Skip to main content
Download PDF

Systematic analysis of bZIP gene family in Suaeda australis reveal their roles under salt stress

BMC Plant Biology volume 24, Article number: 816 (2024) Cite this article

Abstract

Background

Suaeda australis is one of typical halophyte owing to high levels of salt tolerance. In addition, the bZIP gene family assumes pivotal functions in response to salt stress. However, there are little reports available regarding the bZIP gene family in S. australis.

Results

In this study, we successfully screened 44 bZIP genes within S. australis genome. Subsequently, we conducted an extensive analysis, encompassing investigations into chromosome location, gene structure, phylogenetic relationship, promoter region, conserved motif, and gene expression profile. The 44 bZIP genes were categorized into 12 distinct groups, exhibiting an uneven distribution among the 9 chromosomes of S. australis chromosomes, but one member (Sau23745) was mapped on unanchored scaffolds. Examination of cis-regulatory elements revealed that bZIP promoters were closely related to anaerobic induction, transcription start, and light responsiveness. Comparative transcriptome analysis between ST1 and ST2 samples identified 2,434 DEGs, which were significantly enriched in some primary biological pathways related to salt response-regulating signaling based on GO and KEGG enrichment analysis. Expression patterns analyses clearly discovered the role of several differently expressed SabZIPs, including Sau08107, Sau08911, Sau11415, Sau16575, and Sau19276, which showed higher expression levels in higher salt concentration than low concentration and a response to salt stress. These expression patterns were corroborated through RT-qPCR analysis. The six differentially expressed SabZIP genes, all localized in the nucleus, exhibited positive regulation involved in the salt stress response. SabZIP14, SabZIP26, and SabZIP36 proteins could bind to the promoter region of downstream salt stress-related genes and activate their expressions.

Conclusions

Our findings offer valuable insights into the evolutionary trajectory of the bZIP gene family in S. australis and shed light on their roles in responding to salt stress. In addition to fundamental genomic information, these results would serve as a foundational framework for future investigations into the regulation of salt stress responses in S. australis.

Peer Review reports

Background

Coastal wetlands play crucial roles in climate change mitigating, carbon sequestering, and biodiversity support [1, 2]. Unfortunately, over the past few decades, more than one-third of coastal wetlands have experienced a decline in their ecological functionality [3]. The presence of plants is crucial for the preservation of ecological function in coastal wetlands, particularly in terms of enhancing biodiversity [4]. However, substantial variations in salinity levels within coastal wetlands can directly impair plant capabilities, leading to inhibited growth or, in severe cases, plant mortality [5].

Suaeda australis belonging to the genus Suaeda (Chenopodiaceae), is mainly distributed in the subtropical and tropical coastal areas of China, Japan and Oceanica [6]. Pharmacological studies showed that antioxidant and antibacterial activities were found in the extracts of S. australis leaves [7]. Owing to the high nutritional value, the leaves of S. australis have long been used as food ingredient and supplementary feed [8]. Especially, S. australis is a typical halophyte, which can improve the coastal marsh saline-alkali land, reduce pollutants, and protect the coastline [9]. However, the growth zone of S. australis gradually moved from intertidal zone with higher salt concentration to suprapatid zone with lower salt concentration, which is caused by extreme fluctuations in salinity in coastal wetlands. More severely, the amounts of S. australis of suprapatid zone had fallen sharply in the past decades. Hence, it is of great scientific significance and ecological value to investigate the salt tolerance mechanism of S. australis in depth for the conservation of S. australis, utilization of saline soil resources, improvement of coastal salt-alkali environment, and development of marine agricultural economy. The assembled genome size of S. australis was 437.17 Mb and 408.45 Mb (93.43%) of sequences were integrated into 9 pseudo-chromosomes with 24,371 annotated protein-coding genes.

Many valuable genes associated with stress responses need to be identified to enhance stress tolerance effectively for S. australis. As one of the greatest family encoding transcription factors (TFs), the basic leucine zipper (bZIP) gene family plays fundamental roles in environmental and developmental signalling as well as stress response in plants [10]. Previous studies reported that bZIP TFs play pivotal roles in salt stress response [11]. The bZIP TFs typically consist of 60–80 amino acids, containing a bZIP domain characterized by a highly conserved basic region and a variable leucine zipper region [12]. bZIP TFs were categorized into different subgroups based on conserved domains [13]. In addition, the bZIP genes involved in salinity stress varied among different plants. For example, the OsbZIP12, OsbZIP72 and OsbZIP46 genes confer stronger salt tolerant than other members in rice bZIP family [14], while GmbZIP 62, GmbZIP44, and GmbZIP 78 genes of soybean are significantly upregulated in reaction to salt stress [15,16,17].

So far, little reports on the functional characterization of bZIP TFs are available. To identify potential bZIP genes involved in regulation of salt stress responses, this study systematically identified and characterized all bZIP genes in S. australis. We performed a detailed analysis of genome distribution, gene structure, and expression patterns in response to salt stress. Finally, we identified the candidate bZIP genes that exhibit potential responsiveness to salt stress through transcriptome analysis and quantitative real-time PCR (qRT-PCR) evaluation.

Results

Genome-wide identification of the SabZIP gene family in S. australis

SabZIP genes were identified in S. australis genome based on HMM program and the local BLASTP analyses. As a result, 44 identified SabZIP genes were serially named SabZIP01 through SabZIP44 for convenience (Supplementary Table S1). Additionally, it was observed that substantial variations existed among the SabZIP genes in terms of mRNA transcript length and the protein sequences they encoded. Additionally, the results indicated considerable diversity among SabZIP genes in terms of mRNA transcript lengths and the corresponding protein sequences. The mRNA products (CDS) of 44 SabZIP genes exhibited lengths spanning from 420 to 2352 bp, and the corresponding translated protein sequences displayed variations in amino acid counts ranging from 140 to 784. Among these sequences, SabZIP23 featured the shortest amino acid sequence, comprising only 143 amino acid residues, whereas SabZIP20 was characterized by the longest sequence, encompassing 784 amino acid residues. The theoretical isoelectric point (PI) values ranged from 4.91 (SabZIP14) and 9.69 (SabZIP15), and the molecular mass of bZIP members ranged from 16.12 (SabZIP11) to 86.06 kDa (SabZIP20) (Supplementary Table S1).

Chromosome location and synteny analysis of SabZIP genes

Except for SabZIP44, all SabZIP genes in S. australis were mapped onto nine chromosomes (Chrs), as illustrated in Fig. 1. The highest count of SabZIP genes per chromosome was observed on Chr8, where nine SabZIP genes were located. Seven genes per chromosome were located on Chr3 and Chr6; five were found on Chr5, Chr7, and Chr9; two genes each were located on Chr2 and Chr4. Chr1 and Scaffold_136 contained the lowest counts of SabZIP genes, with only one each. For genome mining for tandem duplicated genes of SabZIP involved in salt response. Based on GFF (General Feature Format) annotation files, tandem duplicated genes of SabZIP were identified in analysis on the basis of the following criteria: the distance between two adjacent SabZIP genes should be less than 1 Mb [18]. Among SabZIP genes in S. australis, a total of four pairs of tandem duplicated genes (Sau08107 and Sau08108, Sau11415 and Sau11436, Sau16553 and Sau16575, Sau23047 and Sau23067) were observed, as illustrated in Fig. 1.

