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Genome-wide identification of alcohol dehydrogenase (ADH) gene family in oilseed rape (Brassica napus L.) and BnADH36 functional verification under salt stress

BMC Plant Biology volume 24, Article number: 1013 (2024) Cite this article

Abstract

Background

Alcohol dehydrogenase (ADH) is an enzyme that binds to zinc, facilitating the interconversion of ethanol and acetaldehyde or other corresponding alcohols/aldehydes in the pathway of ethanol fermentation. It plays a pivotal role in responding to environmental stress. However, the response of the ADH family to abiotic stress remains unknown in rapeseed.

Result

In this study, we conducted a comprehensive genome-wide investigation of the ADH family in rapeseed, encompassing analysis of their gene structure, replication patterns, conserved motifs, cis-acting elements, and response to stress. A total of 47 ADH genes were identified within the rapeseed genome. Through phylogenetic analysis, BnADHs were classified into four distinct clades (I, II, IV, V). Prediction of protein domains revealed that all BnADH members possessed a GroES-like (ADH_N) domain and a zinc-bound (ADH_zinc_N) domain. Analysis of promoter sequences demonstrated that BnADHs contained numerous cis-acting elements associated with hormone and stress responses, indicating their widespread involvement in various biological regulatory processes. Expression profiling under different concentrations of salt stress treatments (0%, 0.4%, 0.8%, 1.0% NaCl) further highlighted the significant role played by the BnADH family in abiotic stress response mechanisms. Overexpression of BnADH36 in rapeseed significantly improved the salt tolerance of rapeseed.

Conclusion

The features of the BnADH family in rapeseed was comprehensively characterized in this study, which could provide reference to the research of BnADHs in abiotic stress response.

Peer Review reports

Introduction

Alcohol dehydrogenase (ADH), an enzyme that binds to zinc, plays a crucial role in the ethanol fermentation pathway by facilitating the exchange of ethanol and acetaldehyde, as well as other relevant alcohols/aldehydes [1]. Moreover, ADH exhibits reduced toxicity towards organisms and possesses the ability to regenerate nicotinamide adenine dinucleotides (NADH to NAD+), thereby ensuring the maintenance of glycolysis metabolism [1]. Structurally, ADH comprises two conserved domains: a Groes-like domain (ADH_N) and a zinc-bound domain (ADH_zinc_N). Notably, previous researches reported that catalytic active regions were formed within the Groes-like domains [2, 3]. In recent years, ADH or ADH-like genes have been identified in numerous plant species, such as Arabidopsis thaliana [4], Cucumis melo [5], Solanum lycopersicum [6], Pyrus bretschneideri [7, 8], and Taxodium distichum [9]. In tomato fruits, Le-ADH2 was implicated in the biosynthesis of flavor volatiles and its overexpression significantly elevated the level of C6 alcohol [6]. During fruit ripening in melon, fruit-specific genes Cm-ADH1 and Cm-ADH2 were upregulated and primarily functioned as reductive dehydrogenases to promote specific substrates [5]. Pear exhibited enhanced ADH transcriptional activity under hypoxic conditions [10], with multiple ADHs contributing to aroma generation during fruit ripening [8]. Overall, ADH plays a pivotal role in plant growth and resisting environmental stressors.

Salinity represents a significant abiotic stress factor that profoundly impacts the growth of crop plants hence their global yields [11, 12]. It was estimated that approximately 1.125 billion hectares of land worldwide suffered from soil salinization [13]. The rate of soil salinization was about 3 ha/min [4], posing a formidable challenge to irrigated agriculture [11, 14, 15]. Salinity imposed three primary constraints on plants, including osmotic stress, ion imbalance/toxicity, and oxidative stress [11]. Previous studies have demonstrated a correlation between ADH activity and crop osmotic stress. For instance, in maize seedlings, both roots and shoots exhibited an increase in ADH activity when subjected to osmotic stress, while the activity of roots were twice that of the tender shoots [16]. Furthermore, manipulation ADH gene expression in Arabidopsis thaliana revealed that seeds lacking mutations in the ADH gene displayed a reduced germination rate under various stresses such as osmosis, low temperature, or waterlogging [17]. Similarly, tobacco plants overexpressing sysr1-OX exhibited significantly heightened ADH activity and improved tolerance towards salt stress conditions. Additionally, accumulation of ADH mRNA has been observed under salt stress in soybean, grass bean, and Arabidopsis [18,19,20].

