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Article

Genome-Wide Identification Reveals That BZR1 Family Transcription Factors Involved in Hormones and Abiotic Stresses Response of Lotus (Nelumbo)

by
Ping Zhou
1,
Huiyan Jiang
1,
Jingwen Li
2,
Qijiang Jin
1,
Yanjie Wang
1 and
Yingchun Xu
1,*
1
Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
2
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(8), 882; https://doi.org/10.3390/horticulturae9080882
Submission received: 13 July 2023 / Revised: 28 July 2023 / Accepted: 29 July 2023 / Published: 3 August 2023
(This article belongs to the Section Biotic and Abiotic Stress)
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Abstract

:
The brassinazole-resistant (BZR) transcription factors (TFs) are key components of brassinosteroid (BR) signaling, which play an important role in regulating plant growth and development as well as responding to abiotic stress. However, a functional study of BZR transcription factors in lotuses has not been reported. A total 10 BZR1 genes (four NnBZR1 and six NlBZR1) were identified from the genomes of two lotus species (Nelumbo nucifera and Nelumbo lutea). The construction of the phylogenetic tree showed that the 10 BZR1 genes of the lotus were divided into four groups; the NnBZR1s and NlBZR1s were unevenly distributed on three and four chromosomes, respectively. Gene structure analysis showed that motif 1 and motif 9 are highly conserved in the lotus BZR1 protein, which might be related to the conserved domain BES_N of BZR1. The analysis of promoter cis-acting elements showed that the promoters of most of the BZR1 genes in the lotus contained elements related to light-responsive, ABA-responsive and abiotic stress-responsive factors, indicating that the BZR1 gene of the lotus played an important role in its response to abiotic stress. The responses of BZR1 genes to BR, ABA and four abiotic stresses (Cold, PEG6000, Cd and NaCl) were analyzed by qRT-PCR. The qRT-PCR results further verified that the lotus BZR1 genes play an important role in responding to hormone signals and resisting abiotic stress. This study laid the foundation for further research on the function of lotus BZR1 genes and provided a theoretical basis for future breeding and horticultural applications.

1. Introduction

Brassinosteroid (BR) is considered as a plant growth hormone due to its ability to affect the elongation and division of plant cells [1,2], and it is the sixth hormone discovered after auxin (IAA), cytokinin (CTK), ethylene (ETH), gibberellins (GA) and abscisic acid (ABA) [3]. Subsequent studies have shown that BR is involved in regulating many aspects of plant growth and development, such as seed germination, plant structure, vascular differentiation, stomata formation, flowering, male fertility, senescence, and stress resistance, etc. [3,4,5,6,7,8].
Over the past few decades, the BR signaling pathway has been extensively explored using molecular genetics, biochemistry, genomics, bioinformatics, and structural biology, and the use of the BR signal transduction pathway and regulatory network from cell membrane receptors to transcription factors in the nucleus to regulate the expression of downstream target genes was established [9]. The phosphorylation of protein kinase and phosphorylation and dephosphorylation of transcription factors are important biochemical regulatory mechanisms of BR signal transduction [10]. BR signaling is perceived by leucine-rich repeat (LRR) receptor-like kinase (brassinolide-insensitive 1, BRI1 and its co-receptor BRI1-related receptor kinase 1, BAK1) at the cell surface [11,12,13,14]. After BR recognition, the BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) are activated and translocated to the nucleus, where they regulate the transcription of downstream target genes by binding to BR response elements in their promoters (BRREs, CGTGT/CG) and E-box element (CANNTG) s [15,16].
BZR1/BES1 transcription factors play an important role in BR signal transduction. After responding to BR signaling, they are able to regulate downstream target genes at the transcriptional level to participate in various physiological processes and the growth regulation of plants [17]. BZR1/BES1 transcription factors are considered to belong to the same transcription factor family due to their amino acid sequence similarity of 88% and DNA-binding domain sequence identity of up to 97% [18,19]. BES1/BZR1 have two conserved lysine residues, K280 and K320, and they are also coupling sites for small ubiquitin-like modifications (SUMOs); these modifiers are able to alter the function of BZR1/BES1 by post-translational modification [20]. There are six members of the BZR1/BES1 gene family in Arabidopsis, and studies have shown that BES1 is a major factor in the transcriptional activation or repression of multiple target genes in Arabidopsis [21]. Through chromatin immunoprecipitation microarray (ChIP-chip) technology, a large number of putative target genes of BES1/BZR1 have been identified, reaching 1609 and 3410, respectively. Among the target genes of BES1/BZR1, many target genes are involved in phytohormones and stress signals, such as ABA, salt and cold, etc. [22,23]. BES1/BZR1 are not only master regulators of the BR signal; they also play key roles in other regulatory networks. For example, the dominant mutants of bzr1-D and bes1-D exhibited delayed flowering, wider dark green leaves, and up-regulated expression of BR-responsive genes in Arabidopsis [19,24]. In Arabidopsis, BZR1 positively regulates plant frost tolerance through CBF-dependent and CBF-independent pathways [25]. BZR can also regulate the expression of drought-responsive glutathione s-transferase 1 (GST1) and interact with RD26 and WRKY transcription factors to modulate plant responses to drought, high temperature and freezing stress [25,26].
As a theme plant in the layout of waterscape gardens, lotuses (Nelumbo) can purify the sediment and water environment, improving water quality, and it has huge ecological and environmental benefits [27]. Therefore, lotuses are often used to restore polluted water resources and improve the ecological environment of lakes. Abiotic stresses such as flood, high temperature, salt, low light and heavy metals are the main stresses faced by the lotus during its growth and development [27]. BZR1/BES1 transcription factors have been reported to be involved in the regulation of plant growth, development and response to abiotic stresses in many plants. During the study of BZR1/BES1 gene function, the identification of the BZR1/BES1 gene family and understanding of its regulatory mechanisms in plants have become a hot topic of research in recent years. However, the BES/BZR transcription factor family has only been identified and characterized in limited plant species at present, such as Arabidopsis [19,24], maize (Zea mays L.) [28], but it has not been reported in lotus. Therefore, the exploration of the BZR1 family in the whole genome of lotuses can fill the gap in the study of BR signaling in lotuses. At the same time, it can further understand the regulatory mechanism of the lotus during growth, development and response to abiotic stresses, and it can provide insight for selecting lotus cultivars with excellent agronomic traits.
The BZR1 gene family plays an important regulatory role in response to abiotic stresses. However, there is a lack of studies in lotuses. In order to fill the gap of the BZR1 gene family in lotus research, this study identified six NlBZR1 and four NnBZR1 gene family members in the genomes of two lotus species (Nelumbo nucifera and Nelumbo lutea), respectively. Their gene maps, phylogenetic relationships, conserved protein motif structures and cis-acting regulatory elements in promoters were analyzed. The response of BZR1 genes to different hormonal and abiotic stress treatments were further analyzed. The results of this study provide valuable information for future studies on the functions of the BZR1 gene family in lotuses, and they help to further understand the molecular mechanisms of BZR1 to regulate the response of lotuses to abiotic stresses.