Fig. 1
figure 1

Chromosome distribution of SabZIP genes. S. australis chromosome number is indicated at the base of each chromosome with different colors. The SabZIP genes are marked at the approximate position on the chromosomes

Full size image

To examine the evolutionary relationships among the bZIP genes in these three species (Arabidopsis thaliana, Beta vulgaris, and Oryza sativa), syntenic analysis was performed (Fig. 2). A total of 32, 41, and 22 orthologous gene pairs of orthologous SabZIP genes were identified between S. australis and A. thaliana, S. australis and B. vulgaris, and S. australis and O. sativa, respectively (Fig. 2). In contrast, 19 collinear gene pairs initially identified between S. australis and A. thaliana were also found in the comparison between S. australis and B. vulgaris. Similarly, 11 collinear gene pairs initially associated with S. australis and A. thaliana were also observed when comparing S. australis with O. sativa. Furthermore, all 10 collinear gene pairs initially detected between S. australis and O. sativa were likewise identified when comparing S. australis with B. vulgaris (Fig. 2). Additionally, it's worth noting that each of the following species: S. australis, A. thaliana, B. vulgaris, and O. sativa shared 9 common collinear gene pairs (Fig. 2).

Fig. 2
figure 2

Synteny analysis of SabZIP genes between S. australis and three plant species. The collinear blocks between S. australis and other three plant genomes are shown as the gray lines in the background, while the syntenic SabZIP gene pairs are highlighted with red lines

Full size image

Phylogeny, structural, and conserved motifs analysis of SabZIP genes

To investigate the evolutionary relationships among SabZIP genes, we constructed a neighbor-joining (NJ) phylogenetic tree based on the full-length protein sequences (Fig. 3). SabZIP genes were categorized into 12 subfamilies from S. australis (Fig. 3). The A subfamily containing eight SabZIP family members, was the most extensive clade. In contrast, subfamilies F and J were the smallest, each comprising only one member. Furthermore, subfamilies H, C, K, B, E, I, G, D, and S consisted of 2, 2, 2, 2, 2, 5, 4, 7, and 7 members, respectively.

Fig. 3
figure 3

Phylogenetic analysis of SabZIP proteins in S. australis. A neighbor-joining (NJ) tree was constructed using 44 SabZIP sequences. The tree further clustered into 12 subfamilies, which are shown in different colors

Full size image

The gene structure analysis of the 44 SabZIP genes in S. australis is illustrated in Fig. 4. Our observations revealed notable variability in the gene structure of SabZIP members, with exon numbers ranging from 1 to 13. Notably, members of the G subfamily, namely Sau10955, Sau14074, and Sau19276 (except Sau07938), displayed a maximum of 13 exons, a distinctive feature distinguishing them from members of other subfamilies, where exon numbers ranged from 1 to 12. By contrast, the Sau23745 of B subfamily contained 11 exons, but the other member Sau21199 contained only two exons. Additionally, some SabZIP members within the same group exhibited similar gene structures. For instance, the exon numbers of H subfamily and S members were both 1 and lacked introns. All of C, K, and E subfamily members had 6, 4, and 4 exons, respectively. These results suggested that the structural features of SabZIP genes exhibit noticeable variations, even when considering members in the same gene subfamily.

Fig. 4
figure 4

The phylogenetic relationship, gene structure, and motif compositions of SabZIP proteins. A The phylogenetic relationships of bZIP proteins based on the NJ method. The various colors characterize the 12 subfamilies. B Gene structure of the SabZIP genes family was generated on the basis of the gene Structure Display Server. Exons (blue rectangles) and introns (black lines) are marked along with their sequence length. C Motif in SabZIP proteins were predicted by MEME. The conserved motifs are represented by different colored boxes

Full size image

A total of ten different motifs were identified in SabZIP gene family of S. australis (Fig. 4). All of SabZIP genes contained a bZIP domain (PF00170) represented by motif 1. Motif 7 and motif 8 were specific to the A subfamily of SabZIP family. Moreover, motif 2, motif 3, motif 4, and motif 6 occurred only in D subfamily. Each member belonging to S subfamily and H subfamily exhibited a shared pattern of two motifs (motif 1 and motif 10) within their gene structures. Motif 5 was shared only by two subfamilies (E and I). This collective evidence provided support for the notion that members in the same subfamily exhibit identical conserved motifs.

Estimation of nonsynonymous (Ka) and synonymous (Ks) substitution rates and Ka/Ks values

To investigate the selection pressures acting on SabZIP genes following duplication during their evolutionary history, we computed the nonsynonymous (Ka) to synonymous (Ks) substitutions for each paralogous pair, as presented in Table 1. A total of four pairs of tandem duplications in S. australis were identified. The Ka/Ks ratio spanned from 0.7221 (Sau23047 and Sau23067) to 1.0019 (Sau23047 and Sau23067) with an average ratio of 0.8925. The Ka/Ks ratios of three paralogous SabZIP gene pairs (Sau08107 and Sau08108, Sau11415 and Sau11436, and Sau23047 and Sau23067) were less than 1, indicating that these genes are subject to purifying selection. Conversely, the Ka/Ks value of (Sau16553 and Sau16575) was slightly greater than 1, suggesting positive selection following duplication. Collectively, these results imply that the majority of these gene pairs have been subjected to purifying selection pressure, and the duplication events may contribute significantly to the expansion of the SabZIP family in S. australis.

Table 1 Analysis of tandem duplication events of SabZIP gene pairs in Suaeda australis
Full size table

Promoter region analysis of SabZIP genes

To discover cis-acting elements associated with mechanisms pertaining to the response to salt stress, we analyzed the promoter sequences of S. australis SabZIP genes. In S. australis, the cis-acting elements in promoters’ regions of majority of SabZIP genes (more than 35 members) were mainly associated with anaerobic induction, transcription start, and light responsiveness (Fig. 5 and Table 2). In contrast, we also identified several cis-elements (RY-element, WUN-motif, TATC-box, HD-Zip 3, MBSI, ABRE, A-box, AACA_motif), which were present in a minority of members (less than 6 members) (Table 2). Phytohormone response elements, including abscisic acid, auxin, gibberellin, and MeJA, were also widely observed. Moreover, SabZIP members were also related to low-temperature responsiveness, circadian control, flavonoid biosynthetic genes regulation, drought-inducibility, seed-specific regulation, and stress responsiveness, suggesting their role in responding to various stresses and plant growth. These results offer valuable insights into the regulatory roles of SabZIP gene family during both stress responses and plant development.

