65 belonging to the tan module (Figure S5, Table S12). Of the eight genes whose expression levels were significantly down-regulated, five DoANKs (DoANK19, DoANK31, DoANK41, DoANK45, and DoANK47) belonged to the turquoise module, two DoANKs (DoANK68, and DoANK75) belonged to the brown module, and DoANK54 belonged to the purple module (Figure S5, Table S12).

Discussion

The variation of ANK gene number in different plants may be due to genome size, different plant evolutionary patterns and gene duplication differences, etc

Ankyrin repeat-containing proteins participate in numerous vital physiological pathways in plants, including light regulation [13], cell differentiation and development [14, 15], plant morphogenesis and organogenesis [16, 17], response to organisms [18, 19], and abiotic stress [20]. With the complete genome of Dendrobium now published [9,10,11], there has been a significant advancement in the analysis and research of gene families within this genus. The ANK gene sequence is known for its relative conservation across different plant species. However, the number of ANK family genes varies widely among diverse plant lineages. For example, Arabidopsis thaliana contains 105 ANK family genes [21], while Oryza sativa (rice) has 175 ANK genes [22]. In contrast, our study identified 78, 77, and 58 ANK family genes in the Dendrobium species D. officinale, D. nobile, and D. chrysotoxum, respectively. These differences in gene numbers are likely attributed to variations in genome sizes among these plants. The Arabidopsis genome, at 125 Mb, contains only 105 ANK genes [21], compared to rice’s 175 [22]. D. officinale’s genome measures 1.21Gb [9], roughly equivalent to those of D. nobile and D. chrysotoxum [10, 11]. Despite the similarity in genome sizes between D. officinale and D. nobile, they have nearly the same number of ANK genes, whereas D. chrysotoxum has fewer ANK genes. This discrepancy can be attributed to various factors, including gene duplication events, which significantly contribute to the diversity of ANK gene family members in different plants. Our study revealed 8 pairs of homologous genes in DoANKs, 9 in DnANKs, and only 4 in DcANKs. Despite their smaller genome, Arabidopsis has more ANK genes than the Dendrobium species, indicating that dicotyledons, which possess a greater number of ANK genes, may exhibit a higher degree of evolutionary advancement than monocotyledons. Gene duplication is known to facilitate plant evolutionary processes [23]. In gene families that are large and evolving rapidly, tandem duplications are prevalent, whereas in those that are slow to evolve and more conserved, segmental duplications predominate [24]. Consequently, the ANK gene family in Dendrobium represents a rapidly evolving lineage with a substantial number of members, highlighting the dynamic nature of this gene family in contributing to the evolutionary adaptability of Dendrobium species.

Role of DoANKs in hormone signal transduction pathway and immune system

In the analysis of cis-acting elements of members of the ANK gene family of three species of Dendrobium, the number of cis-acting elements related to the hormone response category, light response category, and stress response category was very rich. It was confirmed that members of ANK gene family participate in multiple metabolic pathways during plant growth and have extensive gene functions. According to the gene annotation analysis results obtained by GO and KEGG annotation under GA3 and ABA treatment, DoANK14 and DoANK19 in ANK gene family of D. officinale participated in biochemical process under GA3 treatment and were enriched in NPR1 transcription factor. Under ABA treatment, DoANK19 and DoANK47 participated in the biochemical process and were enriched in NPR1 transcription factor. Plants have two types of innate immune systems to defend against pathogens: effector-triggered immunity (ETI) and patterns triggered immunity (PTI) systems [25,26,27]. PTI was induced by pattern recognition receptors (PRRs) on the surface of pathogenic microorganisms, such as polysaccharide and flagellin, which could lead to non-specific defense response (basic defense response) in plants [28]. ETI was triggered by the recognition of effector proteins produced by pathogenic microorganisms by plant disease resistance proteins (R proteins), which could make plants produce specific defense responses [29, 33]. During the long-term evolution process, pathogens co-evolved with host plants and established close relationships. When plants were invaded by pathogens, ETI promotes programmed cell death (PCD) at the infection site, thereby limiting the expansion of pathogens [34]. The plant immune system could be activated by signal molecules, such as hormones, reactive oxygen species (ROS), and calcium (Ca2+) [35, 36]. SA in plant hormones is one of the main signal molecules regulating the plant immune system [37]. NPR1 was a major regulator of many genes in plants, such as the antibacterial PR gene, so as to protect plants from pathogens and diseases [38, 39]. The three ANK genes (DoANK14, DoANK19, and DoANK47) of D. officinale screened in this study enable the NPR1 transcription factor in the ETI system to be indirectly activated by SA and regulate the antibacterial PR gene, which was of great value in the screening and breeding of D. officinale disease resistance genes. The expression of DoANK19 gene was up-regulated under GA3 treatment (Fig. 10B), but down-regulated under ABA treatment (Fig. 10C). It is speculated that GA3 can act as a positive signaling molecule of plant immune system, and ABA could act as a negative signaling molecule of plant immune system, inducing NPR1 regulatory factors and activating antibacterial PR gene to protect plants from pathogens.

