Comparative analysis of IRE1s in plants: insights into heat stress adaptation in Triticum aestivum

Abstract

Background

The unfolded protein response (UPR) pathway serves as a crucial mechanism enabling plants to perceive, respond to, and shield themselves from adverse environmental conditions. Inositol-requiring enzyme 1 (IRE1) is one of the key players of the UPR, and resides in the endoplasmic reticulum (ER) within the cell. This study provides a comprehensive analysis of 195 IRE1 genes across 90 diverse plant species, with a focus on their identification and characterization.

Results

To decipher the functions of IRE1 family members, we investigated the evolution and spread of IREs in plants and analysed their structural and localization characteristics. Our detailed cis-element analysis revealed unique IRE1 regulation patterns in different plant species. Furthermore, gene expression analysis revealed tissue-specific and heat stress-responsive expression patterns of TaIRE1s, which were subsequently confirmed via quantitative gene expression analysis. TaIRE1-6A was upregulated in response to dithiothreitol (DTT) treatment as well as heat stress. This finding suggests that IRE1 might play a role in linking the UPR pathway and the heat stress response (HSR).

Conclusions

Our findings provide a comprehensive understanding of the evolution and expansion of IRE1 genes in different plant species. These findings provide a foundation for further in-depth research on the functional diversity of IREs in nutritious crops following polyploidization. By linking the UPR with HSR, IRE1 could be a key contributor to wheat's resilience against heat stress. Additionally, this connection offers important insights for future functional studies in other crops. Thus, this knowledge could be used for engineering climate resilience in crops such as wheat.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05785-z.

Background

The unfolded protein response (UPR) is one of the most conserved responses in eukaryotes and is elicited by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Environmental stresses such as heat stress often disturb protein folding in the ER, thereby leading to ER stress [1]. This leads to the activation of UPR, as the accumulation of misfolded proteins inside the ER results in an increased demand for correct folding which exceeds the capacity for protein folding in the ER. Notably, inositol-requiring enzyme1 (IRE1) is highly conserved throughout multiple kingdoms of life, including fungi, plantae, and animalia. These findings suggest that IRE1 plays a crucial role in survival across diverse organisms on Earth. In Arabidopsis, the UPR signalling network is composed of two branches: one branch involves the IRE1-basic leucine zipper 60 (IRE1-bZIP60) signalling pathway, whereas the other branch involves the membrane transcription factors bZIP17 and bZIP28 [2, 3]. IRE1 functions as an ER transmembrane sensor, that activates the UPR to maintain ER homeostasis inside the cell [4]. In response to stress, the IRE1 protein undergoes activation, which leads to the splicing of a specific target mRNA in the cytoplasm that encodes a stress-response transcription factor, i.e., bZIP60. However, under certain excessive stress conditions, IRE1 loses its selectivity and indiscriminately degrades several mRNAs on the ER membrane encoding secreted proteins in a process called regulated IRE-dependent decay (RIDD) [5–7]. This mechanism can significantly impact plant physiology by alleviating the load of misfolded proteins during stress, effectively reducing the production of non-essential proteins. Consequently, RIDD plays a crucial role in the plant's stress response, helping to ensure survival by reallocating resources to vital cellular processes [7].

IRE1 spans throughout the ER membrane, with its N-terminus facing the ER lumen and its C-terminus protruding into the cytoplasm. The bifunctional protein kinase/ribonuclease activity of IRE1 is attributed to its C-terminus, while the N-terminus serves as a sensor domain [2]. Upon the sensing of misfolded proteins in the ER, the luminal N-terminal domain oligomerizes and brings the cytosolic kinase domains into juxtaposition, allowing their activation via trans-autophosphorylation and cofactor binding [8–10]. This kinase activation, in turn, induces certain conformational alterations that activate the RNase domain of the enzyme. Activation of the RNase domain is followed by splicing, which involves cleavage of the two loops of the substrate RNA (bZIP60 mRNA in plants), removal of the intervening intron, and ligation by tRNA ligase [11, 12]. bZIP60 further upregulates other proteins, such as binding protein 3 (BIP3), which are required in the UPR [11].