Fig. 5
figure 5

Cis-acting components of SabZIP genes in Suaeda australis. All promoter sequences (2000 bp) were analyzed. The SabZIP genes are shown on the left. Scale bar at the base indicates length of promoter sequence. The functions of cis-acting element can be found in Table 2

Full size image
Table 2 Cis-element and corresponding functions of SabZIP genes in Suaeda australis
Full size table

Expression profiles of SabZIP genes under different salt concentrations

RNA-seq was performed on the Illumina HiSeqTM2000 system. The leaves under two salt treatment levels (ST1 and ST2) were used for transcriptomic expression profiles. In total, more than 27 million raw reads were obtained from each samples. After quality evaluation and filtration, the number of clean reads ranged from 27,179,940 to 30,399,735 with an average of 28,486,258 and the average value of Q30 was 95.82%, indicating that the clean reads were of high quality. After alignment with the S. australis genome sequence, more than 95% of the clean reads were mapped to the genome and paired-end reads were aligned to 24,371 S. australis annotated gene models. A pairwise comparison between ST1 and ST2 samples identified 2,434 DEGs. Among them, 1,568 and 866 genes were respectively up-regulated and down-regulated in ST2 compared to ST1. GO and KEGG enrichment analysis revealed that the 2,434 DEGs were significantly enriched in some primary biological pathways, such as response to reactive oxygen species, response to hypoxia, regulation of cytokine production, photosynthesis, biosynthesis of nucleotide sugars, starch and sucrose metabolism, which were closely related to salt response-regulating signaling pathways (Fig. 6).

Fig. 6
figure 6

GO (a) and KEGG (b) enrichment of 2,434 DEGs in ST1 and ST2 samples (ST2_vs_ST1)

Full size image

To investigate the potential functions of SabZIP genes among S. australis under salt stress, we conducted RNA-seq analysis to investigate expression patterns of 44 SabZIP genes. We used FPKM values to build a heatmap (Fig.S1). Of 44 SabZIP genes, 35 were expressed in ST1 samples and 39 were expressed in ST2 samples (FPKM > 0). Among different samples, SabZIP genes showed differences in expression patterns (Fig.S1). For instance, Sau08911, Sau11415, Sau08107, Sau19276, and Sau16575 showed higher expression levels under higher salt concentration stress (ST2) than ST1 leaves. These results indicated that SabZIP genes may play a significant role in the salt tolerance of S. australis. Further, we found that the expression levels of subfamily S members (Sau08107, Sau19742, Sau08108, Sau00495, Sau15737, Sau13274) except for Sau08911, were lower than the other 11 subfamily members. However, the S subfamily Sau08107 gene was highly expressed among two salt treatments of S. australis leaves. Furthermore, the majority of gene pairs undergoing tandem duplications exhibited distinct expression patterns. For example, Sau11436 was strongly expressed in ST2 leaves, but Sau11415 was weakly expressed in them (Fig. 7).

Fig. 7
figure 7

Comparisons of expression levels of six SabZIP genes obtained by qRT-PCR analysis among ST1 and ST2 leaves of Suaeda australis. The left Y-axis indicates the relative expression level, right Y-axis indicates the FPKM, and the X-axis represents different samples after salt stress treatment taken for expression analysis; the mean ± standard error measurement (SEM) value with three replications (n = 3) is displayed. * indicates p < 0.05

Full size image

In addition, a total of six SabZIP genes, namely, S subfamily SabZIP09 (Sau08107) and SabZIP11 (Sau08911) genes; I subfamily SabZIP14 (Sau11415) gene; D subfamily SabZIP26 (Sau16575) gene; G subfamily SabZIP31 (Sau19276) gene; and A subfamily SabZIP36 (Sau21103) gene, were chosen for qRT-PCR analysis (three replicates for each gene). In general, the relative expression levels of selected genes were in agreement with FPKM values obtained from RNA-seq (Fig. 7), verifying the reliability of our transcriptomic expression profiles.

Effect of overexpression of selected SabZIP genes under different salt concentrations

To examine the subcellular localization of above six proteins (SabZIP09, SabZIP11, SabZIP14, SabZIP26, SabZIP31, and SabZIP36), vectors expressing each of above selected genes fused to GFP were constructed and used for a transient expression assay in N. benthamiana leaf cells. All six SabZIP-GFP fusion proteins exclusively co-localized with H2B-mCherry in the nuclei (Fig. 8), suggesting that all six SabZIPs are nuclear proteins, consistent with their potential function as transcriptional regulators. Among six SabZIP proteins, SabZIP14, SabZIP26, and SabZIP36 showed the higher Percent Identity (> 60% based on BLASTP with 1e-5 E-value) to known bZIP family proteins in tobacco, indicating potential functional conservation. To further investigate the mechanism by which SabZIPs regulate the downstream salt response-regulating genes at the transcriptional level, the expression of six stress responsive genes under the three independent transient overexpression lines (SabZIP14, SabZIP26, and SabZIP36) were measured by qRT-PCR (Fig. 9). In tobacco, VRN1 and PRE6, ATG8B and ATG8E, DGAT1 and CAT1 could been regulated by transcription factor RF2b (orthologous gene of SabZIP14), TGA9 (SabZIP26), and ABI 5 (SabZIP36), respectively. The results showed that after salt treatment for 24 h, the expression levels of VRN1, PRE6, ATG8B, ATG8E, DGAT1, and CAT1 in the experimental group were significantly induced compared with the WT N. benthamiana. Interestingly, the expression of PRE6 was only greatly decreased in the SabZIP14-overexpressing transient expressed line compared with the WT, whereas the transcript levels of other above five genes were significantly increased in transgenic lines. These results suggested that SabZIPs may enhance the resistance to salt stress by effectively activating the transcription of the downstream stress-related gene.

Fig. 8
figure 8

Transient expression and subcellular localization of SabZIP09, SabZIP11, SabZIP14, SabZIP26, SabZIP31, and SabZIP36. The colocalization of SabZIP09-GFP, SabZIP11-GFP, SabZIP14-GFP, SabZIP26-GFP, SabZIP31-GFP, and SabZIP36-GFP, and nuclei was determined by the GFP signal and H2B-mCherry histone. GFP was used as a negative control. Scale bars, 10 μm

Full size image
Fig. 9
figure 9

Expression levels of VRN1, PRE6, ATG8B, ATG8E, DGAT1, and CAT1 in the WT N. benthamiana and transient SabZIPs (SabZIP14, SabZIP26, and SabZIP36) overexpression lines as indicated. **P < 0.01

Full size image

SabZIPs bind the promoter of downstream salt stress-related genes and activate their transcription

To explore whether SabZIP14, SabZIP26, SabZIP36 directly activate the expression of VRN1 and PRE6, ATG8B and ATG8E, DGAT1 and CAT1, respectively in N. benthamiana, yeast one-hybrid (Y1H) assays were carried out (Fig. 10). The results showed that GAD-SabZIP14 fusion protein, instead of GAD (GAL1 transcriptional activation domain, AD), respectively activated the LacZ reporter gene driven by the VRN1 and PRE6 promoter (Fig. 10a and b), indicating that SabZIP14 can bind to VRN1 and PRE6 promoter directly. In addition, the interaction of SabZIP26 or SabZIP36 with the promoter of ATG8B and ATG8E or DGAT1 and CAT1 in vitro were also confirmed by Y1H assays (Fig. 10c-f).