Relationship between plant hormone ABA and secondary metabolites of D. officinale

Plant hormone is a key factor affecting plant growth and development, and it is closely related to ANK transcription factor [40,41,42,43]. The application of different exogenous hormones can drive the production of secondary metabolites in various plant species, including flavonoids, anthocyanins, and anthraquinones [44, 45]. At the same time, secondary metabolites were a critical class of substances, often the primary pharmacodynamic agents of medicinal plants [46]. For example, in Nitraria tangutorum Bobr, ABA was significant for the accumulation and regulation of anthocyanin secondary metabolites and flavonoids [45]. In grape, ABA could affect the biosynthesis of secondary metabolites such as quercetin, kaempferol and calcareol [47]. Cis-acting regulatory elements could bind to some transcription factors to control gene transcription. Cis-acting elements have been identified and classified in the 1500 bp promoter region. There were 9 cis-acting elements associated with hormonal response: ABRE, TCA-element, ERE, GARE-motif, P-box, TATC-box, TGACG-motif, TGA-element, and CGTCA-motif (Fig. 6; Table S6) contains at least one in each DoANKs. It is particularly noteworthy that the expression of certain candidate genes, such as DoANK10 and DoANK42, showed significant changes after 24 h of ABA treatment. This indicates that these genes have substantial potential for involvement in the synthesis and regulation of secondary metabolites in D. officinale. The presence of these cis-acting elements and the responsiveness of DoANK genes to ABA treatment underscore the potential regulatory role of ANK transcription factors in the hormonal control of secondary metabolite biosynthesis. This further suggests that manipulating these pathways could enhance the production of valuable secondary metabolites in D. officinale, with implications for both plant physiology and medicinal applications.

DoANK32responds to vesicle transport and auxin polar transport

Polargrowth is a common phenomenon in plant cells. Development and organogenesis in dicotyledonous and monocotyledonous plants are highly dependent on polar austerin transport, which requires the growth tip to secrete large amounts of membrane and cell wall material to maintain rapid growth [48]. Physiology and molecular genetics have demonstrated that this process is regulated by auxin input vector and output vector activity [49, 50]. Vesicle transport can affect the distribution of auxin input vectors, thereby regulating the growth and development of plant organs [51,52,53]. ARF-GAP has a repetitive ANK domain that regulates auxin signaling through the trans-Golgi network (TGN) mediated vesicle transport system and plays a role in the formation and development of plant organs [52]. By GO enrichment analysis of transcriptome data under GA3 treatment, DoANK32 belonged to the category of cellular component (Fig. 9A). KEGG enrichment analysis showed that DoANK32 was located in ArfGAP gene in endocytosis pathway (K12489), and the expression of DoANK32 gene was significantly increased after GA3 treatment (Fig. 9B). However, such results were not obtained under ABA treatment. It is well known that auxin and GA3 have antagonistic effects, and ArfGAP gene can affect the polar transport of auxin by regulating intima transport. In response to GA3, the DoANK32 gene can influence vesicle transport and the polar transport of auxin in D. officinale, thereby affecting the growth and development of its organs and tissues. This highlights the importance of DoANK32 in mediating the effects of GA3 on plant development, potentially offering new insights into the complex regulatory networks governing plant growth and the interplay between different hormonal signals.

Conclusion

In this study, we identified members of the ankyrin repeat (ANK) gene family in the genomes of three Dendrobium species: D. officinale (78 members), D. nobile (77 members), and D. chrysotoxum (58 members). These ANK genes were dispersed randomly across various chromosomes and were classified into 10 distinct subfamilies based on the results of phylogenetic analysis.