Rapeseed (Brassica napus L.), being one of the most significant oil crops, is cultivated in a wide range of areas all over the world. Moreover, rapeseed possesses remarkable salt tolerance capabilities that contribute to enhancing saline-alkali soil conditions and augmenting the production of edible oil [21,22,23]. However, there is still a gap in comprehensive research on ADH gene families concerning salt tolerance in rapeseed.

In this study, a systematic identification of ADH gene family members in B. napus were conducted. Additionally, the evolutionary history of the ADH gene family were fully elucidated through comprehensive analysis of phylogeny, conserved domains, selection pressure, homology relationships, and gene replication events. Furthermore, we employed transcriptome data and qRT-PCR analysis to investigate the expression patterns and trends of ADH family genes under varying concentrations of salt stress in B. napus. Consequently, several candidate genes that are closely associated with salt tolerance in B. napus were identified. We conducted overexpression of BnADH36 in rapeseed, resulting in significant alterations in response to salt stress. The findings demonstrated that the overexpression of BnADH36 could effectively enhance the tolerance to salt stress during germination. These findings provide valuable insights into the evolution and salt tolerance mechanisms underlying ADH family genes in B. napus.

Method

Identification and chromosome localization of BnADH family members

Sequence files for B. napus Zhongshuang11 (ZS11), B. rapa, and B. oleracea were obtained from the BRAD database (BRAD: http://brassicadb.cn/#/, accessed on 25 April 2024) [24]. The ADH_N (PF08240) and ADH_zinc_N (PF00107) domain files were downloaded from the Pfam database (http://www.hmmer.org/, accessed on 25 April 2024) [25] and searched using HMMER software (http://www.hmmer.org/) to obtain the BnADH hypothetical protein sequences with an e-value of 1e − 10. These protein sequences were then uploaded to the NCBI website's CDD tool (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 25 April 2024) for further refinement, resulting in a final assessment of 47 members within the BnADH gene family. The BnADH gene was designated as BnADH1-BnADH47 based on its chromosomal position sequence. The physicochemical properties of the BnADH protein were predicted using the Protparam function available on the ExPASy website (https://web.expasy.org/protparam/, 25 April 2024) [26], while subcellular localization predictions were obtained from the Plant-mPLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, 25 April 2024) [27]. The secondary structure of the BnADH protein was determined using the SMOPA online tool (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html/, 24 April 2024) [28]. Furthermore, chromosome location information for the BnADH gene was retrieved from an annotation file and visualized using TBTools 1.047 [29].

Phylogenetic analysis

The ADH protein sequences of Arabidopsis Thaliana and Cucumis melo were retrieved from the TAIR (TAIR: https://www.arabidopsis.org/, accessed on 25 April 2024) and NCBI databases, respectively. The neighbor joining method in MEGA 11 software was employed with adjusted parameters for phylogenetic analysis. Phylogenetic trees were constructed using NJ algorithm along with 1000 boot-strap replicates. Subsequently, these trees were visually enhanced using the online tool iTOL (https://itol.embl.de/, accessed on 25 April 2024) [30, 31].

Prediction of gene structure, protein conserved motifs and promoter cis-acting elements

The proteins’ structure information and CDS/UTR region of BnADH gene family members were acquired from the NCBI-CDD website and the gff3 annotation files of B. napus. The conserved motif of BnADH protein was obtained using MEME 5.5.1 website (https://meme-suite.org/meme/meme_5.5.3/doc/download.html/., acessed on 25 April 2024)[32], while the prediction of cis-acting elements for a 2000 bp upstream region of the BnADH gene promoter was conducted through PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, acessed on 25 April 2024) [33]. All data visualization was performed using TBTools.