Fig. 10
figure 10

Yeast onehybrid analysis, using pB42AD-SabZIP14 as the prey, ProVRN1-pLacZ and ProPRE6-pLacZ as the baits, respectively; pB42AD-SabZIP26 as the prey, ProATG8B-pLacZ and ProATG8E-pLacZ as the baits, respectively; pB42AD-SabZIP36 as the prey, ProDGAT1-pLacZ and ProCAT1-pLacZ as the baits, respectively; pB42AD-p53 and p53-pLacZ as the positive controls

Full size image

Subsequently, the transient expression assays were then performed using the dual-luciferase reporter system in N. benthamiana leaves. The results demonstrated that a strong LUC signal could be observed only in the leaves co-expressing 35S:SabZIP14 and proPRE6-LUC, 35S:SabZIP14 and proVRN1-LUC, 35S:SabZIP26 and proATG8B-LUC, 35S:SabZIP26 and proATG8E-LUC, 35S:SabZIP36 and proDGAT1-LUC, 35S:SabZIP36 and proCAT1-LUC (Fig. 11). The results indicated that SabZIP14, SabZIP26, SabZIP36 proteins could directly bind to the promoter region of VRN1 and PRE6, ATG8B and ATG8E, DGAT1 and CAT1, respectively to activate genes expression.

Fig. 11
figure 11

The luciferase signal was detected in N. benthamiana leaves

Full size image

Discussion

bZIP transcription factors represent a prominent protein family in flowering plants engaged in stress responses, pathogen defence, light signaling, flower development, and seed maturation [13]. In this study, a genome-wide identification of the bZIP protein family was carried out in S. australis. The 44 identified bZIP proteins in S. australis were categorized into 12 distinct subfamilies, which correspond to the classification and identification results in A. thaliana [10]. Upon comparing the subfamily composition, we found that seven, two, five, and one SabZIP members in subfamilies S, C, I, and F were less than ten, four, seven, and three in A. thaliana, respectively (Fig. 3). In contrast, subfamily A and D exhibited a higher count of members, with eight and seven SabZIP members compared to seven and five in A. thaliana, respectively (Fig. 3). It is worth noting that previous studies have documented the identification of bZIP genes in numerous other plant species, such as 92 members in Oryza sativa [19], 125 members in Zea mays [20], 114 members in Malus domestica [21], and 86 members in poplar [22]. Similar to the S. australis family, the number of bZIP family in Ziziphus jujuba (45 bZIPs) and Prunus mume (49 bZIPs) show the relatively low level [23, 24], indicating the contraction of bZIP family during evolution. Furthermore, the assembled genome size of S. australis (437.17 Mb) was notably larger than that of A. thaliana (119.67 Mb) [25], indicating that there was no substantial correlation between genome size and the quantity of members. In a word, comparison of the bZIP genes number among various plants showed that the number of various species are also different.

Except for minority of subfamilies (S and H), the gene structures of SabZIP members exhibited visible variations, even in the same gene subfamily, particularly in terms of exon length and quantity (Fig. 4). Similar findings have been documented in cucumber [26] and Cyclocarya paliurus [27]. An analysis of motifs (Fig. 5) revealed the presence of 10 motifs in S. australis, designated as motif 1 through motif 10. Besides, motif compositions remained consistent within the same subfamily but exhibited variations among different subfamilies, indicating that subfamily-specific motifs may influence the functional divergence of SabZIP genes. These findings were consistent with previous study of Carthamus tinctorius [28] and Glycyrrhiza uralensis [29]. The diversifcation of subfamily-specific motifs provide ideas for investigating the function of bZIPs, and the diversifcation of functions enables plants to enhance their salt tolerance to adapt to the environment. Subfamily-specific motifs can lead to differences in the regulatory and binding properties of bZIP transcription factors, thereby influencing gene expression patterns under stress conditions. In Arabidopsis, most members belonging to group A contained subfamily-specific motif participate in ABA biological pathway and regulate plant responses to salt stress [30]. In peanut, group B bZIP proteins have a transmembrane domain and a specific domain motif at the C-terminus, also are important to the salt stress response via endoplasmic reticulum stress signaling [31]. Therefore, the SabZIP genes in group A and B could have similar functions to Arabidopsis and peanut. Moreover, the analysis of the promoter region revealed that a majority of the cis-element components in SabZIP genes were associated with temperature responsiveness, circadian control, flavonoid biosynthetic genes regulation, drought-inducibility, seed-specific regulation, and stress responsiveness (Fig. 5).

Based on the expression profiles analysis, it was evident that genes in the same subfamily exhibited diverse expression patterns among two salt treatment samples, but small amounts of subfamily genes (Sau08107, Sau19742, Sau08108, Sau00495, Sau15737, and Sau13274) showed the similar expression profiles. The study suggested that genes in the same subfamily displayed distinct and varied expression patterns throughout the evolutionary process. In addition, these results could be attributed to the intrinsic requirement for paralogous genes of the same origin to prevent functional redundancy while generating subfunctions and novel functions [32]. As a typical halophyte, S. australis possesses a special structure with succulent leaves, which may effectively store water, reduce the concentration of salt ions in leaf cells, maintain the osmotic balance of leaf cells, and heavily contribute to salt-stress adaptability [33]. In our study, five bZIP DEGs containing Sau08107, Sau08911, Sau11415, Sau16575, and Sau19276 showed higher expression levels in ST2 leaves than ST1. Among them, Sau08107 and Sau08911 belonging to group S, which could be directly activated via the ABA-dependent pathway, and form heterodimeric complexes with other members of the bZIP family under the salt stress in rice [34]; group I gene (Sau11415) is involved in cell cycle regulation and salt stress response [10]; Sau16575 belongs to group D, which can regulate the growth and development and decrease the sensitivity to salt stress in plants [35]; group G bZIP gene (Sau19276), has been demonstrated as an inhibitor of salt stress tolerance in tomato [36].

The molecular mechanism of plant stress resistance has been a hot topic in biological research. Previous studies have reported that bZIPs can participate in abiotic stress responses, but no studies have been reported in S. australis. In this study, we aimed to fully understand the function of bZIP genes in S. australis. We found all six differentially expressed SabZIPs are nuclear proteins and were positively activate the VRN1 [37], PRE6 [38], ATG8B [39], ATG8E [40], DGAT1 [41], and CAT1 [42] involved in the salt stress response. We speculate that these SabZIPs may have the same expression pattern as their significantly related transcription factors. This research provides information for research on SabZIPs functions. In conclusion, our results demonstrated the significant roles of the SabZIP gene family in salt stress and plant growth.

