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Article

Genome-Wide Identification and Expression Profiling of ABA-Stress-Ripening (ASR) Gene Family in Barley (Hordeum vulgare L.)

by
Jie Ren
1,2,†,
Kangfeng Cai
1,3,†,
Xiujuan Song
1,3,†,
Wenhao Yue
1,3,
Lei Liu
1,3,
Fangying Ge
1,3,
Qiuyu Wang
1,4 and
Junmei Wang
1,3,*
1
Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2
Agricultural Technology Extension Center, Deqing Bureau of Agriculture and Rural Affairs, Deqing 313200, China
3
National Barley Improvement Centre, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
4
College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(6), 970; https://doi.org/10.3390/plants14060970
Submission received: 9 February 2025 / Revised: 14 March 2025 / Accepted: 17 March 2025 / Published: 19 March 2025
(This article belongs to the Special Issue Cell Physiology and Stress Adaptation of Crops)
Browse Figures
Versions Notes

Abstract

:
Abscisic acid (ABA)-stress-ripening, or ABA-, stress-, and ripening-induced (ASR) proteins play an important role in responses to environmental stimuli. A total of ten barley HvASRs were identified in this study, which were unevenly distributed on three chromosomes. ASRs from barley, wheat, Brachypodium distachyon, rice, maize, foxtail millet, and tomato were classified into two distinct clusters based on phylogenetic analysis. Notably, ASRs from Poaceae were evenly distributed between these two clusters. HvASRs contained a typical ABA/WDS domain, and exhibited similar motif arrangements. Two gene pairs of tandem duplicates (HvASR4/5/6/7 and HvASR8/9) were identified among HvASRs. Cis-acting elements involved in hormone and stress responses, including ABRE, MYB, ARE, and STRE, were consistently identified in the promoters of HvASRs. The expression of HvASRs was substantially influenced by salt, osmotic, and ABA treatments in the roots and leaves of barley seedlings. HvASR2 acts as a transcriptional repressor, whereas HvASR3 serves as a transcriptional activator. These results enhance our understanding of the HvASR family and provide a foundation for further functional characterization.

1. Introduction

Abscisic acid (ABA)-stress-ripening, or ABA-, stress-, and ripening-induced (ASR) genes encode plant-specific, low-molecular-weight, and highly hydrophilic proteins possessing conserved abscisic acid/water deficit stress (ABA/WDS) domains. ABA/WDS is intrinsically disordered, allowing its conformation to adapt in support of the diverse functions of ASRs [1]. Since the initial identification of an ASR gene from a tomato fruit cDNA library in 1993 [2], more and more ASRs have been identified among gymnosperms, monocots, and dicots [3]. However, ASRs are notably absent in Brassicaceae species, including the model plant Arabidopsis.
ASRs exhibit a dual nature, functioning as either transcription factors or chaperones. The unstructured form of tomato SlASR1 has been reported to display chaperone-like activity in the cytosol, effectively preventing protein denaturation during repeated freeze–thaw cycles [4]. Similar results have also been observed in ASRs from plantain [5] and lily [6]. On the other hand, SlASR1 exhibits zinc-dependent DNA-binding activity, and the overexpression of SlASR1 significantly enhances salt tolerance in transgenic tobacco plants [7]. BdASR1 from Brachypodium distachyon acts as a transcription factor and confers drought tolerance by enhancing antioxidant capacity [8].
ASRs play crucial roles in responses to external stimuli. OsASR1 is induced by low temperature, and overexpression of OsASR1 improves photosynthetic capacity and cold stress tolerance in transgenic rice plants [9]. OsASR1 also has been verified to be a positive regulator of the ABA signaling pathway and enhance the drought and salinity stress tolerance of transgenic plants by improving osmolyte accumulation, stomatal closure modulation, and transpiration rates [10]. OsASR5 acts as a chaperone-like protein and improves drought tolerance via regulating the ABA pathway and promoting stomatal closure [11]. OsASR6 serves predominantly as a functional protein and enhances salt tolerance through the modulation of reactive oxygen species (ROS) homeostasis and ABA signaling [12]. The overexpression of SiASR1 significantly enhances tolerance to drought and oxidative stresses in tobacco plants [13]. SiASR4, as the target gene of SiARDP, enhances drought and salt tolerance in transgenic Arabidopsis and foxtail millet plants through ROS- and ABA-dependent pathways [14]. Constitutive overexpression of TaASR1-D improves tolerance to multiple stresses, including oxidative, osmotic, drought, and salinity stress, by enhancing the antioxidant system and ABA signaling [15]. Heterologous expression of TaASR2D in Brachypodium distachyon increases the expression of stress-related and ABA-responsive genes, and endogenous levels of ABA and hydrogen peroxide, and increases stomatal closure under drought stress, thereby leading to improved drought tolerance [16]. ZmASR3 positively regulates drought tolerance by maintaining water content, increasing ABA content and stomatal closure, reducing ROS accumulation, and inducing the expression of genes involved in ROS-related, stress-responsive, and ABA-dependent pathways [17]. Overexpression of BdASR4 leads to enhanced drought tolerance by activating the antioxidant defense system and inducing the expression of genes involved in stress- and ABA-response pathways [18]. SlASR1 [19], SbASR-1 [20,21], PheASR2 [22], ThASR3 [23], and HaASR2 [24] have also been demonstrated to enhance abiotic stresses in transgenic plants. In addition, ASR genes were also found to function in cadmium tolerance [25] and aluminum tolerance [26].
The ASR family has been identified in many species such as rice [27], maize [25,28], wheat [15,29,30], Brachypodium distachyon [8], foxtail millet [13,31], and tomato [32,33]. However, such identification has not been conducted in barley. This work identified a total of 10 HvASRs in barley, and subsequently conducted comprehensive analyses. The response patterns of HvASRs to salt, osmotic, and ABA treatments in barley seedling roots and leaves were examined, and their transcriptional activation capacity was investigated using a Y2HGold yeast system. The results will establish a solid foundation for the functional characterization of HvASRs.

