Next Article in Journal
A Study on the Effect of Energy on the Development of Silkworm Embryos Using an Estrogen-Related Receptor
Next Article in Special Issue
Genome-Wide Identification of CHYR Gene Family in Sophora alopecuroides and Functional Analysis of SaCHYR4 in Response to Abiotic Stress
Previous Article in Journal
MicroRNAs and Nonalcoholic Steatohepatitis: A Review
Previous Article in Special Issue
Seed Priming with MeJa Prevents Salt-Induced Growth Inhibition and Oxidative Damage in Sorghum bicolor by Inducing the Expression of Jasmonic Acid Biosynthesis Genes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 19
10.3390/ijms241914483
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

OsBTBZ1 Confers Salt Stress Tolerance in Arabidopsis thaliana

by
Triono B. Saputro
1,2,
Bello H. Jakada
1,
Panita Chutimanukul
3,
Luca Comai
4,
Teerapong Buaboocha
5,6 and
Supachitra Chadchawan
1,6,*
1
Center of Excellence in Environment and Plant Physiology, Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
2
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Luang, Pathumthani, Bangkok 12120, Thailand
4
Genome Center and Department of Plant Biology, UC Davis, Davis, CA 95616, USA
5
Center of Excellence in Molecular Crop, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
6
Omics Science and Bioinformatics Center, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(19), 14483; https://doi.org/10.3390/ijms241914483
Submission received: 14 August 2023 / Revised: 11 September 2023 / Accepted: 15 September 2023 / Published: 23 September 2023
(This article belongs to the Special Issue Molecular Research in Abiotic Stress Tolerance)
Browse Figures
Review Reports Versions Notes

Abstract

:
Rice (Oryza sativa L.), one of the most important commodities and a primary food source worldwide, can be affected by adverse environmental factors. The chromosome segment substitution line 16 (CSSL16) of rice is considered salt-tolerant. A comparison of the transcriptomic data of the CSSL16 line under normal and salt stress conditions revealed 511 differentially expressed sequence (DEseq) genes at the seedling stage, 520 DEseq genes in the secondary leaves, and 584 DEseq genes in the flag leaves at the booting stage. Four BTB genes, OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7, were differentially expressed under salt stress. Interestingly, only OsBTBZ1 was differentially expressed at the seedling stage, whereas the other genes were differentially expressed at the booting stage. Based on the STRING database, OsBTBZ1 was more closely associated with other abiotic stress-related proteins than other BTB genes. The highest expression of OsBTBZ1 was observed in the sheaths of young leaves. The OsBTBZ1-GFP fusion protein was localized to the nucleus, supporting the hypothesis of a transcriptionally regulatory role for this protein. The bt3 Arabidopsis mutant line exhibited susceptibility to NaCl and abscisic acid (ABA) but not to mannitol. NaCl and ABA decreased the germination rate and growth of the mutant lines. Moreover, the ectopic expression of OsBTBZ1 rescued the phenotypes of the bt3 mutant line and enhanced the growth of wild-type Arabidopsis under stress conditions. These results suggest that OsBTBZ1 is a salt-tolerant gene functioning in ABA-dependent pathways.

1. Introduction

Rice (Oryza sativa L.) is one of the most important primary food resources worldwide. In Asia, rice production is a principal factor for improving food security. Salinity is a major limiting factor for plants such as rice, decreasing their growth and productivity [1]. Moreover, salt toxicity adversely affects the grain yield, panicle length, spikelet number per panicle, seed weight per panicle, and 1000-grain weight [2,3]. Soil salinity can occur naturally or can be induced by human activities, such as constant irrigation with low-quality groundwater [4]. High salt concentrations can adversely affect plant physiology through ion toxicity in the plant cells, which reduces the photosynthesis rate and growth of plants [5].
Salt stress tolerance is a polygenic trait controlled by multiple genes in the rice genome. Various efforts have been implemented to determine the genes or genomic regions responsible for this trait. Molecular markers for salt-tolerant phenotypes have been identified by several research groups [6,7,8,9,10,11,12,13,14,15,16]. After the development of the omics sciences, genomics, transcriptomics, and proteomic approaches have been used to identify the genes/proteins involved in salt tolerance, including their functions in ion transport regulation [17]. A greater understanding of the salt tolerance mechanisms initiated by other genes has also been elucidated. Recently, the negative regulator [18] and protein with a role in absorbed light energy dissipation [19] were reported to have a role in salt tolerance.
A genome-wide association study (GWAS) was conducted on the salt-tolerance traits in rice at the germination [20,21], seedling [18,22,23,24], early vegetative [25,26], and flowering stages [27,28]. In response to various environmental stress conditions, plants alter their gene expression to deal with the negative effects of environmental signals. Transcriptomics is a prominent method for identifying the genes that potentially regulate salt tolerance in rice [29]. Transcriptomics analysis can be used in combination with genomic data to predict salt tolerance genes in rice. Lv et al. [30] performed a GWAS using 3.82 million SNPs associated with the standard evaluation score (SES) of visual salt injury and then combined them with the differentially expressed genes between cultivars 93-11 and PA64s under normal and salinity stress conditions to predict 30 candidate salt-tolerant genes.
The chromosome segment substitution line 16 (CSSL16) is a salt-tolerant line. Based on the DEseq data of CSSL16, along with the genomic comparison between CSSL16 and its original genomic background, ‘Khao Dawk Mali 105 (KDML105)’, Chutimanukul et al. [31] analyzed the transcriptome data of ‘KDML105’ and CSSL16 rice, using a gene co-expression network (GCN), a weighted gene co-expression network (WGCN), and clustering analysis and predicted 92 candidate salt-tolerant genes. Then, this information was combined with a genomic comparison between CSSL16 and KDML105 and revealed nine candidate genes, seven of which were in the salt-tolerant QTL previously reported by Kanjoo et al. [32,33]. In this research, we report the validation of the OsBTBZ1 gene, which is one of the nine candidate salt-tolerant genes predicted by the combined methods of transcriptomic analysis via GCN, WGCN, and CC and genomic comparison.
OsBTBZ1 (LOC_Os01g66890) was predicted as an important gene responsible for the salt tolerance characteristics of CSSL16 [31]. It is a Bric-a-Brac, Tramtrack, and Broad Complex BTB domain with a TAZ zinc finger and Calmodulin-binding domains. BTB proteins have been studied for many crops and play various roles, mainly in plant growth and in responses to abiotic stimuli. For example, the expression of the CsBT1 gene in cucumber plants notably decreased under salt stress [34], whereas the CaBPM4 gene in pepper was induced after 8 h under salt and drought exposure and 12 h after exposure to cold stress [35]. Moreover, the expression in Arabidopsis thaliana of the IbBT4 gene from sweet potato enhanced drought tolerance [36].
In this research, to understand the function of OsBTBZ1 in salt stress conditions, the OsBTB gene family expression at both the seedling and booting stages was investigated. Phylogenetic analysis of the OsBTB gene family was performed. The cis-elements in the promoters of OsBTB genes induced by salt stress were compared to support the salt-responsive expression of the genes. Based on the amino acid sequence of the OsBTBZ1 protein, it is predicted to be a transcription factor and to regulate other genes of the salt-tolerant phenotype [31]. This gene was reported to be involved in plant growth regulation; however, its function during salt exposure has not yet been characterized fully. AtBT3 is the OsBTBZ1 ortholog in Arabidopsis. The Atbtbz1 (Atbt3) mutant, a null mutant of the AtBT3 gene, is more susceptible to salt stress [31]. In this study, the OsBTBZ1 expression cassette was transferred to Arabidopsis wild-type (WT) plants for ectopic expression and to the Atbt3 mutant for a complementation study. The homozygous T3 plants were used to investigate the salt, ABA, and mannitol responses and clarify the function of the OsBTBZ1 protein in these abiotic stresses.

2. Results

2.1. Only Four BTB Genes Were Expressed in CSSL16 under Salt Stress

In order to investigate whether all OsBTB genes can be induced by salt stress, 182 genes containing the BTB domains were retrieved from the Phytozome database [37], and the differentially expressed genes at seedlings and booting stages were explored. These two stages, the seedling and booting stages, of rice were selected for transcriptome analysis because they are susceptible to salt stress [18,28]. Moreover, the flag leaves and second leaves of rice at the booting stage are important for grain-filling. Under salt stress, 511 genes were differentially expressed at the seedling stage, whereas 520 and 584 differentially expressed genes were found in the second and flag leaves, respectively. Among them, only four BTB genes, LOC_Os01g66890 (OsBTBZ1), LOC_Os01g68020 (OsBTBZ2), LOC_Os02g38120 (OsBTBN3), and LOC_Os03g41350 (OsBTBN7), were differentially expressed in salt-stressed CSSL16, while OsBTBZ1 was the only BTB gene expressed at the seedling stage, and the other genes were expressed at the booting stage in both second and flag leaves (Figure 1A). Furthermore, OsBTBZ1 and OsBTBZ2 contained the BTB and Transcription Adaptor putative zinc finger (TAZ zF or zF-TAZ) domains, whereas OsBTBN3 and OsBTBN7 contained the non-phototropic hypocotyl3 (NPH3) domain (Figure 1B).
Phylogenetic analysis of the genes containing the BTB domain in rice and Arabidopsis was conducted using the JTT model, which used a substitution model calculated from the nearest-neighbor proteins with more than 85% similarity. OsBTBZ1 and its ortholog in Arabidopsis AT1G05690 (AtBT3) belong to the same cluster. Both OsBTBZ1 and OsBTBZ2 are in the same cluster and contain the zf-TAZ domain. Moreover, OsBTBN3 and OsBTBN7, which contain an NPH3 domain, were clustered together (Figure 2A). The similar domains in OsBTBZs and OsBTBNs suggest a similar function for these proteins.
The salt-tolerant quantitative trait locus (QTL) identified on chromosome 1 and located between RM1003 and RM3362 contained four BTB genes: OsBTBZ1, OsBTBZ2, OsBTBM1 (LOC_Os01g70670), and OsBTBA3 (LOC_Os01g72020). OsBTBN3 and OsBTBN7 were located on chromosomes 2 and 3, respectively. Furthermore, chromosomes 8 and 10 contained dense clusters of BTB genes, with 34 and 46 BTB genes, respectively (Figure 2B).