2. Materials and Methods

2.1. Plant Materials and Abiotic Stress Treatment

The plant material is an ancient lotus seedling preserved by the Longtan Experimental Base of the Aquatic Flower Research Group in Nanjing Agricultural University, China. The lotus seedlings were transplanted into the soil, and the suitable growth conditions were set to 16/8 h light–dark cycle, 32 day/26 °C night and 60% relative humidity, and stress treatment was carried out when the seedlings grew 6–7 floating leaves. For the hormone treatment, seedlings were immersed in 5 μM BR solution or 30 μM ABA solution. For the low-temperature treatment, the seedlings were placed in a low-temperature environment at 4 °C. For drought treatment, the 25% PEG6000 solution was used to simulate drought stress-like conditions. For Cd treatment, seedlings were immersed in 40 mM CdCl2 water for medium Cd treatment. For salt treatment, seedlings were soaked in 400 mM NaCl solution. Seedings were collected at 6, 12, 24 and 48 h after stress treatment, respectively. The seedings were collected (at least three different seedlings were taken as replicates), immediately frozen in liquid nitrogen (LN2), and stored at −80 °C.

2.2. Identification of BZR1 Genes in N. nucifera (Nelumbo nucifera) Genome and N. lutea (Nelumbo lutea) Genome

The BZR1 gene sequences of N. nucifera and N. lutea were queried using hmmer-3.3.2 software based on the BES1_N (PF05687) conserved domain model, which was downloaded from the Pfam database (http://pfam.xfam.org accessed on 20 December 2022). The genome information of N. nucifera and N. lutea was downloaded from http://nelumbo.cngb.org/nelumbo/home (accessed on 20 December 2022) and NCBI (https://www.ncbi.nlm.nih.gov/ accessed on 20 December 2022) under BioProject number PRJNA747731, respectively. The 6 AtBZR1 genes in the Arabidopsis genome were downloaded from the database (https://www.arabidopsis.org/ accessed on 20 December 2022) with the corresponding gene IDs (At1G19350, At1G75080, At3G50750, At4G36780, At4G18890 and At1G78700). Based on the 6 BZR1 gene sequences from Arabidopsis thaliana, all BZR1 genes in the two genomes of lotus were further identified using BLASTp-2.13.0 software. To ensure the validity of the identified BZR1 genes, the conserved domain analysis was performed on the amino acid sequence of the extracted lotus BZR1 genes using the Simple Modular Architecture Research Tool (SMART: http://smart.embl-heidelberg.de accessed on 21 December 2022).

2.3. Predicted Subcellular Localization of BZR1 Protein

The Cell-PLoc 2.0 database (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ accessed on 22 December 2022) [29] was used to predict the subcellular localization of the BZR1 genes based on the BZR1 protein sequence.