Conclusions

Australis is a typical halophyte distributed in subtropical and tropical coastal areas. Research has mainly focused on increasing yield, quality, and stress tolerance in S. australis. The bZIP gene family is significantly associated with plant growth, development, and the tolerance to salt stress. In this study, we identified 44 members of bZIP gene families in S. australis. Expression patterns analyses clearly discovered the role of several SabZIPs including Sau08107, Sau08911, Sau11415, Sau16575, and Sau19276, which showed higher expression levels in higher salt concentration than low concentration and obviously response to salt stress. Moreover, SabZIP14, SabZIP26, and SabZIP36 proteins could respectively bind to the promoter region of VRN1 and PRE6, ATG8B and ATG8E, DGAT1 and CAT1 and activate their expressions. These results improve our understanding of the role of bZIPs in developmental processes and in salt stress and provide a theoretical basis for further studies aimed at exploring the regulation of salt stress responses in S. australis.

Materials and methods

Plant materials, cDNA synthesis, and transcriptome sequencing

In the present study, the plants of S. australis were grown in Dinghai District, Zhoushan City, Zhejiang Province, China (E122°11′, N30°02′) and then transplanted to Fishery College, Zhejiang Ocean University. Voucher specimens were identified by Yinquan Qu, and deposited in Molecular Ecology Lab of Zhejiang Ocean University (Voucher code: 2022–1-ZS). In addition, we treated plants with salt treatment (ST) with two levels (ST1: the salt concentration of the soil was 10 g/kg; ST2: 18 g/kg) [43]. The leaves were sampled in reproductive development stage (24 October 2022) from S. australis individuals with one year old. The leaves were immediately collected and rapidly frozen in liquid nitrogen, followed by storage at − 80 °C. Each leaf sample consisted of three distinct biological replicates, each of which consisted of tissues from three individual plants. Total RNA extraction was performed employing the E.Z.N.A Plant RNA Isolation Kit (OMEGA, USA). Concentration and purity of RNAs were assessed using the NanoDrop spectrophotometer 2000C (Thermo Fisher Scientific, Waltham, MA, USA), while confirmation of RNA integrity was achieved through 1.0% agarose gel electrophoresis. The RNAs with OD260/OD280 valus within the range of 1.8–2.0 were subsequently subjected to reverse transcription into cDNA using PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, China), following the manufacturer's protocols. The resulting cDNAs were subsequently diluted at a 1:10 ratio with RNase-free water and stored at -20 °C for future qRT-PCR analyses. The cDNA libraries were constructed using the NEBNext® UltraTM RNA Library Prep Kit following the manufacturer's instruction. All cDNA libraries were sequenced using an Illumina HiSeqTM2000 system, generating reads with a length of 2 × 100 bp. Any sequences of low quality (defined by bases with Q-scores < 10%), sequences below 50 bp in length, primer sequences, and adapter sequences were rigorously filtered and excluded from the initial raw data by fastp [44].

Identification of SabZIP genes in S. australis

The whole protein sequence of S. australis was obtained from the Genome Warehouse (GWH) at the National Genomics Data Center, Beijing Institute of Genomics (https://ngdc.cncb.ac.cn/gwh, accessed on 10 July 2023). To identify bZIP proteins within S. australis, we initiated our search using Arabidopsis bZIP protein sequences as our query [10]. We initiated a comprehensive search for potential bZIP proteins using the local alignment search tool (BLASTP) [45] with a stringent E-value threshold of less than 1e-5. Following this preliminary screening, we acquired the HMM (hidden Markov model) profile corresponding to the Pfam bZIP domain (PF00107) from the Pfam database (http://pfam.xfam.org/, accessed on 10 July 2023). Subsequently, we employed this HMM profile to perform a search against the S. australis protein dataset using HMMER v3.3.2 [46] with default parameters. The acquired sequences underwent a rigorous filtration process based on two specific criteria: (1) incomplete sequences were eliminated if their lengths fell below that of the conserved motif (33 bp). (2) sequences containing more than 10 consecutive 'N' bases were excluded from further analysis. This culling process was employed to ensure data quality and conformity to research standards.

Chromosome location and synteny analysis of SabZIP genes

The precise chromosome locations of S. australis bZIP genes were determined using the MapGene2Chrom wb v2 tool (http://mg2c.iask.in/mg2c_v2.0/, accessed on 10 July 2023) on the basis of genome annotation obtained from the GWH database. The CDS sequences and genome feature file of Arabidopsis thaliana (genome assembly TAIR10.1) [47], Beta vulgaris (genome assembly EL10.2) [48], and Oryza sativa (genome assembly IRGSP-1.0) [49] were downloaded from https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001735.4/, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_026745355.1/, and https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001433935.1/, respectively. To explore the synteny relationship of homologous bZIP genes in S. australis and above three species (A. thaliana, B. vulgaris, and O. sativa), we employed the Multiple Collinearity Scan toolkit (MCScanX) [50].

Prediction of cis-regulatory elements of promoter region

The 2000 bp sequences located upstream of SabZIP genes were obtained from the whole-genome sequence. To predict a variety of cis-acting regulatory elements, these sequences were subjected to analysis using the default parameters of the PlantCARE promoter analysis tool, accessible at http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ as of 10 July 2023. This analysis aimed to predict various cis-acting regulatory elements [51].

Estimation of Ka/Ks values

MCScanX was used to perform gene duplication analyses for S. australis genome [50]. BLASTP software was applied to search for candidate homologous gene pairs (E-value < 1e-5) from the S. australis genome [45]. To identify syntenic chains, the candidate homologous gene pairs were analyzed using MCScanX with default parameters. MCScanX was used to further distinguish among segmental and tandem duplication events in bZIP gene family [50]. Subsequently, the Ka and Ks substitution rates were computed through the utilization of the KaKs_Calculator toolkit based on verified duplicated gene pairs of the bZIP family [52].

Expression analysis of selected SabZIP genes

After quality control and adapter trimming of the RNA-seq reads, the paired-end short reads were aligned to S. australis genome using HiSAT2 with its default parameters. Subsequently, the calculation of the expected number of FPKM fragments mapped was performed employing the StringTie program [53] with default parameters. Moreover, the DESeq2 R package [54] was applied to identify the differentially expressed genes (DEGs). In multiple analyses, the threshold of the p-value was calculated by the false discovery rate (FDR). If the FDR was less than 0.05, and the absolute value of log2(fold change) was more than 1, the gene expression differences were considered to be significant. In addition, the Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the clusterProfiler R package [55].