We performed comprehensive bioinformatic analyses on these ANK gene families, which included evaluations of gene structure, motif composition, cis-acting regulatory elements, collinearity, protein-protein interaction networks, and expression patterns. Through these analyses, we identified eight pairs of homologous genes within the D. officinale ANK genes (DoANKs), nine pairs within the D. nobile ANK genes (DnANKs), and four pairs within the D. chrysotoxum ANK genes (DcANKs). These findings provide valuable insights into the evolutionary paths of ANK genes in these three Dendrobium species.

Moreover, our study implicated three specific ANK genes from D. officinale—DoANK14, DoANK19, and DoANK47—in the Effector-Triggered Immunity (ETI) system. These genes appear to play a role in the activation of NPR1, a transcription factor that is indirectly stimulated through salicylic acid (SA) to regulate the expression of pathogenesis-related (PR) genes with antibacterial functions [8]. In addition, we identified DoANK32 as interacting with the ArfGAP gene within the endocytosis pathway, and found that it is expressed in both the cell membrane and nucleus. This interaction suggests that DoANK32 is involved in vesicle trafficking and auxin polar transport, thereby influencing organogenesis and overall development in D. officinale.

Furthermore, the expression levels of DoANK genes were significantly altered following hormonal treatments, indicating their crucial role in the plant’s hormonal response mechanisms. These genes may modulate the growth and development of D. officinale by influencing plant hormones such as gibberellic acid (GA3) and abscisic acid (ABA).

In summary, this study enhances our understanding of the functional roles of ANK genes and their potential impacts on the growth and development of Dendrobium species. By elucidating these roles, we can better understand the molecular mechanisms underlying the physiological processes and stress responses in these important orchid species.

Materials and methods

Identification of protein sequences containing ANK domain in Dendrobium Sw. Genome

In order to identify candidate members of the ANK gene family in the genome of Dendrobium, the Hidden Markov Model (HMMs) map of Ank (PF00023) was downloaded from the Protein family database (PFam) (https://pfam.xfam.org/). The ANK protein sequence of Arabidopsis 23 was downloaded from TAIR database, and all candidate ANK proteins of 3 Dendrobium species (D. officinale, D. nobile, and D. chrysotoxum) were searched using BLASTP. Integrate all candidate ANK protein sequences identified by these two methods. All ANK candidate sequences were submitted to Pfam (https://pfam.xfam.org/), SMART (http://smart.embl-heidelberg.de/), and NCBI-CDD3 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to eliminate false positives. ExPASy software was used to calculate the protein molecular weight (kDa) and theoretical isoelectric point (pI) of ANK protein sequence [54]. The position of genes on chromosomes was visualized with TBtools v1.6 software [55].

Sequence alignment and phylogenetic relationship analysis

Firstly, multiple amino acid sequences of three Dendrobium ANK proteins were compared using ClustalX 2.1 software [56]. Then, MEGA 7 software was used to construct the phylogenetic tree using the neighbor joining method with the following parameters: poisson correction, pairwise deletion and 1,000 bootstrap replicates [57].

Gene structure and motif analysis

The sequences were submitted to MEME platform (https://meme-suite.org/meme/) to determine the conserved motifs of ANK sequences.The number of motifs was set to 15, and other parameters were defaulted. The exon and intron gene structure of each sequence was visualized using GSDS online software (http://gsds.gao-lab.org/index.php). The phylogenetic trees, conservative motifs and gene structures of ANK sequences of each Dendrobium species were integrated using TBtools v1.6 software [55].

Gene replication and collinearity analysis

MCScanX software was used to identify gene repetition events in Dendrobium plants based on DNA sequence comparison results of ANK gene, and Circos software was used to visualize these gene repetition events. At the same time, MCScanX (cscore ≥ 0.7) was used to detect and display the collinearity between the genomes of three species of Dendrobium and Arabidopsis. KaKs_ Calculator 2.0 software [58] was used to calculates non-synonymous (Ka), synonymous (Ks), and ω (= Ka/Ks). When Ka/Ks > 1, it means positive selection, Ka/Ks < 1 means negative selection, Ka/Ks = 1 means neutral selection [58].

