Genome-wide identification of the glutamate receptor-like gene family in Vanilla planifolia and their response to Fusarium oxysporum infection

ABSTRACT

Glutamate receptor-like genes (GLRs) are essential for plant growth and development and for coping with environmental (biological and non-biological) stresses. In this study, 13 GLR members were identified in the Vanilla planifolia genome and attributed to two subgroups (Clade I and Clade III) based on their physical relationships. Cis-acting element analysis and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations indicated the GLR gene regulation’s complexity and their functional diversity. Expression analysis revealed a relatively higher and more general expression pattern of Clade III members compared to the Clade I subgroup in tissues. Most GLRs showed significant differences in expression during Fusarium oxysporum infection. This suggested that GLRs play a critical role in the response of V. planifolia to pathogenic infection. These results provide helpful information for further functional research and crop improvement of VpGLRs.

KEYWORDS:

• Vanilla planifolia
• glutamate receptor-like genes
• expression pattern
• Fusarium oxysporum
• biotic stress

1. Introduction

Ionotropic glutamate receptor (iGluR) genes are widely studied in mammals. Their encoded proteins function as glutamate-activated ion channels in rapid synaptic transmission and may regulate various neurological, mental, and emotional disorders^1,2. Unlike mammalian iGluRs, glutamate receptor-like genes (GLRs) were later discovered in plants. Upon completing the Arabidopsis thaliana genome sequencing project, GLR was discovered in the plant (A. thaliana) in 1998 for the first time³, which was a homolog of mammalian iGluR. There are 20 AtGLR genes in A. thaliana and they are subdivided into three clades^4,5. GLR regulates pollen tube growth and morphogenesis in tobacco and Arabidopsis⁶. OsGLR3.4 regulates rice root growth and promotes nitrate absorption by the roots⁷. AtGLR1.1 and AtGLR3.7 participate in abscisic acid signal transduction and regulate seed germination^8,9. AtGLR1.2 and AtGLR1.3 are positive regulators of cold resistance and promote jasmonate accumulation to cope with cold stress¹⁰. Overexpression of OsGLR1 or OsGLR2 confers enhanced drought tolerance in Oryza sativa and A. thaliana¹¹. GLR also plays a vital role in plant responses to biotic stress¹². Triple mutant GLR2.7/GLR2.8/GLR2.9 Arabidopsis exhibited a more susceptible phenotype in response to Pseudomonas syringae pv. tomato DC3000 infection¹³. GLR3.3 and GLR3.5 were involved in the defense response of tomatoes against Botrytis cinerea through electrical signaling¹⁴. According to previous studies, GLRs are essential for plant growth and development, and associated with various physiological and biochemical processes, including disease resistance.

V. planifolia is a tropical vine of the Orchidaceae family. It contains a precious natural flavoring renowned for its application in spices¹⁵. As the only edible spice in the Orchidaceae family¹⁶, it has been widely applied in food, medicine, cosmetics, and other related industries^17,18. However, its production is severely restricted by pathogenic infections in some producing areas¹⁹. A common disease is root and stem rot (RSR) caused by Fusarium oxysporum; this can drive the necrosis and decay of roots and stems and lead to significant economic losses for V. planifolia planting^20,21. It is unclear if GLR plays a role in V. planifolia defense against pathogens.

This study identified VpGLR through genome-wide analysis and comprehensive analysis of the characteristics of VpGLR. Meanwhile, the expression pattern of VpGLRs in various tissues and after infection by F. oxysporum were assessed. This study aimed to provide helpful information to further explore the function of VpGLR and offer new options for improving the fungal resistance of V. planifolia.

2. Materials and methods

2.1 Identification and phylogenetic analysis of VpGLRs

Genomic sequences of V. planifolia, 20 AtGLRs, and 13 OsGLRs were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/genome/), TAIR (https://www.arabidopsis.org/), and Phytozome (https://phytozome-next.jgi.doe.gov/), respectively. They were used as decoys to retrieve VpGLR family members at the genome-wide level by BLASTP with an e-value cutoff of 1e⁻⁵. The hidden Markov model (HMM) profiles of the GLR domain (PF00060, PF00497, PF01094, PF01609, and PF10613) were acquired from the Pfam database (http://pfam.xfam.org/). They were used to search for possible VpGLR family members of V. planifolia using HMMER v3.0, with an e-value cutoff of 1e⁻⁵. The two queries were merged, followed by verification of the VpGLR genes using SMART (http://smart.embl-heidelberg.de/) with an e-value <1 × 10⁻⁵.