2.4. Phylogenetic Analysis and Classification

In order to analyze the evolutionary relationship and origin of BZR1 transcription factors in N. colorata (Nymphaea colorata), N. nucifera, N. lutea, Vitis vinifera and Arabidopsis, the full-length amino acid sequences of transcription factors were aligned using muscle 2022 software under default parameters, and neighbor-joining (neighbor-joining NJ) sequences were constructed using MEGA 7.0 software [30]; the phylogenetic tree had a bootstrap replication number of 1000. The BZR1 transcription factors were divided into different groups according to the topology of the phylogenetic tree.
In order to study the expression characteristics of BZR1 genes in N. nucifera and N. lutea, the sequence of 2000 bp upstream of the initiation codon (ATG) of BZR1 genes was obtained as the promoter region. The phylogenetic tree of the promoter sequences was constructed using MEGA 7.0 software, and 1000 bootstrap replications were performed.

2.5. Chromosomal Locations and Gene Structures of BZR1 Genes

The length and position information of the lotus BZR1 genes were extracted from the gff files of the N. nucifera and N. lutea genomes. The molecular weight (MW) and isoelectric point (pI) of the BZR1 genes were calculated using the ProtParam tool in ExPASy Server (https://web.expasy.org/protparam/ accessed on 20 December 2022). For the structural, location, and conserved component analysis of NnBZR1 and NlBZR1 genes, the MEME online tool was used to detect protein motifs in each subgroup, identify conserved motifs shared by BZR1 proteins, download the MAST xml files, and visualize by TBtools 2022 software. The gene structures (including introns and utrs) of NnBZR1s and NlBZR1s were analyzed using TBtools, and the chromosomal distribution of the genes was visualized.

2.6. Structural Analysis of Promoter Cis-Reactions

The PlantCARE database (http://bioinformatics.psb.ugent accessed on 25 December 2022) was used to predict the cis-acting regulatory elements in the promoter region of the BZR1 genes, and TBtools was used to visual according to the database analysis results.

2.7. Covariance Analysis

The python version of MCscan (JCVI v1.1.7) was used to align and analyze the genomes of 4 plants, and the collinearity module with the BZR1 genes of lotus was highlighted.

2.8. RNA Isolation, cDNA Synthesis and Quantitative Real-Time PCR Analysis

Total RNA was extracted using a FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotechnology, Nanjing, China) according to the instructions. The integrity of total RNA was checked by agarose gel electrophoresis, and the concentration was determined by NanoDrop 1000 (Thermo, Waltham, MA, USA). The first-strand cDNA was synthesized by HiScript III RT SuperMix for a qPCR (gDNA wiper) kit (Vazyme Biotechnology, Nanjing, China), with 2 μg of total RNA per 40 μL reaction. The cDNA product was diluted 10-fold with deionized water before use. qRT-PCR was performed on the CFX96 Touch™ Real-Time PCR Detection System (BIORAD, Hercules, CA, USA) using the ChamQ SYBR qPCR Master Mix kit (Vazyme Biotechnology, Nanjing, China). A total of 2 μL cDNA was used per reaction. Other reaction components and conditions were performed according to the manufacturer’s instructions. Relative expression was calculated using the 2−ΔΔCt method [31]. All data were generated from the mean of three independent replicates. There were three biological replicates per treatment, and the data were provided as mean ± standard error (SE). Two-way analysis of variance (ANOVA) was performed on the data using SPSS 2022 software. Data were subjected to analysis of variance; p < 0.05 and p < 0.01 was considered statistically significant using Student’s t-test when appropriate. All primers are listed in Additional File: Table S1.

3. Results

3.1. Identification of BZR1 TFs in Lotus

Based on protein sequence homology alignment and the HMM domain model file, the BZR1 proteins were identified from two lotus genomes. The conserved domains were verified by CD-Search and SMART. Finally, four NnBZR1 genes (N. nucifera) and six NlBZR1 genes (N. lutea) were identified. The BZR1 genes were named according to their genome location information: NnBZR1.1, NnBZR1.2, NnBZR1.3, NnBZR1.4 and NlBZR1.1, NlBZR1.2, NlBZR1.3, NlBZR1.4, NlBZR1.5, and NlBZR1.6 (Figure 1 and Table 1). The NnBZR1 genes are distributed on three of the eight chromosomes of N. nucifera: one on chr1, two on chr3, and one on chr6 (Figure 1). The NlBZR1 genes are distributed on four chromosomes, which are chr1, chr3, chr5 and chr6 (Figure 1). The predicted physical and chemical properties of the BZR1 protein were characterized in lotus. The results showed that the BZR1 protein length of lotus ranged from 233 (NlBZR1.6) to 710 aa (NlBZR1.1). The molecular weight ranges from 24,827.91 (NlBZR1.6) to 80,357.63 Da (NlBZR1.1). The theoretical isoelectric constant ranges from 5.64 (NnBZR1.4) to 9.47 (NlBZR1.6). The instability index ranged from 39.86 (NlBZR1.5) to 76.33 (NlBZR1.4), the aliphatic amino acid index ranged from 50.3 (NlBZR1.6) to 74.22 (NnBZR1.4), and the GRAVY ranged from −0.66 (NlBZR1.4) to −0.467 (NlBZR1.1). Prediction of the subcellular localization of the identified BZR1 gene revealed that the BZR1 gene is essentially located in the nucleus, which is consistent with the characteristics of most transcription factors. All the features of the predicted lotus BZR1 protein are provided in Table 1.

