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Article

Genome-Wide Identification and Analysis of R2R3-MYB Genes Response to Saline–Alkali Stress in Quinoa

by
Yuqi Liu
†,
Mingyu Wang
†,
Yongshun Huang
,
Peng Zhu
,
Guangtao Qian
,
Yiming Zhang
and
Lixin Li
*
Key Laboratory of Saline-Alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Sciences, Northeast Forestry University, Harbin 150040, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(11), 9132; https://doi.org/10.3390/ijms24119132
Submission received: 20 April 2023 / Revised: 17 May 2023 / Accepted: 19 May 2023 / Published: 23 May 2023
(This article belongs to the Special Issue Molecular Regulatory Mechanisms of Salinity Tolerance in Plants)
Browse Figures
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Abstract

:
Soil saline–alkalization inhibits plant growth and development and seriously affects crop yields. Over their long-term evolution, plants have formed complex stress response systems to maintain species continuity. R2R3-MYB transcription factors are one of the largest transcription factor families in plants, widely involved in plant growth and development, metabolism, and stress response. Quinoa (Chenopodium quinoa Willd.), as a crop with high nutritional value, is tolerant to various biotic and abiotic stress. In this study, we identified 65 R2R3-MYB genes in quinoa, which are divided into 26 subfamilies. In addition, we analyzed the evolutionary relationships, protein physicochemical properties, conserved domains and motifs, gene structure, and cis-regulatory elements of CqR2R3-MYB family members. To investigate the roles of CqR2R3-MYB transcription factors in abiotic stress response, we performed transcriptome analysis to figure out the expression file of CqR2R3-MYB genes under saline–alkali stress. The results indicate that the expression of the six CqMYB2R genes was altered significantly in quinoa leaves that had undergone saline–alkali stress. Subcellular localization and transcriptional activation activity analysis revealed that CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62, whose Arabidopsis homologues are involved in salt stress response, are localized in the nucleus and exhibit transcriptional activation activity. Our study provides basic information and effective clues for further functional investigation of CqR2R3-MYB transcription factors in quinoa.

1. Introduction

Soil salinization and alkalization inhibit plant growth and development and seriously affect crop yields. The increasing population worldwide has led to an increasing demand for food. Therefore, improving saline–alkali land to become reserve farmland has profound significance, which is beneficial for food security and ecological security, as well as sustainable development of agriculture [1,2]. Saline–alkali stress results in ion toxicity and osmotic stress, as well as inhibition of nutrient absorption [3]. These effects lead to disrupted metabolism and ion homeostasis, impaired photosynthesis, and subsequent severely delayed plant growth and development [4,5,6]. Over their long-term evolution, plants have gained complex stress response systems to preserve the continuity of the species.
As part of the transcription factors that participate in stress regulatory network in plants, MYB proteins are one of the largest families related to abiotic stress responses in plants [7,8,9]. The MYB family is characterized by a highly conserved MYB_DNA-binding domain, which generally consists of up to four repeats (R) composing about 52 amino acids (aa). Each repeat contains three α–helices. The second and third helix of each repeat build a helix–turn–helix (HTH) structure with three regularly spaced tryptophan (or hydrophobic) residues, forming a hydrophobic core in the 3D HTH structure [10]. According to number of the repeats, MYB TFs are divided into 1R-MYB, R2R3-MYB, 3R-MYB, and 4R-MYB [11]. R2R3-type MYB proteins are the largest MYB subfamily related to abiotic stress responses (e.g., drought, dehydration, heat, and salinity) in plants [12].
Many studies have documented that plant R2R3-MYB genes regulate abiotic stress response by thickening leaf cuticular waxes, controlling stomatal aperture, and regulating the ABA signaling pathway [13,14,15]. For example, OsMYB2, a R2R3-MYB TF, is involved in various abiotic stress in rice. OsMYB2-overexpressing plants were more tolerant to salt, cold, and dehydration stresses, and more sensitive to ABA than wild-type plants [16]. AtMYB44 positively regulates ABA-induced stomatal closure and inhibits the expression of protein phosphatase 2C (PP2C) in Arabidopsis [17]. SbMYBHv33 negatively regulates salt tolerance, ion and ROS homeostasis, and sorghum biomass [18]. R2R3-MYB TFs have been demonstrated to regulate secondary metabolism (e.g., biosynthesis of flavonoids) and cell wall formation [19,20]. EbMYBP1 is clarified to regulate the accumulation of flavonoids in Erigeron breviscapus. It can increase the total flavonoid contents in plants by binding to the promoters of flavonoid biosynthesis genes to activate their expression [21]. Arabidopsis MYB4 and its homologues MYB7 and MYB32 interact with bHLH TFs TT8, GL3, and EGL3, thereby interfere with the transcriptional activation activity of the MBW complex. In addition, MYB4 can also inhibit the accumulation of flavonoids by inhibiting the expression of Arogenate dehydratase 6 (ADT6), which catalyzes flavonoid biosynthesis [22]. Overexpression of MYB6 in transgenic poplar promotes the accumulation of anthocyanins and proanthocyanidins. It also interacts with KNAT7 to inhibit the development of secondary cell walls [23].
Quinoa (Chenopodium quinoa Willd.) is an annual dicotyledonous herbaceous crop of the Amaranthaceae family [24]. Quinoa is considered to be a complete food because it is rich in all essential amino acids with a good balance, and also contains a variety of vitamins, a large number of minerals, unsaturated fat acids, dietary fiber, and is free of gluten and cholesterol. Moreover, quinoa contains numerous secondary metabolites with broad spectra of bioactivities. In the past 40 years, at least 193 secondary metabolites in quinoa have been identified, including flavonoids, phenolic acids, terpenoids, steroids, and nitrogen-containing compounds [25]. Compared with widely cultivated staple crops such as rice, wheat and corn, the nutritional composition of quinoa makes it a leader in healthy foods. On the other hand, quinoa exhibits high tolerance to adverse climate and soil conditions such as drought, salinity, and frost. For example, quinoa can adapt to drought environment through its high water-use efficiency [26]. The high tolerance to harsh environment makes it a favorable candidate for agronomic expansion [27,28,29,30]. Therefore, identification and application of quinoa candidate genes for improving stress resistance in marginal lands is very meaningful, and this has further enhanced the position of quinoa in global foods.
In this study, we identified the R2R3-MYB family genes in quinoa, and performed a comprehensive analysis including phylogenetic tree, gene structure, and motif composition. Transcriptome analysis revealed the expression profiles of CqR2R3-MYB family genes in quinoa leaves that had undergone saline–alkali stress. Our study provides basic information and valuable clues for future investigations aiming at the functional characterization of the CqR2R3-MYB genes and can be utilized in the genetic improvement of quinoa.