All GLR proteins from A. thaliana, O. sativa, and V. planifolia were aligned using MAFFT²², and a maximum likelihood tree (ML tree) was constructed with 1000 guided replications using IQtree (v2.2)²³. The result was visualized by MEGA Χ²⁴.

2.2 Molecular characteristics, chromosomal localization, and selection pressure analysis

ExPASY (https://www.expasy.org/) was used to analyze the amino acid number, molecular weight (MW), isoelectric point (pI), instability index, aliphatic index, and grand average hydropathicity (GRAVY)²⁵. WoLF PSORT (https://wolfpsort.hgc.jp/) was used to predict the subcellular localization of the proteins²⁶. TMHMM-2.0 (https://services.healthtech.dtu.dk/ services/TMHMM-2.0/) was used to determine the presence of transmembrane helices (TMHs) in proteins²⁷. MG2C (http://mg2c.iask.in/mg2c_v2.1/) was used to analyze the chromosomal location of VpGLR genes²⁸. Multiplex linear scanning (MCScan X) was used to detect segmental and tandem duplication genes in the VpGLRs²⁹. The nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and Ka/Ks ratio for each pair of duplicated genes were calculated using MEGA X software.

2.3 Conserved motif, gene structure, cis-regulatory elements, and function annotation analysis

Ten conserved motifs of VpGLR proteins were analyzed using the MEME tool (https://meme-suite.org/meme/) with default parameters³⁰. The exon-intron distributions of VpGLR genes were analyzed using the Gene Structure Display Server 2.0 (GSDS 2.0) (http://gsds.gao-lab.org/) according to the Generic Feature Format Version 3 (GFF3) genome annotation files³¹. Two kb sequences up-stream of the start codon of VpGLR genes were extracted as their promoter sequences, which were identified using the PlantCARE tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)³². The key elements were further classified, summarized, and mapped using the GSDS 2.0. Gene Ontology (GO) and KEGG functional annotations of VpGLRs was performed using eggnog-MAPPER (http://eggnog-mapper.embl.de/)³³ and visualized by Tbtools³⁴.

2.4 GLR gene expression analysis by RNA-seq data

RNA-Seq data (accession numbers SRP043630 and SRP214274) were obtained from the Sequence Read Archive (SRA) database to investigate the expression patterns of VpGLR genes in different tissues and biotic stress processes after infection with F. oxysporum. The details of growth conditions and treatment conditions were as described by Rao et al.³⁵ and Solano-De et al.²⁰ respectively. The expression pattern of different tissues was obtained at the age of six months: leaf, stem, mesocarp, placental laminae, hair cells, and seeds. Biotic stress analysis obtained RNA-Seq data after the roots were infected for two days and ten days compared with uninfected plants after 12 weeks of cultivation at room temperature. Trimmomatic, SAMtools, and StringTie were performed for data quality control, creation of the BAM file, and abundance calculation. The GLR expression spectrum was normalized (fragments per kilobase of exon per million reads, FPKM+1) by log2 transformation and then visualization by Tbtools³⁴. A change in expression by a factor of ≥ 2 fold indicates significant differences between the groups.

3 Results

3.1 Protein properties and phylogenetic analysis of VpGLRs

Thirteen VpGLR genes were identified from the genome of V. planifolia and named according to their chromosomal location information and phylogenetic relationships (Table 1 and Figure 1). In short, the number of amino acids was between 438 (VpGLR3.2) and 981 (VpGLR1.2), and the molecular weight (MW) was between 48.36 kDa (VpGLR3.2) and 108.50 kDa (VpGLR1.1). VpGLR3.3 and VpGLR1.4 possessed the highest (8.96) and lowest (5.12) isoelectric points, respectively. VpGLR1.7 had the highest number (6) of transmembrane domains, while VpGLR3.1 and VpGLR3.2 had none. Most were predicted to be localized in the plasma membrane except for VpGLR1.6 (endoplasmic reticulum), VpGLR3.1 (chloroplast), and VpGLR3.2 (located in the mitochondria). Five out of thirteen were classified as hydrophilic (grand average of hydropathicity (GRAVY) index<0).