In Arabidopsis, IRE1 exists in two full-length isoforms, viz. IRE1a and IRE1b [13], and one truncated isoform, IRE1-c, that lacks a luminal domain [14]. Interestingly, rice has only one IRE1 gene [15]. Both Arabidopsis IRE1a and IRE1b have some overlapping and specific functions. For example, IRE1a is highly expressed in embryos and seeds and is involved in response to pathogens [16]. A study by Liu et al. (2012) revealed that IRE1b was the connecting bridge between long-standing ER stress and autophagy in Arabidopsis seedlings [17]. The truncated isoform IRE1c, in association with IRE1b, helps in the process of gametogenesis but has no specific role on its own [14]. However, both vegetative and reproductive development are affected in double and triple mutants of ire1a, ire1b, and bzip28 under stress conditions [18]. In fact, in the same study, it was observed that the RNase activity of IRE1b is responsible for promoting root elongation and shoot growth under stress conditions and that both protein kinase and RNase activities are involved in promoting root elongation under unstressed conditions. Interestingly, the IRE1- bZIP60 pathway has been reported to be crucial in conferring fungal resistance to Nicotiana attenuata against its fungal pathogen Alternaria alternata [19]. These studies indicate that the role of IRE through the UPR is imperative for overall plant development.

Globally, wheat (Triticum aestivum L.) is an essential cereal crop that feeds over 35% of the world's population [20, 21]. Particularly in underdeveloped nations where food security is a major concern, it is an important source of calories and vital nutrients [22]. However, wheat production has been severely affected over the years by several abiotic stresses, particularly heat and drought stresses [23, 24]. Heat stress is one of the major abiotic stresses affecting wheat production. High-temperature stress negatively impacts many physiological and yield components in wheat [25, 26]. Therefore, it is essential to elucidate the developmental processes of wheat and its mechanisms for responding to heat stress. Heat stress activates both HSR and UPR in plants, which helps them acclimatize to stress conditions. HSR involves activation of the heat shock transcription factors (HSFs) which regulate the expression of various heat stress-responsive genes such as HSPs and ROS scavenging enzymes [27]. Remarkably, studies by [28, 29] emphasized the function of bZIP60 and TaHsfA6b in connecting UPR to HSR, even though HSR and UPR function in distinct compartments. Given that IRE is a likely candidate implicated in the UPR pathway as a critical component of the stress response, investigating whether it contributes to heat stress response and is involved in crosstalk between these two response pathways is interesting. This study aims to identify IRE genes across various species, with a special emphasis on wheat. To analyze the biological functions of IRE genes comprehensively, we predicted their functional properties, such as structure and localization, to reveal more about their roles within the cell. To gain insight into the transcriptional regulation of IRE genes, we analyzed their promoter sequences for various cis-regulatory elements. The expression analysis revealed their specific roles in different abiotic stresses, particularly under heat stress. The possible involvement of the TaIRE1-6A gene in heat stress response (HSR) is highlighted by its differential expression pattern in heat-tolerant and heat-sensitive cultivars under heat stress and dithiothreitol (DTT) treatment. As such, this study offers a foundation for further research into the function of IRE1 in the connection between the UPR and HSR. Using this information to improve stress responses in crops such as wheat would be beneficial.

Material and methods

Identification and sequence retrieval of IREs across plant species

The Arabidopsis IRE1a and IRE1b were searched on UniProt [30] and their key Pfam domains were identified. These Pfam IDs (PF00069 and PF06479) were used individually to pinpoint the plant proteins that harbored these two domains. The two searches were separately made in the 90 distinctly available plant species on BioMart (accessed via Ensembl Plants) [31]. The proteins in both searches were identified and designated as IREs and their sequences were downloaded. Next, the gene IDs and chromosomal locations of all the selected IRE proteins were noted down from Ensembl Plants database [32].

Phylogenetic analysis and classification of the found IREs

To understand the evolutionary relationships among the selected IREs, we constructed a phylogenetic tree. This process involved aligning 195 full-length IRE protein sequences from 90 plant species using the MUSCLE algorithm within MEGA Version X software [33]. The alignment parameters used were set to default. Subsequently, this alignment was utilized for constructing the neighbor-joining phylogenetic tree with 1000 bootstrap values in MEGA X software. The phylogenetic tree was appropriately edited and colored categorically in the iTOL v6 online software [34]. The species were categorized based on their broad differentiating group. Additionally, to study the divergence patterns of the species under consideration, an evolution timeline figure was prepared using TimeTree [35].

Selection of representative plant species from all categories

For further analysis, we selected plant species from each category to serve as the representative species such as Cyanidioschyzon merolae from algae, Physcomitrium patens from bryophytes, Amborella trichopoda from basal angiosperms, Triticum aestivum and Oryza sativa from monocots and Arabidopsis thaliana, Glycine max, and Solanum tuberosum representing eudicots.