3.2. Phylogenetic Classification and Analysis of BZR1 TFs in Lotus

Phylogenetic trees were constructed by comparing the BZR1 protein sequences of five species (N. colorata, N. nucifera, N. lutea, Vitis vinifera and Arabidopsis) using MEGA 7.0 software. The results showed that BZR1 proteins could be divided into four subgroups (Figure 2). The four NnBZR1 proteins of N. nucifera were distributed in groups 1, 2 and 3, and no distribution was seen in group 4. The six NlBZR1 proteins of N. lutea were distributed in four groups. However, the six BZR1 proteins of Arabidopsis were only distributed in the third and fourth groups. The BZR1 proteins, which were distributed in group 2, all have the conserved structural domain Glyco_hydro_14, which is a conserved structural domain belonging to the alpha amylase family. The four NnBZR1 proteins of N. nucifera were aggregated with the four NlBZR1 proteins of N. lutea, while NlBZR1.1 and NlBZR1.5 separated from each other in the lotus.

3.3. The Structure, Protein Motif, and Cis-Element Analysis of BZR1 TFs in Lotus

The protein conserved motif analysis showed that there were 10 highly conserved motifs in the BZR1 gene family of lotus. Among them, Motif1 and Motif9 are highly conserved in all BZR1 gene families. It is speculated that they are related to the conserved domain BES_N of the BZR1 gene family. Combined with phylogenetic tree analysis, the clustered proteins had extremely similar conserved motifs, and their gene structures also had great similarities (Figure 3A).
The results of gene structure analysis showed that NlBZR1.1 and NlBZR1.5 had high specificity, and no genes similar to them were found in NnBZR1s. In addition, the two proteins NnBZR1.4 and NlBZR1.6 clustered together in the phylogenetic tree.
The conserved structural domains of the BZR1 gene family in lotuses were identified using the SMART database based on their full-length amino acid sequences. Model analysis showed that these 10 BZR1 proteins share a unique model, BES1_N. It is located at the N-terminal end of these BZR1 proteins (Figure 3B,C). BES1_N is considered to be the most conserved functional motif in the BES/BZR family. BES1_N specifically binds to BR response elements and E-box sequences in many BR regulatory promoters; it is considered a key conserved pattern for receiving BR signals [15,16,22,23]. Five BZR1 protein sequences contain another unique motif of a glycoside hydrolase family [32,33]: glycol_hydro_14, which is present in the C-terminal region of the protein (Figure 3B). Motif analysis revealed that BZR1 transcription factors share similar motif features. This suggests that they may have similar functions, further supporting the reliability of the phylogenetic classification of BZR1 gene family members.
The upstream 2000 bp sequences of BZR1 genes were analyzed, a large number of cis-acting elements were revealed (Figure 4A,B). The most numerous of these cis-acting elements are motifs associated with light response as well as elements associated with response to hormones such as auxin, abscisic acid, MeJA, salicylic acid, and gibberellin. BZR1 family genes in lotuses are also predicted to have cis-acting elements directly related to low temperature, anaerobic induction and defense. This indirectly suggests that BZR1 TFs may be directly involved in the response of lotus to abiotic stresses, including cold, hypoxia, etc. In addition, the cis-acting elements related to plant growth and development include those involved in plant circadian cycle regulation, meristem expression, metabolic regulation, endosperm expression and light response, indicating that BZR1 genes may be involved in multiple processes of plant growth and development.

3.4. Collinearity Analysis of NnBZR1 Genes and NlBZR1 Genes

In order to further understand the evolution of the lotus BZR1 gene family, the python version of MCScanX (JCVI v1.1.7) software was used to perform collinearity analysis on N. nucifera, N. lutea, N. colorata and Arabidopsis. It was found that there were six homologous gene pairs between N. colorata and N. lutea, and three homologous gene pairs with N. nucifera. Compared with Arabidopsis, there were seven homologous gene pairs between N. lutea and Arabidopsis, and there were two homologous gene pairs between N. nucifera and Arabidopsis (Figure 5A). The result suggested that both the contraction and expansion of BZR1 gene family members may have occurred during evolution from N. colorata to N. lutea, whereas expansion of the BZR1 gene family occurred from N. lutea to Arabidopsis.
The results of synteny analysis of the BZR1 gene family of two lotus species revealed that there were six homologous gene pairs between N. nucifera and N. lutea (Figure 5B). However, N. nucifera has only four BZR1 genes, and N. lutea has six. We extracted the sequences of two other homologous gene pairs for comparative analysis. It was found that the NlBZR1.1 and NlBZR1.5 genes that existed specifically in N. lutea had similar sequences of Nn1g01524.2 and Nn5g29512.5 in N. nucifera. Interestingly, the two similar sequences in N. nucifera are very similar at the C-terminus of the sequence (Figure 6A); they are only missing the conserved domain of BES_N (Figure 6B). Further RT-PCR experiments showed that Nn1g01524.2 and Nn5g29512.5 lacked the BES_N structural domain compared with their homologs NlbZR1.1 and NlBZR1.5 (Figure 6C and Table S2). This may explain why N. nucifera has two fewer BZR1 genes than N. lutea.