Cis-acting elements and protein–protein interaction network analysis

Firstly, 1500 bp DNA sequences upstream of all ANK genes were extracted to speculate promoters. And then, submit them to the online website of the PlantCare database to analyze cis-acting elements (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The identified cis acting elements were mainly divided into four categories: hormone response, light response, stress response, and plant growth, and the results were visualized with TBtools v1.6 software [55]. STRING online website (http://string-db.org/) was used to predict the relationship between ANK protein sequences, and Cytoscape v3.7.2 software [59] was used to visualize the results.

Plant treatment

Healthy one-year-old D. officinale tissue culture seedlings were selected as experimental materials. The roots were treated with 150 µM GA3 and 100 µM ABA, with individuals treated with pure water serving as the control. Three independent replicates were set in the control group and the experimental group. 24 h later, plants were collected from the experimental group and the control group, and immediately frozen in liquid nitrogen, and stored at 80℃ until use.

RNA extraction and sequencing

First, the total RNA of D. officinale was extracted with an EASY spin Plant RNA Kit (Aidlab, China). RNA sequencing was performed using Illumina HiSeq2500, a high-throughput sequencing platform. Clean reads obtained from RNA-Seq were mapped to the genome of D. officinale and then assembled using Hisat2 and Stringtie, respectively. The DEseq2 software package was used to identify DEGs [60]. The adjusted P-value standard was 0.05, and the folding change was greater than 1.5× [61].

Function annotation of DEGs and DoANKs

Based on the public databases GO and KEGG, the functional annotation of DEGs was given and KEGG database was used to analyze the functional annotation of 78 DoANKs. ClusterProfiler (3.14.3) was used for GO and KEGG enrichment analysis [62].

Subcellular localization of DoANKs

The CDS of DoANK32 was cloned into the plant expression vector pCambia1300, and the 35 S: DoANK32: GFP fusion construct was constructed. Then, the empty vector and the fusion plasmid were expressed instantaneously in tobacco leaves, respectively. The tobacco (Nicotiana tabacum L.) plants were first grown in the dark for 12 h, then penetrated for 3 days, and the GFP fluorescence was observed by confocal microscopy.

Expression patterns of DoANKs

To analyze the expression pattern of DoANKs in D. officinale, the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra) was searched for RNA-sequence data of root (SRR2014227 and SRR2014230), stem (SRR1917040, SRR1917041, SRR1917042, and SRR1917043), leaf (SRR2012297 and SRR2014325), and flower (SRR2012396 and SRR2014476) four different tissues, seedling (SRR1909494) and mature (SRR1909493) plant at two development stages [63]. The downloaded RNA-sequence data were converted tofastq format using the viafastq-dump function of SRA toolkit.3.0.0 software. The clean reads were compared with the genome of D. officinale, and Hisat2 v2.2.1 was used for localization. Convert data from sam to bam using SAMtools v1.14. The FPKM value of DoANKs was calculated using StringTie v2.2.0, and the results were visualized using TBtools v1.6 [55] heat maps.

Quantitative real-time PCR

The treatment concentration and treatment time of GA3 and ABA were the same as those of the above methods. The extraction method of total RNA is the same as above. And then using HiScript® III-RT SuperMix synthetic qPCR (Vazyme, China), and the primers used therein were designed by Snapgene software, and The real-time PCR detection system was connected with ABI-7500 for qRT-PCR. With 1 µl template in a reaction volume of 20 µl, cDNAs were diluted to 200ng and three technical repetitions were performed. PCR amplification procedure was as follows: 95℃ 30s, 95℃ 10s, 60℃ 30s, 60℃ 15s cycle. The expression data was calculated by 2−∆∆CT method [64]. The gene GAPDH was used as the internal reference gene. Primer sequences were presented in Table S8

Weighted gene co-expression network analysis

Two groups of differentially expressed genes (DEGs) in transcriptome data were analyzed by weighted gene co-expression network (WGCNA). Assuming that the expression of genes with related functions may have a similar spectrum [65]. The parameters were as follows: maxBlockSize: 2000, minModuleSize: 30, deepSplit: 2.

Data availability

All of the raw sequence reads used in this study have been deposited in NCBI (BioProject accession number: PRJNA1088935, website: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1088935).