2. Results

2.1. Genome-Wide Identification of R2R3-MYB Family Genes in Quinoa

MYB transcription factors (TFs) can be divided into four classes based on the number of adjacent repeats (one, two, three, or four). In this study, we screened 204 MYB proteins according to the Hidden Markov Model (HMM) profile (PF00249) [11,31] in quinoa (Chenopodium quinoa Willd.). From them, we identified 65 R2R3-MYB proteins depending on the adjacent repeating sequences and named them as CqMYB2R1-CqMYB2R65 according to the Gene IDs. The CqR2R3-MYB proteins harbor two adjacent repetitive MYB_DNA binding domains (R) at N-terminus (Figure 1). Their basic information of CqR2R3-MYB TFs is summarized in Table 1. The average protein length is 336 amino acid (aa) residues, the average molecular weight is 37.8 kDa, and most CqR2R3-MYBs belong to acidic proteins with pI < 7. Prediction of subcellular localization indicates that most CqR2R3-MYB proteins are localized in the nucleus, except for CqMYB2R35, with a dual-localization of nucleus and cytoplasm.

2.2. Phylogenetic Analysis of CqR2R3-MYB Family Members

To assess the evolutionary relationship of R2R3-MYB TFs in Chenopodium quinoa Willd. and Arabidopsis thaliana (L.), the phylogenetic tree of R2R3-MYB families was constructed using protein sequences of AtR2R3-MYBs [32] and CqR2R3-MYBs. Based on the phylogenetic tree, the AtR2R3-MYB and CqR2R3-MYB family members were divided into 31 subfamilies (Figure 2). CqR2R3-MYB proteins are found in 26 subfamilies, except for S6, S8, S10, S12, and S15 subfamilies, which only contain AtR2R3-MYBs.

2.3. Primary Structures of Genes and Proteins of CqR2R3-MYBs

To gain more insight into the evolutional and structural diversity of CqR2R3-MYBs, we analyzed conserved motifs in CqR2R3-MYB proteins using the MEME suits. A total of 10 distinct and highly conserved motifs were captured (Figure 3A; Supplementary Figure S1). Motifs 1, 3, and 9 are identified as MYB domains, whereas the function of the other motifs (2, 4, 5, 6, 7, 8, and 10) is unknown. The motif distribution pattern in most CqR2R3-MYB proteins is highly conserved, which contains motif 1, motif 2, motif 3, and motif 5. In contrast, some motifs displayed specificity; for example, motif 8 only appears in CqMYB2R05 and CqMYB2R13, and motif 9 only appears in CqMYB2R04, CqMYB2R20, CqMYB2R24, CqMYB2R33, and CqMYB2R35, all of which belong to S28 (Figure 2 and Figure 3A). To further explore the structural diversity of CqR2R3-MYB genes, the intron–exon organization of each gene was analyzed. As shown in Figure 3B, the exon number varied from 1 to 12, and most of the CqR2R3-MYB genes have 2–4 exons, except for 4 genes having 1 exon and CqMYB2R13 having 12 exons.

2.4. Cis-Acting Elements in Promoters of CqR2R3-MYB Genes

The sequences of 2000 bp upstream of the start codon (ATG) were selected as CqR2R3-MYB promoters for cis-regulatory elements analysis. A total of 20 elements were identified in the promoter by PlantCARE software and the score of each element in each promoter is displayed digitally (Figure 4) [33]. The cis-acting elements are classified into four categories, including light response, plant hormone, plant growth, and stress response. The plant hormone group contains the most elements which are involved in the abscisic acid responsiveness (ABRE), gibberellin-responsive element (GARE-motif, P-box), salicylic acid responsiveness (TCA-element), auxin-responsive element (TGA-element), gibberellin-responsive (TATC-box), and MeJA-responsiveness (CGTCA-motif, TGACG-motif), suggesting that the CqR2R3-MYB genes are regulated by multiple hormones, similar to R2R3-MYB genes in many other plant species. These results indicate that CqR2R3-MYB genes are widely involved in various physiological and biochemical activities in plants, and responses to environmental stimuli and stress.