3.5. Expression Pattern of BZR1 Genes under BR and ABA Treatments

In order to understand the response of BZR1 genes to hormones, the lotus seedlings were treated with 30 μM ABA and 5 μM BR. The qRT-PCR results showed that the gene expression levels of NnBZR1.1, NnBZR1.2, NnBZR1.4 and Nn1g01524.2 were significantly up-regulated by ABA. NnBZR1.3 was down-regulated at 6 h after ABA treatment and then re-up-regulated to the control level, while Nn5g29512.5 was up-regulated first, then down-regulated, and then, it returned to the control level. After treatment with BR, NnBZR1.1, NnBZR1.2, and NnBZR1.4 could be up-regulated. Gene expression of NnBZR1.3 was down-regulated after 6 h of BR treatment and then returned to the control level. However, the gene expression levels of Nn1g01524.2 and Nn5g29512.5 did not change after BR treatment. The gene expression levels of Nn1g01524.2 and Nn5g29512.5 were not changed after BR treatment. For NlBZR1s, the expression of NlBZR1.1, NlBZR1.2, NlBZR1.3 and NlBZR1.6 could be up-regulated by ABA activation. NlBZR1.4 and NlBZR1.5 showed a tendency to be down-regulated by ABA treatment. When BR was treated, the expression levels of NlBZR1.1, NlBZR1.2, NlBZR1.3, NlBZR1.4 and NlBZR1.6 were significantly up-regulated, while NlBZR1.5 showed a down-regulation trend after BR treatment. Interestingly, as homologous genes, Nn1g01524.2 and Nn5g29512.5 appeared different from NlBZR1.1 and NlBZR1.5 in response to BR. Nn1g01524.2 and Nn5g29512.5 appeared to be insensitive to BR, whereas the expression levels of NlBZR1.1 and NlBZR1.5 were able to be affected by BR. It is may be caused by the deletion of the BES_N domain (Figure 7).

3.6. Expression Pattern of BZR1 Genes (under Different Stress Conditions)

To further understand the response of the BZR1 gene family to abiotic stress, the gene expression levels BZR1 genes were investigated under different abiotic stresses. It was found that the expression levels of the remaining six NnBZR1 genes could be induced by the four abiotic stresses of low temperature, drought, Cd and NaCl (Figure 8A). The most pronounced response to these four stresses was NnBZR1.2, which was followed by NnBZR1.1, NnBZR1.3 and NnBZR1.4. Nn1g01524.2 and Nn5g29512.5 showed a high sensitivity to these four abiotic stresses, which may be related to the response to ABA signals (Figure 8A). NlBZR1s also showed sensitivity to these four stress treatments, and the gene expression levels of all six NlBZR1s could be affected by these four stress treatments. In addition, the gene expression of all five NlBZR1s could be induced to be up-regulated by these four stresses except for NlBZR1.5, whose expression could only be up-regulated under NaCl treatment (Figure 8B).