Abbreviations

ANK:

Ankyrin

R genes:

Plant Disease Resistance Genes

R proteins:

Resistance Proteins

ABA:

Abscisic Acid

GA3 :

Gibberellic Acid 3

PTI:

Patterns Triggered Immunity

ETI:

Effector-Triggered Immunity

PRRs:

Pattern Recognition Receptors

PCD:

Programmed Cell Death

ROS:

Reactive Oxygen Species

qPCR:

Quantitative Real-Time PCR

WGCNA:

Weighted Gene Co-Expression Network Analysis

DEGs:

Differentially Expressed Genes

TGN:

Trans-Golgi Network

References

  1. Xiang XG, Schuiteman A, Li DZ, Huang WC, Chung SW, Li JW, Zhou HL, Jin WT, Lai YJ, Li ZY, Jin XH. Molecular systematics of Dendrobium (Orchidaceae, Dendrobieae) from mainland Asia based on plastid and nuclear sequences. Mol Phylogenet Evol. 2013;69(3):950–60.

    Article  PubMed  Google Scholar 

  2. Liu JJ, Liu ZP, Zhang XF, Si JP. Effects of various processing methods on the metabolic profile and antioxidant activity of Dendrobium Catenatum Lindley leaves. Metabolites. 2021;11(6):351.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Sedgwick SG, Smerdon SJ. The ankyrin repeat: a diversity of interactions on a common structural framework. Trends Biochem Sci. 1999;24(8):311–6.

    Article  PubMed  CAS  Google Scholar 

  4. Breeden L, Nasmyth K. Similarity between cell-cycle genes of budding yeast and fission yeast and the notch gene of Drosophila. Nature. 1987;329(6140):651–4.

    Article  PubMed  CAS  Google Scholar 

  5. Bae W, Lee YJ, Kim DH, Lee J, Kim S, Sohn EJ, Hwang I. AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis. Nat Cell Biol. 2008;10(2):220–7.

    Article  PubMed  CAS  Google Scholar 

  6. Seong ES, Cho HS, Choi D, Joung YH, Lim CK, Hur JH, Wang MH. Tomato plants overexpressing CaKR1 enhanced tolerance to salt and oxidative stress. Biochem Biophys Res Commun. 2007;363(4):983–8.

    Article  PubMed  CAS  Google Scholar 

  7. Yan J, Wang J, Zhang H. An ankyrin repeat-containing protein plays a role in both disease resistance and antioxidation metabolism. Plant J. 2002;29(2):193–202.

    Article  PubMed  CAS  Google Scholar 

  8. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell. 1997;88(1):57–63.

    Article  PubMed  CAS  Google Scholar 

  9. Niu Z, Zhu F, Fan Y, Li C, Zhang B, Zhu S, Hou Z, Wang M, Yang J, Xue Q, Liu W, Ding X. The chromosome-level reference genome assembly for Dendrobium officinale and its utility of functional genomics research and molecular breeding study. Acta Pharm Sin B. 2021;11(7):2080–92.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Xu Q, Niu SC, Li KL, Zheng PJ, Zhang XJ, Jia Y, Liu Y, Niu YX, Yu LH, Chen DF, Zhang GQ. Chromosome-scale assembly of the Dendrobium nobile genome provides insights into the molecular mechanism of the biosynthesis of the medicinal active ingredient of Dendrobium. Front Genet. 2022;13:844622.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Zhang Y, Zhang GQ, Zhang D, Liu XD, Xu XY, Sun WH, Yu X, Zhu X, Wang ZW, Zhao X, Zhong WY, Chen H, Yin WL, Huang T, Niu SC, Liu ZJ. Chromosome-scale assembly of the Dendrobium chrysotoxum genome enhances the understanding of orchid evolution. Hortic Res. 2021;8(1):183.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Koonin EV, Rogozin IB. Getting positive about selection. Genome Biol. 2003;4(8):331.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Zhang H, Scheirer DC, Fowle WH, Goodman HM. Expression of antisense or sense RNA of an ankyrin repeat-containing gene blocks chloroplast differentiation in Arabidopsis. Plant Cell. 1992;4(12):1575–88.

    PubMed  PubMed Central  CAS  Google Scholar 

  14. Albert S, Després B, Guilleminot J, Bechtold N, Pelletier G, Delseny M, Devic M. The EMB 506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos. Plant J. 1999;17(2):169–79.