Six SabZIP DEGs (Sau08107, Sau08911, Sau11415, Sau16575, Sau19276, and Sau21103) related to salt tolerant were screened for qRT-PCR analysis. The online software Primer3Plus (https://www.primer3plus.com/index.html) was used to design primers, and the primers were synthesized by Beijing Qinke Xinye Biotechnology Ltd. (Beijing, China) (Supplementary Table S2). qRT-PCR was conducted using SYBR qPCR Master MIX (Vazyme) [56] and performed on StepOnePlus (Applied Biosystem, USA) [57]. 18S ribosomal RNA as an internal control was carried out. The methods for calculating relative gene expression were introduced by previous study [58].

Vector construction and subcellular localization

The full-length coding sequence (CDS) of Sau08107, Sau08911, Sau11415, Sau16575, Sau19276, and Sau21103 genes harboring Kpn I and BamH I restriction sites were inserted into pCAMBIA2300-35S-eGFP via in-fusion cloning. Above six recombinant pCAMBIA2300-35S-SabZIP-eGFP vectors were further introduced into the GV3101 Agrobacterium tumefaciens strain for subcellular localization and transient expression in tobacco (Nicotiana benthamiana). We cloned the nuclear-located AtH2B gene from A. thaliana [59]. Then we constructed recombinant pCAMBIA2300-35S-AtH2B-Mcherry plasmid as a positive control, and was also transformed into Agrobacterium GV3101. The engineered Agrobacterium of 35S::SabZIP-eGFP and positive control (35S::AtH2B-Mcherry) were cultured with 30 ml LB medium at 28℃ for 16 h, respectively. Centrifuging the culture at 6000 ×g for 5 min, and then re-suspending the pellet in infiltration buffer (containing sterile deionized water, 10 mM MES, 150 uM AS, and 10 mM MgCl2) with an OD600 of 1.0. Finally, we mixed the interest suspensions with equal volume proportion of positive control culture, respectively. The mixed suspension cultures were placed for 3 hours in the darkness. Tobaccos were grown in the greenhouse with a light/dark photoperiod of 16/8 h at 25℃. The leaves were infiltrated with infiltration buffer containing recombinant strains and were stored in a growth chamber during 60 h. All images were visualized by Leica TCS SP8X DLS laser confocal microscope. Meanwhile, RNA extraction and cDNA synthesis for qRT-PCR were performed on the infiltrated tobacco leaves after 24 hours of salt treatment.

Yeast one-hybrid assay

For Y1H system, the CDS of three SabZIPs (SabZIP14, SabZIP26, and SabZIP36) were individually were fused to the GAL1 AD in the pB42AD vectors to generate the prey vectors (pB42AD-SabZIP14, pB42AD-SabZIP26, and pB42AD-SabZIP36). The promoter fragments of VRN1, PRE6, ATG8B, ATG8E, DGAT1, and CAT1 were individually constructed into pLacZi vectors to construct the baits. pB42AD-SabZIP14, pB42AD-SabZIP26, and pB42AD-SabZIP36 vectors were individually transformed into the EGY48 yeast strains. After selecting the transformants on SD/-Trp plates, the NcoI-cut bait vectors were respectively introduced into the EGY48 yeast strains harbouring pB42AD–SabZIP14, pB42AD-SabZIP26, and pB42AD-SabZIP36. Positively co-transformed cells were screened on SD/-Trp/-Ura medium and cultured at 30 °C for 3 days. The resultant transformants were tested for β-galactosidase activity on selective media (SD/-Trp/-Ura/BU salt/X-gal). A positive (pB42AD-p53 + p53-LacZi) control was processed in the same manner.

Dual-luciferase assay

Dual-luciferase assays were conducted as the method outlined previously [60]. The promoter regions of VRN1, PRE6, ATG8B, ATG8E, DGAT1, and CAT1 were each cloned into pGreenII-0800-LUC reporter vectors, resulting in constructs such as proVRN1-LUC, proPRE6-LUC, proATG8B-LUC, proATG8E-LUC, proDGAT1-LUC, and proCAT1-LUC. The coding sequences (CDS) of SabZIP14, SabZIP26, and SabZIP36 were individually inserted into the pGreenII 62-SK vector under the control of the CaMV35S promoter, generating the effector constructs (35S::SabZIP14, 35S::SabZIP26, and 35S::SabZIP36). After co-infiltrating the effector and reporter plasmids into N. benthamiana leaves via Agrobacterium for 48 h, the leaves were treated with 1mM D-Luciferin potassium salt and subjected to fluorescence imaging using a living imaging system.

Availability of data and materials

A total of 6 transcriptome raw datasets produced by HiSeqTM2000 system were deposited in the Genome Sequence Archive (SRA) database (https://ngdc.cncb.ac.cn/gsa/browse/CRA011892) under the accession number CRA011892. The genome sequences of S. australis was from the Genome Warehouse in National Genomics Data Center Beijing Institute of Genomics (https://ngdc.cncb.ac.cn/gwh/Assembly/64339/show#, accession number GWHDOOL00000000).

References

  1. Villa JA, Bernal B. Carbon sequestration in wetlands, from science to practice: An overview of the biogeochemical process, measurement methods, and policy framework. Ecol Eng. 2018;114:115–28.

    Article  Google Scholar 

  2. Xi Y, Peng S, Ciais P, Chen Y. Future impacts of climate change on inland Ramsar wetlands. Nat Clim Change. 2021;11:45–51.

    Article  Google Scholar 

  3. Sapkota Y, White JR. Carbon offset market methodologies applicable for coastal wetland restoration and conservation in the United States: a review. Sci Total Environ. 2020;701:134497.

  4. Guo JM, Chen YY, Lu PZ, Liu M, Sun P, Zhang ZM. Roles of endophytic bacteria in Suaeda salsa grown in coastal wetlands: plant growth characteristics and salt tolerance mechanisms. Environ Pollut. 2021;287:117641.

  5. Herbert ER, Boon P, Burgin AJ, Neubauer SC, Franklin RB, Ardon M, Hopfensperger KN, Lamers LPM, Gell P. A global perspective on wetland salinization: ecological consequences of a growing threat to freshwater wetlands. Ecosphere. 2015;6:206.

    Article  Google Scholar 

  6. Ou YX, Sheng Y, Hu XJ, Leng DJ, Huang JF, Hu ZY, Bai LQ, Deng ZX, Kang QG, Wu YY. Nonomuraea nitratireducens sp. nov., a new actinobacterium isolated from Suaeda australis Moq. rhizosphere. Int J Syst Evol Micr. 2020;70(9):5026–31.

    Article  CAS  Google Scholar 

  7. Kim H, Park GN, Jung BK, Yoon WJ, Chang KS. Antibacterial activity of Suaeda australis in halophyte. Journal of the Korean Applied Science and Technology. 2016;33(2):278–85.

  8. Huang XK, Huang XD, Bian MJ. Study on the flavonoids compounds of Suaeda australis contents and its antioxidant activity in vitro. J Anhui Agric Sci. 2010;38(3):1432–34, +1542. (in Chinese)

  9. Alam MR, Tran TKA, Stein TJ, Rahman MM, Griffin AS, Yu RMK, MacFarlane GR. Accumulation and distribution of metal(loid)s in the halophytic saltmarsh shrub, Austral seablite, Suaeda australis in New South Wales. Australia Mar Pollut Bull. 2021;169:112475.