2. Results

2.1. Barley HvASRs Identification

In total, 10 barley HvASR genes were identified (Table 1). The amino acid lengths of HvASRs varied from 97 amino acids (aas) to 282 aas. Their isoelectric point (pI) and molecular weight (MW) were 5.21–9.99 kDa and 10.65–29.95 kDa, respectively (Table 1). The instability index varied from 28.89 (HvASR11) to 50.62 (HvASR4). The instability indices of HvASR3/5/6/8/9/10 were <40, suggesting that these proteins are relatively stable [34]. The aliphatic indices and grand average of hydropathicity (GRAVY) of HvASRs were 18.05–67.24 and −1.68–−0.78, respectively, indicating that HvASRs were all hydrophilic proteins. Disordered amino acids of HvASRs varied from 59.57% to 89.34%. HvASRs were predicted to localize in the nucleus, except HvASR2, which was localized in the chloroplast (Table 1). HvASR10 was previously designated as HvASR1 in an earlier study [35]. In a separate investigation, HvASR10 was functionally characterized and named HvASR5 for its similarity to OsASR5 in rice [36].

2.2. Phylogenetic Relationships and Gene Structures of HvASRs

The amino acid sequences of 6 OsASRs in rice, 10 ZmASRs in maize, 36 TaASRs in wheat, 5 BdASRs in Brachypodium distachyon, 6 SiASRs in foxtail millet, 5 SlASRs in tomato, and 10 HvASRs were used for phylogenetic analysis (Figure 1). These 78 ASRs could be classified into two clusters, Cluster I and Cluster II. Cluster I contained 41 members, and Cluster II contained 37 members. SlASRs were all in Cluster I, whereas 60% of HvASRs (6) were in Cluster I. The ASRs of the remaining species were distributed almost evenly between Cluster I and Cluster II.
All HvASRs possessed an ABA/WDS domain, and HvASRs from the identical cluster exhibited comparable motif configurations (Figure 2a,b). The amino acid sequences of motif 1 to motif 5 were “HKIKEKLAALGAVAAGGYALHEHHEAKKD”, “TMYYNTTTEECFDSGKQGHGY”, “GYKKSGGDDED”, “QECRMPVHNSYCN”, and “RPMSYSNTEECFD”, respectively (Figure 2d). The ABA/WDS domain consisted of two of motif 1; therefore, all HvASRs contained two motif 1 elements. In contrast, motif 3, motif 4, and motif 5 exhibited gene-specific distribution and were present only in specific HvASR members. Introns are classified into three kinds based on the positional relationship relative to codons, namely phase 0, phase 1, and phase 2. Phase 0 introns are situated between two codons, whereas phase 1 introns and phase 2 introns are located after the first and second positions within a codon, respectively [37]. Intriguingly, all HvASRs contained one phase 0 intron (Figure 2c).

2.3. HvASRs Distribution and Duplication

Ten HvASRs were unevenly distributed across three out of seven chromosomes, with eight genes on chromosome 3, and one gene each on chromosome 2 and 4, respectively (Figure 3). Gene duplication serves as a primary mechanism driving gene family expansion [38]. Tandem duplicated genes refer to adjacent homologous genes located on the same chromosome, with no more than one intervening gene [39]. Two gene pairs of tandem duplicates (HvASR4/5/6/7 and HvASR8/9) were identified among HvASRs. However, no segmental duplication events were detected.