    Article  PubMed  CAS  Google Scholar 

  15. Hemsley PA, Kemp AC, Grierson CS. The TIP GROWTH DEFECTIVE1 S-acyl transferase regulates plant cell growth in Arabidopsis. Plant Cell. 2005;17(9):2554–63.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Garcion C, Guilleminot J, Kroj T, Parcy F, Giraudat J, Devic M. AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis. Plant J. 2006;48(6):895–906.

    Article  PubMed  CAS  Google Scholar 

  17. Huang J, Chen F, Del Casino C, Autino A, Shen M, Yuan S, Peng J, Shi H, Wang C, Cresti M, Li Y. An ankyrin repeat-containing protein, characterized as a ubiquitin ligase, is closely associated with membrane-enclosed organelles and required for pollen germination and pollen tube growth in lily. Plant Physiol. 2006;140(4):1374–83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Lu H, Rate DN, Song JT, Greenberg JT. ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response. Plant Cell. 2003;15(10):2408–20.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Lu H, Liu Y, Greenberg JT. Structure-function analysis of the plasma membrane- localized Arabidopsis defense component ACD6. Plant J. 2005;44(5):798–809.

    Article  PubMed  CAS  Google Scholar 

  20. Nodzon LA, Xu WH, Wang Y, Pi LY, Chakrabarty PK, Song WY. The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis. Plant J. 2004;40(6):996–1006.

    Article  PubMed  CAS  Google Scholar 

  21. Becerra C, Jahrmann T, Puigdomènech P, Vicient CM. Ankyrin repeat-containing proteins in Arabidopsis: characterization of a novel and abundant group of genes coding ankyrin-transmembrane proteins. Gene. 2004;340(1):111–21.

    Article  PubMed  CAS  Google Scholar 

  22. Huang J, Zhao X, Yu H, Ouyang Y, Wang L, Zhang Q. The ankyrin repeat gene family in rice: genome-wide identification, classification and expression profiling. Plant Mol Biol. 2009;71(3):207–26.

    Article  PubMed  CAS  Google Scholar 

  23. Moore RC, Purugganan MD. The early stages of duplicate gene evolution. Proc Natl Acad Sci USA. 2003;100(26):15682–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004;4:10.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Han GZ. Origin and evolution of the plant immune system. New Phytol. 2019;222(1):70–83.

    Article  PubMed  Google Scholar 

  26. Spoel SH, Dong X. How do plants achieve immunity? Defence without specialized immune cells. Nat Rev Immunol. 2012;12(2):89–100.

    Article  PubMed  CAS  Google Scholar 

  27. Zipfel C. Plant pattern-recognition receptors. Trends Immunol. 2014;35(7):345–51.

    Article  PubMed  CAS  Google Scholar 

  28. Dangl JL, Horvath DM, Staskawicz BJ. Pivoting the plant immune system from dissection to deployment. Science. 2013;341(6147):746–51.

    Article  PubMed  CAS  Google Scholar 

  29. Césari S, Kanzaki H, Fujiwara T, Bernoux M, Chalvon V, Kawano Y, Shimamoto K, Dodds P, Terauchi R, Kroj T. The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance. EMBO J. 2014;33(17):1941–59.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Luo S, Peng J, Li K, Wang M, Kuang H. Contrasting evolutionary patterns of the Rp1 resistance gene family in different species of Poaceae. Mol Biol Evol. 2011;28(1):313–25.

    Article  PubMed  CAS  Google Scholar 

  31. Song W, Wang B, Li X, Wei J, Chen L, Zhang D, Zhang W, Li R. Identification of immune related LRR-Containing genes in maize (Zea mays L.) by genome-wide sequence analysis. Int J Genomics. 2015;2015:231358.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Yang X, Wang J. Genome-wide analysis of NBS-LRR genes in sorghum genome revealed several events contributing to NBS-LRR gene evolution in grass species. Evol Bioinform Online. 2016;12:9–21.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Shao ZQ, Xue JY, Wu P, Zhang YM, Wu Y, Hang YY, Wang B, Chen JQ. Large-scale analyses of angiosperm nucleotide-binding site-leucine-rich repeat genes reveal three anciently diverged classes with distinct evolutionary patterns. Plant Physiol. 2016;170(4):2095–109.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Bruggeman Q, Raynaud C, Benhamed M, Delarue M. To die or not to die? Lessons from lesion mimic mutants. Front Plant Sci. 2015;6:24.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Musetti R, Buxa SV, De Marco F, Loschi A, Polizzotto R, Kogel KH, van Bel AJ. Phytoplasma-triggered ca(2+) influx is involved in sieve-tube blockage. Mol Plant Microbe Interact. 2013;26(4):379–86.