4. Discussion

In order to regulate and adapt to complex environments during growth and development, plants often induce large amounts of gene expression to help them to respond to complex living environments [34]. As a transcription factor highly responsive to the BR signal, the BZR1 gene family has been shown to play an important role in regulating growth, development and response to abiotic stress of plants in the past decades [35]. Using bioinformatics methods to analyze gene families can help understand gene structure and predict gene function [36]. To date, the BZR1 gene family has been genome-wide identified in many species, such as Brassica rapa [37], Brassica napus [33], apple (Malus pumila Mill.) [38], tomato (Solanum lycopersicum L.) [39] and other horticultural plants. However, as an important medicinal and ornamental aquatic plant, the function of the BZR1 gene family in lotus has not been reported. This has seriously hindered the understanding of the molecular regulation mechanism during the growth and development of lotus, and it has limited the progress of the molecular breeding of lotus. Therefore, based on the lotus genome, the BZR1 gene family was comprehensively identified. The structure, functional characteristics, promoter cis-elements and expression pattern of this gene were analyzed more systematically. Our results are important for understanding the functions of the BZR1 gene family and breeding lotus cultivars with high-quality agronomic traits.
Here, we used bioinformatics to identify the BZR1 gene family in the lotus genome, and we performed a systematic analysis of the identified 10 BZR1 genes (four NnBZR1 genes and six NlBZR1 genes) (Table 1). Chromosomal mapping analysis showed that the four NnBZR1 genes and the four NlBZR1 genes were relatively similar in chromosome distribution. Compared with N. lutea, N. nucifera only lacked one BZR1 gene on chr1 and chr5 chromosomes (Figure 1). In order to study the evolutionary relationship of the BZR gene family among different species, we aligned the protein sequences of the BZR genes of N. colorata, N. nucifera, N. lutea, Vitis vinifera and Arabidopsis and constructed a phylogenetic tree. It was found that the BZR1 family members of lotus were divided into four groups: NnBZR1s were distributed in group 1, group 2 and group 3, and NlBZR1s were distributed in all four groups (Figure 2). It is worth mentioning that the NnBZR1 and NlBZR1 proteins distributed in group 2 contain two conserved domains, BES_N and Glyco_hydro_14 (Figure 3B). This is a similar phenomenon in tomato, cabbage and cotton [33,39,40], while the BZR1 gene family in maize, rice and Arabidopsis has only one conserved domain, BES_N [28,41]. In addition, the BZR1 gene family is relatively stable during the evolution process, and it seems that there is no large-scale expansion and contraction, which can be concluded from the collinearity analysis of the four species (Figure 5A). Interestingly, N. nucifera shared six syntenic pairs with N. lutea, which is two more than the number identified for NnBZR1 (Figure 5B). Further analysis found that the sequences of NlBZR1.1 and NlBZR1.5 have one more BES_N conserved domain than the homologous genes Nn1g01524.2 and Nn5g29512.5. Deletion of the BES_N domain resulted in two fewer NnBZR1 genes than NlBZR1 genes (Figure 6). This phenomenon may be caused by the long-term separation of the two species of lotus to adapt to the local environment. According to the subcellular localization prediction of the Cell-PLoc 2.0 website, most of NnBZR1 and NlBZR1 are localized in the nucleus, while some BZR1s are also predicted to be localized in both cytoplasm and nucleus, such as NnBZR1.1 and NnBZR1.4 (Table 1). In addition, the BZR1s are also predicted to be localized in various organelles, such as the cytoplasm, mitochondria, chloroplast, etc. [32]. It seems to have a certain relationship with the Glyco_hydro_14 conserved domain. This suggests that the BZR1 genes may play a role in signaling within the cell.
Gene structure is a typical feature of gene families that represents the process of gene evolution [42,43,44]. Gene structure often affects gene function, and closely related genes often have similar gene structure and conserved motifs [43,44]. Analysis of the structure of the NnBZR1 and NlBZR1 genes showed that genes in the same group were highly conserved in terms of the type, number, gene length and gene structure of their conserved motifs. This suggests that BZR1 genes in the same group may have similar biological functions.
Gene expression is a key step in the generation of gene function, and gene expression is directly affected by promoters [45]. Among the 10 BZR1 gene family members in lotuses, each member has light-responsive elements and cis-acting elements related to plant growth and development (Figure 4A,B). In addition, except for NlBZR1.6, the promoter regions of the remaining nine BZR1 genes are distributed with cis-acting elements related to stress resistance and response elements related to hormones. It means that the BZR1 gene family may play an important role in regulating lotus growth, development and responding to abiotic stress. This also suggests that the BZR1 gene family is a key factor in crosstalk between BR signal and other hormone signals. To further understand the response of lotus BZR1 to hormone and abiotic stress, we investigated the different expression patterns of NnBZR1s and NlBZR1s under BR, ABA hormone treatment and abiotic stress (salt stress, drought, cold and Cd). The results showed that the expression levels of all BZR1 genes were induced and changed under BR treatment, while the expression levels of Nn1g01524.2 and Nn5g29512.5 showed a stable state (Figure 7). However, after ABA treatment, the gene expression levels of NnBZR1.2, NnBZR1.4, NlBZR1.3 and NlBZR1.6 were strongly induced (Figure 7), which may be related to the presence of more ABA-responsive elements in the promoters of these two genes (Figure 4B). The BZR family is a core transcription factor for BR signal transduction. When BR is absent, the BZR proteins are phosphorylated by the negative regulator BRASSINOSTEROIDINSENSITIVE 2 (BIN2), and the phosphorylated BZR protein is mainly distributed in the cytoplasm. When BR is present, BIN2 is inhibited, while BZR proteins are activated by dephosphorylation and enter the nucleus to produce effects [46,47,48,49]. The conserved domain BES_N of the BZR protein plays an important role in the process of dephosphorylation and nuclear import of BZR protein [15,16,22,23]. After BR treatment, Nn1g01524.2 and Nn5g29512.5 may lose their response to BR hormones. However, their homologs, NlBZR1.1 and NlBZR1.5, can both respond to the BR signal. This may be due to the deletion of the BES_N structural domain in the Nn1g01524.2 and Nn5g29512.5 genes that impairs the sensitivity of Nn1g01524.2 and Nn5g29512.5 to BR signals. Under different abiotic stresses, the expression levels of BZR1 genes were significantly induced by these four abiotic stresses, especially NnBZR1.2 and NlBZR1.3 (Figure 8). This result seems to be related to the number of ABA and stress-related response elements in the promoter. Compared with other BZR1 genes, the promoter of NnBZR1.1 and NlBZR1.2 lacks cis-response elements related to ABA and abiotic stress (Figure 4B), which makes the expression level of NnBZR1.1 difficult to be induced by ABA and abiotic stress.
To date, the research on the regulatory mechanism of BZR1 gene is limited to the BR signaling pathway and a few herbaceous plants, with the most research in Arabidopsis. However, in the lotus, there are no reports about BR regulating its growth, development and response to abiotic stress. This study provides valuable information for the study of the BZR1 gene family in lotus, including its genomic features, protein functional characteristics and expression patterns in different tissues, as well as its response to plant hormones and stresses. These results demonstrate that BZR1 plays an important role in the growth, development, and response to abiotic stresses in lotus. Our results provide a basis for further studies on the function of BZR1 genes in lotus and for understanding various aspects of their regulatory network.

