Systematic identification and characterization of the soybean (Glycine max) B-box transcription factor family

Abstract In plants, the B-box (BBX) transcription factors (TFs) are a subfamily of zinc-finger TFs that act to regulate diverse plant growth and development processes. The BBX TFs have been the subject of considerable attention, and are well characterized in diverse plant species, including in rice (Oryza sativa) and Arabidopsis thaliana, but less so in the economically important soybean (Glycine max). In this work, we systematically identified and characterized 57 soybean BBX genes (GmBBX1 to GmBBX57). These genes were mapped to all 20 soybean chromosomes and were divided into five clades with high intra-clade intron–exon similarity. The majority of GmBBX gene promoter cis-acting elements were responsive to light, abscisic acid, salicylic acid and methyl jasmonate, as well as a diverse array of other stimuli. Quantitative RT-PCR indicated that several GmBBX genes exhibited tissue-specific and phytohormone- and abiotic stress-responsiveness. The results of this study will be useful in the continued characterization of soybean BBX gene functions and provide new ideas for soybean breeding.


Introduction
Transcription factors (TFs), which bind to specific regions of target gene promoters to activate or inhibit expression, are important regulators of plant growth and development [1,2].Studies on the structure and function of TFs have shown that they are composed of a nuclear localization signal, oligomerization site, transcription activation domain and DNA binding site [3,4].One large family of TFs, the zinc-finger TFs, is comprised of several subfamilies which all act to regulate different aspects of development and growth in plants [5].Among the subfamilies of the zinc-finger family, the B-box (BBX) family is particularly important [6].In plants, BBX TFs contain one or two N-terminal conserved domains, and sometimes a C-terminal TOC1 (CCT), CONSTANS (CO) or CONSTANS-LIKE (COL) domain [7,8].BBX TFs are crucial for not only plant development and growth, but also for plant response to external stimuli [8], including stress responses [9] and flower induction [10,11], among others.
BBX proteins also act as abiotic stress response regulators in plants.Early studies revealed that Arabidopsis AtBBX2 could enhance yeast (Saccharomyces cerevisiae) salinity tolerance [29].In Arabidopsis, AtBBX24 overexpression improves the growth of salt-stressed plants [30].The overexpression of apple MdBBX10 has also been found to enhance tolerance to salinity in Arabidopsis [31].Furthermore, AtBBX18 acts to negatively regulate heat stress tolerance in Arabidopsis [32].Grapevine VvZFPL is homologous to Arabidopsis BBX32, and overexpression in transgenic Arabidopsis enhances tolerance to cold stress [33].Finally, uV-B radiation tolerance in Arabidopsis is enhanced through overexpression of AtBBX31 [34].
Soybean (Glycine max) is currently the world's leading protein source and second leading source of vegetable oil [35,36].However, soybean yields are negatively impacted by pests, disease, drought, extreme temperatures and salinity, among other abiotic and biotic stressors.After the successful sequencing of the soybean genome, many soybean gene families have been identified and analyzed, such as the bZIP transcription factors (bZIPs) [37], stress-associated proteins (SAPs) [38] and AT-Hook Motif Nuclear Localized (AHL) genes [39], etc.As outlined above, BBX proteins are crucially important for regulating plant stress response.The BBX TF family has been studied extensively in a diverse array of plants, including A. thaliana [40], tomato (Solanum lycopesicum) [41], rice (O.sativa) [42], peanut (Arachis duranensis) [7], grapevine (V.vinifera) [6], pepper (Capsicum annuum) [43] and apple (Malus domestica) [44].However, there has been no comprehensive evaluation of this TF family in soybean.To address this pressing need, this study was conducted to characterize the soybean GmBBX genes.We investigated GmBBX gene structures, phylogenetic relationships, chromosomal localizations and subcellular localizations, as well as tissue-and stressor-specific expression patterns.The results of this study will be useful for the continued characterization of soybean BBX gene functions.