    Article  PubMed  CAS  Google Scholar 

  36. Wang L, Liu S, Gao M, Wang L, Wang L, Wang Y, Dai L, Zhao J, Liu M, Liu Z. The crosstalk of the salicylic acid and jasmonic acid signaling pathways contributed to different resistance to phytoplasma infection between the two genotypes in Chinese jujube. Front Microbiol. 2022;13:800762.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Verhage A, van Wees SC, Pieterse CM. Plant immunity: it’s the hormones talking, but what do they say? Plant Physiol. 2010;154(2):536–40.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Clarke SM, Mur LA, Wood JE, Scott IM. Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. Plant J. 2004;38(3):432–47.

    Article  PubMed  CAS  Google Scholar 

  39. Olate E, Jiménez-Gómez JM, Holuigue L, Salinas J. NPR1 mediates a novel regulatory pathway in cold acclimation by interacting with HSFA1 factors. Nat Plants. 2018;4(10):811–23.

    Article  PubMed  CAS  Google Scholar 

  40. Zhang FJ, Xie YH, Jiang H, Wang X, Hao YJ, Zhang Z, You CX. The ankyrin repeat-containing protein MdANK2B regulates salt tolerance and ABA sensitivity in Malus domestica. Plant Cell Rep. 2021;40(2):405–19.

    Article  PubMed  CAS  Google Scholar 

  41. Lopez-Ortiz C, Peña-Garcia Y, Natarajan P, Bhandari M, Abburi V, Dutta SK, Yadav L, Stommel J, Nimmakayala P, Reddy UK. The ankyrin repeat gene family in Capsicum spp: genome-wide survey, characterization and gene expression profile. Sci Rep. 2020;10(1):4044.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Yuan X, Zhang S, Liu S, Yu M, Su H, Shu H, Li X. Global analysis of ankyrin repeat domain C3HC4-type RING finger gene family in plants. PLoS ONE. 2013;8(3):e58003.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Stone SL, Williams LA, Farmer LM, Vierstra RD, Callis J. KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell. 2006;18(12):3415–28.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Loreti E, Povero G, Novi G, Solfanelli C, Alpi A, Perata P. Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis. New Phytol. 2008;179(4):1004–16.

    Article  PubMed  CAS  Google Scholar 

  45. Zhang J, Cheng K, Liu X, Dai Z, Zheng L, Wang Y. Exogenous abscisic acid and sodium nitroprusside regulate flavonoid biosynthesis and photosynthesis of Nitraria Tangutorum Bobr in alkali stress. Front Plant Sci. 2023;14:1118984.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Sohn SI, Pandian S, Rakkammal K, Largia MJV, Thamilarasan SK, Balaji S, Zoclanclounon YAB, Shilpha J, Ramesh M. Jasmonates in plant growth and development and elicitation of secondary metabolites: an updated overview. Front Plant Sci. 2022;13:942789.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Berli FJ, Moreno D, Piccoli P, Hespanhol-Viana L, Silva MF, Bressan-Smith R, Cavagnaro JB, Bottini R. Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultraviolet-absorbing compounds, antioxidant enzymes and membrane sterols. Plant Cell Environ. 2010;33(1):1–10.

    PubMed  CAS  Google Scholar 

  48. Du C, Chong K. ARF-GTPase activating protein mediates auxin influx carrier AUX1 early endosome trafficking to regulate auxin dependent plant development. Plant Signal Behav. 2011;6(11):1644–6.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Zhuang X, Jiang J, Li J, Ma Q, Xu Y, Xue Y, Xu Z, Chong K. Over-expression of OsAGAP, an ARF-GAP, interferes with auxin influx, vesicle trafficking and root development. Plant J. 2006;48(4):581–91.

    Article  PubMed  CAS  Google Scholar 

  50. Chong K, Zhuang X. ARF-GTPase as a molecular switch for polar auxin transport mediated by vesicle trafficking in root development. Plant Signal Behav. 2007;2(2):101–2.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Naramoto S, Kyozuka J. ARF GTPase machinery at the plasma membrane regulates auxin transport-mediated plant growth. Plant Biotechnol (Tokyo). 2018;35(2):155–9.