2.2. OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 Promoters Contain Multiple Cis-Elements Related to the Water Stress Response

An investigation of the cis-element at the promoter regions of OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 was performed to reveal the salt-responsive elements located in these OsBTB genes. Two thousand base pair sequences upstream of the OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 transcription start sites were analyzed using New PLACE to identify the regulatory cis-elements located in the promoter regions of these genes (Figure 3). Table 1 summarizes the cis-elements related to abiotic stress. Multiple MYCCONSENSUSAT elements, which are water stress-responsive elements, were found in all the tested promoters. More than 10 of these elements were located on the OsBTBZ1, OsBTBZ2, and OsBTBN3 promoters, which is consistent with the upregulation pattern under salt stress conditions. Moreover, other cis-elements related to dehydration stress, such as ACGTATERD1, DPBFCOREDCDC3, and MYBCORE, were found in all four promoters. Interestingly, ABA-responsive elements were found only in the + strand of OsBTBZ1. The GAGA-binding protein binding site (GAGAGMGSA1), which is specific to the CSSL16 allele, but not the ‘KDML105′ allele, was detected only in OsBTBZ1 (+ strand) (Table 1 and Figure 3).

2.3. OsBTBZ1 and OsBTBZ2 Are in the Same Protein-Protein Interaction (PPI) Network

The STRING database was used to investigate the protein-protein interaction (PPI) network of the four BTB proteins: OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 to select the best candidate OsBTB protein, which showed the highest number of connections with other proteins, for further characterization. OsBTBZ1 and OsBTBZ2 were connected, whereas OsBTBN3 and OsBTBN7 were dissociated. Furthermore, OsBTBZ1 and OsBTBZ2 were linked to other proteins, including CARM1, OS07T0626600-01, GCN5, and OS01T0884500-01. CARM1, OS07T0626600-01, and GCN5 are involved in chromatin remodeling. Modifying the chromatin architecture is necessary for the epigenetic control of gene expression, which does not involve alterations in DNA sequences, while increased chromatin compaction results in distinct higher-order structures [38]. OS01T0884500-01 is a zP (CCCH-type) protein. Contrastingly, OsBTBZ1 is connected to OS03T0216600-01, a glucan 1,3-alpha-glucosidase, and is also connected with OsJ_06167, OsWRKY39, and OsJ_25984, a protein kinase [39], which mediates pathogen-associated molecular pattern (PAMP)-triggered responses (Figure 4).
OsBTBN7 was associated with three uncharacterized proteins: OS03T0695500-01, OS04T0258900-01, and OS04T0283600-00, whereas OsBTBN3 was not associated with any other protein (Figure 4). Therefore, the functions of these two proteins were not investigated further.
According to the PPI network prediction using STRING, OsBTBZ1 was predicted to interact with more proteins. Therefore, it was selected for further characterization.

2.4. OsBTBZ1 Is Expressed in All Plant Tissues, Especially in Younger Leaf Sheaths

Because OsBTBZ1 showed differential expression owing to salt stress at the seedling stage, 15-day-old seedlings were selected for monitoring the expression of this gene. High gene expression was detected in the young leaves and root tissues, and a relatively higher level of expression was detected in the young (first leaf) leaf sheath, compared to the young leaf blades. However, the expression levels decreased in older leaves. In the oldest leaf of the 15-day-old seedlings, OsBTBZ1 expression was lower in the leaf sheath than in the leaf blade. At the reproductive stage, OsBTBZ1 was highly expressed in the leaf sheaths of flag leaves, as well as in the peduncles and spikelets (Figure 5).

2.5. OsBTBZ1 Is Localized in the Nucleus, Suggesting the Role of the Transcription Factor

To analyze the subcellular localization of the OsBTBZ1 protein, the CDS of OsBTBZ1 was fused with GFP and transiently expressed in the epidermal cells of Allium cepa. OsBTBZ1-GFP expression resulted in fluorescence in the nucleus, whereas the GFP fluorescence of the control was observed outside the nuclear region (Figure 6). IbBT4, a BTB gene in Ipomoea, is also localized in the nucleus [36]. A similar result was reported by Weber and Hellmann [40], who examined the BPM1, BPM2, and BTB proteins in Arabidopsis. This is consistent with a transcription factor role for OsBTBZ1, which may regulate other genes that are responsible for salt tolerance.

2.6. Ectopic Expression of OsBTBZ1 Could Revert the NaCl and ABA Susceptibility of the Atbt3 Arabidopsis Mutant at the Germination Stage

Chutimanukul et al. [31] reported that the Atbt3 Arabidopsis mutant was more susceptible to salt stress. Therefore, to investigate the role of OsBTBZ1 an ortholog to AtBT3, we fused it to the constitutive CaMV35S promoter and transformed it into the WT Arabidopsis and Atbt3 mutant. We generated two lines (REV1 and REV2) in the Atbt3 mutant background to test for complementation of salt sensitivity, along with two lines, OE1 and OE2, in the WT background to test for the effect of overexpression.
Germination tests were performed to investigate the effects of NaCl, ABA, and mannitol stress on Arabidopsis seed germination. The responses are shown in Figure 7A–E. Under normal conditions, the seeds of all lines, comprising the WT, Atbt3 mutant (bt3), REV1, REV2, OE1, and OE2, displayed no significant difference in germination rate (Figure 7A,B).
Expression of OsBTBZ1 could enhance the germination rate of bt3 mutant lines. When 150 mM NaCl was applied to 0.5× MS medium, the WT germination percentage was reduced to 88%, whereas the bt3 mutant line showed only 54% germination after 6 d of germination. The ectopic expression of OsBTBZ1 in the bt3 mutants reversed this effect. Both REV1 and REV2 had germination rates of 70% after 9 d of germination. Moreover, the OE1 and OE2 lines, which expressed OsBTBZ1 in the WT, had a germination rate similar to that of the WT (Figure 7A,C).
The application of ABA to the medium delayed seed germination in all lines. On 0.5× MS medium supplemented with ABA, no germination occurred in the WT, bt3 mutant, and REV2 after 4 d of germination (Figure 7A,D). Later, after 5 d of incubation, the germination rate of all lines increased and reached a maximum after 10 d. A 100% germination rate was detected in REV1 and OE2, while WT showed an 86% germination rate. The lowest germination rate under ABA was found in the bt3 mutant (73%). The ectopic expression of OsBTBZ1 could revert ABA susceptibility in the bt3 mutant line by increasing the germination rate to over 80% in both REV1 and REV2. In the WT background, OsBTBZ1 expression enhanced germination, and, after 5 d of germination, approximately 40% germination was detected in both OE1 and OE2, whereas WT showed a germination rate of <20%. Although WT and OE1 showed similar levels of germination (73%) after 10 d of germination, OE2 showed 100% germination (Figure 7A,D).
To discern the precise function of the OsBTBZ1 gene, it is essential to evaluate its role in germination under mannitol-induced conditions, which will help in determining whether it is solely responsive to salt or has a broader function within the osmotic regulation mechanisms. Therefore, 150 mM mannitol was added to the MS medium as a germination test. A slight reduction in the WT seed germination percentage was detected. The mutation of AtBt3 increased the sensitivity to mannitol stress, as shown by the significantly lower germination percentage of the Atbt3 mutant. However, the ectopic expression of OsBTBZ1 did not reverse the inhibition of mannitol-induced germination (Figure 7A,B,E).
Based on these results, we can conclude that OsBTBZ1 can reverse the Atbt3 mutation under salt and ABA stress, but not under drought stress that is induced by mannitol.

2.7. OsBTBZ1 Enhanced the Salt and ABA Tolerance in Transgenic Arabidopsis

To investigate whether OsBTBZ1 enhanced the stress tolerance of plants, the fresh weight, root length, and photosynthetic pigment content under stress conditions were measured. At the beginning of the experiment (0 d), seedlings of the WT, bt3 mutant, REV1, REV2, OE1, and OE2 lines showed similar fresh weights (Supplementary Figure S1). After 6 d of the experiment, the bt3 mutant had a significantly lower fresh weight than the WT. Ectopic expression could reverse the phenotype of the bt3 mutant to a fresh weight similar to that of the WT under normal conditions, as shown by the phenotypes of REV1 and REV2 (Figure 8A,B). Moreover, the root lengths of all the lines were similar under normal conditions (Figure 8A,C).
Salt stress and ABA treatments reduced the fresh weight and root length of all lines (Figure 8D–I). The fresh weight of WT was reduced by 50%, whereas that of the Atbt3 mutant decreased by more than 60%. This indicated that the Atbt3 mutant was more susceptible to salt stress. The revertant lines, REV1 and REV2, could reverse NaCl susceptibility by showing a significantly higher fresh weight than the Atbt3 mutant after 6 d of the NaCl treatment. OE1 and OE2 also had higher fresh weights than WT (Figure 8E). A similar response could also be detected via the root-length response under salt stress (Figure 9F). The effect of 1 mM of ABA on Atbt3 growth was not as strong as that of the NaCl treatment. Approximately 50% of the fresh weight was reduced in the ABA-treated Atbt3 mutant after 6 d, whereas a similar reduction in the root length of the WT and mutant was detected. The ectopic expression of OsBTBZ1 in both the WT and mutant backgrounds reversed these effects (Figure 8G–I).
Mannitol reduced the fresh weight and root length of all lines; however, no significant difference in fresh weight was detected (Figure 8J–L). Conversely, the ectopic expression of OsBTBZ1 enhanced the root length (Figure 8L).
We also compared the photosynthetic pigment levels. On the first day of the experiment, all lines had similar levels (Supplementary Figure S2). Under normal conditions, after 6 d, OsBTBZ1 ectopic expression significantly enhanced the Chl b content in the Atbt3 mutant, showing a tendency to enhance the Chl a and carotenoid contents in the WT (Figure 9A–C).
Salt stress (150 mM NaCl) reduced the Chl a, Chl b, and carotenoid contents in all lines. Strong reductions in the Chl a and carotenoid contents were detected in the Atbt3 mutant. However, these effects were reversed by OsBTBZ1 expression, as shown by the REV1 and REV2 pigment contents. The ectopic expression of OsBTBZ1 in the WT did not show significant levels of Chl a, Chl b, and carotenoid contents when compared to the pigment content of the WT (Figure 8D–F).
ABA significantly decreased the Chl a, Chl b, and carotenoid contents in all lines (Figure 8G–I), with a much stronger effect on the Chl b content (Figure 8B,E,F). The Atbt3 mutant was more susceptible to ABA treatment in terms of the photosynthetic pigment content. An approximately 60–75% reduction in Chl a and Chl b content was detected in the Atbt3 mutant, respectively, after ABA treatment, whereas in the WT, an approximately 40–60% reduction in Chl a and Chl b contents was found (Figure 8G,H), along with a reduction in the carotenoid content (Figure 8I). The ectopic expression of OsBTBZ1 reversed these effects.
Furthermore, treatment with 150 mM of mannitol caused a similar reduction in the photosynthetic pigment content in all lines (Figure 8J–L).
Based on the earlier investigation into growth and photosynthetic pigment contents, the expression of OsBTBZ1 in the WT and Atbt3 mutant background could confer abiotic stress tolerance under salt and ABA stress and showed fewer effects under drought stress when treated with 150 mM of mannitol.