2.5. Expression Pattern of CqR2R3-MYB Family Genes in Quinoa Leaves under Saline–alkali Stress

To investigate the potential functions of the CqR2R3-MYB TFs under saline–alkali stress, we performed transcriptome analysis using quinoa leaves that had undergone 150 mM carbonate (100 mM NaHCO3 and Na2CO3 mixture) treatment. The results indicate that the six genes with the greatest changes in expression levels (log2 Fold Change (FC) > 1 or <−1) were CqMYB2R43 (log2FC = −2.4181), CqMYB2R45 (log2FC = −1.3827), CqMYB2R49 (log2FC = −1.2214), CqMYB2R16 (log2FC = −1.2183), CqMYB2R29 (log2FC = −1.0544), and CqMYB2R42 (log2FC = 1.48797) (Figure 5A,B). In order to verify the transcriptome data, we performed RT-qPCR analysis, and the results were consistent with the transcriptome data (Figure 5C). The significant changes of expression of these CqR2R3-MYB genes suggest that they were involved in saline–alkali stress response.

2.6. GO Enrichment Analysis of Differentially Expressed CqR2R3-MYB Genes

To achieve a broader functional characterization, the CqR2R3-MYB genes were subjected to GO enrichment analysis. As a result, the CqR2R3-MYB genes were categorized into 129 subcategories belonging to three main categories: 104 subcategories in Biological Processes (BP), 10 in Cellular Components (CC), and 15 in Molecular Functions (MF) (Figure 6A). The CqR2R3-MYB genes are widely involved in the biosynthesis and metabolic processes of metabolites (e.g., organic cyclic compound biosynthetic process (GO:1901362), cellular aromatic compound metabolic process (GO:0006725), and primary metabolic process (GO:0044238) etc.), development, and response to stimulus (Figure 6B). These results indicate that CqR2R3-MYB TFs are closely related to plant growth and development, and environmental stimuli response.

2.7. Subcellular Localization and Transcriptional Activation Activities of Four CqR2R3-MYBs

The subcellular location of CqR2R3-MYB TFs is related to their roles in the transcriptional regulatory network. Therefore, to validate their subcellular localization, we selected four CqR2R3-MYBs: CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62, whose Arabidopsis homologous are related to salt stress response [13,34,35,36] (Supplementary Figure S2). Since the nucleus localization signals (NLSs) are in the middle of these TFs, we cloned the constructs with GFP fused to N-terminal of CqR2R3-MYBs. The transient assay analysis indicated that all the four GFP-CqR2R3-MYB fusion proteins exhibited nucleus localization which was identical to the prediction (Figure 7A). Then, we investigated the autoactivation activity of these four CqR2R3-MYBs. The recombinant plasmids, pGBKT7-CqMYB2R09, pGBKT7-CqMYB2R16, pGBKT7-CqMYB2R25, and pGBKT7-CqMYB2R62, were transformed into yeast strain AH109 with pGADT7 vector, respectively. The transformants grew on SD/-Leu-Trp-His-Ade medium (Figure 7B), indicating that the four CqR2R3-MYBs have transcriptional activation activity. The above results indicate that CqR2R3-MYBs, CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62 have the characteristics of TFs.

3. Discussion

3.1. Identification and Evolution of the Quinoa R2R3-MYB Gene Family

The MYB gene family is one of the largest families of transcription factor in plants, among which R2R3-MYB TF is the most abundant type [37]. With the development of sequencing technology and the improvement of genomic database of more species, the identification of R2R3-MYB genes is becoming more accurate; for example, 126 R2R3-MYB genes were identified in Arabidopsis [38], 244 in soybean [39], 157 in corn [40], and 88 in rice [41]. In this study, we systematically identified 65 R2R3-MYB members in the quinoa genome. Compared with Arabidopsis thaliana, the number of R2R3-MYB genes in quinoa is significantly lower than that in Arabidopsis. The number of R2R3-MYB genes in plants does not depend entirely on the size of the genome or the phylum of plants [32]. Variability in the number of R2R3-MYB genes might be attributed to the ploidy levels of species and gene duplication events during evolution [42].
The genome of quinoa is an allotetraploid (2n = 4x = 36) with an estimated genome size of approximately 1.5 Gb, but it contains only 65 R2R3-MYB genes, whereas the Arabidopsis genome is only 125 M, but contains 126 R2R3-MYB genes [29,43]. In fact, we have identified a total of 204 MYB genes in quinoa, of which 130 belong to the R1-MYB family, twice the number of R2R3-MYB genes. This is different from most species which have more R2R3-MYBs than R1-MYBs [44].
The quinoa R2R3-MYB TFs were phylogenetically clustered into 26 subgroups. The biological functions of most CqMYB2R TFs have not been characterized, whereas more than 90% of the CqMYB2R proteins are clustered with the known functions of Arabidopsis homologues [7]. For example, in S14, S15, and S21, the Arabidopsis members are involved in abiotic stress response and cell wall biosynthesis [45]. Consequently, phylogenetic analysis aids in the prediction of CqMYB2R gene functions.
Although some motifs are common to all members of CqMYB2Rs, there are significant differences in motif type, number, and alignment among different subfamilies, indicating a functional division. For example, motif 8 only appears in two proteins, motif 9 and motif 10 only appear in five proteins, respectively (Figure 3), suggesting a potential functional specificity of these TFs. Interestingly, although motif 1, motif 3, and motif 9 belong to the MYB domain, motif 9 only exists in five TFs (CqMYB2R04, CqMYB2R20, CqMYB2R24, CqMYB2R33 and CqMYB2R35) in the S28 subfamily, whereas motif 1 and motif 3 appear in all other TFs, emphasizing the functional specificity of the five TFs. All R2R3 motifs are consistently highly concentrated at the N-terminus, whereas the sequences at the C-terminus are variable, which endows CqR2R3-MYB TFs with a diversity of functions, e.g., activation or repression of transcription activities, etc. This is identical to the R2R3-MYB family proteins in many other species [11].
In short, this is the first identification and preliminary analysis of quinoa R2R3-MYB gene function, which provides basic information for further investigation.