Collinearity analysis of ADH gene

The MCScanX software was employed to scrutinize the collinearity relationship among B. napus, B. rapa, and B. oleracea, and ADH genes in the genome of B. napus [34]. The TBTools were utilized for visualizing collinear information. Furthermore, the Ka/Ks value was calculated using KaKs_calculator 3.0 software [35].

Transcriptome expression pattern analysis

The BnADH gene expression levels in the leaves and roots of B. napus ZS11 under various stresses and exogenous hormones were obtained from the BnTIR database (TPM: transcripts per million reads), and subsequently, expression heat maps were generated using TBTools software.

Growth conditions, stress treatment, RNA extraction and real-time fluorescence quantitative PCR of plant materials

10 mL of NaCl solution of different concentrations (0.0%, 0.4%, 0.8%, and 1.0% (m/v), NaCl concentrations were selected based on our previous research results [21]) was added to a culture dish with a diameter of 9 cm, which two layers of filter paper at the bottom and evenly spread 30 full and disease-free ZS11(ZS11 was derived from the offspring of ZS11 bred in the laboratory) seeds onto the culture dish. Seed germination performed in a plant growth chamber at 20 to 22 °C and 65% humidity under a long-day condition (16-h-light/ 8-h-dark cycle). Each treatment includes three biological replications. Seedlings were harvested immediately frozen in liquid nitrogen and stored at -80 °C for RNA extraction.

Total RNA was extracted using the RNA simple Total RNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. cDNA was synthesized with 1 µg RNA from each sample with HiScript® II Q Select RT SuperMix with gDNA wiper (Vazyme, Nanjing, China). Gene-specific primers used for quantitative real-time PCR (qRT-PCR) listed in (Table S5). qRT-PCR was run on the AriaMx real-time PCR system (Agilent Technologies). The following cycling parameters were used: initial denaturation at 95 °C for 5 min; 40 amplification cycles consisting of denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s; The melting curve was then tested at 65–95 °C. The internal standard was the B. napus actin gene (BnaA01g27090D). Three biotic replicates were performed for each sample, and each replicate contained three technical replicates. Relative expression levels were calculated according to the 2 − ΔΔCt method [36]. PCR primers used for qRT-PCR in this study were show in Table S5.

Generation of transgenic plants overexpressing BnADH36 in rape

Primers OE-ADH36-F and OE-ADH36-R were used to amplify the CDS sequence of BnaC06G0262300ZS (The normal name BnADH1 has been renamed to BnADH36 in this study) in Z11 by PCR. The amplified fragment was ligated into pCAMBIA2300 vector. The resulting vector pCAMBIA2300-Pro35S:: BnADH36 was transformed into Agrobacterium strain GV3101. The vector was transferred into rapeseed variety Z11 by using rapeseed transformation technology [37]. The regenerated plants were identified as transgene positive by OE-ADH36-F/P48 primer, and the expression levels were identified by ADH36F/ADH36R. After identification, two lines of OE-ADH36-1 and OE-ADH36-5 were selected for overexpression phenotype verification.

Measurement of plant growth index

After the underground roots were extracted, the surface impurities were removed and washed with deionized water several times. The roots were scanned by root scanner (EPSON PERFECTION V850 PRO) and analyzed by WinRHIZO software to obtain total root length (RL) and shoot length (SL).

Result

Identification and chromosome mapping of BnADH gene family

The HMMER software was utilized to identify the putative BnADH protein with its corresponding structure. Subsequently, any incomplete domain sequences were eliminated using the NCBI-CDD tool, resulting in a final determination of 47 BnADH members. The genes were designated as BnADH1-BnADH47 based on their sequential arrangement on the chromosome. These 47 members were assigned to 17 distinct chromosomes (Table S1). Chromosome A09 harbored six BnADH genes, while chromosomes A01, A07, C01, C03, C05 and C07 each contained four BnADH genes. Additionally, three BnADH genes were localized on chromosomes A06, C06 and C08, whereas two BnADH genes resided on chromosomes A08 and C09. Notably, a single BnADH gene was identified on chromosomes A02, A03, A05, A10 and C02; however no putative BnADH gene was detected on chromosomes A04 and C04.