3. Discussion

The BTB domain genes are part of a large gene family that has various roles in plant responses to abiotic stresses, ubiquitination, and development. In the present study, OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 were induced by salt stress at various stages, suggesting roles in the salt stress response in both the seedling and booting stages. The BTB genes in other species have also been reported to be involved in salt stress. For example, the A. thaliana stress-induced BTB protein 1 (AtSIBP1) is a positive regulator of salinity responses in Arabidopsis [41]. CaBPM4 (Capsicum annuum BTB-POZ and MATH domain protein) from pepper is upregulated during salt-stress exposure [35]. The SlBTB18 in tomatoes contains the TAZ domain and its expression increases dramatically under cold, salt, and oxidative stress [42]. The AtBt3 gene, which was in the same cluster as OsBTBZ1, has been reported to play a crucial role in gametophyte development in Arabidopsis [43]. Chutimanukul et al. [31] demonstrated that the Atbt3 mutant lines were more susceptible to salt stress than the WT. Moreover, over two dozen different protein domains were associated with the BTB, five of which (MATH, Kelch, NPH3, ion transport, and the zF domains) were much more frequent than the others [44]. Furthermore, the combination of the BTB domain with the TAZ domain is only observed in plants [45].
The other motif was NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), which was present in OsBTBN3 and OsBTBN7. The BTB–NPH3 proteins, also called NPH3/RPT2-like (NRLs) proteins, are plant-specific BTB/POZ proteins18. NRLs contain an N-terminal BTB domain and a C-terminal NON-PHOTOTROPHIC HYPOCOTYL 3 (NPH3) domain; some members contain an additional C-terminal coiled-coil domain [46]. NPH3, a BTB NPH3 family member in Arabidopsis, functions as a CRL3 substrate adaptor and regulates the ubiquitylation of phototropin1 (phot1) in response to different blue-light intensities. The dephosphorylation of NPH3 when stimulated by blue light may also be crucial for phot1-dependent phototropism [47]. Proteins containing NPH3 are abundantly localized in the plasma membrane and interact with phototropins and blue light receptor kinases [48].
Evidence from a putative cis-element analysis and PPI showed that OsBTBZ1 and OsBTBZ2 are related to the stress response. CARM1, GCN5, and OS07T0626600-01 are involved in post-translational modifications such as phosphorylation, glycosylation, acetylation, succinylation, carbonylation, S-nitrosylation, and Tyr-nitration, which can alter the epigenetic status of plants [49]. CARM1 histone-arginine methyltransferase, methylates (mono- and asymmetric dimethylation), and the guanidino nitrogens of arginyl residues in several proteins are involved in DNA packaging, transcription regulation, and mRNA stability. They can also be recruited to the promoters upon gene activation to methylate histone H3 and activate transcription via chromatin remodeling. Protein arginine methyltransferases (PRMTs), which are equivalent to CARM1 in mammals, were reported to be related to salt tolerance in Arabidopsis. Prmt5 and prmt4a;4b Arabidopsis mutants display an alteration in salt-stress tolerance [50]. Both OsBTBZ1 and OsBTBZ2 were predicted to directly interact with protein arginine methyltransferases, suggesting that both OsBTBZs are involved in salt-stress tolerance. The histone acetyltransferase GCN5 functions in acetylation of histone H3, which provides a specific tag for epigenetic transcriptional activation. GCN5 operates in concert with certain DNA-binding transcriptional activators that act via the formation of large multiprotein complexes to modify chromatin. Zheng et al. [51] reported that GCN5 plays an important role in cell wall integrity and salt tolerance in Arabidopsis. The wheat TaGCN5 gene can complement the Atgcn5 mutation, leading to the restoration of the salt-tolerant phenotype in the mutant line. This information suggested that OsBTBZ1 and OsBTBZ2 could be involved in chromatin remodeling and epigenetic regulation in the salt-tolerant phenotype in rice. It was also reported that the histone deacetylase, HDA710, regulated salt tolerance in rice via ABA signaling [52]. This is consistent with our results in this research that the complementation of OsBTBZ1 in the Atbt3 mutant background could restore the susceptible phenotypes of the Atbt3 mutant under ABA treatment (Figure 7, Figure 8 and Figure 9). OS07T0626600-01 is a putative MYST-like histone acetyltransferase 1 (histone acetyltransferase), which may be involved in transcriptional activation. The involvement of regulation via acetylation and chromatin remodeling is consistent with an earlier report, which stated that the acetylation levels of histone H3 at K9 in maize increase during salt stress [53]. OS01T0884500-01 is another protein in the PPI of OsBTBZ1 that belongs to a group of protein-like zF (CCCH-type). In general, zinc fingers are of the C2H2-type or CCCC-type, being grouped by the configuration of cysteine and histidine. The less prevalent CCCH zF proteins are crucial for controlling plant stress responses [54].
OsBTBZ1 is also associated with OS03T0216600-01, a defense response protein elicited by PAMPs. This suggests an interaction between the responses to biotic and abiotic stresses. OsJ_06167, a putative WRKY transcription factor (TF), is one of the TFs that are involved in many biotic and abiotic stress regulations. Epigenetic, retrograde, and proteasome-mediated regulations enable WRKYs to attain dynamic cellular homeostatic reprogramming [55]. Li et al. [56] reported that the overexpression of SpWRKY1 in tobacco resulted in enhanced salt and drought stress tolerance by reducing lipid peroxidation, enhancing antioxidant enzyme activity, and maintaining photosynthesis. The promoter of OsBTBZ1 also had a motif that is associated with the WRKY protein. WRKY71OS and WBOXATNPR1 are involved in various stress responses and are mediated by gibberellic acid, ABA, and salicylic acid [57]. In rice, WRKY13 binds to multiple cis-elements to regulate abiotic and biotic stress [58]. OsJ_25984 belongs to the protein kinase superfamily. Kinases are necessary for signal transduction in many aspects of cellular regulation and metabolism. The regulation of plant growth and development and the plant’s responses to stress conditions involve protein kinases such as mitogen-activated protein kinase cascades, receptor-like kinases, sucrose nonfermenting1-related protein kinases, and calcium-dependent protein kinases [59].
The putative cis-element analysis also showed interesting results, summarizing the function of OsBTBZ1. The ABA-responsive element (ABRE) is the most conserved cis-element in plants [60,61]. The ABRE cis-elements control the transcriptional regulation of several genes in response to cytosolic Ca2+ [62]. CAMTA12 enhances drought tolerance in soybean and Arabidopsis by binding to the ABRE cis-elements [63]. Many TFs regulate both abiotic and biotic stress, such as cold, drought, heat, and salinity, by binding to different cis-elements [64]. The OsBTBZ1 promoter contains putative MYB-binding sites that are attached to the MYB transcription factor. MYB cis-elements, including MYBCORE, and MYB1AT, were detected in the promoter regions of OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7; however, the number of elements varied. They play essential roles in the regulation of many genes related to biotic and abiotic stresses [65]. ABRE and MYC play important roles in the ABA-induced activation of biotic and abiotic genes [66]. MYC also responds to drought stress [28]. For example, AtMYC2 and AtMYB2 specifically interact with MYB recognition sites to regulate the ABA genes related to biosynthesis and signaling [67]. In another study, a chromatin immunoprecipitation assay and effector-reporter coexpression assays of Nicotiana tabacum confirmed the relationship of MYB and WRKY cis-elements with the promoters of peroxidase, superoxide dismutase, and phenylalanine ammonia-lyase to regulate abiotic stress [68]. Additionally, MYB- and WRKY-related cis-elements were found to regulate the transcription of auxin-regulated genes [69]. Similarly, the dehydration-responsive element (DRE) is also an essential cis-element that regulates the drought response and other abiotic stresses in plants. DREB-responsive genes are regulated by the DREB cis-elements to mediate stress responses in plants [70,71]. In banana plants, DREBs mitigate heat and drought stress [71], whereas in soybean plants, the cis-elements of DREBs are important for proline accumulation to mediate the plant’s response to salt stress [72].
OsBTBZ1 was selected to confirm its role in salt tolerance by generating the revertant lines REV1 and REV2 in the Atbt3 Arabidopsis mutant. Upon adding 150 mM of mannitol, the expression of the OsBTBZ1 gene could increase the tolerance of the transgenic lines to salt stress more than the mutant line, but not to drought stress (Figure 7, Figure 8 and Figure 9). This suggested that the function of OsBTBZ1 was more specific to the salt stress response than to osmotic stress. Phenotypic complementation of the mutant line by the expression of OsBTBZ1 was also observed under the ABA treatment, suggesting that the mechanism via the ABA-dependent pathway is involved in OsBTBZ1 functioning, which is consistent with the model proposed by Chutimanukul et al. [31].