    Article  CAS  PubMed  Google Scholar 

  10. Dröge-Laser W, Snoek BL, Snel B, Weiste C. The Arabidopsis bZIP transcription factor family-an update. Curr Opin Plant Biol. 2018;45:36–49.

    Article  PubMed  Google Scholar 

  11. Wang LF, Zhu JF, Li XM, Wang SM, Wu J. Salt and drought stress and ABA responses related to bZIP genes from V. radiata and V. angularis. Gene. 2018;651:152–60.

    Article  CAS  PubMed  Google Scholar 

  12. Joo H, Baek W, Lim CW, Lee SC. Post-translational modifications of bZIP transcription factors in abscisic acid signaling and drought responses. Curr Genomics. 2021;22(1):4–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jakoby M, Weisshaar B, Dröge-Laser W, Vicente-Carbajosa J, Tiedemann J, Kroj T, Parcy F. bZIP transcription factors in Arabidopsis. Trends Plant Sci. 2002;7:106–11.

    Article  CAS  PubMed  Google Scholar 

  14. Mukherjee K, Choudhury AR, Gupta B, Gupta S, Sengupta DN. An ABREbinding factor, OSBZ8, is highly expressed in salt tolerant cultivars than in salt sensitive cultivars of indica rice. BMC Plant Biol. 2006;6:18.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Lu G, Gao CX, Zheng XN, Han B. Identification of OsbZIP72 as a positive regulator of ABA response and drought tolerance in rice. Planta. 2009;229:605–15.

    Article  CAS  PubMed  Google Scholar 

  16. Hossain MA, Cho JI, Han MH, Ahn CH, Jeon JS, An GH, Park PB. The ABRE-binding bZIP transcription factor OsABF2 is a positive regulator of abiotic stress and ABA signaling in rice. J Plant Physiol. 2010;167:1512–20.

    Article  CAS  PubMed  Google Scholar 

  17. Hossain MA, Lee YJ, Cho JI, Ahn CHH, Lee SK, Jeon JS, Kang H, Lee CH, An GH, Park PB. The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice. Plant Mol Biol. 2010;72:557–66.

    Article  Google Scholar 

  18. Liu GE, Ventura M, Cellamare A, Chen L, Cheng Z, Zhu B, Eichler EE. Analysis of recent segmental duplications in the bovine genome. BMC Genom. 2009;10:1–16.

    Article  Google Scholar 

  19. Guedes Corrêa LG, Riaño-Pachón DM, Guerra Schrago C, Vicentini dos Santos R, Mueller-Roeber B, Vincentz M. The role of bZIP transcription factors in green plant evolution: adaptive features emerging from four founder genes. PloS One. 2008;3(8):e2944.

  20. Wei KF, Chen J, Wang YM, Chen YH, Chen SX, Lin YN, Pan S, ZhongXJ, Xie DX. Genome-wide analysis of bZIP-encoding genes in maize. DNA Res. 2012;19(6):463–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Li YY, Meng D, Li MJ, Cheng LL. Genome-wide identification and expression analysis of the bZIP gene family in apple (Malus domestica). Tree Genet Genomes. 2016;12:82.

    Article  Google Scholar 

  22. Zhao K, Chen S, Yao WJ, Cheng ZH, Zhou BR, Jiang TB. Genome-wide analysis and expression profile of the bZIP gene family in poplar. BMC Plant Biol. 2021;21:122.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang Y, Gao WL, Li HT, Wang YK, Li DK, Xue CL, Liu ZG, Liu MJ, Zhao J. Genome-wide analysis of the bZIP gene family in Chinese jujube (Ziziphus jujuba Mill). BMC Genom. 2020;21:483.

    Article  CAS  Google Scholar 

  24. Li P, Zheng TC, Li LL, Wang J, Cheng TR, Zhang QX. Genome-wide investigation of the bZIP transcription factor gene family in Prunus mume: Classification, evolution, expression profile and low-temperature stress responses. Hortic Plant J. 2022;8(2):230–42.

    Article  CAS  Google Scholar 

  25. Sloan DB, Wu ZQ, Sharbrough J. Correction of persistent errors in Arabidopsis reference mitochondrial genomes. Plant Cell. 2018;30(3):525–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Baloglu MC, Eldem V, Hajyzadeh M, Unver T. Genome-wide analysis of the bZIP transcription factors in cucumber. PLoS ONE. 2014;9(4):e96014.

  27. Tao YT, Chen LX, Jin J, Du ZK, Li JM. Genome-wide identification and analysis of bZIP gene family reveal their roles during development and drought stress in Wheel Wingnut (Cyclocarya paliurus). BMC Genom. 2022;23:743.

    Article  CAS  Google Scholar 

  28. Li HY, Li LX, ShangGuan GD, Jia C, Deng SN, Noman M, Liu YL, Guo YX, Han L, Zhang XM, Dong YY, Ahmad N, Du LN, Li HY, Yang J. Genome-wide identification and expression analysis of bZIP gene family in Carthamus tinctorius L. Sci Rep. 2020;10(1):15521.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Han YX, Hou ZN, He QL, Zhang XM, Yan KJ, Han RL, Liang ZS. Genome-Wide characterization and expression analysis of bZIP gene family under abiotic stress in Glycyrrhiza uralensis. Front Genet. 2021;12:754237.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Finkelstein RR, Lynch TJ. The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell. 2000;12(4):599–609.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang Z, Yan L, Wan L, Huai D, Kang Y, Shi L, Jiang H, Lei Y, Liao Y. Genome-wide systematic characterization of bZIP transcription factors and their expression profiles during seed development and in response to salt stress in peanut. BMC Genom. 2019;20:1–14.

    Google Scholar 

  32. Kong WL, Gong ZY, Zhong H, Zhang Y, Zhao GQ, Gautam M, Deng XX, Liu C, Zhang CH, Li YS. Expansion and evolutionary patterns of glycosyltransferase family 8 in gramineae crop genomes and their expression under salt and cold stresses in Oryza sativa ssp. japonica. Biomolecules. 2019;9(5):188.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Robinson SP, Downton WJS. Potassium, sodium and chloride ion concentrations in leaves and isolated chloroplasts of the halophyte Suaeda australis R. Br Funct Plant Bio. 1985;12(5):471–9.

    Article  CAS  Google Scholar 

  34. Liu CT, Mao BG, Ou SJ, Wang W, Liu LC, Wu YB, Chu CC, Wang XP. OsbZIP71, a bZIP transcription factor, confers salinity and drought tolerance in rice. Plant Mol Biol. 2014;84:19–36.

