Abstract
Background
Acyl-CoA-Binding proteins (ACBPs) function as coenzyme A transporters and play important roles in regulating plant growth and development in response to abiotic stress and phytohormones, as well as in membrane repair. To date, the ACBP family has not been a comprehensively characterized in barley (Hordeum vulgare L.).
Results
Eight ACBP genes were identified in the barley genome and named as HvACBP1–8. The analysis of the proteins structure and promoter elements of HvACBP suggested its potential functions in plant growth, development, and stress response. These HvACBPs are expressed in specific tissues and organs following induction by abiotic stressors such as drought, salinity, UV-B exposure, temperature extremes, and exposure to exogenous phytohormones. The HvACBP7 and HvACBP8 amino acid sequences were conserved during the domestication of Tibetan Qingke barley.
Conclusions
Acyl-CoA-binding proteins may play important roles in barley growth and environmental adaptation. This study provides foundation for further analyses of the biological functions of HvACBPs in the barley stress response.
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Background
Lipids are essential components of cell membranes and play pivotal roles in the cellular framework [1,2,3]. They provide energy for intracellular activity and sites for physiological processes such as photosynthesis, signal recognition, and transmission. They also act as signaling molecules in growth, development, and stress responses [4,5,6]. Key proteins involved in non-vesicular lipid transportation include lipid transfer proteins, ATP-binding cassette transporters, and acyl-coenzyme A-binding proteins (ACBPs) [7]. Compared with lipid transfer proteins and ATP-binding cassette transporters, ACBPs exhibit a wider range of lipid capabilities, allowing for broader subcellular localization. In addition, they can interact with long acyl-coenzyme A (acyl-CoA) moieties [8].
ACBPs are classified into four subfamilies (Class I–IV) based on their functional domains and relative molecular masses. Class I (small ACBPs) consists of low-molecular-weight ACBPs with only one ACBP domain. Class II (ANK-ACBPs) consists of higher-molecular-weight ACBPs and N-terminal ACBP domains coupled with C-terminal ankyrin domains [9, 10]. Class III (large ACBPs) includes various higher-molecular-weight ACBPs with one ACBP domain [11]. Class IV (Kelch-ACBPs) comprises higher-molecular-weight ACBPs with N-terminal ACBP domains and multiple C-terminal Kelch structural domains [12, 13].
As with other lipid transport proteins, the functions of ACBPs are influenced by their subcellular localization. All Class I ACBPs, including AtACBP6 [14], OsACBP1–3 [15], ChACBP1 [16], and HbACBP1 [9], are localized in the cytoplasm. Class II ACBPs, including AtACBP1/2 [17] and OsACBP4 [15], are localized in the cell membrane and endoplasmic reticulum, whereas HbACBP2 is localized in the endoplasmic reticulum [9]. The subcellular localization of Class III ACBPs is diverse: AtACBP2 is found in the peroxisomes, cell membranes, and endoplasmic reticulum around the Golgi apparatus complex [18]; OsACBP5 is localized in the peroxisomes [15]; and VfACBP3A and VfACBP3B are localized in the endoplasmic reticulum [19]. Among Class IV ACBPs, AtACBP4 and AtACBP5 are localized in the cytoplasm [20], whereas OsACBP6 is localized in the peroxisome [15].
In Arabidopsis, AtACBP expression varies widely among the different organs (roots, cotyledons, flowers, fruits, seedlings, and mature plants) [13]. AtACBP1–2 expression is highest in the siliques and mature seeds [21,22,23], whereas AtACBP3 is highly expressed in the pistils [11, 24, 25]. AtACBP5 and AtACBP6 are highly expressed in pollen, microspores, and trichome cells [2, 3, 14, 20, 26]. In rice, ACBPs are expressed in the roots, stems, leaves, and seeds, with the highest expression in the leaves and the lowest expression in the roots and stems [27, 28].
ACBPs have been studied for their roles in plant growth, development, and stress responses in model plants such as Arabidopsis and rice. In Arabidopsis, overexpression of AtACBP6 enhances cold tolerance by upregulating the expression of phospholipase Dδ and increasing the levels of phosphatidic acid. AtACBP6 has binding affinity for various types of phosphatidylcholines, suggesting its role in phosphatidylcholine transport [14, 29]. AtACBP6 knockout affects the seed oil content and lipid composition, leading to a reduced seed weight [30]. Similarly, in rice, overexpression of OsACBP2 results in an increased grain size and oil content [31]. The expression of OsACBP1–3 is suppressed under cold stress [27]. In Arabidopsis, AtACBP1 and AtACBP2 respond to cold, drought, hypoxia, and heavy metal stresses by maintaining a pool of membrane-associated acyl groups that are essential for early embryonic development [17, 20, 32, 33]. AtACBP3 regulates autophagy-mediated leaf senescence and hypoxia responses, and its overexpression upregulates pathogenesis-related proteins and the salicylic acid pathway, thus resulting in improved pathogen resistance [25, 34]. Mutations in AtACBP4–6 alter acyl-CoA expression, affecting both seed germination and pollen tube growth [2, 30]. AtACBP4 responds to heavy metals, ethylene, methyl jasmonate (MeJA), and pathogen infections [35]. In rice, OsACBP4 is responsive to salt and drought stress, and its overexpression significantly improves salt tolerance [27, 31]. OsACBP5 overexpression enhances resistance to various pathogen types via the jasmonic acid and salicylic acid pathways [36, 37]. OsACBP6 responds to physical damage, influences growth, participates in indole-3-butyric acid oxidation, and contributes to jasmonic acid biosynthesis [15, 27, 38]. In soybeans, GmACBP3 and GmACBP4 overexpression enhances lipoxygenase activity and salt tolerance [39]. These studies emphasize the critical role of ACBPs in plant biology, lipid metabolism, signaling pathways, and adaptive stress responses. However, there is a lack of research on the ACBP family of barley.
We comprehensively characterized the ACBP family in barley (Hordeum vulgare L.) and identified HvACBPs in barley using genome-wide bioinformatics analysis. Predicted the promoter elements to determine the changes in the expression of HvACBPs in response to abiotic stresses (temperature extremes, elevated salinity, drought, and UV exposure) and phytohormones indole-3-acetic acid (IAA), abscisic acid (ABA), and MeJA. This study provides a basis for the further in-depth exploration of the functional roles of HvACBPs to understand their complex functions in plant growth, development, and responses to environmental stimuli.
Results
Identification of barley ACBP gene family and phylogenetic and collinearity analysis
An extensive search for prior studies of ACBP in rice and Arabidopsis was conducted using public and private barley genomic and transcriptomic databases. Eight HvACBP genes were identified in the Morex reference genome. ACBP genes are distributed on all barley chromosomes, except for 3 H and 6 H. Additionally, HvACBP3 and HvACBP4 are tandem repeat genes on 2 H.
To analyze the evolutionary relationships of ACBP genes among different species, 59 ACBP protein sequences from seven crops were used to construct a phylogenetic tree. The genes were classified into the four ACBP Classes (Fig. 1a). Based on comparison with genes in rice and Arabidopsis, HvACBP1/2/6/8 were classified into Class I (small ACBPs), HvACBP3/4 into Class II (ANK-ACBPs), HvACBP5 into Class III (large ACBPs), and HvACBP7 into Class IV (Kelch-ACBPs).
Collinearity analysis revealed the presence of collinear blocks in the barley and Arabidopsis genomes, although no collinear pairs of ACBP genes were identified. In contrast, comparisons of the barley, rice, and wheat genomes revealed four pairs of collinear ACBP genes (Fig. 1b).
Gene structure, conserved motif, and domain analysis
Gene structure analysis revealed that most of the genes contained upstream regulatory regions, and all contained exons, introns, and a conserved ACBP domain (Fig. 2a, b and d), with similar exon numbers and positions. The ACBP protein motifs and conserved domains were examined in Arabidopsis, rice, wheat, maize, and barley. The structural motifs of ACBPs proteins contained ten protein motifs (Motifs 1–10) of 0.21–50 amino acids in length. Motif 1 and Motif 2 are highly conserved in the ACBP protein family. Motif 9 was identified only in Class II, and Motifs 3–6 and 8 were only identified in Class IV (Fig. 2c). All ACBP proteins contained an ACBP domain. All ANK-ACBPs contained an ANKYR domain at the C-terminus, and all Kelch-ACBPs had an unequal number of Kelch domains.
HvACBP protein properties, secondary and tertiary structure
We analyzed the physical and chemical properties of HvACBP proteins. Their sizes ranged from 93 (HvACBP8) to 669 amino acids (HvACBP7), with molecular weights of 10.24 to 72.62 kDa (Table 1). The theoretical isoelectric point ranged from 4.23 (HvACBP5) to 6.22 (HvACBP1). Their instability index ranged from 30.54 (HvACBP2) to 52.97 (HvACBP3); those of HvACBP1, HvACBP3, HvACBP4, HvACBP5, and HvACBP7 exceeded 40, indicating that these proteins are unstable. Aliphatic index ranged from 66.33 (HvACBP1) to 84.07 (HvACBP5), suggesting relatively consistent thermal stability. The GRAVY (Grand average of hydropathicity) were negative, indicating that they are hydrophilic proteins (positive values indicate that proteins are more inclined towards hydrophobicity, whereas negative values indicate that proteins are more inclined towards hydrophilicity). The proteins were all predicted to localize in the cytoplasm.
Predictive analysis to predict HvACBP secondary and tertiary structures prediction revealed notable differences among the four gene Classes (Fig. 3). Class I ACBP proteins contained a structural motif characterized by 3–5 α-helical segments, accounting for 32.31–67.74% of the overall amino acid content (Table 1; Fig. 3b). HvACBP5 showed a similar structure to that of Class I. Class II HvACBPs exhibited more α-helical than random coil structures in terms of their total amino-acid composition. In contrast, for Class IV, most of the amino acids exhibited a random coil conformation. The Class IV tertiary structure showed substantial segments of β-fold conformations because of the prevalence of Kelch structures.
Cis-element analysis of HvACBP promoters
Cis-elements within eight HvACBP gene promoters were classified into three categories. The first category included elements closely associated with phytohormones, including auxins, MeJA, salicylic acid, ABA, gibberellin, and ethylene. The second category included elements closely associated with responses to environmental stressors, including drought, cold, wounding, anaerobic conditions, other stressors, and with defense, The third category included elements associated with plant growth and development, including meristem-specific and seed-specific expression, zein metabolism regulation, and transcription factor binding sites (specifically MYB and MYC transcription factors).
All eight HvACBP promoters contained elements associated with drought, light regulation, MYB binding, and stress responses; these elements participate in hormonal signaling, stress responses, and transcription factor binding. Our findings strongly suggest that HvACBPs influence hormonal signaling pathways and environmental responses, and regulate plant growth and development (Fig. 4).
Tissue-specific expression of HvACBPs
HvACBPs showed tissue-specific expression. HvACBP6 expression was negligible in all the examined tissue types. HvACBP2 expression was moderate in the ROO2, EPI, SEN, and EMB tissue types. HvACBP1 was expressed predominantly during grain development, with minimal expression observed in the other tissue types. HvACBP4 was few expressed in all detected tissues. Expression of the other four HvACBPs varied significantly within tissues.
HvACBP8 exhibited the highest expression across all tissue types, particularly in the young root, shoot, and grain tissues (LEA, ROO1, CAR5, and EMB). In all tissue types, HvACBP3 and HvACBP4 (in the ANK-ACBP subfamily) exhibited similar expression patterns, although HvACBP3 expression substantially higher than that of HvACBP4, suggesting its importance in barley. Classes III and IV each included one member (HvACBP5 and HvACBP7, respectively) that was abundantly expressed across all tissue types. The robust expression of HvACBP3/4/5/7/8 across all barley tissues throughout the growth and developmental phases indicated the conservation and pivotal roles of ACBP genes in physiological processes in barley (Fig. 5).
HvACBP responses to hormonal stress induction
Bioinformatics analysis of the promoter elements revealed the presence of cis-regulatory elements for IAA, ABA, and MeJA in the upstream region of HvACBP (Fig. 4). The expression of HvACBP1/6 was not detected. Treatment of barley seedlings with IAA, ABA, and MeJA induced HvACBP expression to varying degrees (Fig. 6f-h). Following IAA treatment, all genes, except for HvACBP7, exhibited robust upregulation at 1 and 3 h post-induction. However, at 6 h post-induction, HvACBP expression began to decline toward (or below) basal levels, ultimately returning to their baseline expression at 12–24 h post-induction (Fig. 6f).
Compared with the effects of ABA, MeJA caused longer and more sustained induction of HvACBPs (Fig. 6g, h). At 3 h post-treatment, the expression of HvACBPs was still elevated, although it had declined at 6, 12, and 24 h. In contrast, at 3 h post-ABA induction, HvACBP expression had reverted to pre-induction levels.
HvACBP expression under abiotic stress
We examined the regulation of HvACBP expression following exposure to abiotic stressors (salinity, drought, temperature variation, and UV radiation) (Fig. 6). HvACBP1 and HvACBP6 expression was not detected. Under salt stress, six of the HvACBP genes exhibited varying degrees of upregulation; in contrast, at 6 h, HvACBP2 was slightly downregulated (Fig. 6a). HvACBP4 exhibited significant upregulation (5.2-fold) after 6 h of salt stress, whereas HvACBP3 exhibited a moderate response. HvACBP5 exhibited significant upregulation after 12 h of salt stress. Six HvACBPs exhibited rapid upregulation within 1 h of drought induction (Fig. 6b), although their expression declined over time. HvACBP2 expression was lower than that at baseline after 6 and 12 h of drought stress.
HvACBP expression was differentially regulated under high and low temperature stress (Fig. 6c, d). Following exposure to 4 °C, HvACBP expression was rapidly upregulated, with HvACBP4 exhibiting a remarkable 12-fold increase after 1 h. Heat stress induced significant upregulation of most of the HvACBPs, particulary for HvACBP5, which exhibited 20-fold upregulation after 24 h.
Six of the HvACBPs were differentially regulated in response to UV radiation, with upregulation at 1 h post-exposure (Fig. 6e), followed by a decline and subsequent increase to a peak at 12 h. HvACBP2 and HvACBP7 showed the most significant responses to UV stress, with upregulation of 6.6- and 5.4-fold, respectively. These findings suggest that HvACBPs are crucial in mediating abiotic stress-tolerance responses.
Haplotype variations analysis of HvACBP7 and HvACBP8 between barley and Tibetan barley
To investigate the role of the ACBP genes in environmental adaptation in barley, we analyzed the haplotypes of ACBP members showing high expression levels. Ten haplotypes were identified in HvACBP7. Among them, Hap_10 was the most prevalent, present in 101 samples, whereas Hap_2 was the rarest, present in only 8 (Fig. 7; Table 2). Wild barley predominantly exhibited Hap_5 (23.7%) and Hap_1 (52.6%), whereas nearly all Tibetan barley samples exhibited Hap_7–10 (mostly HAP_10, 80.4%). Haplotype network analysis revealed a distant genetic relationship between Hap_7–10 and Hap_1–2, indicating that HvACBP7 gathered in some new haplotypes during the domestication of Tibetan barley. Protein sequences analysis (Additional Data_2) revealed a substitution event (replacement of tyrosine with phenylalanine) in Hap_10 at amino acid 199 of HvACBP7.
Nine haplotypes were identified in HvACBP8 (Supplementary Data 3). Hap_9, Hap_8, and Hap_6 were the dominate haplotypes. Among them, Tibetan barley mainly contained Hap_6, Hap7, Hap_8, and Hap_9. Remarkably, wild barley had a higher distribution proportion of these haplotypes. Protein sequence analysis revealed no amino acid variations between haplotypes, suggesting that the function of HvACBP8 is highly conserved in function.
Discussion
ACBPs are structurally conserved and colinear in gramineous crops
The functions of ACBPs have been extensively studied in higher plants, including Arabidopsis, rice [27], wheat [40], rapeseed [41], maize [42], and soybeans [43], but not in barley. Here, we comprehensively characterized ACBP sequences in barley, by comparing them with those of Arabidopsis, rice, maize, wheat, soybeans, and sorghum. All taxa were contained all four ACBP subfamilies, which shared a common ACBP structural domain and had a similar exon number and position, consistent with prior findings for maize [42]. This finding suggests that ACBP was conserved during plant evolution. Within subfamilies, different plants contain varying numbers of members, which may be related to genome size and gene copy number during evolution, such as the tandem repeats of HvACBP3 and HvACBP4.
In addition, although the four subfamilies were conserved, and the collinearity of ACBP between the dicotyledonous plants, Arabidopsis and soybean, and barley was lower than that between monocotyledonous plants. This result suggests that, although homologous ACBPs with identical functional structural domains are common across plant species and that the four subfamilies evolved before the divergence of monocotyledonous and dicotyledonous plants, ACBP sequences widely varied during plant diversification, further indicating their importance and conserved roles, as well as highlighting the functional divergence among HvACBPs.
ACBP regulates responses to exogenous hormones and modulates plant growth and development
ACBPs perform critical roles in plant growth and development, as indicated by their tissue-specific expression and involvement in diverse phytohormone pathways [13, 44].
Previous studies revealed functional redundancy among ACBP members. For example, AtACBP1 and AtACBP2 have similar expression patterns, with the loss of one gene leading to compensatory expression of the other gene. The double mutant of atacbp1 and atacbp2 can cause embryonic death in seeds [45]. HvACBP3 and HvACBP4, which are tandem repeat genes, show similar expression patterns, suggesting their conserved and redundant functions in regulating seed development. These similar expression profiles suggest that HvACBPs are involved in barley growth and development. For example, the small ACBP, AtACBP6, of Arabidopsis is highly expressed in pollen, microspores, and trichome cells [14], whereas OsACBP1/2 in rice is highly expressed in developing grains [13, 28]. The variation in AtACBP6 affects lipid accumulation in Arabidopsis seeds [30]. The specific high expression of HvACBP1/8 in grains suggests their function in regulating seed development.
Analysis of cis-elements indicated that HvACBPs are involved in the cross-talk of phytohormones, and the effects of hormone treatment also confirmed that HvACBPs are involved in the plant hormone response, which is crucial for various aspects of plant growth, development, and stress responses, these results highlighting the potential regulatory functions of HvACBPs in barley growth, development, and environmental adaptation.
ACBPs show functional conservation and sub-functionalization in paralogs in responses to abiotic stress
ACBPs are vital in plant responses to various stressors. In Arabidopsis, overexpression of AtACBP1 enhances the sensitivity to salt stress by activating ABA expression via AtAREB1 [46]. In soybeans, salt tolerance is mediated by GmACBP3 and GmACBP4 (Class II) via alternative splicing [39]. The expression level of the OsACBP4 gene in rice rapidly increases under high-salt treatment. In barley, HvACBP4 responds strongly to salt stress, suggesting a role for this gene salt tolerance, whereas HvACBP3 reacts more slowly, indicating distinct roles for these duplicated genes. In Arabidopsis, AtACBP2 is closely associated with drought response via regulation of ABA-dependent guard-cell closure to improve drought resistance [47,48,49,50]. AtACBP3 and AtACBP4 are associated with drought responses [51,52,53]. OsACBP4 and OsACBP5 in rice; as well as GhACBP1, GhACBP3, and GhACBP6 in cotton, are induced by drought [27, 54, 55]. In barley, the six detected HvACBPs upregulated under salt treatment, suggesting that they are related to drought tolerance in barley. AtACBP6 overexpression enhances stability under cold stress, whereas AtACBP1 knockout enhances freezing resistance in Arabidopsis [29, 56]. Under cold stress, all six OsACBPs were downregulated [27]. In cotton, GhACBP1, GhACBP3, and GhACBP6 respond to low-temperature stress, whereas GhACBP6 exhibits significant regulation under heat stress [54]. We found that the HvACBPs exhibited varied responses to heat and cold but responded more strongly to heat than to cold. Therefore, HvACBPs respond rapidly and in diverse manners to temperature stress, highlighting their importance in temperature adaptation in barley. UV radiation induces membrane damage [57]. Our findings reveal that ACBPs participate in repairing UV damage in barley.
The ACBP gene shows sub-functionalization in paralogs. Among Ankry-ACBP subfamily genes, in Arabidopsis, AtACBP1 is only expressed in the trichomes, whereas AtACBP2 is only expressed in guard cells, indicating that AtACBP2 is associated with stomatal opening and the drought response [21, 22, 52, 47]. In barley, HvACBP3 and HvACBP4 are tandem repeat genes; HvACBP3 expression was higher during growth and breeding, whereas HvACBP4 is more strongly responsive to abiotic stress. HvACBP2, a small ACBP in barley, showed greater sensitivity to UV-B than did HvACBP8. These results indicate that ACBPs underwent sub-functionalization in paralogs during species evolution.
The Tibetan Plateau, the predominant cultivation region for Tibetan barley, which was domesticated from eastern domesticated barley, is characterized by harsh environmental conditions, including low temperatures, substantial diurnal temperature fluctuations, low latitudes, high elevations, low-density air, intense solar radiation, and high UV-B exposure [48]. ACBPs constitute a well-conserved family of proteins in eukaryotes that are important in stress responses and development [49]. The chromosomal region containing HvACBP7 was under selective pressure during the domestication of barley in the Tibetan region [50]. Analysis of HvACBP7 and HvACBP8 haplotypes showed that during the domestication of barley, HvACBP7/8 were selected to adapt to the unique environment of the Tibetan Plateau, but their amino acid sequences remained conserved, suggesting the importance and conservation of ACBPs in the growth, development, and environmental adaptation of barley; however, the variation of the cis-elements in the promoter requires further analysis.
Conclusions
HvACBPs respond to multiple stressors, underscoring their crucial role in stress resilience in barley, as well as revealing the importance of ACBPs in plant growth, development, and stress adaptation, particularly abiotic stress. Our results provide a foundation for the further functional analysis of HvACBP and theoretical basis for improving barley via breeding. We determined mechanisms of plant ecological adaptation in extreme environments.
Materials and methods
Plant material, growth, and abiotic stress conditions
Barley seeds (H. vulgare L., Morex) were provided by the Chinese Academy of Agricultural Sciences. The seeds were disinfected with 0.1% NaClO and 75% ethanol, rinsed with distilled water, and placed on moist filter paper into dark for 24 h. The germinated seeds were transferred to hydroponic containers and grown under 24 °C with a 14 h light/10 h dark cycle. The nutrient solution (1/2 Hoagland solution) was replaced every 3 d. Different treatments were applied after 4 weeks.
The 4-week-old seedlings were subjected low-temperature stress (4 °C), high-temperature stress (42 °C), 200 mM NaCl application, 20% polyethylene glycol (PEG 6000) treatment, UV radiation at 30 µW/cm2, 0.15 µmol/L IAA treatment, 100 µmol/L ABA treatment, and 100 µmol/L MeJA treatment. The seedlings were harvested at 0, 1, 3, 6, 12, and 24 h post-treatment, immediately frozen in liquid nitrogen, and stored at − 80 °C for subsequent experiments.
Sequence retrieval and identification of ACBPs
We performed a BLASTP search using the ACBP family from Arabidopsis (https://www.arabidopsis.org/) in the barley genome database (http://barlex.barleysequence.org/) with an e-value of 10− 10. We also downloaded the ACBP domain file (PF00887) from the Pfam website (http://pfam.xfam.org/) and uploaded it to the HMM website (https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch) to search for genes in the barley genome potentially containing this conserved domain. Duplicate genes were removed by comparing the results of the two different searches. The remaining genes were validated for the presence of the ACBP-type domain using the CD search (https://www.ncbi.nlm.nih.gov/cdd/) and SMART (http://smart.embl-heidelberg.de/) websites.