4. Materials and Methods

4.1. Transcriptome Analysis

A transcriptome study was conducted during the seedling and booting stages of the CSSL16 line. It is a chromosome substitution salt-tolerant line with a ‘KDML105’ genetic background from BC5F4, originally taken from a cross between ‘KDML105’ and DH105, which was selected as an abiotic stress-tolerant double haploid line. After the cross, F1 progeny was backcrossed to ‘KDML105’ for 5 generations and then, self-fertilized to create BC5F4. Marker-assisted selection was used to select those CSSLs with the abiotic stress-tolerant regions from DH105 [31,32]. Transcriptomic data were retrieved from the database of the National Center for Biotechnology Information under the BioProject IDs PRJNA507040 and PRJNA659381 [31]. Briefly, the CSSL16 line plants were grown under normal and salt stress conditions (75 mM of NaCl treatment). The total RNA from 21-day-old seedling leaves was extracted after 0 and 48 h of salt stress treatment, whereas the total RNA from the flag and second leaves at the booting stage was extracted at 0 h and 72 h after salt stress, respectively, using a plant RNA purification reagent (Invitrogen, USA). Genomic DNA was extracted using DNase I (Invitrogen, Waltham, MA, USA). A KAPA stranded RNA-Seq library preparation kit (Illumina, San Diego, CA, USA) was used to synthesize the cDNA libraries, which were sequenced using Illumina next-generation sequencing (Illumina, USA). The differentially expressed genes were identified using the DESeq tool, version 1.24.0 [73]. Genes with significantly different expressions were considered those with a p-value of < 0.01. The PC, sequencing data matrix, and box plot showing the quality of the transcriptome data are shown in Supplementary Figures S3–S5.

4.2. Phylogenetic Analysis and an In Silico Analysis of BTB Proteins in Oryza sativa

The amino acid sequences from the Rice Genome Annotation Project database were subjected to a motif search (https://www.genome.jp/tools/motif/ accessed on 10 October 2022) to predict the protein motifs [74]. All BTB proteins were retrieved from the Phytozome database, available at https://phytozome.jgi.doe.gov/pz/portal.html [75], accessed on 25 October 2022. Orthologous BTB proteins from A. thaliana were retrieved from the TAIR database (https://www.arabidopsis.org/ accessed on 25 October 2022). In total, 209 proteins (182 from rice and 27 from A. thaliana) containing the BTB domain were used to construct a phylogenetic tree. All BTB protein sequences were aligned in MEGAX, while for the maximum likelihood, the Jones–Taylor–Thornton (JTT) method was employed for phylogenetic tree construction. The phylogenetic tree thus obtained from MEGAX was visualized using iTOL, which is available at https://itol.embl.de/ [76] accessed on 3 November 2022. All the BTB genes obtained from the Phytozome database were subsequently subjected to the Oryzabase-integrated rice science database http://viewer.shigen.info/oryzavw/maptool/MapTool.do [77] (accessed on 28 October 2022).

4.3. Putative Promoter Analysis

A promoter analysis of BTBZ1 was carried out on a sequence retrieved from the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 1 November 2022) [37], from 0 to −2000 bps, and entered into the New Plant cis-acting regulatory DNA elements (New PLACE) website (https://www.dna.affrc.go.jp/PLACE/?action=newplace, accessed on 7 November 2023). The positions of stress-related cis-regulatory elements were visualized using the TBtools software, and the functions of these elements were mainly obtained from the New PLACE database and the published literature [78].

4.4. Protein-Protein Interaction (PPI) Based on the STRING Database

The PPI was predicted via STRING [39]. The LOC_Os01g66890 (OsBTBZ1), LOC_Os01g68020 (OsBTBZ2), LOC_Os02g38120 (OsBTBN3), and LOC_Os03g41350 (OsBTBN7) proteins have been named according to the Rice Genome Annotation project (http://rice.uga.edu, accessed on 20 November 2022). However, the STRING website recognizes the Rice Annotation Project Database (RAP-DB) ID. To facilitate this analysis, the locus numbers based on the Rice Genome Annotation Project were converted into RAP-DB IDs using the following tool (https://rapdb.dna.affrc.go.jp/tools/converter/run, accessed on 20 November 2022). The RAP-DB id of four BTB genes are as follows: Os01g0893400 (OsBTBZ1), Os01g0908200 (OsBTBZ2), Os02g0594700 (OsBTBN3), and Os03g0609800 (OsBTBN7). The interactome was produced using a full-string network based on automated text mining, high-throughput experiments, prior stored databases, and co-expression sources. False-positive and false-negative results were reduced using a high confidence score (0.700). The other parameters were set to the default values. Enrichment detection was used to predict a network that covered all the mapped proteins and their interconnections.

4.5. Detection of OsBTBZ1 Gene Expression

A quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate the expression profile of OsBTBZ1 in various tissues of Nipponbare rice varieties under normal conditions. Various tissues were selected at specific time points, as follows. In 15-day-old seedlings, the leaf blade and leaf sheath of the first fully expanded leaf, the second-youngest leaf, and the oldest leaf, including the root tissues, were collected for the gene expression study. Then, in 30-day-old plants, only the leaf blade of a fully expanded leaf was collected. At the reproductive stage, the leaf blade and leaf sheath of the flag leaf, the panicle, and the spikelets were collected to investigate gene expression. Three biological replicates were used for each analysis. Total RNA from all tissues was extracted using the GENEzolTM reagent GZR100, following the manufacturer’s protocol, and then treated with DNase I. cDNA synthesis was performed using the Accupower RT premix (Bioneer Inc., Alameda, USA) and using oligoDT(T)18 as a primer to produce 40 ng/μL cDNA. The synthesized cDNA was used as a template and qRT-PCR was conducted on a Luna Universal qPCR master mix M3003L (New England Biolabs Inc., Ipswich, MA, USA). The qRT-PCR conditions were: 95 °C for 60 s, followed by 39 cycles at 95 °C for 15 s, 61.5 °C for 30 s, and 95 °C for 5 s; furthermore, the melt curve and plate read were at 60–94 °C, along with an increase in temperature of 5 °C per 5 s. A qRT-PCR was conducted in triplicate for each sample. The negative control was performed without a template and those reactions containing OsEF-1α primers (Table 2) were used as the internal reference genes [79]. The coding sequence obtained from the rice genome annotation database [37] was used to design primers that were specific to OsBTBZ1 (Table 2). The average cycle threshold (Cq) values of the gene were normalized with the level of the OsEF-1α reference gene in the same sample and were then used to measure the relative gene expression using the method described by Pfaffl [80]. Gene expression was analyzed using the relative expression levels. The statistical program SPSS was used to conduct an analysis of variance (ANOVA, p < 0.05) and the means were compared using Duncan’s multiple range test.

4.6. Generation of Complementation and Over-Expression of Arabidopsis Lines with the OsBTBZ1 Gene

A full-length OsBTBZ1 cDNA clone, J023077N08, obtained from the NARO DNA Bank was cloned into the Escherichia coli DH5α strain and cultured onto LB semisolid medium (10 g tryptone, 5 g yeast extract, 10 g NaCl, and 7.5 g agar per liter of water) along with 100 µg/mL of ampicillin. Single colonies were selected and then cultured into the LB broth with ampicillin for 16 h at 37 °C and 200 rpm. The plasmid was extracted using the Presto mini plasmid kit according to the manufacturer’s protocol (Geneaid, Taiwan), and the sequence of the inserted fragment was determined using the M13 (-20) forward primer to validate the correct sequence of the OsBTBZ1 gene.
The expression vector was constructed using the Gateway system. The OsBTBZ1 gene was added with the CACC adaptor in 5′ ends, inserted into the pENTR D-TOPO plasmid (Thermo Fisher Scientific, Waltham, MA, USA) as a donor vector, and then cloned to the TOP10 E. coli strain using the heat shock method. The cells were then plated on an LB medium, supplemented with kanamycin (50 µg/mL), and were subsequently incubated at 37 °C overnight. The correct sequence of the plasmid host was utilized in the LR clonase reaction to switch the OsBTBZ1 gene from the donor vector pENTR to the pGWB512 and pGWB505 plasmids as the destination vectors. The destination vector was transferred to the E. coli strain DH5α and cultured on LB medium, supplemented with 50 µg/mL spectinomycin. Plasmids with the correct sequence were transferred to Agrobacterium tumefaciens GV3101-competent cells using the cold-shock method. To identify colonies with the inserted fragment, a polymerase chain reaction (PCR) was performed using the CaMV35S forward primer and the reverse primer in the OsBTBZ1 gene to identify the correct clone for plant transformation.

4.7. Transformation of A. thaliana

An OsBTBZ1 cDNA in the pGWB512 construct was inserted into Arabidopsis plants using the floral dip transformation [81]. The resulting T1 plants were positive for the OsBTBZ1 gene and were grown in soil to obtain T2 seeds, which were acquired from each transgenic line and then germinated on Murashige and Skoog (MS) medium, supplemented with 25 mg/L hygromycin. The 3:1 segregation ratio of resistance: sensitivity to hygromycin was determined to identify transgenic lines with a single insertion. The selected lines were then grown in soil to obtain homozygous T3 seeds, which were used for further characterization [82].

4.8. Subcellular Localization in Onion Inner Epidermal Cells

The agroinfiltration of onion (Allium cepa) inner epidermal cells was performed to observe the subcellular localization of OsBTBZ1. The Agrobacterium GV3101 harboring the CaMV35S::OsBTBZ1-GFP construct in pGWB505 was cultured into 5 mL LB, supplemented with the appropriate antibiotics (50 µg/mL spectinomycin, 25 µg/mL rifampicin, and 50 µg/mL gentamicin) at 28 °C for 1 d. Later, 250 µL of the culture was inoculated in 25 mL LB, supplemented with 10 mM MES (pH 5.6, 100 µM acetosyringone, and antibiotics), and grown at 28 °C to obtain an optical density (OD) at 600 of 0.8. Subsequently, the culture was centrifuged at 5000 rpm for 5 min. The cell pellet was resuspended in MMA liquid medium (10 mM MgCl2, 10 mM MES (pH 5.6), and 100 µM acetosyringone) to a final OD600 of 0.8–1.0 [83]. The mixture was incubated at room temperature (25–26 °C) for 3 h and then infiltrated using a 5 mL needleless syringe, with only 500 µL injected per spot in the onion epidermal cells [84]. The onions were incubated in dim light or in dark conditions at 22 °C under high humidity for 48 h. The Agrobacterium GV3101, containing CaMV35S::GFP, was used as a control and was treated in the same manner. The onion epidermal cell layers were cut into 1 × 1 cm2 squares, peeled, and transferred directly to glass slides. Subsequently, 40 µL of 1 µg/mL 4′,6-diamidino-2-phenylindole, or DAPI dye was added to the epidermal cell sections. The green fluorescence protein (GFP) signal was observed under a Zeiss microscope (ZEISS Axio 10, Göttingen, Germany) at an excitation wavelength of 488 nm.