2.4. Cis-Acting Elements

Seventy cis-acting elements were detected in HvASR promoters. These elements were classified into six classes (Figure 4; File S1). The “Stress response” category contained the most elements (21, 30.0%), followed by “light response” (18, 25.7%), “hormone response” (13, 18.6%), “development/tissue specificity” (12, 17.1%), and “promoter/enhancer element” (5, 7.1%). The “Circadian control” category had only one element. A TATA box and CAAT box were present in all HvASR promoters, suggesting their pivotal roles in transcription regulation. In the “hormone response” category, the ABA responsiveness-related element, ABRE, was widely distributed in HvASR promoters. G-box in the “light response” was also prevalent in the promoters of HvASRs (Figure 4). Furthermore, elements in “stress response”, such as ARE, MYB, and STRE, were also ubiquitously present in the promoters of all HvASRs. However, other elements were comparatively gene-specific. Thus, HvASRs may play crucial roles in external stimuli responses.

2.5. Syntenic Relationships of ASRs Between Barley and Other Species

The synteny of ASRs between barley and other plant species were investigated (Figure 5; File S2). There was only one pair of orthologs each, observed between barley and tomato and between barley and rice, namely HvASR1/SlASR3 and HvASR10/OsASR5, respectively. A comparison of barley to maize, foxtail millet, and Brachypodium distachyon showed the presence of two pairs of orthologous genes, namely HvASR10/ZmASR1 and HvASR10/ZmASR2, and HvASR10/SiASR1 and HvASR10/SiASR2, as well as HvASR2/BdASR1 and HvASR10/BdASR4, respectively. Wheat is more closely related to barley than the other five plant species; thus, the comparison between these two species led to substantially more orthologous gene pairs (15 pairs). Each of HvASR1/2/8/10 was orthologous to three TaASRs, and each of HvASR4/5/7 was orthologous to one TaASR gene. HvASR3, HvASR6, and HvASR9 had no orthologs within the wheat genome.

2.6. Tissue-Specific Expression of HvASRs

The expression levels of HvASRs displayed dramatic variation among 14 different tissues, and HvASRs in Cluster I exhibited comparably higher expression levels than those in Cluster II (Figure 6; File S3). The expression of HvASR2 and HvASR10 within Cluster I was detected across all tissue types and developmental stages, whereas the expression of the other HvASRs was relatively tissue-specific and of low abundance. In addition, the expression of HvASR8 was detected in roots from seedlings at the 10 cm shoot stage, whereas expression was absent in roots at 28 days after pollination, suggesting the growth stage also matters.

2.7. Responses of HvASRs to Salt and Osmotic Stress and Exogenous ABA Treatment

The expression of HvASRs in response to salt and osmotic stresses, as well as exogenous ABA treatment, were further examined in the roots and leaves of barley seedlings (Figure 7 and Figure 8). HvASRs were significantly induced or inhibited in response to stress treatments, albeit to varying degrees. In roots, the expression of HvASR1 was progressively inhibited by salt stress, with the degree of inhibition intensifying as the treatment duration extended (Figure 7a). HvASR2 was slightly induced by salt stress at 0.5 h, and thereafter progressively inhibited as treatment duration extended. HvASR2/4/5/7/8/9 were dramatically induced (9.0-fold to 46.2-fold of control), and their expression levels peaked after salt treatment for 1 d. HvASR6 was also induced by salt stress, but its expression level peaked at 3 h and 6 h. However, the expression of HvASR10 was relatively stable. The response of HvASRs to osmotic stress and ABA treatment were considerably less pronounced in comparison to salt stress (Figure 7a,b). HvASR2 was the gene that was induced the most; the expression level of which reached 9.7-fold of control after osmotic stress treatment for 1 d. Interestingly, the expression of HvASR4/5/7/8/9 was significantly decreased upon the initiation of osmotic stress, then increased and peaked after treatment for 1 d, and subsequently dramatically decreased after treatment for 3 d. The expression of HvASR1/3 alternated between induction and suppression, and that of HvASR10 was significantly induced only after osmotic stress for 3 d. The expression pattern of HvASR2 in response to ABA treatment was analogous to its response to osmotic stress, with the highest induction and peak expression occurring after a 1 day of ABA treatment (Figure 7c). The response patterns of HvASR8/9 after ABA treatment resembled that under osmotic stress conditions (Figure 7b,c). HvASR4 was consistently suppressed during the whole period of ABA treatment, whereas HvASR1/3/10 were significantly inhibited at the late stage of treatment (6 h to 3 d).
The expression levels of HvASR1 and HvASR3 in leaves were induced by salt stress, exhibiting opposite response patterns compared with those in roots (Figure 7a and Figure 8a). The expression of the other HvASRs was dynamic, alternating between induction and suppression (Figure 8a). The expression of HvASR1 was continuously upregulated, whereas that of HvASR8 and HvASR9 was steadily downregulated in response to osmotic stress (Figure 8b). The expression levels of the other HvASRs fluctuated over time. The expression of HvASR1 was induced, whereas that of HvASR8 was continuously suppressed after exogenous ABA treatment (Figure 8c). The expression of HvASR2/3/4/5/6/7/9/10 was relatively dynamic.