HvACBP structure and conserved motif analysis
The structures of the HvACBPs were deduced from the coding and genomic sequences using a gene structure displayer (https://gsds.gao-lab.org/). Conserved motifs in each HvACBP were analyzed using MEME Suite (https://meme-suite.org/meme/tools/meme).
Analysis of cis-acting elements in HvACBP promoters
A region approximately 2000 bp upstream of the start codon (ATG) was investigated from the reference genome sequence of MorexV3, Cis-acting elements were predicted using the PlantCare website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Expression patterns of HvACBP family in different tissues
HvACBPs transcriptome data (fragments per kilobase of transcript per million mapped fragments) were downloaded from the barley genome database (http://barlex.barleysequence.org/). Transcriptome data were normalized by logarithms, and the expression heat maps were drawn using R4.2.1 software.
Phylogenetic analysis of HvACBPs
To calculate the correlations between HvACBPs and their counterparts in various species, the ACBP protein sequences of Arabidopsis, rice, maize, soybean, sorghum, wheat, and barley were subjected to multiple sequence alignment using the Clustal W tool. A phylogenetic tree was constructed using the maximum likelihood (ML) method via MEGA 7, employing the WAG + G model. The resultant phylogenetic tree was further refined and visualized using iTOL v6 (https://itol.embl.de/).
Protein physical and chemical properties and prediction of secondary and tertiary structures
We used ExPASy (https://web.expasy.org/protparam/) to compute the amino acid content, isoelectric point (pI), molecular weight (average), instability coefficient, aliphatic index, and average hydrophobicity index. The SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) and PredictProtein (https://predictprotein.org/) websites were used to predict the secondary structure of the proteins. The tertiary structure of HvACBP proteins was predicted using the SWISS-MODEL Interactive Workshop (https://www.swissmodel.expasy.org/interactive).
RNA isolation, cDNA synthesis, and quantitative real-time PCR analysis
Total RNA extraction was performed using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). RNA was reverse-transcribed into cDNA using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was conducted using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The internal reference gene used for normalization was HvGAPDH (HORVU.MOREX.r3.7HG0703580). Each sample contained 10 µL of a reaction mixture composed of 5 µL of 2×PCR mix, 1 µL of primer mixture (10 µmol L-1 each upstream and downstream primers), 1 µL of cDNA, and 3 µL of deionized water. The PCR thermal cycling conditions were as follows: initial denaturation at 95 °C for 30 s followed by 35 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 95 °C for 15 s. A final extension step was performed at 60 °C for 60 s, followed by melt curve analysis at 95 °C for 15 s. The experiment was conducted in triplicate. The primers are listed in Supplementary Data.
Haplotype analysis of HvACBP7 and HvACBP8
All single-nucleotide polymorphisms and insertions–deletions used for haplotype analysis were obtained by resequencing of Tibet barley [55]. Haplotype identification was performed using DnaSP_V5 and haplotype network analysis was performed using Arlequin.
Data availability
All datasets supporting the results of this study are included within the article and its supplementary information.
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Acknowledgements
We would like to thank Dr. Zhaoyan Chen and Yanling Ma (Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, ICS-CAAS, China) for discussions and help with the manuscript.
Funding
This work was supported by the Modern Agro-Industry Technology Research System of Shanxi Province, China (grant number 2023CYJSTX03-19), National Laboratory for Minor Crops Germplasm Innovation and Molecular Breeding, China (in preparation) (grant number 202204010910001-06), and the China Agriculture Research System of MOF and MORA (CARS-05).
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HC performed the bioinformatics analysis. HC and MM performed qRT-PCR analyses and drafted the manuscript. MG, SL, and ML planted the crops and collected the materials. GG and GX supervised the research and modified the manuscript. All authors have read and approved the final manuscript.
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Chang, H., Ma, M., Gu, M. et al. Acyl-CoA-binding protein (ACBP) genes involvement in response to abiotic stress and exogenous hormone application in barley (Hordeum vulgare L.). BMC Plant Biol 24, 236 (2024). https://doi.org/10.1186/s12870-024-04944-6
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DOI: https://doi.org/10.1186/s12870-024-04944-6