Prediction of physical and chemical properties, structure and subcellular localization of BnADH protein

Through predictive analysis, we have determined that the amino acid range of BnADH protein spans from 296 to 523. Additionally, the relative molecular mass (MW) ranges from 32.08 to 57.82 kDa, while the theoretical isoelectric point (PI) varies between 5.26 and 8.90. Furthermore, the average hydrophobicity (GRAVY) ranges from -0.222 to 0.149 (Table S1). Subcellular localization predictions indicate that all BnADH proteins are likely present in the cytoplasm, with potential presence in the nucleus for BnADH16, BnADH40, and BnADH46 as well.

The results of protein secondary structure prediction revealed that all BnADH proteins exhibited α-helix (Hh), extended backbone (Ee), and random coil (Cc) structural elements (Table S1). Notably, the random coil structure (Cc) predominated throughout the entire protein architecture, accounting for a proportion ranging from 44.44% to 55.45%. The α-helical structure (Hh) occupied an intermediate position with proportions varying from 22.41% to 35.98%. Conversely, the extended backbone structure (Ee) constituted the smallest fraction, ranging between 16.36% and 25.50% (Table S1).

Phylogenetic analysis of rapeseed ADH family

To investigate the phylogenetic relationships among ADH proteins, we constructed a comprehensive phylogenetic tree comprising 47 rapeseed ADH protein members, along with 7 Arabidopsis and 13 counterparts (Fig. 1). Based on their genetic affinities, we classified the ADH proteins into four distinct groups (Group I, II, III, and IV), which aligns with classification of three types: long chain, medium chain, and short chain [38]. The analysis of the evolutionary tree revealed varying protein counts across different branches. Notably, Group IV encompassed a majority of the proteins (43 in total). Following this is Group III consisting of 19 BnADH proteins exclusively. Group II contained only 3 members while Group I had the fewest representatives with just 2; notably not clustered within BnADH (Fig. 1). Amongst the identified 46 BnADH proteins, solely BnADH33 was categorized as a long-chain ADH protein whereas all others were classified as medium-chain ADH variants.

Fig. 1
figure 1

Phylogenetic tree of ADH proteins in B. napus, A. Thaliana and C. melo. Phylogenetic tree of the ADH family in B. napus, A. Thaliana and C. melo. The neighbor-joining tree was generated through the MEGA11 program using the amino acid sequences of the ADH proteins by the neighbor-joining (NJ) method, with 1000 bootstrap replicates

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Analysis of ADH family structure, conserved motif and promoter cis-acting elements in rapeseed

After analyzing the Motif structure of BnADH, conserved Motif 1 and Motif 2 were identified in the entire BnADH protein family (Fig. 2A). It is noteworthy that Group II exhibits uniqueness with only 3 or 4 motifs. In contrast, Group III displays seven motifs present in all proteins except for BnADH14. Similarly, Group IV contains eight motifs in all proteins except for BnADH22, BnADH47, and BnADH32. The characterization of the protein structure revealed that all proteins possess ADH_N and ADHZinc_N domains (Fig. 2B). Furthermore, analysis of promoter cis-acting elements indicated that the BN-ADH motif may respond to stress, plant hormone signaling, and photosynthesis (Fig. 2C). Additionally, examination of mature mRNA structures demonstrated the presence of coding sequence regions ranging from three to fourteen (Fig. 2D). Moreover, the structure of BnADH mRNA includes one to two untranslated regions.

Fig. 2
figure 2

Gene structure analysis of ADH family in B. napus. A Conserved motifs of BnADH family proteins; B Structure of BnADH family protein Pfam; C Promoter cis-acting element of BnADH family; D The mRNA structure encoded by the BnADHs

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Collinearity analysis of ADH family

To investigate the collinearity of the ADH gene, we conducted a comprehensive mapping analysis involving B. napus, B. rapa, and B. oleracea genomes (Fig. 3). The results revealed the presence of 32 pairs of collinear genes between rape and cabbage, as well as 74 pairs between rape and cabbage (Table S2). Furthermore, an in-depth examination within the rape genome unveiled 47 collinear gene pairs associated with BnADH (Fig. 4 and Table S3). In order to elucidate the protein-coding sequence affinity exhibited by these BnADH collinear gene pairs, we performed KaKs calculations to explore their selection pressure relationship (Table S4). Encouragingly, all these 47 collinear gene pairs displayed Ka/Ks values < 1, indicating their susceptibility to purification selection pressures.