    Article  CAS  PubMed  Google Scholar 

  35. Li B, Liu Y, Cui XY, Fu JD, Zhou YB, Zheng WJ, Lan JH, Jin LG, Chen M, Ma YZ, Xu ZS, Min DH. Genome-wide characterization and expression analysis of soybean TGA transcription factors identified a novel TGA gene involved in drought and salt tolerance. Front Plant Sci. 2019;10:549.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Pan YL, Hu X, Li CY, Xu X, Su CG, Li JH, Song HY, Zhang XG, Pan Y. SlbZIP38, a tomato bZIP family gene downregulated by abscisic acid, is a negative regulator of drought and salt stress tolerance. Genes. 2017;8(12):402.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Liu J, Yao YY, Xin MM, Peng HR, Ni ZF, Sun QX. Shaping polyploid wheat for success: Origins, domestication, and the genetic improvement of agronomic traits. J Integr Plant Biol. 2022;64(2):536–63.

    Article  PubMed  Google Scholar 

  38. Fan S, Jia Y, Wang R, Chen X, Liu W, Yu H. Multi-omics analysis the differences of VOCs terpenoid synthesis pathway in maintaining obligate mutualism between Ficus hirta Vahl and its pollinators. Front Plant Sci. 2022;13:1006291.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Wang P, Nolan TM, Yin Y, Bassham DC. Identification of transcription factors that regulate ATG8 expression and autophagy in Arabidopsis. Autophagy. 2020;16(1):123–39.

    Article  CAS  PubMed  Google Scholar 

  40. Magen S, Seybold H, Laloum D, Avin-Wittenberg T. Metabolism and autophagy in plants-a perfect match. FEBS Lett. 2022;596(17):2133–51.

    Article  CAS  PubMed  Google Scholar 

  41. Kong Y, Chen S, Yang Y, An C. ABA-insensitive (ABI) 4 and ABI5 synergistically regulate DGAT1 expression in Arabidopsis seedlings under stress. FEBS Lett. 2013;587(18):3076–82.

    Article  CAS  PubMed  Google Scholar 

  42. Collin A, Daszkowska-Golec A, Szarejko I. Updates on the role of ABSCISIC ACID INSENSITIVE 5 (ABI5) and ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTORs (ABFs) in ABA signaling in different developmental stages in plants. Cells. 2021;10(8):1996.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Chen LH, Zhang H, Yao YT, Zhang C, Zheng JH, Zhang FG. Effects of Suaeda salsa covering on soil physicochemical properties in coastal beach. J Plant Resour & Environ. 2021;30(2):19–27.

    Google Scholar 

  44. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884–90.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Lavigne R, Seto D, Mahadevan P, Ackermann HW, Kropinski AM. Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools. Res Microbiol. 2008;159(5):406–14.

    Article  CAS  PubMed  Google Scholar 

  46. Prakash A, Jeffryes M, Bateman A, Finn RD. The HMMER web server for protein sequence similarity search. Curr Protoc Bioinformatics. 2017;60(1):3–15.

    Article  Google Scholar 

  47. Naish M, Alonge M, Wlodzimierz P, Tock AJ, Abramson BW, Schmücker A, Henderson IR. The genetic and epigenetic landscape of the Arabidopsis centromeres. Science. 2021;374(6569):eabi7489.

    Article  PubMed  PubMed Central  Google Scholar 

  48. McGrath JM, Funk A, Galewski P, Ou S, Townsend B, Davenport K, Dorn KM. A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.). DNA Res. 2023;30(1):dsac033.

    Article  PubMed  Google Scholar 

  49. Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, Matsumoto T. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice. 2013;6:1–10.

    Article  Google Scholar 

  50. Wang YP, Tang HB, Debarry JD, Tan X, Li JP, Wang XY, Lee TH, Jin HZ, Guo H, Kissinger JC, Paterson AH. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30(1):325–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhang Z. KaKs_Calculator 3.0: calculating selective pressure on coding and non-coding sequences. Genom Proteom Bioinf. 2022;20(3):536–40.

    Article  CAS  Google Scholar 

  53. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT. StringTie and Ballgown Nat Protoc. 2016;11(9):1650–67.

    Article  CAS  PubMed  Google Scholar 

  54. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21.

    Article  Google Scholar 

  55. Yu GC, Wang LG, Han YY, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics: a j integrat biol. 2012;16(5):284–7.

    Article  CAS  Google Scholar 

  56. Zheng HH, Zhang SJ, Cui JT, Zhang J, Wang LY, Liu F, Chen HY. Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR. Mol Cell Probe. 2020;50:101524.

  57. Cao Y, Sivaganesan M, Kinzelman J, Blackwood AD, Noble RT, Haugland RA, Griffith JF, Weisberg SB. Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods. Water Res. 2013;47(1):233–41.

    Article  PubMed  Google Scholar 

  58. Qu YQ, Chen XL, Mao X, Huang P, Fu XX. Transcriptome analysis reveals the role of GA3 in regulating the asynchronism of floral bud differentiation and development in heterodichogamous Cyclocarya paliurus (Batal.) Iljinskaja. Int J Mol Sci. 2022;23(12):6763.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Jiang D, Borg M, Lorković ZJ, Montgomery SA, Osakabe A, Yelagandula R, Axelsson E, Berger F. The evolution and functional divergence of the histone H2B family in plants. PLoS Genet. 2020;16(7):e1008964.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Fan L, Wang Y, Xu L. A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L. Hortic Res. 2020;7(1):164.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was funded by the Province Key Research and Development Program of Zhejiang (2021C02047). This work was also supported by the Youth Project of Zhejiang Natural Sciences Foundation (LQ24D060004). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data in writing the manuscript.

Author information

Author notes

  1. Yinquan Qu and Ji Wang contributed equally to this work.

Authors and Affiliations

  1. Fishery College, Zhejiang Ocean University, Zhoushan, 316022, Zhejiang, China

    Yinquan Qu, Tianxiang Gao, Caihui Qu, Xiaoyun Mo & Xiumei Zhang

  2. School of Teacher Education, Nanjing Xiaozhuang University, Nanjing, 211171, Jiangsu, China

    Ji Wang

Authors
  1. Yinquan Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ji Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tianxiang Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Caihui Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoyun Mo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiumei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YQ, TG, JW, and XZ planed and designed the experiments. YQ, XM, CQ, and JW collected the samples and performed experiments. YQ analyzed and interpreted the sequencing data. YQ and JW wrote the manuscript. XZ provided funding for sequencing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xiumei Zhang.

Ethics declarations

Ethics approval and consent to participate

The materials of S. australis in this experiment were collected from S. australis germplasm resources bank (E122°11′, N30°02′) of the Fishery College, Zhejiang Ocean University, located in Molecular Ecology Laboratory of Zhejiang Ocean University, Zhoushan, Zhejiang Province, China. The collection of plant material complies with institutional guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qu, Y., Wang, J., Gao, T. et al. Systematic analysis of bZIP gene family in Suaeda australis reveal their roles under salt stress. BMC Plant Biol 24, 816 (2024). https://doi.org/10.1186/s12870-024-05535-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-024-05535-1

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us