5. Conclusions

In this study, 10 BZR1 genes (four NnBZR1s and six NlBZR1s) were identified from two lotus genomes. Subsequently, the chromosomal location, conserved amino acid residues within the BES1-type structural domain, evolutionary relationships, gene structure, conserved motifs and cis-elements of this family were systematically analyzed. The expression patterns of BZR1 genes in response to hormones and four abiotic stresses were further investigated, showing that BES_N is a key structural domain of the BZR1 gene family in response to the BR signal. It was demonstrated that BZR1s play a key regulatory role in the growth, development and response to abiotic stresses in lotus. Our results provide a valuable resource for future studies on the function of the lotus BZR1 gene family in biological processes as well as a solid foundation for the selection and breeding of stress-resistant lotus cultivars. In order to further deepen our understanding of the function of BZR1s, our future direction will focus on the study of the specific regulatory mechanisms of BZR1s in regulating stress tolerance in lotus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9080882/s1, Table S1: List of primers used in this study, Table S2: Sequence alignment analysis of NnBZR1s and NlBZR1s.

Author Contributions

P.Z.: Conceptualization and writing. H.J.: Methodology. J.L.: Validation. Q.J.: Software. Y.W.: Formal analysis. Y.X.: Editing and Reviewing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (Grants No’s 31971710 and 32071829), Fundamental Research Funds of the Central Government (Special Project of Lotus Germplasm Resources) (KYZZ2021003).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genomic data of Nelumbo nucifera used in this article are available in the database of Nelumbo (http://nelumbo.cngb.org/nelumbo/home accessed on 20 December 2022). The genomic data of Nelumbo lutea used in this article are available in the database of NCBI (https://www.ncbi.nlm.nih.gov/genome/9878 accessed on 20 December 2022). The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Localization of BZR1 genes in two lotus genomes.
Figure 1. Localization of BZR1 genes in two lotus genomes.
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Figure 2. The phylogenetic tree of BZR1 genes in 4 species of plants. MEGA7.0 was used to construct the phylogenetic tree (the boot program value was 1000 repeats), and the boot program value showed on each branch. I–IV referred to the phylogenetic tree clusters.
Figure 2. The phylogenetic tree of BZR1 genes in 4 species of plants. MEGA7.0 was used to construct the phylogenetic tree (the boot program value was 1000 repeats), and the boot program value showed on each branch. I–IV referred to the phylogenetic tree clusters.
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Figure 3. The structure and conserved domain analysis of BZR1 genes. The motif and the basic structure analysis of BZR1 genes (A). The conserved domains of BZR1 genes (B). Multiple sequence alignment of BES1-type domain (marked with underline) of BZR1 genes in lotus (C).
Figure 3. The structure and conserved domain analysis of BZR1 genes. The motif and the basic structure analysis of BZR1 genes (A). The conserved domains of BZR1 genes (B). Multiple sequence alignment of BES1-type domain (marked with underline) of BZR1 genes in lotus (C).
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Figure 4. Phylogenetic analysis and cis-acting elements analysis in the promoter regions of BZR1 genes from lotuses. Distribution of cis-reactive elements on the promoter (A). Classification of promoter cis-response elements with statistical heat map (B). Left panel: Unrooted phylogenetic tree reconstructed on the basis of promoter sequences of BZR1 genes in lotuses using MEGA version 7.0 with the neighbor-joining method (bootstrap value = 1000). Boxes in different colors represent the cis-regulatory elements. Scale bar at the bottom indicates length of promoter sequence.
Figure 4. Phylogenetic analysis and cis-acting elements analysis in the promoter regions of BZR1 genes from lotuses. Distribution of cis-reactive elements on the promoter (A). Classification of promoter cis-response elements with statistical heat map (B). Left panel: Unrooted phylogenetic tree reconstructed on the basis of promoter sequences of BZR1 genes in lotuses using MEGA version 7.0 with the neighbor-joining method (bootstrap value = 1000). Boxes in different colors represent the cis-regulatory elements. Scale bar at the bottom indicates length of promoter sequence.
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Figure 5. The synteny analysis of BZR1 genes. The synteny analysis of BZR1 genes in lotuses and other plants (A). The synteny analysis of BZR1 genes in two lotus species (B). The gray line represents the collinearity module between the genomes of different species, and the blue line represents the collinearity module of the BZR1 genes of different species. Among the collinear modules of the N. nucifera BZR1 genes, the two extra collinear modules were framed in red.
Figure 5. The synteny analysis of BZR1 genes. The synteny analysis of BZR1 genes in lotuses and other plants (A). The synteny analysis of BZR1 genes in two lotus species (B). The gray line represents the collinearity module between the genomes of different species, and the blue line represents the collinearity module of the BZR1 genes of different species. Among the collinear modules of the N. nucifera BZR1 genes, the two extra collinear modules were framed in red.
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Figure 6. Sequence alignment analysis of NlBZR1.1 and NlBZR1.5 collinear modules in lotus. Sequence alignment analysis (A). Conserved domain analysis (B). RT-PCR to verify the integrity of NlBZR1.1 and NlBZR1.5 gene (C).
Figure 6. Sequence alignment analysis of NlBZR1.1 and NlBZR1.5 collinear modules in lotus. Sequence alignment analysis (A). Conserved domain analysis (B). RT-PCR to verify the integrity of NlBZR1.1 and NlBZR1.5 gene (C).
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Figure 7. Expression analysis of BZR1 genes in lotus under hormone treatments. The result data were the average (±SD) of 3 independent experiments. The asterisk in the figure represented a significant difference calculated by Student’s t-test (* p < 0.05).
Figure 7. Expression analysis of BZR1 genes in lotus under hormone treatments. The result data were the average (±SD) of 3 independent experiments. The asterisk in the figure represented a significant difference calculated by Student’s t-test (* p < 0.05).
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Figure 8. The expression of BZR1 genes after stress treatment in lotus. The result data were the average (±SD) of 3 independent experiments. Response of NnBZR1s to four stresses (A). Response of NlBZR1s to four stresses (B). The asterisk in the figure represented significant difference calculated by the student’s t-test (* p < 0.05).
Figure 8. The expression of BZR1 genes after stress treatment in lotus. The result data were the average (±SD) of 3 independent experiments. Response of NnBZR1s to four stresses (A). Response of NlBZR1s to four stresses (B). The asterisk in the figure represented significant difference calculated by the student’s t-test (* p < 0.05).
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Table 1. The detailed characteristics of BZR1 genes identified in lotuses.
Table 1. The detailed characteristics of BZR1 genes identified in lotuses.
SpeciesGene NameGene IDChromosome LocationProtein Length (aa)MW (Da)pIAIGRAVYPredicted Location(s)
Nelumbo nuciferaNnBZR1.1Nn1g09318.4chr1:204,191,887-204,230,451(+)68977,942.66.3670.94−0.548Cytoplasm, Nucleus
NnBZR1.2Nn3g20241.1chr3:74,526,375-74,527,263(−)25727,833.279.450.54−0.661Nucleus
NnBZR1.3Nn3g21067.3chr3:94,419,885-94,424,008(−)33335,471.428.5956.61−0.578Nucleus
NnBZR1.4Nn6g33434.9chr6:30,291,446-30,348,832(+)64071,770.735.6474.22−0.507Cytoplasm, Nucleus
Nelumbo luteaNlBZR1.1Al05735chr1:38,701,447-38,713,085(−)71080,357.635.6573.62−0.467Cytoplasm, Nucleus
NlBZR1.2Al21137chr1:211,166,400-211,207,570(+)69878,611.415.5773.51−0.518Cytoplasm, Nucleus
NlBZR1.3Al22218chr3:76,546,305-76,547,121(−)32734,780.68.4355.26−0.597Nucleus
NlBZR1.4Al12740chr3:96,705,633-96,710,336(−)31634,086.929.2256.58−0.660Nucleus
NlBZR1.5Al13506chr5:65,454,507-65,457,060(+)64572,936.196.4672.29−0.511Nucleus
NlBZR1.6Al00702chr6:30,635,376-30,668,093(+)23324,827.919.4750.3−0.547Nucleus
(+): Forward transcription; (−): Reverse transcription aa: Amino acid; MW: Molecular weight; pI: isoelectric point; GRAVY: Grand average of hydropathicity; AI: Aliphatic index.
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MDPI and ACS Style