3.2. Putative Functions of CqR2R3-MYB Transcription Factors

In this study, we analyzed TF characteristics of four CqMYB2R proteins, CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62, whose Arabidopsis homologues are involved in saline–alkali stress response (Supplementary Figure S2). CqMYB2R09 is a homologous of AtMYB73, which is associated with the SOS pathway and thus involved in plant salt-stress response [30]. CqMYB2R16 is a homologous of AtMYB42s that can activate SOS2 expression [35]. CqMYB2R25 is a homologous of AtMYB49 which regulates the stratum corneum formation in Arabidopsis leaves in response to salt stress [13]. CqMYB2R62 is a homologous of AtMYB30 which regulates expression of AOX1A (alternative oxidase 1A) for plant resistance to salt stress [36]. All of the four CqMYB2R proteins presented strong autoactivation activities and nucleus localization, which is identical to the prediction. Therefore, the biological functions of these four CqMYB2R TFs deserve further investigation.
A total of 64 out of the 65 CqMYB2R proteins were predicted to have a nucleus localization, except for CqMYB2R35 with a dual localization of nucleus and cytoplasm. The R2R3-MYBs in other species also have this type TFs [37]. The function of CqMYB2R35 is also worth further research.
So far, the functional research of R2R3-MYB transcription factors mainly focused on the herbaceous model plant Arabidopsis, and the functions of about 80% of AtR2R3-MYBs have been demonstrated [44]. However, the research on quinoa is still in its early stages. There is currently no report on the functionality of CqR2R3-MYB family members. Identification of the CqMYB2Rs provides basic information for future functional research of CqR2R3-MYBs.
Our transcriptome analysis presented the expression profile of CqMYB2Rs in quinoa leaves that had undergone carbonate treatment (Figure 5). It revealed that six CqMYB2R TFs—CqMYB2R16, CqMYB2R29, CqMYB2R42, CqMYB2R43, CqMYB2R45 and CqMYB2R49—were involved in the saline–alkali stress response. Tomato MYB49, an R2R3-MYB TF, is a homologous of CqMYB2R49 (Supplementary Figure S2). MYB49-overexpressing tomato plants showed stronger tolerance to drought and salt stress [46]. Combined with its significant decrease in expression level under saline–alkali stress (Figure 5), CqMYB2R49 is likely to participate in stress response. It is worth investigating this point.
Usually, the expression of genes is determined by their regulatory elements in promoters. Many cis-acting elements in CqMYB2R promoters are widely involved in response to environmental stimuli and stress, such as MBS, LTR, and TC-rich repeat response to salt tress, ABRE and GCTGA-motif to phytohormone, and G-box and Box4 to light, etc. It is well known that R2R3-MYB TFs regulate flavonoid biosynthesis and other secondary metabolites synthesized in the Phenylpropane biosynthesis pathway [47]. The promoters of six CqMYB2R genes contain MBSI element, which acts as MYB binding sites involved in the regulation of flavonoid biosynthesis [48]. MBS is also a MYB binding site, and the binding is induced by drought stress [49]. TC-rich repeats are involved in defense and stress response [50]. In pear, ABRE-binding factor3 (PpABF3) promotes malate accumulation in response to salinity [51]. The JA signaling pathway participates in not only plant defense against abiotic and biotic stress, but also biosynthesis of flavonoids and anthocyanins, etc. [52,53]. Identification of JA-responsive elements (CGTCA-motif, TGACG-motif) suggests that some CqR2R3-MYB TFs are JA responsive. Interestingly, the Box4, G-box and ABRE in some genes have high scores (10–14), suggesting they may play more important roles in response to saline–alkali stress. However, the possibility of other genes participating in regulation cannot be ruled out.
R2R3-MYB transcription factors have been reported to play important roles in abiotic stress response in Arabidopsis and other species. For example, OsMYBR57 participates in the response to drought stress, and OsMYBR57-overexpressing transgenic rice had higher yields under drought stress [54]. GmMYB14-overexpressing transgenic soybean had better drought resistance and gained higher yields in high planting density under field conditions [55]. Our research also clarified that transcriptional levels of some CqR2R3-MYB genes altered significantly under saline–alkali stress, suggesting they may be involved in stress response. Further investigation will help to understanding their functions, which may provide target genes for molecular design breeding with the goal of generating stress resistant quinoa lines.