Gene structure construction and conserved motif analysis

We employed Gene Structure and Display Server 2.0 (GSDS) [36] to illustrate the structural features of the 16 IRE genes from the 8 representative species (excluding like-type IREs), such as their exon–intron composition and relative position, and conserved elements. Further, we used the Motif Elicitation (MEME) Software [37] to visualize the conserved motifs of wheat IRE proteins using only the default parameters, and the maximum number of motifs was set to 15.

Identification and analysis of the cis-acting regulatory elements (CAREs)

For promoter analysis, we retrieved 1.5 kb upstream sequences for each IRE gene from the representative species using BioMart [31] embedded in Ensembl Plants. These promoter sequences were uploaded into PlantCARE web tools [38] to predict and screen all cis-acting regulatory elements. The analysis was displayed diagrammatically using TBTools [39].

Sub-cellular localization and gene ontology enrichment analysis

The localization of IRE proteins within the cell of various representative species was determined using the webtool Plant-mSubP [40]. Further, a web-based tool, ShinyGO v0.741 [41] was used to understand the enrichment profile and visualize the various ontologies of the IREs from representative species.

In-silico gene expression analysis

The in silico expression analysis of the IRE genes of A. thaliana, G. max and O. sativa under four primary abiotic stresses, namely heat, cold, drought and osmotic stress, was performed using the online Arabidopsis RNA-Seq Database [42].

Domain structure elucidation of IREs

The molecular characteristics (weight, protein length, and isoelectric point) of the IRE1 proteins were obtained from the Ensembl database [32]. The positional and compositional information on the two characteristic domains (Protein Kinase and Ribonuclease) of IREs was retrieved from the Pfam Database [43], while those of Transmembrane and ER Luminal Domains was sourced from TMHMM Server, v. 2.0 [44]. Finally, all the domains were collectively illustrated using the DOG 1.0 Software [45].

In silico expression analysis of TaIRE genes

The in silico expression analysis of the three TaIRE genes was performed with the help of Genevestigator [46]. The gene expression was checked in three aspects: first, differential expression in various plant parts; second, expression levels at different developmental stages of the plant; and last, during various episodes of heat stress.

Plant material, stress treatment and quantitative real-time PCR analysis

TaIRE1 gene expression profiling under control, heat stress, and DTT conditions was investigated in two wheat cvs. HD2329 (heat-sensitive) and HD2985 (heat-tolerant) have differential heat tolerance [47]. The seeds of the two genotypes mentioned above were obtained from the Division of Genetics at the Indian Agricultural Research Institute (IARI) in India. The seeds of these cultivars were surface sterilized with 4% sodium hypochlorite, followed by gentle washing with distilled water, and then germinated for 2 days in plastic trays (375 × 300 × 75 mm) on water-soaked cotton beds (20 mm thick) at 22 °C in plant culture room. The seedlings were supplemented with a half-strength Hoagland solution and maintained under a 16:8 h light/dark photoperiod. For heat stress treatment, one-week-old seedlings were subjected to 42 °C for 6 h [48]. For control, the plants were kept at 22 °C. For DTT treatment, one-week-old seedlings were exposed to 7.5 mM DTT for 1 h and 6 h [29, 49]. Each treatment included three biological and three technical replicates. After each treatment, the seedlings were harvested immediately, and total RNA was isolated by using the TRIzol Reagent (Ambion). The RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) transformed isolated RNA into cDNA. Quantitative real time-PCR (qRT- PCR) was done using the SYBR Green (Applied Biosystem) in step- plus Real-Time PCR System (Applied Biosystems). TaActin (GenBank accession number: AB181991) was used as the housing keeping genes and relative gene expression was calculated by following the 2 − ΔΔCt method [50]. Three biological and three technical replicates were used for the data analysis. The mean values of treatments and controls were compared using Duncan's multiple range test to assess significant differences (P ≤ 0.05).