4.9. Evaluation of the Effect of OsBTBZ1 Gene Expression in Transgenic Arabidopsis Lines

The WT Arabidopsis, Atbt3 mutants, two homozygous complemented lines, REV1 and REV2, and two ectopic expression lines in the WT background, OE1 and OE2, were used for phenotyping. The experiment was performed using a completely randomized design with three replicates. The seeds were germinated by subjecting the seeds to the 0.5× MS medium as a control or 0.5× MS medium supplemented with 150 mM NaCl, 1 µM ABA, or 150 mM mannitol. Germinating seeds were recorded from 0–11 d to calculate the germination rate.
To elucidate the growth responses, 7-day-old Arabidopsis seedlings of each line were grown in 0.5× MS medium and were then subjected to four treatments: 0.5× MS medium as a control, 0.5× MS medium, supplemented with 150 mM NaCl, 0.5× MS medium supplemented with 1 µM ABA, and 0.5× MS medium supplemented with 150 mM mannitol, to investigate the impact on fresh weight, root length, and pigment contents. All parameters were measured 0–6 d after initiating the treatments. Fresh weight was calculated as the weight per plant, while the root length was calculated as the change in root length during the experimental period. The pigments, chlorophyll a, chlorophyll b, and total carotenoid contents, were determined according to the procedures described by Wellburn [85]. All experiments were performed in at least three replicates. An ANOVA was performed using SPSS Statistical Software version 23 (IBM Corp., Armonk, USA), followed by Duncan’s multiple-range test, to compare the means of each parameter.

5. Conclusions

Our study indicates a clear role for OsBTBZ1 in salt tolerance in Arabidopsis. A role in salt tolerance in rice is consistent with the higher expression of this gene in the salt-tolerant line, CSSL16, compared to the original genetic background, ‘KDML105’ rice [31]. It is also consistent with the location of this gene on the salt tolerance QTL on chromosome 1 [31,86]. Therefore, this study can support the use of this gene and QTL for the improvement of salt tolerance in rice.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241914483/s1.

Author Contributions

Conceptualization, S.C., L.C. and T.B.; methodology, S.C. and L.C.; validation, T.B.S., P.C. and B.H.J.; formal analysis, T.B.S., B.H.J., S.C. and T.B.; investigation, T.B.S., P.C. and B.H.J.; resources, P.C. and S.C.; data curation, T.B.S. and B.H.J.; writing—original draft preparation, T.B.S.; writing—review and editing, S.C., L.C. and T.B.; visualization, T.B.S., B.H.J. and S.C.; supervision, S.C. and L.C.; project administration, S.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Network Strengthening Fund—Program 16 (B16F640103). TBS was supported by the RGJ-Asean-PHD Scholarships. BHJ and PC were supported by a C2F postdoctoral scholarship.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The transcriptome sequences are available in the NCBI biotechnology information database under Bioproject IDs: prjna507040 and prjna659381.