3.3. CqR2R3-MYB Genes in Cell Wall Biosynthesis

The R2R3-MYB gene family is closely related to Phenylpropane metabolism and secondary cell wall synthesis. R2R3-MYB TFs binds bHLH and WDR proteins to form MBW complexes regulating plant flavonoid biosynthesis [56]. The AtR2R3-MYBs in S5, S6, and S15 subfamilies, and the R/B-like bHLH TFs in IIIf subgroup, may synergistically regulate biosynthesis of flavonoid, anthocyanin, and proanthocyanidin (PA) [47]. The flavonoid biosynthesis-related cis-acting elements in CqR2R3-MYB promoters suggest the potential functions of CqMYB2Rs in S5, S6, and S15 subfamilies.
In Arabidopsis, a significant number of R2R3-MYB genes are involved in cell wall biosynthesis, e.g., MYB20, MYB42, MYB43, and MYB85 can regulate phenylalanine and lignin biosynthesis during secondary cell wall formation [57]. They can bind to the promoters of ADT6 (Arogenate dehydratase 6) and HTC (quinate hydroxycinnamoyl transferase) and activate their expression [58]. These MYB TFs also directly activate expression of the genes for lignin and phenylpropanoid biosynthesis during secondary wall formation [57]. Whether the quinoa homologous genes of the Arabidopsis R2R3-MYB genes also have these functions is a scientific question worth exploring.
In summary, we identified 65 CqR2R3-MYB family genes, analyzed their protein and gene structure and expression pattern under saline–alkali stress, and verified their subcellular localization and transcriptional activation abilities. Our study provides preliminary evidence that some CqR2R3MYB transcription factors may play important roles in stress response in quinoa.

4. Materials and Methods

4.1. Plant Materials, Growth Conditions, and Stress Treatments

Quinoa (Jiaqi #3) (provided by Jiaqi Agricultural Technology Co., Ltd., Taiyuan, Shanxi, China) plants grew under a 16 h light/8 h dark cycle at 22 °C. Two-week-old quinoa seedlings were treated with a solution containing 100 mM Na2CO3:NaHCO3 = 1:9 once every 5 days, three times in total. The control group was treated with water. The leaves were harvested randomly five days after the third treatment, then sent for RNA sequencing. The datasets used in the current study are deposited in NCBI Sequence Read Archive (SRA) Database as accession numbers PRJNA972512.

4.2. Genome-Wide Identification of R2R3-MYB Family Members in Quinoa (Chenopodium quinoa Willd.)

From Ensemble Plants (http://plants.ensembl.org/index.html, accessed on 23 March 2023), we downloaded the genome file and GFF3 file of quinoa (http://plants.ensembl.org/index.html, accessed on 23 March 2023), and from TAIR (https://www.Arabidopsis.org/, accessed on 23 March 2023), we downloaded Arabidopsis R2R3-MYB protein sequences.

4.3. Bioinformatics Analysis of Quinoa R2R3-MYB Family Genes

4.3.1. Phylogenetic Analysis

The phylogenetic tree of quinoa and Arabidopsis R2R3-MYB families was constructed based on multiple protein sequence alignment using MAGA 11 software [59] and the maximum-likelihood model (bootstrap is 1000). iTOL (http://itol.embl.de/, accessed on 25 March 2023) website was used to optimize the evolutionary tree.

4.3.2. The Physicochemical Properties, Conserved Motif Analysis

The protein length, isoelectric point (pI), and molecular weight (MW) were predicted using ExPASy website (https://www.expasy.org/, accessed on 25 March 2023) [60].
The conserved motifs were retrieved by searching MEME website (https://meme-suite.org/meme/doc/meme.html, accessed on 26 March 2023) [61]. The maximum retrieval value was set to 10, and the other parameters were default. InterProScan software (InterProScan 5.62-94.0, EMBL, Heidelberg, Germany) was used to annotate the motifs.

4.3.3. Gene Structure and Cis-Acting Element Analysis

The gene structure of the R2R3-MYB family genes was analyzed using TBtools combined with the GFF3 gene annotation data, and to plot the exon–intron diagram.
The 2000 bp sequences upstream of start codon of R2R3-MYB genes were screened using TBtools and used as promoter sequences for analysis. The plantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 2 April 2023) [33] was used to investigate the cis-acting elements in promoters to predict the regulatory roles of genes in stress responses.

4.4. RT-qPCR Validation

The total RNA used for RT-qPCR (reverse transcription quantitative PCR) was the same sample as that for RNA sequencing. RT-qPCR was performed following the manufacturer’s instructions for Ultra SYBR Mixture (Low ROX) on the ABI7300 real-time PCR system (Applied Biosystems, Waltham, MA, USA). UBQ9 (AUR62020068) was used as the reference gene for normalizing mRNA transcription [62]. The relative expression level was calculated by the 2−∆∆CT method [63]. All RT-qPCR analyses were set with 3 technical repeats. The primers are listed in Supplemental Table S1.