2.8. Transcriptional Activation Capacity of HvASRs

ASRs function as either chaperones or transcription factors. The transcriptional activation capacity of HvASRs was analyzed using the Y2HGold yeast system (Figure 9). Y2HGold yeast strains expressing pGBKT7 exhibited limited growth on SD/-His/-Trp plates, and were unable to grow on SD/-Ade/-His/-Trp plates. Herpes simplex virus protein VP16 can activate the transcription of target genes, and GAL4-VP16 is a transcriptional activator, which is formed by fusion of the yeast activator GAL4 to VP16 [40]. GAL4-VP16 is capable of activating the expression of HIS3 and ADE2, thereby rescuing the growth of Y2HGold yeast strains on SD/-Ade/-His/-Trp plates. Yeast strains expressing HvASR2 and HvASR3 succeeded in growing on SD/-Ade/-His/-Trp plates, demonstrating that these two genes possess transcriptional activation capacity. The expression of HvASR2 inhibited the growth of yeast strains expressing pGBKT7-VP16, while the expression of HvASR3 enhanced such growth on SD/-Ade/-His/-Trp plates. This suggested that HvASR2 acts as a transcriptional repressor, whereas HvASR3 functions as a transcriptional activator. Notably, the yeast strains expressing pGBKT7-VP16-HvASR5, pGBKT7-VP16-HvASR8, and pGBKT7-VP16-HvASR10 failed to grow on SD/-Trp plates. The authors speculated that the fusion expression of HvASR5, HvASR8, and HvASR10 might have interfered with the function of GAL4-VP16 through an unknown interaction.