Analysis of phylogenetic relationships
To interrogate the phylogenetic relationships between the BBX family genes in both soybean and Arabidopsis, protein sequences were collected from Phytozome v13 (http://www.phytozome.org)[47].MEGA-X was used to align BBX proteins and construct a neighbor-joining (NJ) phylogenetic tree [48], and bootstrapping was conducted using 1000 replications.Each gene was classified in accordance with its distance homology with Arabidopsis genes.

Analysis of chromosomal location, intron-exon structure and conserved motifs
Phytozome v13 (http://www.phytozome.org) was used to map all GmBBX genes to soybean chromosomes.TBtools was used to create a chromosomal location map, extract soybean genomic full-length and coding sequences, generate intron-exon structure diagrams, and draw a conserved motif map [49].The MEME analysis tool (https://meme-suite.org/meme/tools/meme) was utilized to identify the conserved motifs of BBX proteins, allowing for a maximum of 10 motifs and otherwise using the default settings [50].

Plant materials and experimental treatments
Seeds of soybean variety 'Williams 82′ were obtained from Liaocheng university (China).Seeds were sown in pots (10*10 *8.5 cm) filled with grass charcoal (a seed per flowerpot) and grown in a climate-controlled greenhouse at 26 °C using a cycle of 10 h dark and 14 h light.At the V1 stage, each seedling was irrigated with 50 mL, specifically, for drought treatment (10% polyethylene glycol-6000 [PEG-6000]) [52], high salinity (100 mmol/L NaCL) [53], 100 μmol/L abscisic acid (ABA), 100 μmol/L salicylic acid (SA) or 100 μmol/L methyl jasmonate (MeJA) [43].To examine the effects of extreme temperatures, seedlings were kept either at low (4 °C) or high (38 °C) temperatures using climate-controlled growth chambers [54,55].In all cases, leaves were harvested at 0, 3, 6 and 12 h post-treatment, promptly frozen using liquid nitrogen and then ground with a mortar to extract RNA.Three biological replicates were collected per time point per treatment.

Isolation of RNA and analysis of gene expression using real-time quantitative (RT)-qPCR
An RNAprep pure Plant Total RNA Extraction Kit (Tiangen, Beijing, China) was utilized for RNA extraction from leaf samples.Reverse transcription of mRNA was accomplished using HiScript III RT SuperMix (plus gDNA wiper) for qPCR (Vazyme, Nanjing, China).Gene expression was examined using ChamQ universal SyBR qPCR Master Mix (Vazyme, Nanjing, China).A Bio-Rad CFX96 system was used to carry out quantitative real-time PCR utilizing the following protocol: 95 °C for 30 s, 95 °C for 5 s, 56 °C for 30 s for 39 cycles.The 2 −ΔΔCt method was utilized to quantify relative gene expression levels.All qRT-PCR analyses utilized GmACTIN gene and GmTubulin as the internal reference genes.Three technical replicates were carried out per treatment.All primers used in the experiment can be found in Supplemental Table S1.

Systematic identification and characterization of soybean BBX family genes and proteins
In order to determine putative soybean BBX genes, Arabidopsis BBX proteins were searched against the soybean genomic database [40].Then, the BBX domain (Pfam00643) HMM profile was used to search the soybean genome.After removing redundant sequences and identifying the conserved domain, we identified 57 putative soybean GmBBX genes and named them according to their chromosomal location (GmBBX1-57).
These 57 putative genes were predicted to encode proteins ranging from 125 (GmBBX43 and GmBBX34) to 481 (GmBBX22) amino acids in length.These proteins were predicted to exhibit molecular masses ranging from 14.00827 to 53.91340 kDa and pIs ranging from 4.2 to 9.72.All 57 GmBBX proteins were predicted to have nuclear localization (Supplemental Table S2).

Analysis of evolutionary relationships and conserved domains of soybean BBX proteins
To investigate the evolutionary relationships and functional divergences of the GmBBX gene family, we created a phylogenetic tree illustrating the evolutionary relationships of BBX genes between Arabidopsis and soybean (Figure 1).