4.5. Subcellular Localization of CqR2R3-MYBs

The coding region of CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62 were amplified and ligated into pCAMBIA1300 vector with an enhanced green fluorescent protein (GFP) fused in N-terminal. The plasmids were transformed into Agrobacterium tumefaciens (GV3101) and infiltrated to tobacco leaves. After growing under light conditions for 48 h, the tobacco leaves were stained with DAPI [0.1 M sodium phosphate (pH 7.0), 1 mM EDTA, 0.1% Triton X-100 (v/v), and 0.5 mg/mL DAPI], then the subcellular localization of GFP-CqR2R3-MYB fusion proteins were observed by Leica fluorescence microscope (DM4 B, Wetzlar, Germany) (GFP, excitation 488 nm, emission 507 nm; DAPI, excitation 340 nm, emission 488 nm).

4.6. Transcriptional Activation Assay

For transcriptional activation assay, the coding region of CqMYB2R09, CqMYB2R16, CqMYB2R25, and CqMYB2R62 were amplified and fused in-frame downstream of the GAL4 DNA binding domain in pGBKT7 vector. The constructs were transformed with pGADT7 vector into strain AH109 (S. cerevisiae) (Clontech), respectively, and grown on SD/-Leu/-Trp medium. The autoactivation activity was examined on SD/-Leu/-Trp/-His/-Ade medium. The empty pGBKT7 vector and pGBKT7-AtbHLH112 construct were used as negative and positive controls, respectively.

5. Conclusions

In this study, we identified 65 R2R3-MYB genes in the quinoa genome and analyzed their physicochemical properties, evolutionary relationships, conserved domains, and motifs in proteins, gene structure, and cis-regulatory elements in promoters. We also confirmed the subcellular localization and transcriptional activation activity of four CqMYB2R TFs whose Arabidopsis homologues are involved in salt stress response. The results indicate that the TF characteristics are conserved in CqR2R3-MYB family proteins. The transcriptome analysis reveals that some CqR2R3-MYBs are important for response to saline–alkali stress. Our study provides basic information of R2R3-MYB family TFs in quinoa for future functional research.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24119132/s1.

Author Contributions

L.L. conceived and supervised the project. L.L., Y.L., and M.W. designed the experiments. L.L., M.W., Y.H., G.Q., Y.Z., and P.Z. analyzed the data. Y.L., M.W., and Y.H. performed the experiments. L.L., Y.L., and M.W. drafted the manuscript. All authors contributed to manuscript revision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32170279).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are presented in this article.

Acknowledgments

We are grateful for Jiaqi Agricultural Technology Co., Ltd. for providing Jiaqi #3 quinoa seeds.