    Article  PubMed  CAS  Google Scholar 

  52. Koizumi K, Naramoto S, Sawa S, Yahara N, Ueda T, Nakano A, Sugiyama M, Fukuda H. VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation. Development. 2005;132(7):1699–711.

    Article  PubMed  CAS  Google Scholar 

  53. Song XF, Yang CY, Liu J, Yang WC. RPA, a class II ARFGAP protein, activates ARF1 and U5 and plays a role in root hair development in Arabidopsis. Plant Physiol. 2006;141(3):966–76.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol. 1999;112:531–52.

    PubMed  CAS  Google Scholar 

  55. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–202.

    Article  PubMed  CAS  Google Scholar 

  56. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8.

    Article  PubMed  CAS  Google Scholar 

  57. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Wang D, Zhang Y, Zhang Z, Zhu J, Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteom Bioinf. 2010;8(1):77–80.

    Article  CAS  Google Scholar 

  59. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Love MI, Huber W, Anders S. Moderated estimation of Fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Varet H, Brillet-Guéguen L, Coppée JY, Dillies MA, SARTools:. A DESeq2- and EdgeR-based R pipeline for comprehensive differential analysis of RNA-seq data. PLoS ONE. 2016;11(6):e0157022.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16(5):284–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Chen Z, Yuan Y, Fu D, Shen C, Yang Y. Identification and expression profiling of the auxin response factors in Dendrobium officinale under abiotic stresses. Int J Mol Sci. 2017;18(5):927.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402–8.

    Article  PubMed  CAS  Google Scholar 

  65. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank the College of Life Sciences, Nanjing Normal University for supporting this work. We are also grateful to the Jiangsu Provincial Engineering Research Center for Technical Industrialization of Dendrobiums for technical support.

Funding

Our work was funded by the National Natural Science Foundation of China (Grant No. 32070353 and 31670330), Forestry Science and Technology Innovation and Promotion Project of Jiangsu Province (LYKJ[2021]12), Agricultural Science and Technology Independent Innovation Fund Project of Jiangsu Province (CX(22)3147).

Author information

Authors and Affiliations

  1. College of Life Sciences, Nanjing Normal University, Nanjing, 210023, China

    Lingli Li, Jiapeng Yang, Qian Zhang, Qiqian Xue, Meiqian Li, Qingyun Xue, Wei Liu, Zhitao Niu & Xiaoyu Ding

  2. Jiangsu Provincial Engineering Research Center for Technical Industrialization of Dendrobiums, Nanjing, China

    Lingli Li, Jiapeng Yang, Qian Zhang, Qiqian Xue, Meiqian Li, Qingyun Xue, Wei Liu, Zhitao Niu & Xiaoyu Ding

Authors
  1. Lingli Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiapeng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qiqian Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Meiqian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qingyun Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhitao Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xiaoyu Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

XYD and LLL designed the study. LLL performed the experiments. LLL, JPY, QZ, QQX, and MQL analyzed the data. XYD, QYX, WL, and ZTN collected the materials. LLL wrote the manuscript, which was revised by XYD. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Xiaoyu Ding.

Ethics declarations

Ethics approval and consent to participate

This study does not involve any human or animal tissue materials and does not require ethical approval. We declare that the D. officinale individuals used in this study are cultivated species and do not involve the conservation of wild or endangered resources. The D. officinale cultivated seedlings used in this study were collected from Anhui, China. Experimental research with D. officinale species complies with Nanjing Normal University guidelines (http://bwc.njnu.edu.cn/info/1085/1433.htm), preserving the genetic background of the species used. The voucher specimen (LLL202403) was prepared by L.L.L in March 2024 and is stored at the Institute of Plant Resources and Environment, College of Life Sciences, Nanjing Normal University. The authors’ organizations (College of Life Sciences, Nanjing Normal University and Jiangsu Provincial Engineering Research Center for Technical Industrialization of Dendrobiums) approved the publication of this paper.

Consent for publication

not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, L., Yang, J., Zhang, Q. et al. Genome-wide identification of Ankyrin (ANK) repeat gene families in three Dendrobium species and the expression of ANK genes in D. officinale under gibberellin and abscisic acid treatments. BMC Plant Biol 24, 762 (2024). https://doi.org/10.1186/s12870-024-05461-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-024-05461-2

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us