Fig. 3
figure 3

Collinearity analysis of ADH family genes between B. napus, B. rapa and B. oleracea

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Fig. 4
figure 4

Collinearity analysis between BnADH genes. The circles in the figure from inside to outside represent the unknown base N ratios, gene density, GC ratio, GC skew, and chromosome lenth of the B. napus genome

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Expression patterns of exogenous hormones and abiotic stress

In order to investigate the expression of BnADHs under different exogenous hormone and abiotic stress treatments, we downloaded relevant data from rape database website and made expression heat map. The data, after calculating log10 (TPM + 1), were used to generate the gene expression heatmap (Fig. 5). The results showed that the expression of BnADH8, BnADH14, BnADH28, BnADH40, BnADH46 and BnADH47 genes was increased or inhibited in the leaf (L) tissues treated with different hormones (Fig. 5A). In the root (R) tissues, BnADH6, BnADH7, BnADH35, BnADH13 and BnADH38 were significantly up-regulated or down-regulated. The expression levels of BnADH20, BnADH44, BnADH17 and BnADH34 did not change significantly in different tissues. This is consistent with our prediction of promoter region structure and the ADH gene expression pattern in Arabidopsis, maize and alfalfa [39,40,41,42,43]. These results indicate that different ADH genes have different activities in different tissues, respond differently to different exogenous hormones, and have functional redundancy or differentiation.

Fig. 5
figure 5

BnADHs Expression patterns of exogenous hormones and abiotic stress. A Hormone treatments (CK, IAA, ACC, GAs, ABA, TZ, JA, and BL); B Non-biological stress treatments (CK, salt, drought, freezing, cold, heat, and osmotic). L: leaves; R: Roots

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Under abiotic stress, different BnADH genes showed different expression responses (Fig. 5B). For example, BnADH10, BnADH14, BnADH18, BnADH33, BnADH42, BnADH21, and BnADH4 were mainly expressed in the ground after stress, and were significantly responsive to drought, osmosis and cold damage. BnADH6, BnADH13, BnADH38 and BnADH41 were mainly expressed in roots in response to salt, drought and heat stress. The expression levels of BnADH17, BnADH20, BnADH34 and BnADH44 genes did not change significantly under abiotic conditions [19, 20, 44, 45].

Expression of BnADHs under salt stress

In order to further investigate the expression patterns of BnADHs under different stress conditions, we analyzed the samples germinated under NaCl (0, 0.4%, 0.8%, 1.0%) stress conditions for one week by qRT-PCR based on previous research experience in rapeseed (40) (Fig. 6A). After salt stress, the phenotype of germination organisms in rape was significantly changed, for example, the root length of T2 and T3 was significantly reduced compared with the control CK. The root surface area, root volume, and root tip number of the T3 treatment group were significantly lower than those of CK and T1 (Fig. 6B).

Fig. 6
figure 6

Phenotype of ZS 11 under different treatments. A Phenotype of ZS 11 under different treatments. B Physiological change of ZS11 root under salt stress. Student’s t-test was used to determine differences. *, P < 0.05, **P < 0.01, ****P < 0.0001

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The qRT-PCR analysis results revealed that most BnADHs genes exhibited an up-regulated trend (> twofold change) in response to salt stress (Fig. 6B). These findings unequivocally demonstrate the significant responsiveness of the detected BnADHs genes towards abiotic stresses. In addition, the expression levels of all BnADH genes were significantly upregulated in response to a 0.4% salt concentration treatment compared to the control group. Notably, the expression levels of BnADH9, BnADH10, BnADH18, BnADH22 and BnADH28 reached their peak when the salt concentration increased to 0.8% (Fig. 7). However, when the salt concentration increased to 1.0%, the expression of these genes decreased, which is consistent with the results of previous studies [44]. Interestingly, in the 1.0% treatment group, only three genes (BnADH33, BnADH36 and BnADH40) exhibited high levels of expression that were significantly higher than those observed in other treatment groups and the control group (Fig. 7). This observation may be attributed to inhibited germination and reduced biological activity of seeds under high salt concentrations. Overall, our findings demonstrate that all detected BnADHs genes are responsive to salt stress with varying degrees of responsiveness among different genes.