Conflicts of Interest

The authors declare no conflict of interest. The funders have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Figure 1. Conservative domain analysis of CqR2R3-MYB proteins. The green box represents MYB_DNA binding domain.
Figure 1. Conservative domain analysis of CqR2R3-MYB proteins. The green box represents MYB_DNA binding domain.
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Figure 2. Phylogenetic analysis of R2R3-MYB proteins. A maximum-likelihood phylogenetic tree containing 65 CqR2R3-MYB proteins in quinoa and 126 in Arabidopsis thaliana. At, Arabidopsis thaliana; Cq, Chenopodium quinoa. The subgroups were distinguished by different colors. S1, subgroup 1.
Figure 2. Phylogenetic analysis of R2R3-MYB proteins. A maximum-likelihood phylogenetic tree containing 65 CqR2R3-MYB proteins in quinoa and 126 in Arabidopsis thaliana. At, Arabidopsis thaliana; Cq, Chenopodium quinoa. The subgroups were distinguished by different colors. S1, subgroup 1.
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Figure 3. Analysis of conservative motifs and gene structure of CqR2R3-MYB family members. (A) Conservative motif analysis of CqR2R3-MYB proteins. Different colors represent different motifs. (B) Gene structure of CqR2R3-MYBs. UTR, untranslated region.
Figure 3. Analysis of conservative motifs and gene structure of CqR2R3-MYB family members. (A) Conservative motif analysis of CqR2R3-MYB proteins. Different colors represent different motifs. (B) Gene structure of CqR2R3-MYBs. UTR, untranslated region.
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Figure 4. Analysis of cis-acting elements in CqR2R3-MYB promoters. The categorized groups are indicated by color bars. The score which indicates the occurrence frequency of each cis-acting element in each promoter is displayed by number inside the circle, the depth of the circle’s color is proportional to the score, and the corresponding relationship between numbers and colors is shown by a color scale in lower panel.
Figure 4. Analysis of cis-acting elements in CqR2R3-MYB promoters. The categorized groups are indicated by color bars. The score which indicates the occurrence frequency of each cis-acting element in each promoter is displayed by number inside the circle, the depth of the circle’s color is proportional to the score, and the corresponding relationship between numbers and colors is shown by a color scale in lower panel.
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Figure 5. The expression pattern of CqR2R3-MYB genes. (A) The expression levels of all CqR2R3-MYB genes in quinoa leaves that had undergone saline–alkali stress. CK1, CK2, and CK3 are the three replicates of the CK group. Treat1, Treat2, and Treat3 are the three replicates of the carbonate-treatment group. (B) The log2 fold change of the six CqR2R3-MYB genes with the greatest changes in expression levels. (C) RT-qPCR validation of the CqR2R3-MYB genes in (B). UBQ9 (AUR62020068) was used as an endogenous control. Three independent experiments per sample, three replicates per experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t-test.
Figure 5. The expression pattern of CqR2R3-MYB genes. (A) The expression levels of all CqR2R3-MYB genes in quinoa leaves that had undergone saline–alkali stress. CK1, CK2, and CK3 are the three replicates of the CK group. Treat1, Treat2, and Treat3 are the three replicates of the carbonate-treatment group. (B) The log2 fold change of the six CqR2R3-MYB genes with the greatest changes in expression levels. (C) RT-qPCR validation of the CqR2R3-MYB genes in (B). UBQ9 (AUR62020068) was used as an endogenous control. Three independent experiments per sample, three replicates per experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student’s t-test.
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Figure 6. GO enrichment analysis of CqR2R3-MYB genes in quinoa leaves that had undergone saline–alkali stress. (A) Go enrichment analysis of TOP50 of CqR2R2-MYB genes. (B) The enriched GO terms in Biology Process of the differentially expressed CqR2R2-MYBs. Rectangles indicate the most significant terms. Rectangle and oval colors represent the relative significances, dark red indicates the most significant, p < 0.0001.
Figure 6. GO enrichment analysis of CqR2R3-MYB genes in quinoa leaves that had undergone saline–alkali stress. (A) Go enrichment analysis of TOP50 of CqR2R2-MYB genes. (B) The enriched GO terms in Biology Process of the differentially expressed CqR2R2-MYBs. Rectangles indicate the most significant terms. Rectangle and oval colors represent the relative significances, dark red indicates the most significant, p < 0.0001.
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Figure 7. Determination of subcellular localization and autoactivation activity of CqR2R3-MYBs. (A) Subcellular localization of GFP-CqMYBs was detected by transient expression in tobacco leaves. The plasmids harboring GFP-CqR2R3-MYBs were transformed into GV3101 and infiltrated to tobacco leaves. The subcellular localization of GFP-CqR2R3-MYB fusion proteins were observed after DAPI staining. (B) Analysis of autoactivation activity. The pGBKT7 plasmids harboring GFP-CqR2R3-MYBs were transformed with pGADT7 vector into strain AH109, respectively, and grown on SD/-Leu/-Trp medium. The autoactivation activity was examined on SD/-Leu/-Trp/-His/-Ade medium. pGBKT7 empty, a negative control; AtbHLH112, a positive control.