Figure 1. Phylogenetic tree of GLR proteins from Arabidopsis, rice, and soybean. All protein sequences were aligned using MAFFT and the maximum likelihood (ML) tree was constructed using IQtree with 1,000 bootstrap replicates. Different colors indicate distinct subgroups or species.

Table 1. Basic information for the Vanilla planifolia glutamate receptor-like genes VpGLR gene family members.

Gene name	Gene ID	Chromosome location	Size (aa)	MW (kDa)	pI	TD	SL	Instability index	Aliphatic index	GRAVY
VpGLR1.1	Vpl05Ag10477	Chr5:5575246–5584481	965	108.50	5.21	4	plas	37.78	92.99	−0.038
VpGLR1.2	Vpl05Ag10758	Chr5:9299996–9303584	981	107.22	8.48	3	plas	41.94	88.40	0.023
VpGLR1.3	Vpl05Ag12402	Chr5:48784160–48794538	776	86.28	6.98	2	plas	36.88	89.07	−0.061
VpGLR1.4	Vpl08Ag17148	Chr8:27407012–27410142	953	104.77	5.12	3	plas	36.35	87.04	0.049
VpGLR1.5	Vpl08Ag17149	Chr8:27433144–27453280	463	52.24	5.84	3	plas	35.91	93.09	−0.034
VpGLR1.6	Vpl08Ag17155	Chr8:27700702–27709385	869	98.08	8.59	3	ER	37.25	97.15	0.028
VpGLR1.7	Vpl08Ag17156	Chr8:27743951–27747073	751	82.93	5.30	6	plas	40.33	90.61	0.082
VpGLR3.1	Vpl01Ag03715	Chr1:67431928–67471016	649	71.34	5.39	0	chlo	34.69	99.24	−0.033
VpGLR3.2	Vpl01Ag03721	Chr1:67651449–67687932	438	48.36	5.36	0	mito	35.99	97.65	−0.073
VpGLR3.3	Vpl01Ag04134	Chr1:75866947–75877873	906	100.90	8.96	3	plas	38.43	97.40	0.108
VpGLR3.4	Vpl02Ag05107	Chr2:19738624–19745750	956	106.57	5.47	4	plas	42.74	92.02	0.042
VpGLR3.5	Vpl12Ag22538	Chr12:16954665–16957860	844	93.80	5.20	3	plas	43.94	95.39	0.053
VpGLR3.6	Vpl13Ag24036	Chr13:6771823–6783404	792	86.74	6.70	3	plas	42.17	97.11	0.148

Note: MW, pI, TD, SL and GRAVY are molecular weight, isoelectric point, transmembrane domain, subcellular localization and grand average of hydropathicity, respectively; plas, ER, chlo, and mito indicate the plasma membrane, endoplasmic reticulum, chloroplast, and mitochondria, respectively.

We introduced 20 AtGLRs and 13 OsGLRs to construct the evolutionary relationship of VpGLRs more clearly. All the GLRs participating in building the evolutionary tree were divided into two groups (Figure 1). The VpGLRs clustered with Arabidopsis and rice’s Clade I members were designated as VpGLR1.1-VpGLR1.7 following previous naming conventions. In contrast, the remaining VpGLRs (VpGLR3.1-VpGLR3.6) clustered with Clade III members. The evolutionary relationship between the VpGLRs and OsGLRs was closer than that of AtGLRs.

3.2 Conserved motifs and gene structure of VpGLRs

This motif is closely related to the biological function of the protein, and we scanned the motifs in the 13 VpGLR members. As shown in Figure 2, the results revealed ten motifs in VpGLRs, and detailed information of these motifs was shown in Table 2. Two of the ten identified motifs (motif one and motif 10) contained the Lig_chan domain. This represents the ligand-gated ion channel super-family, including four transmembrane regions (M1, M2, M3, and M4). Motifs 2, 5, 8, and 9 belong to the peripheral binding protein type 2 superfamily that contains ligand-binding domain residues. Motifs 3, 4, and 6 contain the ANF receptor domain belonging to the type 1 periplasmic binding-fold superfamily. VpGLR1.1, VpGLR1.2, VpGLR1.4, and VpGLR3.4 had the most motifs, while VpGLR3.2 contained the least motifs. Motif 10 only existed in VpGLR1.1, VpGLR1.2, VpGLR1.4, and VpGLR1.7. Most motifs are widely distributed among VpGLR members. Interestingly, motif 5 appeared twice in VpGLR3.4. VpGLR3.1 (18) and VpGLR3.2 (15) possessed more CDSs than the other members. VpGLR1.1, VpGLR1.2, VpGLR1.4, VpGLR1.6, and VpGLR1.7 had the least number (4) of CDSs.

Figure 2. Schematic diagram of the phylogenetic tree (a); conserved motif (b); and gene structure (c) of VpGlrs. Genes from the same subtribe were indicated by the same color.

Table 2. Multilevel consensus sequences in VpGLP gene family identified by MEME.

Motifs	Domain	Best possible match	Width(aa)	Sites	E-value
1	Lig_chan domain	WIFLKPLTTDLWCVTGAFFIFTGFVVWILEHRINPEFRGPPKDQCGTVFWFSFSTLVFAHREKMLSNLSRFVMIV WVFVVMIIQSSYTASLTSMLTVRQLQPTIRGIDDLIKQGDPVGYQNGSFVPKYL	129	9	5.4e-455
2	Ligand-binding domain	HCCKYTMVGPIYKTDGWGFAFPRDSPLVPDVSRAILKLTES	41	11	1.4e-169
3	ANF receptor domain	QYPYFVRTTQSDSYQMKAIAELIQYFGWREVIPIYEDDEYGRGIVPYLIDALQ	53	10	3.6e-140
4	ANF receptor domain	IDCRIPYKCAIPPQASDDHILEKLYHLKTMQSRVFVVHMTCDLGLRIFHVAHEEGMMTEGYAWIAT	66	9	4.4e-135
5	Ligand-binding domain	KNFDAVVGDITIMANRSHYVDFTQPYTDSGVSMLVPVKNKEE	42	10	1.1e-119
6	ANF receptor domain	TDVKAIIGPQTSEEAHFVAELGNEAQVPIISFAATDPSLSS	41	11	1.2e-113
7	IspD domain	KRMGVSMPKQYLSLLGKPIALYSLFTFSELSEVKEIVVVCEPSYQNLFEDIIEEVNVDIKFAL PGKERQDSVFNGLQEVDGSSELVCIHDSARPLVLSTDVRKVLKDGWLNGAAVLGVPVKATIKEANKD SFVTRTFDRKTLWEMQTPQVIKPDLLRAGFELVNREGLEVTDDASIVEYLSHPVYITEGSYTNIKVTTPD	200	2	1.7e-072
8	Ligand-binding domain	WPGQTTMVPRGWEWPTNGQKLRIGVPNRPSYPQFVSKEKDPDT	43	7	4.5e-070
9	Ligand-binding domain	EYPQDYHNGPSNGGVAAIFDEIPYLKIFM	29	10	7.4e-069
10	Lig_chan domain	DIMMEIERKWFGDQKPCLPQSEGVSSTSLNFSNFGGLFLITGTVSVFALL IYILVFLRKEWDDVRAATLDAGDRGGSFWDKAAALAKHYDSIKEKRGSGTARGN MEQELSCNGIISEGPQTPISISEMSALNCVSQQTVASGRTSTEEMPTEQFIELRG	159	2	1.0e-059

3.3 Chromosomal distribution and gene duplication analysis of VpGLR genes

Thirteen VpGLR genes were unequally distributed on six chromosomes of V. planifolia (except for VpGLR3.4 and VpGLR3.5), while the other members were distributed close to the chromosome ends (Figure 3). Chr08 possessed the most VpGLRs (4) among these six chromosomes, whereas Chr02, Chr12, and Chr13 had only one member.

Figure 3. Chromosomal distribution of GLR genes in Vanilla planifolia. Chromosome size is indicated by its relative length. Chromosome numbers are shown at the left of each chromosome.

Two pairs of tandem duplications and one pair of fragment duplication genes were identified in the VpGLR family (Table 3). These homologous pairing genes were all members of the Clade I subgroup, and no duplication events were found in Clade III. Interestingly, VpGLR1.4 was associated with tandem and segmental duplication events. The collinearity of homologous genetic relationships between VpGLRs, OsGLRs, and AtGLRs was analyzed to further understand the GLR gene family amplification mechanism. Three homologous genes were found between V. planifolia and O. sativa (VpGLR1.4 and OsGLR1.1; VpGLR1.6 and OsGLR1.4; and VpGLR1.6 and OsGLR2.2), and there was no collinearity relationship with Arabidopsis (Figure 4). VpGLR1.4 exhibited collinearity between species, whereas VpGLR1.6 corresponded to two homologous genes in rice. These results suggested that these genes may have played an essential role in the evolution of the GLR gene family. In addition, the Ka/Ks ratios of all homologous gene pairs were below one.

Figure 4. Genome-wide synteny analysis of GLRs between Vanilla planifolia, Oryza sativa, and Arabidopsis thaliana. GLRs and chromosomes from the different species were indicated by the different colors.

Table 3. Ka/Ks analysis for duplicated GLR genes.

Gene ID1	Gene ID2	Ka	Ks	Ka/Ks	Duplication type
VpGLR1.4	VpGLR1.5	0.5223	3.0314	0.1723	Tandem
VpGLR1.6	VpGLR1.7	0.6233	1.4164	0.4401	Tandem
VpGLR1.2	VpGLR1.4	0.6067	0.9883	0.6139	Segmental
VpGLR1.4	OsGLR1.1	0.6524	0.8828	0.7390	Segmental
VpGLR1.6	OsGLR1.4	0.5181	0.8178	0.6335	Segmental
VpGLR1.6	OsGLR2.2	0.6048	1.1692	0.5173	Segmental

3.4 Cis-acting regulatory elements and analysis

The presence of cis-regulatory elements (CAREs) was predicted to further understand the potential transcriptional regulation of VpGLRs. Forty-six cis-acting regulatory elements were identified. They belonged to five main categories: binding site element (3), stress-induced component (6), growth and development component (5), hormone response (8), and light response element (24) (Figure 5).

Figure 5. The distribution of cis-acting regulatory elements in the promoters of the VpGLR gene family members.

Thirty-three Box 4 and thirty-two GT1-motif fragments were present in the promoter region of VpGLRs. These two elements were associated with 11 VpGLRs each, making them the most widely distributed CAREs. VpGLR3.5 had the highest number (32) and type (19) of CAREs, whereas VpGLR1.3 and VpGLR1.4 contained the least number of CAREs (13), and VpGLR1.4 also contained the least number of CAREs (10).

3.5 Enrichment analysis using GO and KEGG

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore the biological function of VpGLR at the molecular level. GO annotation for the VpGLR protein was analyzed using molecular function (MF), cellular component (CC), and biological process (BP). Seven of the 13 VpGLR genes were significantly enriched in 60 terms (p-value<0.05), including 17 MFs, three CCs, and 40 BPs (Figure 6). The enriched VpGLR members were mainly related to ion and protein transport activities in MF ontology. The CC ontology was mainly associated with the components of the cell membrane and the peripheral structure. The BP ontology was mainly involved in regulating cell transport, communication, and reactions.

Figure 6. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of VpGlrs. (a) Highly enriched GO terms in VpGlrs; (b) highly enriched KEGG pathways in VpGlrs.

Further enrichment analysis of KEGG pathways showed that 12 of the 13 members (except for VpGLR3.2) were significantly enriched in two pathways: ion channels and protein families (signaling and cellular processes).

3.6 Expression patterns of VpGlrs

Gene expression analysis was performed for six different tissues (placenta, hair, seed, leaf, stem, and mesocarp) at the age of six months to understand the expression profiles of the GLR gene family in V. planifolia. The expression of different VpGLR members considerably varied in the tissues (Figure 7a). Interestingly, VpGLR expression in the six tissues occurred in two patterns, except for VpGLR1.1. Clade I members had lower overall expression compared with Clade III. Clade I members showed low to moderate expression in hair and seed (except for VpGLR1.1, VpGLR1.4, and VpGLR1.7), and in the leaf, stem, and mesocarp (except for VpGLR1.2). The remaining members had very weak or no expression. All Clade III members were expressed in all six tissues. Among them, VpGLR3.4 possessed the highest expression in placental, hair, seed, and mesocarp, while VpGLR3.3 showed the highest expression level in leaves and stems, although this high expression was essentially the same as in VpGLR3.4. In addition, VpGLR1.1 was highly expressed in the mesocarp and VpGLR3.5 was highly expressed in the placenta, seed, and mesocarp. All the genes involved in high expression may play an essential role in tissue development and metabolism.

Figure 7. Expression profiles of VpGLR genes. (a) Expression pattern of VpGLR genes in different tissues at six months; (b) Expression pattern of VpFAD genes for Fusarium oxysporum-infected roots after 12 weeks of room-temperature culture. “C-2d” denotes untreated by F. oxysporum at two days control group (uninfected group); “C-10d” means untreated by F. oxysporum at ten days control group (uninfected group); “I-2d” represents the infected group at two days after F. oxysporum treatment; “I-10d” indicates the infected group at ten days after F. oxysporum treatment.

Biotic stress was determined by characterizing the expression pattern of the VpGLR gene family using transcriptome data from F. oxysporum-infected roots that were cultured at room temperature for 12 weeks before infection. The expression of VPGLR 1.2, VPGLR 1.4, VPGLR 1.7, and VPGLR 3.3 was significantly increased at two days of infection compared to the uninfected group (Figure 7b). The expression levels of most members (except for VpGLR1.7 and VpGLR3.5) were significantly up-regulated on the 10th day of infection, especially VpGLR1.1, VpGLR1.2, VpGLR1.3, VpGLR1.6, and VpGLR3.2 which increased by at least four-fold.

4. Discussion

In recent decades, plant GLRs were proven to play crucial roles in growth and development, signal transduction, and responses to biotic and abiotic stresses^36–38. However, knowledge of GLRs in V. planifolia is limited. In this study, 13 GLRs from V. planifolia were identified and clustered into two clades based on phylogenetic relationships. Clade I contained seven members and Clade III contained six members. The GLR gene family is divided into three branches in many plants, including Arabidopsis⁴, Solanum lycopersicum³⁹, and Saccharum officinarum¹². However, VpGLRs only had two branches and lacked Clade II. A. thaliana GLRs in Clade I and Clade II are closely related and belong to sister branches⁴⁰. Clade I and Clade II were attributed to the same large subgroup in the GLR of Rosaceae⁴¹. Here, the absence of Clade II members was likely due to the fact that Clade I and Clade II members of V. planifolia showed closer genetic relationships compared with O. sativa GLRs and A. thaliana GLRs. In addition, GLR members belonging to these two subgroups were found in Zea mays⁴² and Brassica rapa⁴³. These results are consistent with those observed in our phylogenetic tree.

Currently, most GLRs are thought positioned on the cell plasma membrane, with little information available on GLR-targeting organelles and related functions. A splicing variant of AtGLR3.5 is located in the endoplasmic reticulum⁴⁴. Its deletion mutation causes a lightweight reduction of calcium ions in the endoplasmic reticulum, and may affect the shape of the endoplasmic reticulum (ER) and lead to the loss of cristae, while promoting the aging of cells⁴⁴. AtGLR 3.1 and AtGLR3.3 are located in the ER-like structures of xylem contact cells and phloem sieve elements, respectively, and play an essential role in calcium signaling triggered by wounds⁴⁵. Spinach iGLR3 is located in the chloroplast, but its function remains unclear⁴⁶. Mutation of chloroplast localized AtGLR3.4 in A. thaliana leads to a slight decrease in photosynthesis⁴⁷. In this study, ten members of 13 VpGLRs were predicted to be located in the plasma membrane. This suggested that most of them play a role in the plasma membrane. VpGLR1.6, VpGLR3.1, and VpGLR3.2 were predicted to be located in the ER, chloroplast, and mitochondria, respectively. Further work is required to determine their actual localization and whether their functions are consistent with the AtGLRs mentioned above.

Gene replication is an essential driver of gene expansion and evolution and plays a significant role in the adaptive evolution of species^48,49. This study identified two tandem and one segmental duplication among the 13 VpGLR genes. The Ka/Ks ratios of these homologous paired genes were all below 1.0. This indicated that they underwent a purification selection process in evolutionary history to maintain their functions⁵⁰. In addition, collinear analysis of VpGLRs among different species showed that VpGLRs have no collinear relationship with GLRs from the dicotyledonous Arabidopsis. However, VpGLRs are homologous with GLRs from monocotyledonous rice plants. This correlated with a previous study showing that GLRs show no collinearity in four dicotyledons and two monocotyledons¹². Therefore, it is speculated that the generation of homologous GLR genes only occurs after the differentiation of dicotyledonous and monocotyledonous plants.

The expression and distribution of GLR in plant tissues was known as early as the beginning of this century. There are no branch-specific organ expression patterns found for 20 GLRs in A. thaliana⁴⁰. Similarly, we are yet to find this in V. planifolia. However, we found that the expression levels in different tissues were branch-specific, and that the expression levels of Clade III members were generally higher than those of GLRs in Clade I. The same situation was observed in sugarcane¹². This suggests that the GLRs of Clade III might have essential roles in plant growth regulation mechanisms. Interestingly, the clustering of Clade I members according to their expression levels in various organizations is surprisingly consistent with the branch they belong to in the evolutionary tree. The aggregation of most Clade I members was entirely consistent with their assemblage in the evolutionary tree except for VpGLR1.1, which had a relatively high expression and aggregated with Clade III members.

Additionally, GLRs are one of the key molecules in the resistance of plants to pathogenic infections. The use of GLR inhibitors can reduce the host’s resistance to pathogenic bacteria. AtGLR3.3 confers resistance to Pseudomonas syringae pv tomato DC3000 and Hyaloperonospora arabidopsidis in Arabidopsis^51,52. Transgenic Arabidopsis seedlings overexpressing small radish RsGluR upregulate the expression of jasmonic acid (JA) biosynthesis-related genes and inhibit the growth of the pathogenic fungus, Botrytis cinerea⁵³. A single nucleotide mutant of GhGLR4.8 (from GhGLR4.8C to GhGLR4.8A) confers cotton resistance to Fusarium oxysporum f. sp. Vasinfectum (fov)⁵⁴. These results suggest that GLR overexpression, heterogeneous expression, and genetic engineering modifications can enhance the beneficial immune properties of the host against the pathogen. In contrast, most VpGLRs were found to be significantly upregulated during F. oxysporum infection in our study, particularly VpGLR1.1, VpGLR1.2, VpGLR1.3, VpGLR1.6, and VpGLR3.2, which may play an essential role in the infection process, and these genes are potential targets for breeding resistant V. planifolia.

5. Conclusions

This study identified 13 members of the GLR family in the V. planifolia genome and analyzed their basic physicochemical properties, chromosomal distribution, and genetic structural characteristics. They were divided into two large subsets based on phylogenetic relationships: Clades I and III possessed seven and six members, respectively. Prediction of cis-acting elements revealed that VpGLR expression may be regulated by multiple categories of factors. Gene Ontology and KEGG analyses demonstrated that the molecular functions of VpGLRs were diverse. In addition, Clade III members were more commonly expressed in various tissues (and in relatively higher amounts) than Clade I members. Most GLRs were significantly upregulated following F. oxysporum infection. In particular, VpGLR1.1, VpGLR1.2, VpGLR1.3, VpGLR1.6, and VpGLR3.2 were significantly upregulated by at least four-fold on day 10 of infection. This suggested that they contribute to V. planifolia-F. oxysporum interaction. These findings provide critical information for the GLR gene family in V. planifolia and offer a basis for further functional research.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Guizhou Education Department Youth Science and Technology Talent Growth Project (Qianjiaohe KY Zi [2022]060) and the Liupanshui Normal University High-level Talents Start-up Fund Project (LPSSYKYJJ201803).