Fig. 7
figure 7

Expression patterns of BnADHs at different salt concentration stresses. Expression patterns of BnADHs under stresses. The y-axis represents relative expression, calculated using the 2 − ΔΔCt formula. The x-axis represents different stress treatments. Expression profiles of BnADHs were obtained under NaCl (0%, 0.4%, 0.8%, and 1.0%) stress conditions, respectively. Data represent the mean ± standard error for three biological experiments. Student’s t-test was used to determine differences. *, P < 0.05, **P < 0.01, ****P < 0.0001

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Overexpression of BnADH36 in rapeseed increased the tolerance to salt stress

In order to further investigate the function of BnADH in stress response, we overexpressed BnADH36 in oilseed rape. The effects of two independent homozygous T2 transgenic rapeseed plants under different stress treatments were studied (Fig. 8A). Phenotypic analysis showed that the shoot length and root length of two OE- BnADH36 plants treated with 1.0% NaCl were significantly higher than those of WT plants (Fig. 8 C, D). Under control conditions, hypocotyl length and root length of transgenic plants had no significant changes compared with WT plants (Fig. 8 C, D). These results indicated that transgenic plants were more tolerant to salt stress than WT plants.

Fig. 8
figure 8

Overexpression of BnADH36 in rapeseed increased the tolerance to salt stress. A Phenotypes of Z11 and OE-ADH36-1 at different salt concentrations. B Expression levels of Z11, OE-ADH36-5 and OE-ADH36-1 at different salt concentrations. C-D Physiological change of Z11, OE-ADH36-5 and OE-ADH36-1 shoot and root length under different salt stress. Data represent the mean ± standard error for three biological experiments. Student’s t-test was used to determine differences. *, P < 0.05, **P < 0.01, ****P < 0.0001

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Discussion

Studies have demonstrated that ADH exhibits catalytic activity in facilitating the interconversion of alcohols and aldehydes, as well as actively participating in the biosynthesis of aromatic compounds and plant stress responses during fruit maturation [45,46,47]. Although the ADH gene family has been extensively investigated in various genome-sequenced plant species, our understanding of ADHs in oilseed rape remains limited. In this study, a total of 47 ADH gene members in B. napus through rigorous evolutionary analysis were identified and classified, which yielded consistent results with previous findings in melon and Arabidopsis (Fig. 1). Notably, it is worth mentioning that the number of ADH gene families varies across different species. For instance, Arabidopsis harbors 9 ADH genes [48], while the wheat genome encompasses 22 [49], and Taxodium distichum chinensis exhibits 15 related genes [9].

In contrast to Arabidopsis, B. napus (including B. rapa and B. oleracea) has undergone genome-wide triploid replication events during its evolutionary history [50]. Theoretically, B. napus should possess more than five times the number of genes as Arabidopsis, which aligns with our findings. B. napus is an autotetraploid plant that originated from a natural hybridization event between B. rapa and B. oleracea [51]. Therefore, a strong genetic relationship exists between these species. Collinearity analysis revealed that nearly all BnADH genes could correspond to both BrADH and BoADH, indicating their inheritance from ancestral species (i.e., B. rapa and B. oleracea) during hybridization. Our findings demonstrated the widespread distribution of numerous BnADHs across the genome, consistent with previous studies on ADH families in plants [4, 5, 7,8,9]. Chromosome localization confirmed the presence of 1–6 members of the BnADH gene family on all chromosomes except A04 and C04 (Fig. 1), suggesting that amplification of the BnADH family primarily occurred through discrete replication within the B. napus genome.