3. Discussion

ASRs were classified into two clusters according to phylogenetic relationships (Figure 1). All SlASRs from tomato were in Cluster I, whereas ASRs from other species including barley, wheat, rice, maize, Brachypodium distachyon, and foxtail millet were almost distributed evenly between Cluster I and Cluster II. The protein lengths of ASRs from Cluster II were substantially longer than those from Cluster I (File S4). In terms of ASRs in barley, HvASR1/2/3/10 were classified into Cluster II with an average length of 228 amino acids, which was 2.28 times the average length of the other HvASRs in Cluster I (Figure 1; File S4). Notably, as the evolutionary distance to barley increased, the length ratio of Cluster II to Cluster I progressively decreased, declining from 2.02 in wheat to 1.34 in maize (File S4).
ZmASR7, ZmASR8, and ZmASR9 in maize were devoid of introns, whereas the other ZmASRs had one intron [28]. Two TaASRs in wheat had two introns, and the remaining TaASRs contained one intron [29,30]. MsASR6 was intron-rich with four introns, whereas the others in Miscanthus had no or only one intron [41]. All HvASRs in barley contained one intron (Figure 2c), which was also observed among BdASRs in Brachypodium distachyon [8] and TtASRs in Tetragonia tetragonoides [42]. Introns may impede prompt regulatory responses and are, therefore, subject to negative selection in genes that necessitate rapid regulation during stress responses, which represents an internal mechanism driving the extensive loss of introns observed in certain eukaryotic lineages throughout evolution. [43]. Based on this perspective, HvASRs might be fast-responding genes upon environmental stimuli.
A total of 23 pairs of tandem duplicated genes and six pairs of segmentally duplicated genes were identified within 29 wheat TaASRs [29]. On the other hand, 33 TaASRs were identified in other research, and 14 pairs of TaASRs were tandem duplicated, and eight TaASR groups were segmentally duplicated [30]. Two pairs of TtASRs were segmentally duplicated in Tetragonia tetragonoides [42]. However, among the HvASRs in barley, only two gene pairs of tandem duplicates (HvASR4/5/6/7 and HvASR8/9) were detected, while no segmental duplication events were observed (Figure 3). These results indicated that plant species took different strategies for ASR gene family expansion during evolution.
The expression of HvASR2 and HvASR10 was detected across all tissues and stages, whereas that of the other HvASRs was comparatively low and tissue-specific (Figure 6; File S3). Furthermore, HvASR10 exhibited a much higher expression level compared to the other HvASR genes across most tissues and developmental stages. Tissue-specific expression of ASRs was also observed in tomato [33], wheat [30], maize [28], and foxtail millet [31]. Cis-acting elements, such as ABRE in the “hormone response” category, G-box in the “light response” category, and ARE, MYB, and STRE in the “stress response” category, were ubiquitous in the promoters of HvASRs (Figure 4). Similar phenomena were also observed in MsASRs from Miscanthus [41]. The widespread occurrence of cis-acting elements associated with hormone signaling and external stimuli suggested a significant role for HvASRs in responding to environmental cues. The responses of HvASRs to salt, osmotic, and ABA treatments in the roots and leaves of barley seedlings were further examined via qRT-PCR (Figure 7 and Figure 8). The expression of HvASRs was significantly induced or inhibited by salt, osmotic, and ABA treatments, albeit to varying degrees (Figure 7 and Figure 8). Several HvASRs displayed opposite response patterns in roots and leaves, such as HvASR1 and HvASR3 under salt stress conditions (Figure 7a and Figure 8a). After 1 d of salt, osmotic, and ABA treatments, HvASR2 was significantly upregulated in roots, whereas it was only slightly induced in leaves without reaching statistical significance (Figure 7 and Figure 8), indicating that tissue type also affected expression patterns. The responses of HvASRs to osmotic stress and ABA treatment were considerably less pronounced in comparison to salt stress (Figure 7 and Figure 8), which might be explained by the complex nature of salt stress comprising osmotic stress, ionic stress, and oxidative stress [44].
Some ASRs were proved to be intrinsically disordered proteins, such as TtASR1 in wheat [35], SbASR-1 in Salicornia brachiata [21], PgASR3 in pearl millet [45], and SlASR in Suaeda liaotungensis [46]. HvASR10 was composed of 84.78% disordered amino acids, and was also proved to be an intrinsically disordered protein in previous research (designated as HvASR1 therein) [35]. HvASR2 and HvASR3 were composed of 59.57% and 67.57% disordered amino acids, respectively, whereas the other HvASRs consisted of >70% disordered amino acids (Table 1). A transcriptional activation assay revealed that the expression of HvASR2 and HvASR3 restored yeast growth on SD/-Ade/-His/-Trp plates, indicating the transcriptional activation capacity of HvASR2 and HvASR3 (Figure 9). Thus, HvASR2 and HvASR3 may function as transcription factors, whereas the remaining HvASRs function as molecular chaperones. HvASR2 and HvASR3 were both in Cluster II and were closer to each other than any other HvASRs based on phylogenetic analysis (Figure 2a). In addition, motif 2, motif 4, and motif 5 were exclusively present in HvASR2 and HvASR3 (Figure 2b), which might lead to their transcriptional activation capacity. Similarly, twelve TaASRs were proved to possess activation activity in yeast cells [29], and BdASR1 was also evidenced to be a transcription factor [8]. In addition, the expression of HvASR2 enhanced the growth of yeast strains expressing pGBKT7-VP16, while the expression of HvASR3 inhibited such growth on SD/-Ade/-His/-Trp plates (Figure 9), indicating that HvASR2 functions as a transcriptional repressor, whereas HvASR3 serves as a transcriptional activator.

4. Materials and Methods

4.1. Identification of HvASRs

The protein sequences of ASRs from tomato [32] and rice [27] were used as a query for a blast search in barley. Meanwhile, “IPR003496” and “PF02496” were used to search ASR candidates in the barley genome in Ensembl Plants (http://plants.ensembl.org/, accessed on 16 October 2024). The putative HvASRs were further validated in InterPro [47]. In the end, 10 HvASR genes were identified.

4.2. Properties and Localizations of HvASRs

The pI, MW, instability indices, aliphatic indices, and GRAVY of HvASRs were predicted using PROTPARAM [48]. Disordered amino acids and subcellular localizations were analyzed using PrDOS [49] and BUSCA [50], respectively.

4.3. Phylogeny, Synteny, and Duplication Analyses

ASR amino acid sequences from barley (10), rice (6), maize (10), Brachypodium distachyon (5), wheat (36), foxtail millet (6), and tomato (5) were aligned using MAFFT [51], and phylogenetic trees were built using MEGA X 1.0 (maximum-likelihood method with 1000 bootstraps) [52]. Syntenic relationships and duplication events were analyzed using TBtools [53,54].

4.4. Sequence, Promoter, and Expression Analyses

Conserved motifs were analyzed on MEME Suite 5.5.2 (classic mode with five motifs and any number of repetitions) [55,56]. Cis-acting elements within the 2 kb upstream regions of HvASRs were identified through PlantCARE [57]. Transcriptomic data (FPKM) were downloaded from BARLEX (Accession: PRJEB14349) [58,59], and visualized using TBtools v2.210 [53].