Investigation of GmBBX gene intron-exon structure and conserved motifs
Representing the same five clades as Figure 1, a NJ tree was created to evaluate the evolution of the soybean BBX family gene structure, comparing both exon-intron structure and conserved motifs (Figure 2).We identified ten conserved motifs, with CCT (Motif 2), B-box2 (Morif 3) and B-box1 (Motifs 1 and 8).Intriguingly, Motifs 5 and 10 were found only in Clade V and Motifs 4 and 9 were found only in Clade II, perhaps contributing to the functional divergence of BBX genes.Additionally, both GmBBX9 and GmBBX18 of Clade IV were composed only of Motifs 6 and 8. Gene structure varied considerably between clades, although intra-clade differences were minimal.Overall, the evaluated BBX gene family members contained between one and five exons.

Analysis of phylogenetic relationships and conserved domains of soybean BBX proteins
TBtools was used to map the 57 GmBBX genes onto the soybean chromosomes (Figure 3).Overall, the genes were found to be unevenly distributed across the twenty chromosomes, ranging from one (chromosomes 01, 03, 14, 15, 16 and 18) to nine (chromosome 13) genes per chromosome.Chromosomes 04, 10, 19 and Figure 1.analysis of phylogenetic relationships of BBX genes between arabidopsis and soybean.mega-X was used to align BBX proteins and construct a neighbor-joining (nJ) phylogenetic tree [48]; bootstrapping was conducted using 1000 replications.20 each possessed three GmBBX genes.Two genes were found on chromosomes 02, 05, 07, 09 and 17.To explore the evolutionary relationships of this gene family across plant species, we examined the collinearity relationships of BBXs of two dicotyledonous plants (A.thaliana and Brassica rapa) and two monocotyledonous plants (Brachypodium distachyon and O. sativa; Figure 4).Higher homology was found between soybean, B. rapa and A. thaliana than between B. distachyon and O. sativa.Furthermore, dicots contained more homologous BBX genes than monocots.

Cis-element analysis for GmBBX gene promoters
Cis-elements are involved in transcriptional regulation and can respond to a variety of stressors.TFs regulate gene transcription by binding to cis-elements present at the promoter regions of target genes.Sequences 2.0 kb upstream of the GmBBX genes were isolated to explore their potential function (Figure 5).We found that the GmBBX gene promoters included cis-elements regulating plant defense, circadian rhythm, cell cycling, phytohormones (e.g.ABA, auxin, gibberellin, MeJA and SA), and response to a wide range of stimuli, including wounding, low temperature, anoxia and light, as well as seed-specific regulation, among others.Of these, cis-elements related to light response and SA, ABA and MeJA phytohormone response were the most common in soybean, although the number of each type of element varied greatly between promoters.gmBBX gene structures and conserved protein motifs.analysis of phylogenetic relationships of soybean BBX genes (a).gene structure of gmBBX (B).conserved motif analysis of gmBBX genes (c).tbtools [49] was used for visualization.

Discussion
The BBX TFs of two model plants, Arabidopsis (dicot) and rice (monocot) have received the most study [12,13,45].Responding to a lack of data concerning the BBX TFs in the economically important soybean, we systematically identified and characterized 57 BBX genes, including their chromosomal position, gene structure, conserved motifs, predicted proteins, subcellular localization, and stress-responsive and tissuespecific patterns of expression.
The majority of the GmBBX promoters were found to contain cis-elements regulating both plant development and responses to diverse stimuli, with the majority of these specifically regulating phytohormones (ABA and MeJa) and light response (Figure 4).ABA and MeJa are critical for regulating the plant response to both biotic and abiotic stress [56].The high number of these cis-regulatory elements found in the GmBBX promoters indicates that this gene family is critical for soybean plant response to challenging, and changing, environmental conditions.GmBBX7, GmBBX12, GmBBX39 and GmBBX49 are highly expressed in various tissues and may play an essential role in soybean growth and development (Supplemental Table S3).
BBX TFs are key mediators of plant responses to diverse stimuli, including both biotic and abiotic stress and phytohormones [8], as indicated by studies in Arabidopsis [26] and other plants.In soybean, the patterns of GmBBX gene expression were markedly different under different abiotic stress treatments (Figure 7).However, the expression dynamics of two particular genes, GmBBX34 and GmBBX43, stood out.These two genes exhibited upregulation under drought stress (Figure 7B), salt stress (Figure 7A), and cold stress (Figure 7D).Both GmBBX34 and GmBBX43 are homologues of AtBBX32 (VvZFP), which enhances tolerance to low temperatures in transgenic Arabidopsis [33].Also of note, both GmBBX1 and GmBBX30, which were upregulated following high temperature stress, clustered with AtBBX18 (Figure 7C), a negative regulator of heat tolerance in Arabidopsis [32].Patterns of GmBBX gene expression were also markedly different between phytohormone treatments (Figure 8).For example, GmBBX43 was strongly induced after MeJa, SA and ABA treatments, while GmBBX51 was repressed in to these treatments.While it is clear that BBX proteins participate in phytohormone signal transduction, the exact BBX gene functions in soybean remain to be determined.
Overall, through bioinformatics analysis and further experimental verification, we believe that GmBBX34 and GmBBX43 can be used as candidate stress-resistance genes for soybean.