The subcellular localization of plant ADH has not been thoroughly investigated. Previous studies have demonstrated that AtADH1 was predominantly found in nuclear and cytoplasmic compartments [34]. Similarly, TdADHs in Taxodium distichum are primarily expressed in the nucleus and cytoplasm [9]. In this study, through subcellular localization analysis, we predict that all BnADH proteins may predominantly localize to the cytoplasm, while BnADH16, BnADH40, and BnADH46 might also be present in the nucleus. Considering that lactic acid fermentation enhances cytoplasmic acidity and stimulates alcoholic fermentation processes [52], it is reasonable to expect ADH expression in the cytoplasm. The presence of ADH at other cellular locations suggests potential unidentified mechanisms associated with its function.

Recent studies have demonstrated the involvement of ADHs in diverse biological and abiotic stress responses [9, 18, 38, 49]. It has been established that cis-acting elements present in the gene promoter region can significantly influence plant growth and development, environmental adaptation, and stress resistance [53]. In this investigation, 47 promoter components of BnADH were thoroughly analyzed and several crucial cis-acting elements were successfully identified. Notably, a majority of these cis-acting elements encompass multiple core components associated with stress response mechanisms, plant hormone signaling pathways, and photosynthesis (Fig. 2). Hence, the pivotal role played by BnADHs in effectively responding to various biological as well as abiotic stresses was implied. Promoters classified as BnADHs within the same taxonomic clade exhibit similar types and quantities of cis-acting elements, implying potential functional similarities among BnADHs within this specific taxonomic group. The transcriptional data of BnADHs under different exogenous hormone and abiotic stress treatments was obtained through the rape transcriptome database, and expression heat maps was made (Fig. 5A, B). The results showed that most BnADH was expressed in response to exogenous hormones and stress, which was consistent with previous research reports [19, 20, 39,40,41,42,43]. Different BnADHs have different expressions in response to different exogenous hormones and abiotic stresses, which shows that different ADH gene expression tissue specificity, expression level activation or inhibition are different. These results are similar to the function of ADH gene in grass bean, soybean, and rice [18, 19, 54, 55]. It was again proved that different ADH genes have different activities in different tissues, show different responses to different abiotic stresses, and have obvious functional redundancy or differentiation.

Due to its crucial role in plant growth and resistance to environmental stresses, the expression of ADHs is typically induced when plants are exposed to biological or abiotic stress. Prior research has demonstrated that ADH mRNA accumulation can be triggered by hormonal changes in cucumber [38], waterlogging stress in Taxodium distichum [9], hypoxia stress in pear [10], as well as salt stress in soybean, grass bean, and Arabidopsis thaliana [18,19,20]. Transcriptome expression analysis of rapeseed stress database revealed that over twenty members of the BnADH family exhibited up-regulated expression under conditions such as cold injury, drought, and salt stress (Fig. 5B). Simultaneously, ZS11 B. napus varieties were subjected to treatments with salt concentrations of 0%, 0.4%, 0.8% and 1.0%. Subsequently, samples were collected for q-PCR verification. The findings revealed a significant increase in the expression levels of BnADH9, BnADH28 and BnADH33 genes, which exhibited higher expression levels as the salt concentration increased.

These results suggested potential functional redundancy and differentiation among these homologous genes. Phenotypic analysis showed that the root length and hypocotyl length of two OE-BnADH33 line were significantly higher than those of WT plants (Fig. 8) under 1.0% NaCl treatment. These results suggested that transgenic plants were more resistant to salt stress than WT plants. Further investigation of the biological functions of these genes would undoubtedly contribute to the improvement of rapeseed yield under salt stress.

Adverse environmental stress (such as drought, waterlogging, salt stress, etc.) can inhibit respiration [22, 56,57,58]. When respiration is inhibited, lactate dehydrogenase (LDH) converts the pyruvate produced during glycolysis into lactic acid. The ADH gene can convert the metabolic pathway of pyruvate synthesis of lactic acid into the pathway of ethanol synthesis (Fig. 9). The toxic effect of ethanol on plants is much smaller than that of lactic acid or the intermediate product acetaldehyde, and is easily diffused. The ethanol fermentation pathway can sustain low levels of sugar metabolism, ATP and NAD+ formation [7, 46]. Similarly, rapeseed may also respond to the impact of salt stress on respiration by upregulating ADH and enhancing its activity (Fig. 9).