4.5. Plant Growth, Treatments, and qRT-PCR

Barley (cv. Morex) seeds were germinated and grown in a 1/5 Hoagland solution for 10 d. Then, salt stress (200 mM NaCl), osmotic stress (20% PEG8000), and ABA treatments (100 μM ABA) were imposed on the seedlings [60]. Seedlings grown in normal nutrient solution were set as a control. After treatments for 0.5 h, 1 h, 3 h, 6 h, 1 d, and 3 d, roots were sampled in three replicates for qRT-PCR [60]. A MiniBEST Plant RNA Extraction Kit (9769, TaKaRa, Shiga, Japan) and PrimeScript RT Master Mix (RR036A, TaKaRa, Shiga, Japan) were used for RNA extraction and cDNA synthesis, respectively. ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) was used for qRT-PCR with an LC480 II (Roche, Basel, Switzerland), with two technical replicates [61]. Relative expression levels of HvASRs were calculated using the 2−ΔΔCT method [62], with actin as the reference gene [54]. The primers used are listed in File S5.

4.6. Transcriptional Activation Capacity Assay

First-strand cDNA synthesis and amplification were performed as described in [61]. The gene-specific primers are listed in File S6. HvASRs were cloned into pYES2-NTB plasmids using EcoRI (R0101V, New England Biolabs, Ipswich, MA, USA) and ClonExpress II One Step Cloning Kit (C112, Vazyme, Nanjing, China). HvASR2 and HvASR8 exhibited high similarity in the 3′ end of the coding sequence to HvASR3 and HvASR9, respectively; thus, they were de novo synthesized into pYES2-NTB plasmids (SUNYA, Hangzhou, China). Subsequently, HvASRs were reconstructed into pGBKT7 and pGBKT7-VP16 plasmids, respectively (File S7). Recombinant vectors were transformed into Y2HGold yeast cells, and the transcriptional activity assay was conducted following instructions (MH102, Coolaber, Beijing, China).

5. Conclusions

A total of ten barley HvASRs were identified. All HvASRs comprised a typical domain of ABA/WDS and displayed conserved motif configurations. Two gene pairs of tandem duplicates (HvASR4/5/6/7 and HvASR8/9) were identified among HvASRs. Hormone- and stress response-related cis-acting elements, including ABRE, ARE, MYB, and STRE, were ubiquitous in HvASR promoters. The expression of HvASRs was significantly affected by salt, osmotic, and exogenous ABA treatments, with varying expression levels observed in roots and leaves. HvASR2 might be a transcriptional repressor, whereas HvASR3 serves as a transcriptional activator, and both warrant further investigation.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14060970/s1: File S1. Cis-acting elements in the promoters of HvASRs; File S2. Orthologous ASR gene pairs in synteny analysis; File S3. Tissue expression patterns of HvASRs; File S4. Amino acid length of HvASRs from different clusters and evolutionary timescale of barley, wheat, Brachypodium distachyon, rice, foxtail millet, and maize; File S5. Primers for qRT-PCR; File S6. Primers for cloning of HvASRs; File S7. Primers for transcriptional activity assay of HvASRs.

Author Contributions

J.R.: investigation, formal analysis, writing—review and editing. K.C.: conceptualization, formal analysis, writing—original draft, writing—review and editing, funding acquisition. X.S.: investigation, resources, formal analysis. W.Y.: formal analysis. L.L.: formal analysis. F.G.: formal analysis. Q.W.: formal analysis. J.W.: writing—review and editing, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Zhejiang Province (LQ23C130004), Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding (2021C02064-3-2), China Agriculture Research System (CARS-05-01A-06), and an open project of the Key Laboratory of Digital Dry Land Crops of Zhejiang Province (2022E10012).

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of ASR family proteins from rice, maize, wheat, Brachypodium distachyon, foxtail millet, tomato, and barley. Amino acid sequences of all ASRs were aligned with MAFFT, and a phylogenetic tree was constructed using MEGA X with the maximum-likelihood method and 1000 bootstraps. Os: Oryza sativa; Zm: Zea mays; Ta: Triticum aestivum; Bd: Brachypodium distachyon; Si: Setaria italica; Sl: Solanum lycopersicum; Hv: Hordeum vulgare.
Figure 1. Phylogenetic tree of ASR family proteins from rice, maize, wheat, Brachypodium distachyon, foxtail millet, tomato, and barley. Amino acid sequences of all ASRs were aligned with MAFFT, and a phylogenetic tree was constructed using MEGA X with the maximum-likelihood method and 1000 bootstraps. Os: Oryza sativa; Zm: Zea mays; Ta: Triticum aestivum; Bd: Brachypodium distachyon; Si: Setaria italica; Sl: Solanum lycopersicum; Hv: Hordeum vulgare.
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Figure 2. Phylogeny and sequence characteristics of HvASRs. (a) Phylogenetic tree of HvASRs, constructed using MEGA X with maximum-likelihood method and 1000 bootstraps. (b) Conserved motifs of HvASRs. (c) Gene structures of HvASRs. (d) Amino acid sequences of motifs; the uppercase letters designate one-letter abbreviation of amino acids.
Figure 2. Phylogeny and sequence characteristics of HvASRs. (a) Phylogenetic tree of HvASRs, constructed using MEGA X with maximum-likelihood method and 1000 bootstraps. (b) Conserved motifs of HvASRs. (c) Gene structures of HvASRs. (d) Amino acid sequences of motifs; the uppercase letters designate one-letter abbreviation of amino acids.
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Figure 3. Chromosomal distribution and tandem duplication of HvASRs. Duplication events were analyzed using Tbtools v2.210. Two tandemly duplicated gene pairs are displayed in red and green, respectively.
Figure 3. Chromosomal distribution and tandem duplication of HvASRs. Duplication events were analyzed using Tbtools v2.210. Two tandemly duplicated gene pairs are displayed in red and green, respectively.
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Figure 4. Cis-acting elements in the promoters of HvASRs. A total of 2 kb of upstream regions of all HvASRs were used for analysis through PlantCARE. The absent elements are displayed in a grey color.
Figure 4. Cis-acting elements in the promoters of HvASRs. A total of 2 kb of upstream regions of all HvASRs were used for analysis through PlantCARE. The absent elements are displayed in a grey color.
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Figure 5. Syntenic relationships of ASRs between barley and six plant species (Solanum lycopersicum, Oryza sativa, Zea mays, Setaria italica, Brachypodium distachyon, and Triticum aestivum). Syntenic relationships were analyzed using TBtools. Syntenic blocks are indicated with grey lines, and syntenic ASR gene pairs are indicated with blue lines. Triangles indicate the positions of HvASRs.
Figure 5. Syntenic relationships of ASRs between barley and six plant species (Solanum lycopersicum, Oryza sativa, Zea mays, Setaria italica, Brachypodium distachyon, and Triticum aestivum). Syntenic relationships were analyzed using TBtools. Syntenic blocks are indicated with grey lines, and syntenic ASR gene pairs are indicated with blue lines. Triangles indicate the positions of HvASRs.
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Figure 6. Expression levels of HvASRs in 14 tissues. Transcriptomic data were downloaded from BARLEX, and FPKM values were normalized using log10(FPKM+1) transformation. The heatmap was drawn using TBtools. ROO1, roots from seedlings (10 cm shoot stage); ROO2, roots (28 DAP); CAR5, developing grain (5 DAP); CAR15, developing grain (15 DAP); LEA, shoots from seedlings (10 cm shoot stage); ETI, etiolated seedling, dark condition (10 DAP); EPI, epidermal strips (28 DAP); INF1, young developing inflorescences (5 mm); INF2, developing inflorescences (1–1.5 cm); RAC, inflorescences, rachis (35 DAP); LEM, inflorescences, lemma (42 DAP); LOD, inflorescences, lodicule (42 DAP); NOD, developing tillers, third internode (42 DAP); SEN, senescing leaves (56 DAP). Grey squares indicate that gene expression was not detected.
Figure 6. Expression levels of HvASRs in 14 tissues. Transcriptomic data were downloaded from BARLEX, and FPKM values were normalized using log10(FPKM+1) transformation. The heatmap was drawn using TBtools. ROO1, roots from seedlings (10 cm shoot stage); ROO2, roots (28 DAP); CAR5, developing grain (5 DAP); CAR15, developing grain (15 DAP); LEA, shoots from seedlings (10 cm shoot stage); ETI, etiolated seedling, dark condition (10 DAP); EPI, epidermal strips (28 DAP); INF1, young developing inflorescences (5 mm); INF2, developing inflorescences (1–1.5 cm); RAC, inflorescences, rachis (35 DAP); LEM, inflorescences, lemma (42 DAP); LOD, inflorescences, lodicule (42 DAP); NOD, developing tillers, third internode (42 DAP); SEN, senescing leaves (56 DAP). Grey squares indicate that gene expression was not detected.
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Figure 7. Expression responses of HvASRs in barley seedling roots after salt stress (a), osmotic stress (b), and ABA treatments (c). Relative expression levels of HvASRs were calculated using the 2−ΔΔCT method. Lowercase letters indicate significance analysis performed using a threshold of p < 0.05.
Figure 7. Expression responses of HvASRs in barley seedling roots after salt stress (a), osmotic stress (b), and ABA treatments (c). Relative expression levels of HvASRs were calculated using the 2−ΔΔCT method. Lowercase letters indicate significance analysis performed using a threshold of p < 0.05.
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Figure 8. Expression responses of HvASRs in barley seedling leaves after salt stress (a), osmotic stress (b), and ABA treatments (c). Relative expression levels of HvASRs were calculated using the 2−ΔΔCT method. Lowercase letters indicate significance analysis performed using a threshold of p < 0.05.
Figure 8. Expression responses of HvASRs in barley seedling leaves after salt stress (a), osmotic stress (b), and ABA treatments (c). Relative expression levels of HvASRs were calculated using the 2−ΔΔCT method. Lowercase letters indicate significance analysis performed using a threshold of p < 0.05.
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Figure 9. Transcriptional activity analyses of HvASR proteins. HvASRs were cloned and inserted into pGBKT7 and pGBKT7-VP16 vectors. Subsequently, pGBKT7, pGBKT7-VP16, and the recombinant vectors were transformed into Y2HGold yeast strains, respectively, and cultured on SD/-Trp, SD/-His/-Trp, and SD/-Ade/-His/-Trp plates for 3 d.
Figure 9. Transcriptional activity analyses of HvASR proteins. HvASRs were cloned and inserted into pGBKT7 and pGBKT7-VP16 vectors. Subsequently, pGBKT7, pGBKT7-VP16, and the recombinant vectors were transformed into Y2HGold yeast strains, respectively, and cultured on SD/-Trp, SD/-His/-Trp, and SD/-Ade/-His/-Trp plates for 3 d.
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Table 1. Characteristics of 10 HvASR genes in barley.
Table 1. Characteristics of 10 HvASR genes in barley.
Gene NameGene IDLength (aas)pIMW (kDa)Instability IndexAliphatic IndexGRAVYDisordered aas (%)Subcellular Localization
HvASR1HORVU.MOREX.r3.2HG01681202725.2129.5941.9518.05−1.6889.34nucleus
HvASR2HORVU.MOREX.r3.3HG03251502826.5929.9550.6235.07−0.8959.57chloroplast
HvASR3HORVU.MOREX.r3.3HG03251702226.4023.8638.1929.14−1.0067.57nucleus
HvASR4HORVU.MOREX.r3.3HG0325200979.8210.7642.8048.45−1.2273.20nucleus
HvASR5HORVU.MOREX.r3.3HG03252101059.5211.1136.3267.24−0.7876.19nucleus
HvASR6HORVU.MOREX.r3.3HG03252301049.4011.0635.5465.10−0.8475.00nucleus
HvASR7HORVU.MOREX.r3.3HG0325240979.8710.6542.0549.59−1.1371.13nucleus
HvASR8HORVU.MOREX.r3.3HG0325300999.9911.0434.6749.60−1.3473.74nucleus
HvASR9HORVU.MOREX.r3.3HG0325310999.9411.0838.9346.67−1.2974.75nucleus
HvASR10HORVU.MOREX.r3.4HG03494501386.1415.4328.8943.33−1.2084.78nucleus
aas: amino acids; pI: isoelectric point; MW: molecular weight; GRAVY: grand average of hydropathicity.
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Ren, J.; Cai, K.; Song, X.; Yue, W.; Liu, L.; Ge, F.; Wang, Q.; Wang, J. Genome-Wide Identification and Expression Profiling of ABA-Stress-Ripening (ASR) Gene Family in Barley (Hordeum vulgare L.). Plants 2025, 14, 970. https://doi.org/10.3390/plants14060970

AMA Style

Ren J, Cai K, Song X, Yue W, Liu L, Ge F, Wang Q, Wang J. Genome-Wide Identification and Expression Profiling of ABA-Stress-Ripening (ASR) Gene Family in Barley (Hordeum vulgare L.). Plants. 2025; 14(6):970. https://doi.org/10.3390/plants14060970

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Ren, Jie, Kangfeng Cai, Xiujuan Song, Wenhao Yue, Lei Liu, Fangying Ge, Qiuyu Wang, and Junmei Wang. 2025. "Genome-Wide Identification and Expression Profiling of ABA-Stress-Ripening (ASR) Gene Family in Barley (Hordeum vulgare L.)" Plants 14, no. 6: 970. https://doi.org/10.3390/plants14060970

APA Style

Ren, J., Cai, K., Song, X., Yue, W., Liu, L., Ge, F., Wang, Q., & Wang, J. (2025). Genome-Wide Identification and Expression Profiling of ABA-Stress-Ripening (ASR) Gene Family in Barley (Hordeum vulgare L.). Plants, 14(6), 970. https://doi.org/10.3390/plants14060970

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