Acknowledgments

The authors would like to thank the Department of Biochemistry, Faculty of Science, Chulalongkorn University, for providing the camera for use in localization studies.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Flower, T.J. Improving crop salt tolerance. J. Exp. Bot. 2004, 55, 307–319. [Google Scholar] [CrossRef] [PubMed]
  2. Khatun, S.; Flowers, T.J. Effects of salinity on seed set in rice. Plant Cell Environ. 1995, 18, 61–67. [Google Scholar] [CrossRef]
  3. Zeng, L.; Shannon, M.C. Effects of salinity on grain yield and yield components of rice at different seeding densities. Agron. J. 2000, 92, 418–423. [Google Scholar] [CrossRef]
  4. Shahid, S.A.; Zaman, M.; Heng, L. Soil salinity: Historical perspectives and a world overview of the problem. In Guideline for Salinity Assessment; Mitigation; and Adaptation Using Nuclear and Related Techniques; Zaman, M., Shahid, S.A., Heng, L., Eds.; Springer International Publishing: Berlin/Heidelberg, Germany, 2018; pp. 43–53. [Google Scholar] [CrossRef]
  5. Roychoudhury, A.; Basu, S.; Sengupta, D.N. Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance. J. Plant Physiol. 2011, 168, 317–328. [Google Scholar] [CrossRef]
  6. Ammar, M.H.M.; Singh, R.K.; Singh, A.K.; Mohapatra, T.; Sharma, T.R.; Singh, N.K. Mapping QTLs for salinity tolerance at seedling stage in rice (Oryza sativa L.). In African Crop Science Conference Proceedings; African Crop Science Society: Kampala, Uganda, 2007; pp. 617–620. [Google Scholar]
  7. Bimpong, I.K.; Manneh, B.; Diop, B.; Ghislain, K.; Sow, A.; Amoah, N.K.A.; Gregorio, G.; Singh, R.K.; Ortiz, R.; Wopereis, M. New quantitative trait loci for enhancing adaptation to salinity in rice from Hasawi, a Saudi landrace into three african cultivars at the reproductive stage. Euphytica 2014, 200, 45–60. [Google Scholar] [CrossRef]
  8. Ghomi, K.; Rabiei, B.; Sabouri, H.; Sabouri, A. Mapping QTLs for traits related to salinity tolerance at seedling stage of rice (Oryza sativa L.): An agrigenomics study of an Iranian rice population. OMICS 2013, 17, 242–251. [Google Scholar] [CrossRef]
  9. Hossain, H.; Rahman, M.A.; Alam, M.S.; Singh, R.K. Mapping of quantitative trait loci associated with reproductive-stage salt tolerance in rice. J. Agron. Crop. Sci. 2015, 201, 17–31. [Google Scholar] [CrossRef]
  10. Koyama, M.L.; Levesley, A.; Koebner, R.M.; Flowers, T.J.; Yeo, A.R. Quantitative trait loci for component physiological traits determining salt tolerance in rice. Plant Physiol. 2001, 125, 406–422. [Google Scholar] [CrossRef]
  11. Lee, S.Y.; Ahn, J.H.; Cha, Y.S.; Yun, D.W.; Lee, M.C.; Ko, J.C.; Lee, K.S.; Eun, M.Y. Mapping QTLs related to salinity tolerance of rice at the young seedling stage. Plant Breed. 2007, 126, 43–46. [Google Scholar] [CrossRef]
  12. Lin, H.X.; Zhu, M.Z.; Yano, M.; Gao, J.P.; Liang, Z.W.; Su, W.A.; Hu, X.H.; Ren, Z.H.; Chao, D.Y. QTLs for Na+ and K+ uptake of the shoots and roots controlling rice salt tolerance. Theor. Appl. Genet. 2004, 108, 253–260. [Google Scholar] [CrossRef]
  13. Yao, M.; Wang, J.; Chen, H.; Zhang, H. Inheritance and QTL mapping of salt tolerance in rice. Rice Sci. 2005, 12, 25–32. [Google Scholar]
  14. Mohammadi, R.; Mendioro, M.S.; Diaz, G.Q.; Gregorio, G.B.; Singh, R.K. Mapping quantitative trait loci associated with yield and yield components under reproductive stage salinity stress in rice (Oryza sativa L.). J. Genet. 2013, 92, 433–443. [Google Scholar] [CrossRef] [PubMed]
  15. Prasad, S.R.; Bagali, P.G.; Hittalmani, S.; Shashidhar, H.E. Molecular mapping of quantitative trait loci associated with seedling tolerance to salt stress in rice (Oryza sativa L.). Curr. Sci. 2000, 78, 162–164. [Google Scholar]
  16. Qiu, X.; Yuan, Z.; Liu, H.; Xiang, X.; Yang, L.; He, W.; Du, B.; Ye, G.; Xu, J.; Xing, D. Identification of salt tolerance-improving quantitative trait loci alleles from a salt-susceptible rice breeding line by introgression breeding. Plant Breed. 2015, 134, 653–660. [Google Scholar] [CrossRef]
  17. Ullah, M.A.; Abdullah-zawawi, M.-R.; Zainal-abidin, R.-A.; Sukiran, N.L.; Uddin, M.I.; Zainal, Z. A Review of Integrative Omic Approaches for Understanding Rice Salt Response Mechanisms. Plants 2022, 11, 1430. [Google Scholar] [CrossRef]
  18. Kojonna, T.; Suttiyut, T.; Khunpolwattana, N.; Pongpanich, M.; Suriya-arunroj, D.; Comai, L.; Buaboocha, T.; Chadchawan, S. Identification of a Negative Regulator for Salt Tolerance at Seedling Stage via a Genome-Wide Association Study of Thai Rice Populations. Int. J. Mol. Sci. 2022, 23, 1842. [Google Scholar] [CrossRef]
  19. Punchkhon, C.; Chutimanukul, P.; Chokwiwatkul, R.; Saputro, T.B.; Grennan, A.K.; Diego, N.D.; Spíchal, L.; Chadchawan, S. Role of LOC_Os01g68450, Containing DUF2358, in Salt Tolerance Is Mediated via Adaptation of Absorbed Light Energy Dissipation. Plants 2022, 11, 1233. [Google Scholar] [CrossRef]
  20. Yu, J.; Zhao, W.; Tong, W.; He, Q.; Yoon, M.Y.; Li, F.P.; Choi, B.; Heo, E.B.; Kim, K.W.; Park, Y.J. A genome-wide association study reveals candidate genes related to salt tolerance in rice (Oryza sativa) at the germination stage. Int. J. Mol. Sci. 2018, 19, 3145. [Google Scholar] [CrossRef]
  21. Duan, Y.; Zheng, H.; Wen, H.; Qu, D.; Cui, J.; Li, C.; Wang, J.; Liu, H.; Yang, L.; Jia, Y.; et al. Identification of Candidate Genes for Salt Tolerance at the Germination Stage in Japonica Rice by Genome-Wide Association Analysis. Agriculture 2022, 12, 1588. [Google Scholar] [CrossRef]
  22. Batayeva, D.; Labaco, B.; Ye, C.; Li, X.; Usenbekov, B.; Rysbekova, A.; Dyuskalieva, G.; Vergara, G.; Reinke, R.; Leung, H. Genome-wide association study of seedling stage salinity tolerance in temperate japonica rice germplasm. BMC Genet. 2018, 19, 2. [Google Scholar] [CrossRef]
  23. Kim, T.-H.; Kim, S.-M. Identification of Candidate Genes for Salt Tolerance at the Seedling Stage Using Integrated Genome-Wide Association Study and Transcriptome Analysis in Rice. Plants 2023, 12, 1401. [Google Scholar] [CrossRef] [PubMed]
  24. Xu, S.; Cui, J.; Cao, H.; Liang, S.; Ma, T.; Liu, H.; Wang, J.; Yang, L.; Xin, W.; Jia, Y.; et al. Identification of candidate genes for salinity tolerance in Japonica rice at the seedling stage based on genome-wide association study and linkage mapping. Front. Plant Sci. 2023, 14, 1184416. [Google Scholar] [CrossRef] [PubMed]
  25. Nayyeripasand, L.; Garoosi, G.A.; Ahmadikhah, A. Genome-Wide Association Study (GWAS) to Identify Salt-Tolerance QTLs Carrying Novel Candidate Genes in Rice During Early Vegetative Stage. Rice (N. Y.) 2021, 14, 9. [Google Scholar] [CrossRef] [PubMed]
  26. Yadav, A.K.; Kumar, A.; Grover, N.; Ellur, R.K.; Bollinedi, H.; Krishnan, S.G.; Bhowmick, P.K.; Vinod, K.K.; Nagarajan, M.; Singh, A.K. Genome-wide association study reveals marker–trait associations for early vegetative stage salinity tolerance in rice. Plants 2021, 10, 559. [Google Scholar] [CrossRef]
  27. Warraich, A.S.; Krishnamurthy, S.L.; Sooch, B.S.; Vinaykumar, N.M.; Dushyanthkumar, B.M.; Bose, J.; Sharma, P.C. Rice GWAS reveals key genomic regions essential for salinity tolerance at reproductive stage. Acta Physiol. Plant 2020, 42, 134. [Google Scholar] [CrossRef]
  28. Lekklar, C.; Pongpanich, M.; Suriya-arunroj, D.; Chinpongpanich, A.; Tsai, H.; Comai, L.; Chadchawan, S.; Buaboocha, T. Genome-wide association study for salinity tolerance at the flowering stage in a panel of rice accessions from Thailand. BMC Genom. 2019, 20, 76. [Google Scholar] [CrossRef] [PubMed]
  29. Wang, X.; Li, N.; Li, W.; Gao, X.; Cha, M.; Qin, L.; Liu, L. Advances in transcriptomics in the response to stress in plants. Glob. Med. Genet. 2020, 7, 30–34. [Google Scholar] [CrossRef]
  30. Lv, Y.; Ma, J.; Wei, H.; Xiao, F.; Wang, Y.; Jahan, N.; Hazman, M.; Qian, Q.; Shang, L.; Guo, L. Combining GWAS, Genome-Wide Domestication and a Transcriptomic Analysis Reveals the Loci and Natural Alleles of Salt Tolerance in Rice (Oryza sativa L.). Front. Plant Sci. 2022, 13, 912637. [Google Scholar] [CrossRef]
  31. Chutimanukul, P.; Saputro, T.B.; Mahaprom, P.; Plaimas, K.; Comai, L.; Buaboocha, T.; Siangliw, M.; Toojinda, T.; Chadchawan, S. Combining genome and gene co-expression network analyses for the identification of genes potentially regulating salt tolerance in rice. Front. Plant Sci. 2021, 12, 704549. [Google Scholar] [CrossRef]
  32. Kanjoo, V.; Jearakongman, S.; Punyawaew, K.; Siangliw, J.L.; Siangliw, M.; Vanavichit, A.; Toojinda, T. Co-location of quantitative trait loci for drought and salinity tolerance in rice. Thai J. Genet. 2011, 4, 126–138. [Google Scholar] [CrossRef]
  33. Kanjoo, V.; Punyawaew, K.; Siangliw, J.L.; Jearakongman, S.; Vanavichit, A.; Toojinda, T. Evaluation of agronomic traits in chromosome segment substitution lines of KDML105 containing drought tolerance QTL under drought stress. Rice Sci. 2012, 19, 117–124. [Google Scholar] [CrossRef]
  34. Zhou, Y.; Li, G.; Zhang, L.; Xu, J.; Hu, L.; Jiang, L.; Liu, S. Comprehensive genomic analysis and expression profiling of the BTB and TAZ (BT) genes in cucumber (Cucumis sativus L.). Czech J. Genet. Plant Breed 2020, 56, 15–23. [Google Scholar] [CrossRef]
  35. He, Y.-M.; Liu, K.-K.; Zhang, H.-X.; Cheng, G.-X.; Ali, M.; Ul Haq, S.; Wei, A.-M.; Gong, Z.-H. Contribution of CaBPM4; a BTB domain-containing gene; to the response of pepper to Phytophthora capsici infection and abiotic stresses. Agronomy 2019, 9, 417. [Google Scholar] [CrossRef]
  36. Zhou, Y.; Zhai, H.; He, S.; Zhu, H.; Gao, S.; Xing, S.; Wei, Z.; Zhao, N.; Liu, Q. The sweet potato BTB-TAZ protein gene; IbBT4; enhances drought tolerance in transgenic Arabidopsis. Front. Plant Sci. 2020, 11, 877. [Google Scholar] [CrossRef] [PubMed]
  37. Ouyang, S.; Zhu, W.; Hamilton, J.; Lin, H.; Campbell, M.; Childs, K.; Thibaud-Nissen, F.; Malek, R.L.; Lee, Y.; Zheng, L.; et al. The TIGR Rice Genome Annotation Resource: Improvements and new features. Nucleic Acids Res. 2007, 35, D883–D887. [Google Scholar] [CrossRef]