Figure 7. Determination of subcellular localization and autoactivation activity of CqR2R3-MYBs. (A) Subcellular localization of GFP-CqMYBs was detected by transient expression in tobacco leaves. The plasmids harboring GFP-CqR2R3-MYBs were transformed into GV3101 and infiltrated to tobacco leaves. The subcellular localization of GFP-CqR2R3-MYB fusion proteins were observed after DAPI staining. (B) Analysis of autoactivation activity. The pGBKT7 plasmids harboring GFP-CqR2R3-MYBs were transformed with pGADT7 vector into strain AH109, respectively, and grown on SD/-Leu/-Trp medium. The autoactivation activity was examined on SD/-Leu/-Trp/-His/-Ade medium. pGBKT7 empty, a negative control; AtbHLH112, a positive control.
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Table
Table 1. Basic information of CqR2R3-MYB proteins. GRAVY, grand average of hydropathicity.
Table 1. Basic information of CqR2R3-MYB proteins. GRAVY, grand average of hydropathicity.
Gene IDNameLength
(aa)		Molecular Weight (KDa)pIInstability
Index		Ali-Phatic IndexGRAVYLocalization Predictor
AUR62000115CqMYB2R0130633.725.450.0261.5−0.606nucleus
AUR62000244CqMYB2R0255660.795.1857.5365.43−0.619nucleus
AUR62000484CqMYB2R03979110.495.1149.3170.91−0.921nucleus
AUR62001688CqMYB2R0430834.546.7245.0262.08−0.776nucleus
AUR62001998CqMYB2R0534138.486.344.5378.97−0.633nucleus
AUR62002080CqMYB2R0636039.796.8646.3677.83−0.506nucleus
AUR62002136CqMYB2R0723526.624.2360.7265.53−0.586nucleus
AUR62003804CqMYB2R0833838.126.2343.8577.93−0.626nucleus
AUR62003939CqMYB2R0930232.848.2755.9965.23−0.526nucleus
AUR62004043CqMYB2R1032236.11653.767.61−0.635nucleus
AUR62004326CqMYB2R1129933.916.2758.6667.22−0.709nucleus
AUR62004628CqMYB2R1232435.118.739.7167.47−0.594nucleus
AUR62005744CqMYB2R1346151.947.1454.9173.64−0.691nucleus
AUR62006590CqMYB2R1455160.215.1559.0565.5−0.602nucleus
AUR62006826CqMYB2R15979110.305.1849.5271.71−0.889nucleus
AUR62007454CqMYB2R1630434.044.9450.2173.78−0.701nucleus
AUR62007558CqMYB2R1732236.266.0155.4164.57−0.692nucleus
AUR62008199CqMYB2R1845651.255.7842.2669.47−0.693nucleus
AUR62008324CqMYB2R1936140.005.9450.6361.39−0.711nucleus
AUR62008966CqMYB2R2025529.025.2365.4961.22−0.687nucleus
AUR62011751CqMYB2R2128733.035.754.0767.28−0.771nucleus
AUR62011870CqMYB2R2228632.265.6153.4863.11−0.772nucleus
AUR62013046CqMYB2R238810.109.153.3277.73−0.716nucleus
AUR62014537CqMYB2R2419922.834.5863.0661.26−0.632nucleus
AUR62014700CqMYB2R2536140.995.5646.8561.61−0.739nucleus
AUR62014701CqMYB2R2638943.665.8545.8874.24−0.713nucleus
AUR62014702CqMYB2R2728932.556.9650.9680.66−0.55nucleus
AUR62015573CqMYB2R2824027.084.2459.9270.29−0.588nucleus
AUR62017171CqMYB2R2931635.166.7637.4969.49−0.618nucleus
AUR62018324CqMYB2R3027531.216.4547.9771.64−0.737nucleus
AUR62018693CqMYB2R3124427.546.2336.9779.18−0.609nucleus
AUR62019989CqMYB2R3215417.6710.1855.7370.26−0.929nucleus
AUR62020094CqMYB2R3330834.546.7246.4563.34−0.783nucleus
AUR62020426CqMYB2R3412313.269.5130.0385.61−0.354nucleus
AUR62020972CqMYB2R3532135.248.8944.3572.02−0.539nucleus/cytoplasm
AUR62021197CqMYB2R3622125.456.3151.5863.53−0.76nucleus
AUR62021199CqMYB2R3728331.598.356.0563.78−0.639nucleus
AUR62022338CqMYB2R3832236.489.5856.3452.14−0.926nucleus
AUR62022709CqMYB2R3932234.988.7738.8969.44−0.534nucleus
AUR62022815CqMYB2R4042446.385.3358.8368.8−0.537nucleus
AUR62022912CqMYB2R4125728.427.0639.970.82−0.866nucleus
AUR62023242CqMYB2R4221023.988.8651.6372.1−0.818nucleus
AUR62023549CqMYB2R4332936.946.4645.570.82−0.696nucleus
AUR62024595CqMYB2R4431135.526.8653.9476.17−0.496nucleus
AUR62024713CqMYB2R4529533.236.2251.2363.83−0.745nucleus
AUR62025096CqMYB2R4660168.368.9437.1977.24−0.598nucleus
AUR62025146CqMYB2R4724127.835.5542.4278.13−0.532nucleus
AUR62025185CqMYB2R4829033.139.1560.2477.97−0.746nucleus
AUR62027278CqMYB2R4930634.274.849.373.63−0.705nucleus
AUR62028989CqMYB2R5028932.969.0561.2981.97−0.697nucleus
AUR62030563CqMYB2R5132836.766.1548.0574.6−0.608nucleus
AUR62030594CqMYB2R5232236.276.0542.5365.47−0.641nucleus
AUR62032801CqMYB2R5330033.568.7576.9755.67−0.755nucleus
AUR62033319CqMYB2R5429933.766.160.7767.26−0.671nucleus
AUR62033340CqMYB2R5529933.766.160.7767.26−0.671nucleus
AUR62033728CqMYB2R5628733.216.1847.2971.67−0.752nucleus
AUR62034368CqMYB2R5728232.159.4956.8655.39−0.983nucleus
AUR62034976CqMYB2R5836240.686.7255.9272.98−0.707nucleus
AUR62036035CqMYB2R5920622.939.1731.0169.71−0.568nucleus
AUR62037317CqMYB2R6035140.105.1660.1870.57−0.778nucleus
AUR62039339CqMYB2R6137341.996.4149.4876.81−0.659nucleus
AUR62039812CqMYB2R6235540.166.7362.1956.34−0.946nucleus
AUR62040017CqMYB2R6333337.296.7547.865.86−0.803nucleus
AUR62041016CqMYB2R6446852.188.2952.965.66−0.709nucleus
AUR62041339CqMYB2R6536942.09964.5471.17−0.801nucleus
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MDPI and ACS Style

Liu, Y.; Wang, M.; Huang, Y.; Zhu, P.; Qian, G.; Zhang, Y.; Li, L. Genome-Wide Identification and Analysis of R2R3-MYB Genes Response to Saline–Alkali Stress in Quinoa. Int. J. Mol. Sci. 2023, 24, 9132. https://doi.org/10.3390/ijms24119132

AMA Style

Liu Y, Wang M, Huang Y, Zhu P, Qian G, Zhang Y, Li L. Genome-Wide Identification and Analysis of R2R3-MYB Genes Response to Saline–Alkali Stress in Quinoa. International Journal of Molecular Sciences. 2023; 24(11):9132. https://doi.org/10.3390/ijms24119132

Chicago/Turabian Style

Liu, Yuqi, Mingyu Wang, Yongshun Huang, Peng Zhu, Guangtao Qian, Yiming Zhang, and Lixin Li. 2023. "Genome-Wide Identification and Analysis of R2R3-MYB Genes Response to Saline–Alkali Stress in Quinoa" International Journal of Molecular Sciences 24, no. 11: 9132. https://doi.org/10.3390/ijms24119132

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