Zhou, P.; Jiang, H.; Li, J.; Jin, Q.; Wang, Y.; Xu, Y. Genome-Wide Identification Reveals That BZR1 Family Transcription Factors Involved in Hormones and Abiotic Stresses Response of Lotus (Nelumbo). Horticulturae 2023, 9, 882. https://doi.org/10.3390/horticulturae9080882

AMA Style

Zhou P, Jiang H, Li J, Jin Q, Wang Y, Xu Y. Genome-Wide Identification Reveals That BZR1 Family Transcription Factors Involved in Hormones and Abiotic Stresses Response of Lotus (Nelumbo). Horticulturae. 2023; 9(8):882. https://doi.org/10.3390/horticulturae9080882

Chicago/Turabian Style

Zhou, Ping, Huiyan Jiang, Jingwen Li, Qijiang Jin, Yanjie Wang, and Yingchun Xu. 2023. "Genome-Wide Identification Reveals That BZR1 Family Transcription Factors Involved in Hormones and Abiotic Stresses Response of Lotus (Nelumbo)" Horticulturae 9, no. 8: 882. https://doi.org/10.3390/horticulturae9080882

APA Style

Zhou, P., Jiang, H., Li, J., Jin, Q., Wang, Y., & Xu, Y. (2023). Genome-Wide Identification Reveals That BZR1 Family Transcription Factors Involved in Hormones and Abiotic Stresses Response of Lotus (Nelumbo). Horticulturae, 9(8), 882. https://doi.org/10.3390/horticulturae9080882

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