  38. Kang, H.; Fan, T.; Wu, J.; Zhu, Y.; Shen, W.H. Histone modification and chromatin remodeling in plant response to pathogens. Front. Plant Sci. 2022, 13, 986940. [Google Scholar] [CrossRef]
  39. Szklarczyk, D.; Gable, A.L.; Nastou, K.C.; Lyon, D.; Kirsch, R.; Pyysalo, S.T.; Doncheva, N.T.; Legeay, M.; Fang, T.; Bork, P. The STRING database in 2021: Customizable protein-protein networks; and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res. 2021, 49, D605–D612. [Google Scholar] [CrossRef]
  40. Weber, H.; Hellmann, H. Arabidopsis thaliana BTB/POZ-MATH proteins interact with members of the ERF/AP2 transcription factor family. FEBS J. 2009, 276, 6624–6635. [Google Scholar] [CrossRef]
  41. Wan, X.; Peng, L.; Xiong, J.; Li, X.; Wang, J.; Li, X.; Yang, Y. AtSIBP1, a novel BTB domain-containing protein; positively regulates salt signaling in Arabidopsis thaliana. Plants 2019, 8, 573. [Google Scholar] [CrossRef]
  42. Li, J.; Su, X.; Wang, Y.; Yang, W.; Pan, Y.; Su, C.; Zhang, X. Genome-wide identification and expression analysis of the BTB domain-containing protein gene family in tomato. Genes Genom. 2018, 40, 1–15. [Google Scholar] [CrossRef]
  43. Robert, H.S.; Quint, A.; Brand, D.; Vivian-Smith, A.; Offringa, R. BTB and TAZ domain scaffold proteins perform a crucial function in Arabidopsis development. Plant J. 2009, 58, 109–121. [Google Scholar] [CrossRef] [PubMed]
  44. Perez-Torrado, R.; Yamada, D.; Defossez, P.A. Born to bind: The BTB protein-protein interaction domain. BioEssays 2006, 28, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  45. Gingerich, D.J.; Hanada, K.; Shiu, S.H.; Vierstra, R.D. Large-scale; lineage-specific expansion of a bric-a-brac/tramtrack/broad complex ubiquitin-ligase gene family in rice. Plant Cell 2007, 19, 2329–2348. [Google Scholar] [CrossRef] [PubMed]
  46. Ban, Z.; Estelle, M. CUL3 E3 ligases in plant development and environmental response. Nat. Plants 2021, 7, 6–16. [Google Scholar] [CrossRef] [PubMed]
  47. Roberts, D.; Pedmale, U.V.; Morrow, J.; Sachdev, S.; Lechner, E.; Tang, X.; Zheng, N.; Hannink, M.; Genschik, P.; Liscum, E. Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-ring E3 ubiquitin ligase CRL3(NPH3). Plant Cell 2011, 23, 3627–3640. [Google Scholar] [CrossRef]
  48. Inada, S.; Ohgishi, M.; Mayama, T.; Okada, K.; Sakai, T. RPT2 is a signal transducer involved in a phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana. Plant Cell 2004, 16, 887–896. [Google Scholar] [CrossRef]
  49. Hashiguchi, A.; Komatsu, S. Impact of post-translational modifications of crop proteins under abiotic stress. Proteomes 2016, 4, 42. [Google Scholar] [CrossRef]
  50. Hernando, C.E.; Sanchez, S.E.; Mancini, E.; Yanovsky, M.J. Genome wide comparative analysis of the effects of PRMT5 and PRMT4/CARM1 arginine methyltransferases on the Arabidopsis thaliana transcriptome. BMC Genom. 2015, 16, 192. [Google Scholar] [CrossRef]
  51. Zheng, M.; Liu, X.; Lin, J.; Liu, X.; Wang, Z.; Xin, M.; Yao, Y.; Peng, H.; Zhou, D.X.; Ni, Z.; et al. Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes. Plant J. 2019, 97, 587–602. [Google Scholar] [CrossRef]
  52. Ullah, F.; Xu, Q.; Zhao, Y.; Zhou, D.-X. Histone deacetylase HDA710 controls salt tolerance by regulating ABA signaling in rice. J. Integr. Plant Biol. 2021, 63, 451–467. [Google Scholar] [CrossRef]
  53. Li, H.; Yan, S.; Zhao, L.; Tan, J.; Zhang, Q.; Gao, F.; Wang, P.; Hou, H.; Li, L. Histone acetylation associated up-regulation of the cell wall related genes is involved in salt stress induced maize root swelling. BMC Plant Biol. 2014, 14. [Google Scholar] [CrossRef] [PubMed]
  54. Han, G.; Qiao, Z.; Li, Y.; Wang, C.; Wang, B. The roles of CCCH zinc-finger proteins in plant abiotic stress tolerance. Int. J. Mol. Sci. 2021, 22, 8327. [Google Scholar] [CrossRef] [PubMed]
  55. Phukan, U.J.; Jeena, G.S.; Shukla, R.K. WRKY transcription factors: Molecular regulation and stress responses in plants. Front. Plant Sci. 2016, 7, 760. [Google Scholar] [CrossRef] [PubMed]
  56. Li, J.B.; Luan, Y.S.; Liu, Z. Overexpression of SpWRKY1 promotes resistance to Phytophthora nicotianae and tolerance to salt and drought stress in transgenic tobacco. Physiol. Plant 2015, 155, 248–266. [Google Scholar] [CrossRef]
  57. Trivedi, D.K.; Ansari, M.W.; Tuteja, N. Multiple abiotic stress responsive rice cyclophilin: (OsCYP-25) mediates a wide range of cellular responses. Commun. Integr. Biol. 2013, 6, e25260. [Google Scholar] [CrossRef]
  58. Xiao, J.; Cheng, H.; Li, X.; Xiao, J.; Xu, C.; Wang, S. Rice WRKY13 regulates cross talk between abiotic and biotic stress signaling pathways by selective binding to different cis-elements. Plant Physiol. 2013, 163, 1868–1882. [Google Scholar] [CrossRef]
  59. Chen, X.; Ding, Y.; Yang, Y.; Song, C.; Wang, B.; Yang, S.; Guo, Y.; Gong, Z. Protein kinases in plant responses to drought; salt; and cold stress. J. Integr. Plant Biol. 2021, 63, 53–78. [Google Scholar] [CrossRef]
  60. Yamaguchi-Shinozaki, K.; Shinozaki, K. Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci. 2005, 10, 88–94. [Google Scholar] [CrossRef]
  61. Nakashima, K.; Yamaguchi-Shinozaki, K. ABA signaling in stress-response and seed development. Plant Cell Rep. 2013, 32, 959–970. [Google Scholar] [CrossRef]
  62. Kaplan, B.; Davydov, O.; Knight, H.; Galon, Y.; Knight, M.R.; Fluhr, R.; Fromm, H. Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. Plant Cell 2006, 18, 2733–2748. [Google Scholar] [CrossRef]
  63. Noman, M.; Jameel, A.; Qiang, W.D.; Ahmad, N.; Liu, W.C.; Wang, F.W.; Li, H.Y. Overexpression of GmCAMTA12 enhanced drought tolerance in Arabidopsis and soybean. Int. J. Mol. Sci. 2019, 20, 4849. [Google Scholar] [CrossRef] [PubMed]
  64. Cheng, M.C.; Liao, P.M.; Kuo, W.W.; Lin, T.P. The Arabidopsis ethylene RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals. Plant Physiol. 2013, 162, 1566–1582. [Google Scholar] [CrossRef] [PubMed]
  65. Ma, D.; Constabel, C.P. MYB repressors as regulators of phenylpropanoid metabolism in plants. Trends Plant Sci. 2019, 24, 275–289. [Google Scholar] [CrossRef] [PubMed]
  66. Li, R.; Zhu, F.; Duan, D. Function analysis and stress-mediated cis-element identification in the promoter region of VqMYB15. Plant Signal. Behav. 2020, 15, 1773664. [Google Scholar] [CrossRef]
  67. Abe, H.; Urao, T.; Ito, T.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 2003, 15, 63–78. [Google Scholar] [CrossRef]
  68. Guo, H.; Wang, L.; Yang, C.; Zhang, Y.; Zhang, C.; Wang, C. Identification of novel cis-elements bound by BplMYB46 involved in abiotic stress responses and secondary wall deposition. J. Integr. Plant Biol. 2018, 60, 1000–1014. [Google Scholar] [CrossRef]
  69. Berendzen, K.W.; Weiste, C.; Wanke, D.; Kilian, J.; Harter, K.; Dröge-Laser, W. Bioinformatic cis-element analyses performed in Arabidopsis and rice disclose bZIP- and MYB-related binding sites as potential AuxRE-coupling elements in auxin-mediated transcription. BMC Plant Biol. 2012, 12, 125. [Google Scholar] [CrossRef]
  70. Lata, C.; Prasad, M. Role of DREBs in regulation of abiotic stress responses in plants. J. Exp. Bot. 2011, 62, 4731–4748. [Google Scholar] [CrossRef]
  71. Baral, A. Bananas tackling drought and heat—With DREBs and more. Physiol. Plant 2019, 165, 128–130. [Google Scholar] [CrossRef]
  72. Nguyen, Q.H.; Vu, L.T.K.; Nguyen, L.T.N.; Pham, N.T.T.; Nguyen, Y.T.H.; Le, S.V.; Chu, M.H. Overexpression of the GmDREB6 gene enhances proline accumulation and salt tolerance in genetically modified soybean plants. Sci. Rep. 2019, 9, 19663. [Google Scholar] [CrossRef]
  73. Anders, S.; Huber, W. Differential expression analysis for sequence count data. Genome Biol. 2010, 11, R106. [Google Scholar] [CrossRef]
  74. Kanehisa, M.; Goto, S.; Kawashima, S.; Nakaya, A. The KEGG databases at GenomeNet. Nucleic Acids Res. 2002, 30, 42–46. [Google Scholar] [CrossRef] [PubMed]
  75. Goodstein, D.M.; Shu, S.; Howson, R.; Neupane, R.; Hayes, R.D.; Fazo, J.; Mitros, T.; Dirks, W.; Hellsten, U.; Putnam, N.; et al. Phytozome: A comparative platform for green plant genomics. Nucleic Acids Res. 2012, 40, D1178–D1186. [Google Scholar] [CrossRef] [PubMed]
  76. Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49, W293–W296. [Google Scholar] [CrossRef] [PubMed]
  77. Kurata, N.; Yamazaki, Y. Oryzabase. an integrated biological and genome information database for rice. Plant Physiol. 2006, 140, 12–17. [Google Scholar] [CrossRef]
  78. Higo, K.; Ugawa, Y.; Iwamoto, M.; Korenaga, T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 1999, 27, 297–300. [Google Scholar] [CrossRef]
  79. Udomchalothorn, T.; Plaimas, K.; Sripinyowanich, S.; Boonchai, C.; Kojonna, T.; Chutimanukul, P.; Comai, L.; Buaboocha, T.; Chadchawan, S. OsNucleolin1-L expression in Arabidopsis enhances photosynthesis via transcriptome modification under salt stress conditions. Plant Cell Physiol. 2017, 58, 717–734. [Google Scholar] [CrossRef]
  80. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29, 2002–2007. [Google Scholar] [CrossRef]
  81. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735–743. [Google Scholar] [CrossRef]
  82. Endo, A.; Egawa, C.; Oohashi, M.; Maoka, A.M.; Shimosaka, E.; Sato, Y. Ectopic expression of mutated type 2C protein phosphatase OsABI-LIKE2 decreases abscisic acid sensitivity in Arabidopsis and rice. Sci. Rep. 2018, 8, 12320. [Google Scholar] [CrossRef]
  83. Liu, Z.Z.; Wang, J.L.; Huang, X.; Xu, W.H.; Liu, Z.M.; Fang, R.X. The promoter of a rice glycine-rich protein gene; Osgrp-2; confers vascular-specific expression in transgenic plants. Planta 2003, 216, 824–833. [Google Scholar] [CrossRef] [PubMed]
  84. Xu, K.; Huang, X.; Wu, M.; Wang, Y.; Chang, Y.; Liu, K.; Zhang, J.; Zhang, Y.; Zhang, F.; Yi, L.; et al. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis. PLoS ONE 2014, 9, e83556. [Google Scholar] [CrossRef] [PubMed]