Results

Global identification of IRE proteins from 90 plant species

To understand the diversity and evolution of IRE proteins, the IRE gene family was identified in 90 different species, including algae, bryophyta, basal angiosperms, dicots and monocots (Table S1). A total of 195 non-redundant IRE genes were identified in these species, represented in the form of an evolution tree (Fig. 1A). Interestingly, a single IRE member was identified in different algae members whereas duplication was observed in the case of Physcomiterella patens, where two IRE genes were identified. The number of both IRE and IRE-like genes were found to increase in eudicots such as Eucalyptus grandis, Camelina sativa, Brassica napus and Glycine max. Similarly, more members were identified in monocots species like Eragrostis tef, Musa acuminata, Saccharum spontaneum, Triticum aestivum, Triticum dicoccoides, Triticum spelta, Triticum turgidum, and Zea mays, which could be attributed to the events of duplication that have occurred over the time of evolution (Fig. 1A). Wheat being hexaploid has three copies of IRE1 genes, which are coded by 6A, 6B, and 6D homeologous chromosomes, and thus the corresponding IRE1 genes are denoted as TaIRE1-6A, TaIRE1-6B, and TaIRE1-6D. Among the three homeologues, TaIRE1-6D had the maximum number of exons (8) with a protein length of 886 amino acids (Table S2). The only IRE1 genes found in algae and bryophyta taxa were grouped in two clades, whereas both IRE and IRE-Like genes were found to be present and grouped in the same clade in dicot as well as in monocot plants (Fig. 1B).

Fig. 1.

A Dated-phylogeny tree and number of IRE1 genes in different species. The species tree represents 195 IRE genes from 90 different species. The divergence time was estimated by molecular clock dating using TimeTree. Stars on the branch indicate Whole Genome Duplication (WGD) events. In the species tree dark green, yellow, purple, blue, green, and red indicate algae, moss, gymnosperms, Amborella trichopoda, monocots, and eudicots respectively. Following the species names are the number of IREs identified in each group as well as in total. Ma, million years ago. B Phylogenetic tree of IRE1 protein from algae, bryophyte, basal angiosperms, monocots and eudicotyledons represented in different colors. Bars indicate length of amino acids. The phylogenetic tree was constructed using iTOL software

Gene structure and domain analysis of IRE1

To decipher more about the IRE1 gene family, the gene structure and the conserved domains of the 16 IRE1 genes representing each taxa (consisting of Cyanidioschyzon merolae, Physcomitrella patens, Arabidopsis thaliana, Oryza sativa, Triticum aestivum, Glycine max, and Solanum tuberosum) were selected for further analysis. Interestingly, only IRE1 from Cyanidioschyzon merolae (CyamIRE1) was found to be intron-less, whereas all the other IRE1 genes were found to have both introns and exons (Fig. 2A). Amongst the selected genes, PhpIRE1 contains a maximum number of exons (8), whereas CyamIRE1 has the least number of exons (1) (Fig. 2B). Moreover, in the case of Triticum aestivum, three homeologous genes of IRE1 (TaIRE1-6A, TaIRE1-6B and TaIRE1-6D) have similar gene structures.

Fig. 2.

Gene structure and motif structure of plant IRE gene family members. A Exon and intron structure of IRE1 genes. Green boxes, exons; lines, introns; pink boxes, upstream/downstream region. The lengths of boxes and lines are scaled according to gene length. B Graph representing length of 5’ and 3’ UTR regions in various IRE1 genes. Green color represents 5’ UTR and blue color represents 3’ UTR. C MEME motif structures. Numbers and different colors were used to represent conserved motifs

The classification and evolutionary relationships between the TaIRE1 members and the other eight species were validated by the IRE1 family's conserved motif analysis. In total 15 motifs were detected in 16 IRE1 genes (Fig. 2C). The highest number of motifs (15) was detected in OsIRE1, TaIRE1-6A, TaIRE1-6B and TaIRE1-6D whereas CyamIRE1 had the fewest conserved motifs (7). Interestingly, motif 13 was only found in monocots i.e. Oryza sativa and Triticum aestivum and also in Glycine max. This indicates that proteins of this group might be involved in specific functions.

Apart from motifs, conserved domains were also identified. All the IRE1 proteins were found to possess ER lumen interface at their N-terminal, followed by a trans-membrane domain, and a cytosolic interface at their C-terminal. The cytosolic interface consisted of the protein kinase domain and the ribonuclease domain, (Figure S1). The domains were found to be well conserved in all the eight species highlighting the conserved functional role of IRE1 protein in plants.

Sub-cellular localization prediction

The localization of any protein plays an important role in determining its function inside the cell. Therefore, in silico protein localization of the selected IRE1 genes was predicted using Plant-mSubP software. All the IRE1 members were predicted to localize in ER, determined by the prediction of the ER lumen interface in these proteins (Table 1).

Table 1.

Subcellular localization prediction of IRE1 genes by using Plant-mSubP software

Protein	Blast-p based subcellular location	Score (bits)	E-value
AtIRE1a	Endoplasmic reticulum membrane; Single-pass	298	7.00E-86
AtIRE1b	Endoplasmic reticulum membrane; Single-pass	299	6.00E-86
GmIRE2	Endoplasmic reticulum membrane; Single-pass	295	2.00E-84
GmIRE4	Endoplasmic reticulum membrane; Single-pass	299	1.00E-85
GmIRE3	Endoplasmic reticulum membrane; Single-pass	304	2.00E-88
GmIRE1	Endoplasmic reticulum membrane; Single-pass	300	5.00E-87
StIRE2	Endoplasmic reticulum membrane; Single-pass	293	3.00E-84
StIRE1	Endoplasmic reticulum membrane; Single-pass	281	2.00E-79
OsIRE1	Endoplasmic reticulum membrane; Single-pass	293	3.00E-84
TaIRE1-6A	Endoplasmic reticulum membrane; Single-pass	299	1.00E-86
TaIRE1-6B	Endoplasmic reticulum membrane; Single-pass	302	1.00E-87
TaIRE1-6D	Endoplasmic reticulum membrane; Single-pass	301	2.00E-87
AtrIRE1	Endoplasmic reticulum membrane; Single-pass	293	3.00E-83
CyamIRE1	Endoplasmic reticulum membrane; Single-pass	260	2.00E-70
PhpIRE2	Endoplasmic reticulum membrane; Single-pass	319	5.00E-93
PhpIRE1	Endoplasmic reticulum membrane; Single-pass	318	2.00E-92

Identification of cis‐acting regulatory element in the promoter

The presence of different cis-acting regulatory elements in the promoter region of any gene indicates their transcriptional regulation and gene function. The 1500 bp promoter regions of IREs were searched for the cis-regulatory elements of the selected genes. Interestingly, most of stress-responsive elements, such as AP-1, CAAT box, CCAAT-box, W-box, and STRE were found to be present in the promoter region of IRE1 genes in all the organisms (Fig. 3). In Physcomitrium patens, Amborella trichopoda, Triticum aestivum, and Arabidopsis thaliana, these constituted nearly 50% of the total cis-elements. Heat stress-responsive elements were also found to be present in all the promoters, with their dominance in Cyanidioschyzon merolae, Physcomitrium patens, and Triticum aestivum (Fig. 3). Apart from stress, various hormone-responsive elements, such as abscisic acid-responsive elements (ABRE), auxin-responsive elements (AuxRR-core), gibberellin-responsive elements (P-box, GARE-motif, TATC-motif), methyl jasmonate-responsive elements (CGTCA and TGACG motif), and salicylic acid-responsive elements (TCA-element) were observed in the promoter regions. IRE1 genes of Glycine max and Oryza sativa had the maximum number of the hormone-responsive cis-elements in their promoter region. Similarly, light-responsive elements such as I-box, G-box, GA-motif, SP-1, chs- CMA2a, Box 4, TCT-motif, ACE and circadian motif were also detected in all the promoter regions, with maximum occurrence in Triticum aestivum IRE1 promoter. Plant development-related cis-elements such as TATA-box, TATA, O2-site, GCN4-motif, F-box, CAT-box, Box III, RY-element, and A-box were found to be present in the upstream region of IRE1 genes. Solanum tuberosum IRE1 gene had the maximum number of development-related cis-elements in its promoter (Fig. 3).

Fig. 3.

Promoter structure analysis and functional annotation of identified cis-regulatory elements. A Putative motifs have been identified using the PlantCARE database and their unique distribution pattern and functional annotation have been depicted in dot plot. Pink and yellow color circles indicate the presence and absence of the corresponding motif in IRE1 gene promoters. Presence of specific motifs has been presented in the red bar and in line (total number), respectively. Stars indicate motifs that get activated by heat stress B Pie chart represents the proportion of plant developmental (purple), light-responsive (green), stress-responsive (red), heat stress-responsive (Sky blue) and hormonal regulation (yellow) elements

Gene ontology annotations of IRE1 genes

To further characterize the function of IRE1 genes, GO enrichment analysis was performed using the ShinyGO tool. The analysis showed that all the IRE1 genes were involved in UPR, nuclease activity, signal transduction, and cellular response to stress in GO biological processes (Fig. 4; Figure S2). The GO molecular processes showed that IRE1 genes are unfolded protein-binding, having protein Ser/Thr kinase activity, and endoribonuclease activity. This annotation indicates that IRE1 genes are involved in ER stress responses, where protein unfolding occurs and plays an important role in the splicing of mRNA in ER.

Fig. 4.

Gene Ontology (GO) analysis using ShinyGO identified enriched biological processes of IRE1 genes of Physomitrium patens, Arabidopsis thaliana, Oryza sativa and Triticum aestivum. The green dots represent the nodes for each GO biological process; while the lines (yellow and grey) represent the interaction between the nodes (minimum of 20% genes common between two connected)

Expression analysis of IRE1 genes in different abiotic stresses

The cis-element analysis and the GO annotation suggested the potential role of IRE1 genes in stress response. The expression of OsIRE1, AtIRE1a, AtIRE1b, GmIRE1, GmIRE2, GmIRE3, and GmIRE4 genes was analyzed under different abiotic stresses (Fig. 5). OsIRE1 showed maximum expression under drought stress. AtIRE1a and AtIRE1b were found to be significantly upregulated under heat, drought, and cold stress conditions. GmIRE1 had maximum expression under heat stress, while GmIRE2 had the highest expression under drought stress. Both GmIRE3 and GmIRE4 were found to express higher transcript levels under drought stress. Thus, it appears that IRE1 genes play an important role in various abiotic stresses.

Fig. 5.

Expression levels of various IREs under different abiotic stresses. Boxplot showing the distribution of expression level (FPKM) of IRE1 genes in Oryza sativa, Arabidopsis thaliana, and Glycine max under different abiotic stresses based on the RNA-seq data (the numbers in brackets represent sample size). Whisker caps represent maximum and minimum values, the box encompasses lower and upper quartiles, the line across the box represents the median, and the asterisk indicates outliers. Heat stress, HS; Drought stress, DS; Cold stress, CS; Salt stress, SS

Expression profiling of TaIRE1 genes in different developmental stages and in different tissues

Out of all the IRE1 members, we focused our attention on IRE1 genes in wheat since little information is available about them. The expression of these TaIRE1s was checked in different developmental stages of wheat by Genevestigator software (Fig. 6). The three homeologous IRE1 genes showed similar expression patterns in all the stages, with TaIRE1-6D showing the maximum expression and TaIRE1-6B showing the minimum expression. Among the different stages, the expression of IRE1 peaked at the anthesis stage, which is one of the most important stages of plant development and is highly sensitive to heat stress (Fig. 6A). Apart from different developmental stages, their expression was also analyzed in various tissues and interestingly a significant difference in their expression was observed. Higher expression levels of TaIRE1-6D were detected in floret, nodal root, sheath, spikelets, awn, glume, roots, pistil, crown and shoot apex. This indicates potential role of TaIRE1-6D, particularly in reproductive developmental processes (Fig. 6B).

Fig. 6.

Expression level of TaIRE1 homeologous genes in different developmental stages and tissues. Scatterplots for the three TaIRE1 genes, the left side in (A) and the top side in (B) represent the change of gene expression using the base 2 logarithm scale. Different TaIRE1 genes are represented by distinct colors as indicated. Error bars represent standard errors. Data were analyzed with the Genevestigator tool

Expression pattern of TaIRE1 genes under heat stress conditions

Heat stress is one of the major stresses that severely affect wheat productivity. Therefore, there has been recent research to understand the mechanism by which plants respond to heat stress and identify the genes associated with heat stress tolerance [51]. Since a lot of heat-responsive elements were found in the promoter region of TaIRE1 genes, the expression pattern of these genes under heat stress was determined using wheat expression data available in the Genevestigator tool and validated by performing the qRT-PCR experiments. The wheat RNA-seq data (available on the Genevestigator tool) of the different stages under heat stress (37 °C–40 °C) was evaluated (Fig. 7). Interestingly, all three TaIRE1 homeologous genes showed a similar heat-responsive expression pattern. All TaIRE1 genes were found to be downregulated at the seed development stage [14 days after anthesis (14 DAA) stage]. Their expression increased and reached its maximum at 6 h of heat in the leaf samples. A rise in temperature, such as 37 °C, also caused upregulation of TaIRE1 homeologues in flag leaf as well as during the seed development stage (10 DAA). Amongst the three IRE1 genes, TaIRE1-6D showed maximum expression in leaf samples after 6 h of heat stress. Interestingly, TaIRE1-6A showed differential expression as compared to TaIRE1-6B and TaIRE1-6D during the seed development stage at 14 DAA. TaIRE1-6A was significantly downregulated in comparison to TaIRE1-6B and TaIRE1-6D. Further, the expression profile of TaIRE1-6A was validated by real-time quantitative PCR under heat stress conditions (Fig. 8). For this purpose, contrasting wheat genotypes (sensitive cv. HD2329 and tolerant cv. HD2985) were used. TaIRE1-6A showed considerable differential expression under heat stress treatment. Its expression was found to be 11-fold higher in the heat-tolerant cv. HD2985 and three-fold higher in the heat-sensitive cv. HD2329 as compared to control under heat stress treatment. The expression of TaHSP70, TaHSP90, and TaHSP17 was also checked under heat stress conditions in both the cultivars as a positive control for this experiment and was found to be increased after heat stress treatment in both heat-sensitive and heat-tolerant cultivars. This in turn confirmed that the upregulation of the TaIRE1-6A gene is because of the heat stress experienced by the plants. As heat stress often leads to UPR, therefore, we also checked the expression of TaIRE1-6A after treating the plants with DTT, a known inducer of UPR. As observed in Fig. 8, the transcript levels of TaIRE1-6A, increased after 1 h and 6 h of DTT treatments in both the cultivars. The level of upregulation was much more in the case of heat-tolerant cv. HD2985 as compared to heat-sensitive cv. HD2329. This indicates that TaIRE1-6A has a role to play under both heat stresses as well as in UPR in plants.

Fig. 7.

Expression profiling of TaIRE1 genes under heat stress conditions. Bar graph representing fold change values of TaIRE1-6A, TaIRE1-6B and TaIRE1-6D genes (represented by different colors) after exposure to different heat stress conditions at different stages. The details of the treatment are mentioned below the graph

Fig. 8.

Transcript profiling of TaIRE1-6A gene by qRT-PCR after heat stress and DTT treatment. The relative expression level of TaHSP17, TaHSP70, TaHSP90 and TaIRE1-6A genes was analyzed in heat-sensitive (HD2329) and heat-tolerant (HD2985) wheat cultivars after heat stress treatment (42 °C for 6 h). Expression of TaIRE1-6A was also analyzed in these cultivars after 1 h and 6 h of DTT treatment. Each bar value represents the means (± SE) derived from three biological. Different lower-case letters indicate significant difference at P ≤ 0.05 by Duncan’s multiple range test

Discussion

IRE1 represents one of the important ER membrane proteins which functions as stress sensors/transducers. Its conserved nature across all taxa, from yeast to mammals, highlights its importance in stress response. However, studies related to comparative analysis of proteins such as IRE1 are still limited. Interestingly, non-vascular plants and many monocots have been reported to have a single copy of the IRE1 gene, whereas dicots such as Brassicaceae family members have been reported to have a greater number of IRE1 genes [3, 13, 15, 52]. This study not only identifies the IRE1 gene across 90 plant species but also offers a comprehensive analysis of its evolutionary trajectory over time. Our findings indicate that lower plants and the majority of monocots possess only a single IRE1 gene, with no IRE-like genes detected. In contrast, the number of IRE1 genes in monocots increased only through gene duplication. Notably, most dicots have multiple IRE and IRE-like genes, highlighting a significant difference compared to monocots (Fig. 1). This implies that the expansion of the IRE1 gene family is related to polyploidation and that the gene family's increased number contributed to plant development and stress tolerance. This aligns with the proliferation of genes like HSFs and GRAS, which are more numerous in polyploid plants [53, 54]. Additional phylogenetic analysis revealed that IRE and IRE-l like genes clustered together, suggesting that gene duplication led to the evolution of IRE-like genes from IRE genes. It is therefore possible to hypothesize that an IRE-like gene, while identical to the IRE gene, may have a more varied role that aids in plant adaption responses. In the case of grasses, rapid functional divergence following small-scale gene duplication has been recently reported [55, 56].

The domain analysis of IRE1 genes showed that the ER lumen interface, the trans-membrane domain, and the cytosolic interface, which has kinase and nuclease domain are well conserved throughout the various species. Interestingly, the motif analysis highlighted the presence of maximum motifs in the cytosolic interface domain and these motifs were found in all the species thus suggesting that the protein kinase and the RNase domain are vital for the functioning of IRE1 genes. The conservation of IRE1 as a protein underscores its vital role in plant development. This is further highlighted by the observation that knockout of the IRE1 gene results in lethality in plants, reinforcing the importance of its structure and function [15, 52, 57]. According to subcellular localization studies, IRE1 proteins mainly localize to the ER, which is in line with their function in the UPR pathway [1]. Interestingly, ER-localized proteins have been shown to contribute to thermotolerance and be implicated in HSR as well. TaOBF1, a heat-responsive bZIP, has been proposed to be involved in UPR after it was found to localize in the ER via its interaction with TaSTI. Plants that overexpressed TaOBF1 showed enhanced resistance to heat stress [58]. Similarly, Arabidopsis protein disulfide isomerase 9 (PDI9) was found to localize to ER and provided pollen thermotolerance [59].

Interestingly, the cis-element analysis revealed a pattern of differential regulation of IRE1 genes in different organisms. In plants such as Arabidopsis thaliana and Triticum aestivum, most of the stress regulatory elements were found to be present in the promoter region. On the other hand, in crops such as Solanum tuberosome and Glycine max, plant development-related and hormone-related elements dominate in the upstream region (Fig. 3). These variations in expression can be attributed to mutational evolution, divergence, duplication, or the transposition of existing cis-elements that regulate the expression of the associated gene [60]. As the core functional structure is highly conserved at the protein level, the differential activity of its family members among organisms might be because of the genomic environment [61].

The GO analysis and the subsequent expression analysis of IRE1 gene family members in various crops under different abiotic stresses highlighted their conserved role in stress responses. However, it is interesting to note that in Oryza sativa, which has a single IRE1 gene, showed maximum upregulation in only drought stress. On the contrary, organisms such as Arabidopsis thaliana, Glycine max, and Triticum aestivum having multiple IRE1 members showed significant upregulation under heat stress and cold stress as well (Fig. 4). This suggests that IRE1 paralogous genes might show a coordinated expression pattern; wherein some isoforms are expressed in one stress while others work in another stress. This is further supported by the study of Lian and co-workers, where differential expression patterns of paralogs in response to environmental stresses led to the development of a relationship between expression levels and sequence divergences [62].

In the case of Triticum aestivum, three homologues of IRE1 genes were identified and found to be heat-responsive, with an almost similar expression pattern under heat stress. The expression of IRE1 peaked at the anthesis stage which is among the most sensitive stages to heat stress leading to loss of floret fertility, decreased seed-set, seed numbers and yield in wheat [63–65]. Further TaIRE1-6A showed more upregulation in heat-tolerant cultivars as compared to heat-sensitive cultivars (Fig. 8). This indicated that TaIRE1-6A might positively regulate thermotolerance. Moreover, its expression was also found to increase after DTT treatment, which hints towards its role in UPR, which occurs in the ER. Heat stress disrupts protein folding in ER, which leads to the activation of UPR [66]. Thus, it is probable that IRE1 plays an important role in linking the heat stress response with the UPR. Previously, TaHsfA6b has also been reported to be upregulated by both heat stress and DTT treatment and was found to link the heat stress response with the UPR [29]. Therefore, it is probable that TaIRE1-6A might function similarly and acts as an important link in maintaining cellular homeostasis by linking these two responses.

Conclusion

This study provides a panoramic view of the evolution and expansion of the IRE genes in various important species. This provides the first step for more detailed investigations of the functional diversity of IREs in nutritional crops after polyploidization. The phylogenetic relationship, cis-element distribution in the promoter region, sub-cellular localization prediction, GO analysis, and expression analysis highlight the important role of IRE1 genes in stress responses. Particularly in wheat, IRE1 might play a significant role in attributing heat stress tolerance by linking UPR with HSR. These findings highlight the significance of selecting the IRE1 gene sequence to create new transgenic plants with enhanced tolerance to heat stress. Thus, this knowledge could be used for future research on crop improvement for abiotic stress tolerance in wheat and other crops.

Authors’ contributions

A.S. Data curation, Investigation, Methodology, Writing – original draft. H.S. Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. A.S. Formal analysis, Methodology, Writing – review & editing. H.G. Formal analysis, Investigation, Methodology. V.J. Formal analysis, Resources, Supervision, Writing – review & editing. I.D. Investigation, Methodology, Writing – review & editing. PV V.D. Resources, Supervision, Writing – review & editing. V.G. Conceptualization, Formal analysis, Funding acquisition, Resources, Supervision, Writing – review & editing.

Funding

This study was supported by the Anusandhan National Research Foundation (earlier SERB), India (SRG/2023/000320).

Data availability

This published article includes all the data generated or analyzed during this study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.