Fig. 9
figure 9

Plant respiration is inhibited by salt damage, leading to ethanol fermentation pathways

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Conclusion

In this study, a comprehensive analysis of the ADH family in rapeseed were conducted. A total of 47 BnADH proteins were identified and classified into four distinct subgroups. Furthermore, phylogenetic analysis, gene structure examination, conserved motif identification, chromosome localization determination, gene duplication assessment, and cis-acting element exploration were thoroughly investigated. The molecular evolutionary mechanism underlying the rapeseed ADH family was elucidated. At the transcriptional level, differential expression patterns of BnADHs were observed and found to be closely associated with pivotal biological processes such as fruit development and stress response. Notably, under salt stress conditions, a significant upregulation in expression levels of most BnADH isoforms was detected, which indicated that abiotic stress stimuli induced the activation of ADH family members in rapeseed. Overexpression of BnADH36 in rapeseed significantly improved the salt tolerance of rapeseed. These findings contribute to an expand knowledge regarding the response mechanisms exhibited by BnADHs towards abiotic stresses, and also would provide a crucial foundation for further unraveling the vital function of ADHs within rapeseed biology.

Data availability

All the data generated or analyzed during this study are included in this published article and its supplementary information files. Zhongshuang 11 was derived from the offspring of Zhongshuang 11 bred in the laboratory, and the overexpressed material was created by our laboratory.

Abbreviations

ADH:

Alcohol dehydrogenase

ZS11:

Zhongshuang 11

OE:

Overexpression

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Acknowledgements

We express our sincere gratitude to the National Key Laboratory of Crop Genetic Improvement , Huazhong Agricultural University  for providing us with the experimental platform. Special thanks are extended to Professor Chuchuan Fan in National Key Laboratory of Crop Genetic Improvement, National Engineering Research Center of Rapeseed, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei province, P.R. China for his invaluable technical guidance and to Professor Zhao Huixia in Hubei Engineering Research Center for Protection and Utilization of Special Biological Resources in the Hanjiang River Basin, School of Life Science, Jianghan University, Wuhan, Hubei province, P.R. China for her meticulous revision of the article.

Funding

This research was financially supported by the Natural Science Foundation of China (U22A20469), Hubei Provincial Natural Science Foundation (2023AFB427), Hubei Hongshan Laboratory research funding (2021HSZD004) and the Fundamental Research Funds for the Central Universities (2662023PY004).

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Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, National Engineering Research Center of Rapeseed, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Hao Zhang, Ouqi Li, Jing Wen, Lun Zhao, Tingdong Fu & Jinxiong Shen

  2. Hubei Engineering Research Center for Protection and Utilization of Special Biological Resources in the Hanjiang River Basin, School of Life Science, Jianghan University, Wuhan, 430056, China

    Shanshan Wang, Changli Zeng, Xiaoyun Liu & Heping Wan

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  1. Hao Zhang
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  2. Shanshan Wang
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  3. Ouqi Li
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  5. Xiaoyun Liu
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  6. Jing Wen
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  7. Lun Zhao
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  9. Heping Wan
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  10. Jinxiong Shen
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Contributions

H. Z., S. W., O. L. and X. L. performed the main experiments; H. W. and J. S. conceived and designed the experiments; C. Z. and X. L. performed the data collection and bioinformatics analysis; H. Z. and S. W. wrote the manuscript; H.W., J.S., J.W., L.Z. and T. F. revised the manuscript. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Heping Wan or Jinxiong Shen.

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Zhang, H., Wang, S., Li, O. et al. Genome-wide identification of alcohol dehydrogenase (ADH) gene family in oilseed rape (Brassica napus L.) and BnADH36 functional verification under salt stress. BMC Plant Biol 24, 1013 (2024). https://doi.org/10.1186/s12870-024-05716-y

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BMC Plant Biology

ISSN: 1471-2229

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