Conclusions
In this study, 57 soybean GmBBX genes, and their associated proteins, were systematically identified and characterized.These 57 genes were distributed, albeit unevenly, across all 20 soybean chromosomes.The majority of the predicted BBX proteins were found to have high homology with Arabidopsis BBX proteins, and GmBBXs were grouped into five clades with high intra-clade exon-intron similarity.The majority of the GmBBX gene promoter cis-acting elements were found to be responsive to light, ABA, SA and MeJA, although other elements were found to respond to a variety of stimuli.In addition, several GmBBX genes exhibited tissue-specific, and abiotic stress-and phytohormoneresponsive expression patterns.The results of this study will be useful in the continued characterization of soybean BBX gene functions and could be explored further for soybean breeding.

Figure 2 .
Figure2.gmBBX gene structures and conserved protein motifs.analysis of phylogenetic relationships of soybean BBX genes (a).gene structure of gmBBX (B).conserved motif analysis of gmBBX genes (c).tbtools[49] was used for visualization.

Figure 3 .
Figure 3. chromosomal distribution of soybean BBX genes.chromosomal mapping was based on the physical position in 20 soybean chromosomes.the scale on the left is in megabases (mb).

Figure 4 .
Figure 4. analysis of collinearity of the BBX gene family between Arabidopsis thaliana, Brassica rapa, Brachypodium distachyon, Oryza sativa and Glycine max. the segmental duplicated genes are indicated in red and connected by lines.orange pillar and green pillar represent chromosome.

Figure 5 .
Figure 5. heatmap of putative cis-elements present at GmBBX gene promoter regions.the 22 elements contained in the BBX gene family include zein metabolism regulation, wound responsive, seed-specific regulation, salicylic acid responsive, myBhv1 binding site, meristem expression, meJa responsive, flavonoid biosynthetic, endosperm-specific negative expression, endosperm expression, defense and stress responsive, circadian control, cell cycle regulation, auxin responsive, gibberellin responsive, at-rich Dna binding domain, light responsive, anoxic-specific inducibility, anerobic induction, low temperature responsive, abscisic acid responsive and 60 K protein binding site.the number of cis-acting elements present in the promoter are represented by different values.

Figure 7 .
Figure 7. qRt-pcR gene expression analysis of GmBBX genes in abiotically stressed soybean plants: high salt stress treatment (a); osmotic stress treatment (B); high temperature stress treatment (c); cold temperature stress treatment (D).Different letters indicate significant differences at the p < 0.05 level.all experiments consisted of three biological replicates.GmACTIN and GmTubulin were used as reference genes.

Figure 8 .
Figure 8. qRt-pcR gene expression analysis of 10 GmBBX genes in phytohormone-treated soybean plants.aBa treatment (a).Sa treatment (B).meJa treatment (c).Different letters indicate significant differences at the p < 0.05 level.all experiments consisted of three biological replicates.the black column indicates the expression level of GmACTIN as an internal reference gene.the grey column indicates the expression level of GmTubulin as an internal reference gene.