  85. Wellburn, A.R. The Spectral determination of chlorophyll a and b; as well as total carotenoids; using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 1994, 144, 307–313. [Google Scholar] [CrossRef]
  86. Chutimanukul, P.; Kositsup, B.; Plaimas, K.; Buaboocha, T.; Siangliw, M.; Toojinda, T.; Comai, L.; Chadchawan, S. Photosynthetic responses and identification of salt tolerance genes in a chromosome segment substitution line of ‘Khao Dawk Mali 105′ rice. Environ. Exp. Bot. 2018, 155, 497–508. [Google Scholar] [CrossRef]
Ijms 24 14483 g001
Figure 1. Venn diagram showing the intersection of DEseq data with the BTB protein in O. sativa (A). BTB, zf-TAZ, and NPH3 domains are present in the differentially expressed BTB genes under salt stress conditions, namely, OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 (B).
Figure 1. Venn diagram showing the intersection of DEseq data with the BTB protein in O. sativa (A). BTB, zf-TAZ, and NPH3 domains are present in the differentially expressed BTB genes under salt stress conditions, namely, OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7 (B).
Ijms 24 14483 g001
Ijms 24 14483 g002
Figure 2. Phylogenetic tree and the position of BTB genes in the rice chromosome. (A) Maximum likelihood phylogenetic tree. The tree was constructed using the amino acid sequences of 27 BTB genes in Arabidopsis and 182 BTB genes in rice. LOC_Os01g66890 (BTBZ1) was marked, along with LOC_Os01g68020, LOC_Os02g38120, and LOC_Os03g41350. (B) The chromosomal location shows the distribution of the BTB gene family in the 12 rice chromosomes.
Figure 2. Phylogenetic tree and the position of BTB genes in the rice chromosome. (A) Maximum likelihood phylogenetic tree. The tree was constructed using the amino acid sequences of 27 BTB genes in Arabidopsis and 182 BTB genes in rice. LOC_Os01g66890 (BTBZ1) was marked, along with LOC_Os01g68020, LOC_Os02g38120, and LOC_Os03g41350. (B) The chromosomal location shows the distribution of the BTB gene family in the 12 rice chromosomes.
Ijms 24 14483 g002
Ijms 24 14483 g003
Figure 3. Prediction of the cis-elements related to stress in OsBTBZ1, OsBTBZ2, OsBTBN3, and the OsBTBN7 promoter.
Figure 3. Prediction of the cis-elements related to stress in OsBTBZ1, OsBTBZ2, OsBTBN3, and the OsBTBN7 promoter.
Ijms 24 14483 g003
Ijms 24 14483 g004
Figure 4. The protein-protein interaction (PPI) network assembly of LOC_Os01g66890 (A: OsBTBZ1), LOC_Os01g68020 (B: OsBTBZ2), LOC_Os02g38120 (C: OsBTBN3), and LOC_Os03g41350 (D: OsBTBN7) proteins, identified using STRING. The lines represent a high confidence PPI score of 0.7.
Figure 4. The protein-protein interaction (PPI) network assembly of LOC_Os01g66890 (A: OsBTBZ1), LOC_Os01g68020 (B: OsBTBZ2), LOC_Os02g38120 (C: OsBTBN3), and LOC_Os03g41350 (D: OsBTBN7) proteins, identified using STRING. The lines represent a high confidence PPI score of 0.7.
Ijms 24 14483 g004
Ijms 24 14483 g005
Figure 5. OsBTBZ1 gene expression in different tissues of “Nipponbare” rice. B = leaf blade; S = leaf sheath; P = peduncle; R = root; Sp = spikelet. The different lowercase letters above the bars mean the significant difference between means analyzed by Duncan’s multiple range test at p < 0.05.
Figure 5. OsBTBZ1 gene expression in different tissues of “Nipponbare” rice. B = leaf blade; S = leaf sheath; P = peduncle; R = root; Sp = spikelet. The different lowercase letters above the bars mean the significant difference between means analyzed by Duncan’s multiple range test at p < 0.05.
Ijms 24 14483 g005
Ijms 24 14483 g006
Figure 6. The subcellular localization of OsBTBZ1-GFP in onion (Allium cepa) epidermal cells. Red arrows point to the location of the nucleus.
Figure 6. The subcellular localization of OsBTBZ1-GFP in onion (Allium cepa) epidermal cells. Red arrows point to the location of the nucleus.
Ijms 24 14483 g006
Ijms 24 14483 g007
Figure 7. Germination tests of the wild-type (WT), mutant, and transgenic lines in several different media. (A) The lines of WT, bt3 mutant, REV1, REV2, OE1, and OE2 in ½ MS basal medium as a control, medium with NaCl 150 mM, ABA 1µM, and mannitol 150 mM (line mark = 1 cm). (B) Germination curve in the control medium (½ MS medium). (C) Germination curve in the ½ MS medium containing 150 mM NaCl. (D) Germination curve in the ½ MS medium containing 1 µM ABA. (E) Germination curve in ½ MS medium containing 150 mM mannitol.
Figure 7. Germination tests of the wild-type (WT), mutant, and transgenic lines in several different media. (A) The lines of WT, bt3 mutant, REV1, REV2, OE1, and OE2 in ½ MS basal medium as a control, medium with NaCl 150 mM, ABA 1µM, and mannitol 150 mM (line mark = 1 cm). (B) Germination curve in the control medium (½ MS medium). (C) Germination curve in the ½ MS medium containing 150 mM NaCl. (D) Germination curve in the ½ MS medium containing 1 µM ABA. (E) Germination curve in ½ MS medium containing 150 mM mannitol.
Ijms 24 14483 g007
Ijms 24 14483 g008
Figure 8. Growth (A,D,G,J) WT, bt3 mutant, and transgenic lines of 7-day-old Arabidopsis seedlings in various conditions: normal (control), supplemented with 150 mM NaCl, 1 mM ABA, or 150 mM mannitol on day 6 of stress exposure. (B,E,H,K): fresh weight of plants; (C,F,I,L): Δ root length of plants. The different letters above the bars represent the significant difference in means at p < 0.05 and NS represents no significant difference.
Figure 8. Growth (A,D,G,J) WT, bt3 mutant, and transgenic lines of 7-day-old Arabidopsis seedlings in various conditions: normal (control), supplemented with 150 mM NaCl, 1 mM ABA, or 150 mM mannitol on day 6 of stress exposure. (B,E,H,K): fresh weight of plants; (C,F,I,L): Δ root length of plants. The different letters above the bars represent the significant difference in means at p < 0.05 and NS represents no significant difference.
Ijms 24 14483 g008
Ijms 24 14483 g009
Figure 9. Contents of pigments, namely, Chl a, (A,D,G,J), Chl b, (B,E,H,K), and carotenoids (C,F,I,L) of 13-day-old Arabidopsis seedlings treated with 150 mM NaCl, 1 mM ABA, or 150 mM mannitol for 6 d. The data were collected from the WT and bt3 complemented lines with the OsBTBZ1 gene in the bt3 mutant background (Rev1 and Rev2) and the OsBTBZ1 ectopic expression line with a WT background (OE1 and OE2). The different letters above the bars represent the significant difference in means at p < 0.05 and NS represents no significant difference.
Figure 9. Contents of pigments, namely, Chl a, (A,D,G,J), Chl b, (B,E,H,K), and carotenoids (C,F,I,L) of 13-day-old Arabidopsis seedlings treated with 150 mM NaCl, 1 mM ABA, or 150 mM mannitol for 6 d. The data were collected from the WT and bt3 complemented lines with the OsBTBZ1 gene in the bt3 mutant background (Rev1 and Rev2) and the OsBTBZ1 ectopic expression line with a WT background (OE1 and OE2). The different letters above the bars represent the significant difference in means at p < 0.05 and NS represents no significant difference.
Ijms 24 14483 g009
Table 1. The number of Stress-related cis-elements detected in OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7.
Table 1. The number of Stress-related cis-elements detected in OsBTBZ1, OsBTBZ2, OsBTBN3, and OsBTBN7.
Factor or Site NameSignal
Sequence		OsBTBZ1OsBTBZ2OsBTBN3OsBTBN7Functions
Strand (+)Strand
(−)		Strand (+)Strand
(−)		Strand (+)Strand (−)Strand (+)Strand (−)
ABREATCONSENSUSYACGTGGC 1-------ABA-responsive element
ABRELATERD1ACGTG33-611-1ERD-related gene (early responsive to dehydration)
ACGTATERD1ACGT55884411Abiotic stresses (drought, salt); response to light
CBFHVRYCGAC 1322-4--Dehydration-responsive element (DRE) binding proteins (DREBs)
CCAATBOX1CCAAT-2-111-2Heat-shock element
CURECORECRGTAC55333355Oxygen-responsive element
DPBFCOREDCDC3ACACNNG43131132Dehydration and ABA response
GAGAGMGSA1GAGAGAGAGAGAGAGAGA2----1--GAGA binding protein (GBP) binding site
GT1GMSCAM4GAAAAA11-15362Plays a role in pathogen- and salt-induced SCaM-4 gene expression
MYB1ATWAACCA 21421112Element for the dehydration-responsive gene in Arabidopsis
MYBCORECNGTTR 56243311Responsive to water stress, induced by dehydration stress
MYCATERD1CATGTG1-2-122-Necessary for ERD1 expression, the binding site of the NAC protein
MYCCONSENSUSATCANNTG16161010121244Element for the dehydration-responsive gene in Arabidopsis
RAV1AATCAACA5421-361Rosette leaves and the root-specific element, growth, and development, hormonal regulation brassinosteroid (BR); metabolism
WBOXATNPR1TTGAC22131121Salicylic acid-induced WRKY DNA binding proteins
WRKY71OSTGAC863118234Involved in gibberellic acid (GA), ABA-mediated pathways, and pathogen-related protein (PR)
Table 2. Primer sequence for qRT-PCR to detect gene expression.
Table 2. Primer sequence for qRT-PCR to detect gene expression.
NameSequence 5′→3′Tm (°C)
qPCR_OsBTBZ1_FWTTCCTGCCTGCAAGGGCATC63
qPCR_OsBTBZ1_REVTCCTTGAAATGCCTACAGAGGGG60
qPCR_OsEF1α_FWATGGTTGTGGAGACCTTC53
qPCR_OsEF1α_REVTCACCTTGGCACCGGTTG60
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Saputro, T.B.; Jakada, B.H.; Chutimanukul, P.; Comai, L.; Buaboocha, T.; Chadchawan, S. OsBTBZ1 Confers Salt Stress Tolerance in Arabidopsis thaliana. Int. J. Mol. Sci. 2023, 24, 14483. https://doi.org/10.3390/ijms241914483

AMA Style

Saputro TB, Jakada BH, Chutimanukul P, Comai L, Buaboocha T, Chadchawan S. OsBTBZ1 Confers Salt Stress Tolerance in Arabidopsis thaliana. International Journal of Molecular Sciences. 2023; 24(19):14483. https://doi.org/10.3390/ijms241914483

Chicago/Turabian Style

Saputro, Triono B., Bello H. Jakada, Panita Chutimanukul, Luca Comai, Teerapong Buaboocha, and Supachitra Chadchawan. 2023. "OsBTBZ1 Confers Salt Stress Tolerance in Arabidopsis thaliana" International Journal of Molecular Sciences 24, no. 19: 14483. https://doi.org/10.3390/ijms241914483

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop