Cloning and Expression Analysis of the BocMBF1c Gene Involved in Heat Tolerance in Chinese Kale

Abstract

Chinese kale (Brassica oleracea var. chinensis Lei) is an important vegetable crop in South China, valued for its nutritional content and taste. Nonetheless, the thermal tolerance of Chinese kale still needs improvement. Molecular characterization of Chinese kale’s heat stress response could provide a timely solution for developing a thermally tolerant Chinese kale variety. Here, we report the cloning of multi-protein bridging factor (MBF) 1c from Chinese kale (BocMBF1c), an ortholog to the key heat stress responsive gene MBF1c. Phylogenetic analysis showed that BocMBF1c is highly similar to the stress-response transcriptional coactivator MBF1c from Arabidopsis thaliana (AtMBF1c), and the BocMBF1c coding region conserves MBF1 and helix-turn-helix (HTH) domains. Moreover, the promoter region of BocMBF1c contains three heat shock elements (HSEs) and, thus, is highly responsive to heat treatment. This was verified in Nicotiana benthamiana leaf tissue using a green fluorescent protein (GFP) reporter. In addition, the expression of BocMBF1c can be induced by various abiotic stresses in Chinese kale which indicates the involvement of stress responses. The BocMBF1c-eGFP (enhanced green fluorescent protein) chimeric protein quickly translocated into the nucleus under high temperature treatment in Nicotiana benthamiana leaf tissue. Overexpression of BocMBF1c in Arabidopsis thaliana results in a larger size and enhanced thermal tolerance compared with the wild type. Our results provide valuable insight for the role of BocMBF1c during heat stress in Chinese kale.

1. Introduction

Chinese kale (Brassica oleracea var. chinensis Lei Syn:Brassica alboglabra Bailey) is a popular vegetable for its health benefits and taste [1]. Chinese kale, belonging to the Brassica genus, is native to China and, currently, is predominantly cultivated in South China and Southeast Asia [1]. Recently, the importance of Chinese kale has generated interest in its nutrition and genetics [2]. In particular, Chinese kale contains high levels of glucoraphanin which may possess cancer prevention properties [3].

Unfortunately, Chinese kale is very sensitive to heat stress, which often leads to decreased yield and quality [4]. Consequently, the main growing season and distribution of Chinese kale is limited to the winter season of South China.

High temperature can disturb the membrane, structure of protein, and chromatin architecture of the plant cells which respond with several signaling pathways to address the stress [5]. Many plant hormones are reported in the heat response of plants, and exogenous application of plant hormones can activate expression of heat stress-related genes thus improving thermal tolerance. For example, applying abscisic acid (ABA) can increase the thermal tolerance level by upregulating ABA responsive transcription factors (ABRFs), heat stress transcription factor (HSF) A2c (FaHSFA2c), and heat shock proteins (HSPs) in tall fescue [6]; treatment with methyl jasmonate (MeJA) stimulates the expression of HSP70 [7]; and exogenous salicylic acid (SA) increases the heat stress resistance of wheat by regulating the accumulation of ethylene (ET) and proline, increasing the efficiency of nitrogen usage [8]. Application of ET and jasmonic acid (JA) can activate thermal stress-related genes by upregulating ethylene response factor (ERF) 1 in Arabidopsis.

In plants, enormous progress has recently been made in understanding of the molecular mechanism of thermal tolerance [9]. In addition to HSF- and HSP-responsive pathways [9], the transcriptional coactivator Multi-protein bridging factor1c (MBF1c) was identified as a critical regulator for thermal tolerance responses in Arabidopsis [10]. In Arabidopsis, heat stress can lead to increased expression of AtMBF1c and triggers the nuclear localization of AtMBF1c proteins. Nuclear AtMBF1c then function as transcription factors to regulate the downstream SA, trehalose, and ET thermal resistance-related pathways [10]. The AtMBF1c is a trans-acting regulatory element which recognizes CTAGA as a potential binding sequence and can regulate 36 genes, including stress-responsive gene dehydration-responsive element (DRE)-binding protein 2A (DREB2A) [11]. In addition, overexpression of AtMBF1c and its ortholog from wheat can improve the performance of host plants under high temperature [11,12]. Therefore, MBF1c is an ideal gene for improving plant thermal tolerance and, thus, increase productivity under heat stress. The expression of the MBF1c gene in Antarctic Moss (PaMBF1c) can be induced by several abiotic stresses, and overexpression of PaMBF1c can enhance heat, cold, and salinity stress tolerance in Arabidopsis [12]. However, to date there have been no studies conducted on the MBF1c of Chinese kale. In this study, we reported the ortholog of AtMBF1c in Chinese kale, termed BocMBF1c, and characterized its potential role in regulating heat tolerance in Chinese kale.

2. Results

2.1. Cloning and Analysis of the BocMBF1c Gene

As the genome sequence of Chinese kale is still unavailable, we used the Brassica rapa MBF1c gene (LOC103828628) as a reference sequence for primer design and then cloned the coding region of MBF1c from Chinese kale (variety “Ai-jiao-xiang-gu”) genomic DNA. The resultant amplicon of 707 bp was sequenced and confirmed to be BocMBF1c by sequence homology analysis (Figure 1a,b). Open reading fragment (ORF) prediction by ORF finder did not find any intron within the BocMBF1c gene, which was then confirmed by agarose gel analysis (Figure S1). Amino acid sequence analysis identified multi-protein bridging factor1 (MBF1) and helix-turn-helix (HTH) domains (Figure 1a). Phylogenetic analysis indicated that BocMBF1c clustered with Arabidopsis MBF1c (Figure 1b, Table S1). Next, we obtained the MBF1c gene and promoter region of Brassica oleracea (Bol013952) by searching the BocMBF1c coding sequence (CDS) using BLASTN (http://brassicadb.org/brad/blastPage.php). The 2005 bp DNA fragment containing the promoter of BocMBF1c was cloned with primers designed from BoMBF1c, from which we used 1409 bp upstream sequence from initiation codon for further analysis. The BocMBF1c gene was submitted to GenBank (Accession number: MH685643).

Sequence analysis of the 1409 bp BocMBF1c promoter revealed multiple stress and hormonal responsive cis-elements, including heat shock elements (HSEs), ethylene responsive elements (EREs), abscisic acid-responsive element (ABRE), MeJA responsive motifs (CGTCA-motif), drought stress and pathogen-responsive TC-rich repeat, and MYB binding sites (MBS). All of those cis-elements are located upstream of the TATA box (Figure 2).

We cloned the coding region of BocMBF1c from genomic DNA, and the deduced BocMBF1c protein contained the MBF1 gene family domain and HTH domain (Figure 1a and Figure S2). The amino acid sequence of BocMBF1c shared 99% and 94% similarity with its ortholog from Brassica napus (CDY39547.1) and Arabidopsis, respectively (NP_189093.1). We queried the BocMBF1 protein sequence using the BLASTP program (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the resulting sequences all belonged to angiosperm. The phylogenetic tree showed BocMBF1c was clustered in one group with MBF1c genes from dicots, while all MBF1c genes from monocots belonged to another group. In addition, BocMBF1c belonged to the MBF1c gene clade from the Brassicaceae family including Brassica napus and Arabidopsis. The AtMBF1a/b were clustered in an individual cluster, implying they belonged to different members of the MBF1 gene family (Figure 1b, Table S1).

2.2. Transient Expression Analysis Showed BocMBF1c Promoter Activity Can Be Induced by Heat Stress

The BocMBF1c promoter contains several HSEs, indicating its role in heat stress response. Thus, we analyzed the activity of the BocMBF1c promoter under heat treatment. We combined the 1409 bp region upstream of the BocMBF1c start code with the CDS of green fluorescent protein (GFP), and then transiently expressed in Nicotiana (N.) benthamiana leaf tissue by agroinfiltration under ambient and heat-stressed conditions. As predicted, the activity of the BocMBF1c promoter was rapidly stimulated under heat stress in Tabaco leaves (Figure 3a,b), which is consistent with previous reports [13].

2.3. Expression Pattern of BocMBF1c

Under a standard growth environment, expression of BocMBF1c was measured in various tissues of Chinese kale (Figure 4a). The results showed BocMBF1c had a comparatively higher expression level in combining sites (CS) and leaf veins (LV) and maintained a comparatively low expression level in other tissues (Figure 4b).

Under heat stress, BocMBF1c expression rapidly increased >250 fold within 0.5 h and maintained this high expression level for at least 8 h in the leaves of Chinese kale (Figure 5a). When the Chinese kale was exposed to chilling conditions, BocMBF1c transcript abundance increased five-fold within 0.5 h, then slowly returned to unstressed levels by 4 h post-treatment (Figure 5b). As illustrated in Figure 2, the BocMBF1c promoter possessed a series of hormone and other related cis-elements. The qPCR analysis confirmed that BocMBF1c was moderately responsive to salinity treatment and several plant hormones including MeJA, ABA, SA, and ET (Figure 5c,d). Interestingly, the temporal response patterns of BocMBF1c expression were distinct among different treatments. Under heat and cold stress, the increase in the BocMBF1c gene reached full extent within 0.5 h. For salinity treatment, the expression level of BocMBF1c reached the maximum after 8 h. When MeJA, ABA, SA, and ethephon (CEPA) were applied, the highest BocMBF1c transcription activities were detected after 2 h, 8 h, 2 h, and 16 h, respectively. Overall, our results indicated that other abiotic factors could also influence the transcript level of BocMBF1c besides responding to high-temperature conditions. These results indicate that BocMBF1c in Chinese kale involves resistance to multiple stresses including heat stress.

2.4. BocMBF1c Protein Localizes to the Nucleus under Heat Stress

Under normal temperature, the GFP-tagged BocMBF1c protein shows no obvious distribution preference when transiently expressed in N. benthamiana leaf tissue (Figure 6b). However, the chimeric BocMBF1c-eGFP protein quickly concentrated into the nucleus when exposed to 37 °C for 1 h (Figure 6c). This translocation is critical for the functionality of this heat responsive transcription coactivator.

2.5. Phenotype and Thermotolerance Analysis of BocMBF1c Overexpression Lines

We selected three T₄ homozygous transgenic lines (Figure S3) overexpressed with the BocMBF1c and β-glucuronidase (GUS) fusion gene driven by the 35S cauliflower mosaic virus (CaMV) promoter (Figure 7a) for further study. We named them BocMBF1c–OE1, BocMBF1c–OE2, and BocMBF1c–OE3. β-glucuronidase activity analyses were performed with 5 day old seedlings to validate the expression of BocMBF1c (Figure 7b). We also examined the growth of 2 week old BocMBF1c-OE plants. The BocMBF1c-OE plants show larger sizes compared to wild type (WT) under normal growth conditions (Figure 7c) which is consistent with a previous report [14]. The five-day-old seedlings grown on MS plates were used for thermal tolerance analysis. After treatment at 46 °C for 2 h, the transgenic lines showed a survival ratio of 21.7% to 30.0%, while only 10.7% of the WT plants survived after heat stress (Figure 7d).

3. Discussion

3.1. BocMBF1c Can Respond to Multiple Stresses and Hormone Treatment in Addition to Heat Stress in Chinese Kale

We previously measured the effects of heat stress among 10 different Chinese kale varieties with different growth parameters, and the variety “Ai-jiao-xiang-gu” was found to be the most tolerable to heat stress [15]. We further measured the expression level of BocMBF1c; “Ai-jiao-xiang-gu” had the highest increase in expression level [15], indicating its important role in heat stress tolerance.

The promoter of BocMBF1c contained several HSEs; moreover, in N. benthamiana cells, the BocMBF1c promoter activity remained steady under ambient temperatures but was activated rapidly by high temperature stress. This is supported by semi-quantitative and qPCR evidence that the transcript abundance of BocMBF1c in planta is strongly and rapidly increased in response to heat stress treatment. Together, these data indicate that the expression of the BocMBF1c gene responds to heat stress via promoter activity. Our results showed that BocMBF1c can respond to cold, salinity, and several hormone treatments thus potentially possessing other functions besides thermal tolerance.

Bioinformatic analysis predicted that the BocMBF1c promoter contained ERE- and ABRE-responding cis-elements, implying ERFs and ABFs (ABRE binding factors)/ABEB could regulate the expression of BocMBF1c. Accumulation of ABA can initiate the expression of stress-responsive genes and participate in the regulation of the network through ABFs/AREB [16]. Ethylene and JA can stimulate the expression of downstream stress-related genes by activating ERF1 [17]. Previous reports showed that AtMBF1c functions upstream of SA and ET during thermotolerance [10]. From our results, BocMBF1c reacted distinctly under different hormone treatments. The expression levels of BocMBF1c both increased after exogenous application of MeJA and ABA although with different temporal response feature. For MeJA and ABA treatment, BocMBF1c expression peaked after 2 h and 4 h, respectively, which is possibly attributed to the difference in activation times for ERF1 and ABFs/AREB. After SA and ET application, the expression level of BocMBF1c both increased after a certain reduction period at the beginning. This indicates that BocMBF1c could possibly be negatively regulated by a high concentration of ET and SA, since the applied hormones would slowly degrade after application. With lower concentrations, ET can activate ERF1 thus stimulating the expression of BocMBF1c.

Overall, the accumulation of ABA, ET, and JA can activate corresponding transcription factors, which will then interact with the cis-elements located at the BocMBF1c promoter. The expressed BocMBF1c protein would then participate in the heat stress regulation network by regulating the downstream SA and ET signaling pathway (Figure 8).

Through analysis of the Arabidopsis public database (http://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpweb.cgi), we found AtMBF1c could be upregulated by heat, salt, and ABA treatment, and that it does not respond to cold and MeJA treatment (Figure S4) (http://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpWeb.cgi) [18]. The different responsive spectrums between BocMBF1c and AtMBF1c implies the BocMBF1c gene could participate in stress-related tolerance for the host plant.

The BocMBF1c protein quickly relocated to the nucleus when the cells were exposed to high temperature, further suggesting that this is a regulator for the expression of many heat stress-responsive genes. However, the mechanism of this nuclear translocation in a high-temperature environment remains unknown and should be the focus of future study. Likewise, downstream genes activated by this transcription factor remain unknown in Chinese kale, and transcriptome analysis by next-generation sequencing would be useful in the identification of candidate downstream genetic elements. Expression of these elements as well as known targets from other species can be further confirmed by qPCR. Earlier studies found the “CTAGA” element as a potential binding sequence for downstream genes; thus, it may be interesting to analyze the orthologs of the downstream genes in Chinese kale. This may reveal the reason for the relative temperature sensitivity of Chinese kale. Thus, it is highly likely that the BocMBF1c gene is necessary for multiple biotic/abiotic stresses in Chinese kale.

3.2. BocMBF1c Can be Applied to Improve the Productivity of Chinese Kale

The high-level conservation of MBF1c sequences (Table S1 and Figure S2) implies that this gene is critical for thermal tolerance and climate adaptation. For Chinese kale, bolting stems are the main edible tissue. The genetic mechanism underlying declined yield and quality of bolting stems under heat stress are still elusive in Chinese kale; cloning of BocMBF1c genes as a key heat tolerance factor would shed light on improving the heat tolerance of Chinese kale.

Transformation of Chinese kale is readily feasible and has been used for improving drought stress tolerance [19]. Our results indicate that the BocMBF1c gene can respond to heat treatment from both transcription and protein localization level. Overexpression BocMBF1c in Arabidopsis result in larger-sized plants with enhanced heat tolerance, while knockout of the BocMBF1c gene will be needed to fully validate its role in the thermal tolerance of Chinese kale, for example, by the CRISPR/Cas9-based gene editing technique [20]. Ectopic expression of AtMBF1c can improve tolerance to bacterial infection, heat, and osmotic stress in Arabidopsis [14]. Further study showed that overexpression of AtMBF1c could improve thermal tolerance without impairing the yield in Arabidopsis [10], soybean [11], and rice [21] under controlled conditions. A recent report showed that overexpression of MBF1c in Antarctic moss could make the plant bigger and enhance tolerance to salinity stress in Arabidopsis [12]. Thus, it will be intriguing to overexpress the BocMBF1c gene as a potential solution to improving the biotic/abiotic stress resistance and productivity of Chinese kale.

4. Materials and Methods

4.1. Plant Growth and Abiotic Treatment Conditions

We collected Chinese kale samples for tissue-specific expression analysis from the greenhouse (22–25 °C) at the South China Agricultural University (Guangzhou, China) [2]. For abiotic treatment, Chinese kale (B. oleracea var. chinensis Lei) variety “Ai-jiao-xiang-gu” were grown in a culture room under controlled conditions at 25 °C, with a 16 h light/8 h dark cycle. Chinese kale seedlings of 4~6 true leaves were transferred to the growth chamber for 24 h with the same conditions, and then the temperature was switched to heat (37 °C) and cold (4 °C) treatments. Leaf tissues were harvested for RNA extraction at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h.

For salinity treatment, each Chinese kale seedling of 4~6 true leaves were sprayed and watered by 100 mL of 100 mM NaCl, and leaf tissues were collected with the timeline of 0 h, 1 h, 2 h, 4 h, 8 h, and 16 h. Similarly, hormone treatments were carried out by applying 50 μM of MeJA solution, 100 μM ABA, 100 μM SA, and 100 ppm CEPA, respectively, and leaf tissues were collected along the same timeline. All samples used in this study were collected from three separated Chinese kale.

Arabidopsis thaliana (ecotype Columbia) were grown in chamber under controlled condition at 22 °C day/18 °C night, 70% relative humidity, and 100 μmol m⁻² s⁻¹ with a 16 h light/8 h dark light cycle. Arabidopsis seeds were germinated on MS plates or soil (Sunshine^® Mix #8, Sun Gro Horticulture) after being sterilized with 10% bleach for 10 min. All seeds were kept at 4 °C for at least 2 days before transfer to the growing chamber.

4.2. Cloning of the BocMBF1c CDS and Promoter

Leaves of Chinese kale were harvested for genomic DNA extraction [22]. We used the MBF1c gene from Brassica rapa (LOC103828628) as a reference sequence (Supplementary B1) to design primers for the CDS of BocMBF1c. The gDNA fragment containing the BocMBF1c CDS was amplified with primer MBF1c-up (5’-TCGAACTCTCCAGAAACTCGT-3’) and MBF1c-dw (5’-CGTTTGCCAGATACGATGT-3’). Next, BocMBF1c promoter was amplified with primer MBF1cP-F (5’-AGAATCCCCATTGACAGCCT-3’) and MBF1cP-R (5’-GGCATCGTCGCTGTTGTTTA-3’), designed from the gene sequence of Brassica oleracea (Supplementary B2). All PCRs were carried out with HiFiTaq PCR StarMix (GeneStar) according to the manufacturer’s protocol.

4.3. Sequence Analysis of the BocMBF1c Gene

The open reading frame (ORF) of BocMBF1c was predicted by the ORF finder (http://www.bioinformatics.org/sms2/orf_find.html), and the amino acid sequence was generated by the Expasy-Translate tool (https://web.expasy.org/translate/). Sequence query and alignment were carried out with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was constructed with the software Mega 6.05, and the conserved sites were identified by DNAMEN.

The cis-elements of the BocMBF1c promoter were predicted by PlantCARE Database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

4.4. Promoter Activity and Subcellular Localization

The putative BocMBF1c promoter of the 1409 bp upstream sequence from the initiation codon was cloned into the binary vector pBI101-GFP with In-Fusion cloning, using primers designed following the manufacturer’s instructions (http://bioinfo.clontech.com/infusion/convertPcrPrimersInit.do), including pBI101 infusion M-P-F (5’-GCAGGTCGACTCTAGAGGTACCGAATAGCTCTCCCC-3’) and pBI101 infusion M-P-R (5’- CTTTACCCATTCTAGAGGCATCGTCGCTGTTGTTTA-3’). Similarly, CDS of the BocMBF1c was inserted into the pBE-eGFP construct by the In-Fusion technique, with primers MBF1-yxb–F (5’-CGCGGGCCCGGGATCCATGCCGAGCAGAT-3’) and MBF1-yxb-R (5’-GGCGACCGGTGGATCCTTGACATGTTT-3’). Xba I was used to digest the pBI101-GFP vector, and pBE-eGFP was digested by BamH I, resulting in pBI101-pBocMBF1c-GFP and pBE-BocMBF1c-eGFP constructions.

Transient expression was carried out in the leaf tissue of Nicotiana benthamiana seedlings with 4 to 6 true leaves, and with Agrobacterium strain GV3101 harboring the pBI101-pBocMBF1c-GFP and pBE-BocMBF1c-eGFP constructs. The GFP signal was observed two days after agro-infiltration under fluorescent microscopy [23].

4.5. Gene Expression Patterns of BocMBF1c by qPCR and Semi-Quantitative RT-PCR

The RNA was extracted using HiPure Plant RNA Kits (Magen), and HiScript^® II Q Select RT SuperMix (Vazyme) was used for cDNA synthase. Gene expression was assessed by real-time PCR using AceQ^® qPCR SYBR^® Green Master Mix (Vazyme) and Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Primers MBF1c-YG1-UP (5’-GTTAACGCGGCTCTCAGAAG-3’) and MBF1c YG1-DW (5’-TCCTCCAGCTTCTTCGTGTT-3’) were used for BocMBF1c; Tubulin8 was used as an internal control with primers Tubulin-F (5’-CTTCTTTCGTGCTCATTTTGCC-3’) and Tubulin-R (5’-CCATTCCCTCGTCTCCACTTCT-3’) [19]. The qPCR conditions: initial denaturation of 95 °C for 5 min, then 40 cycles of 95 °C for 10 s followed by 60 °C for 30 s; melting curves were obtained by gradually increasing the temperature from 60 °C to 95 °C for 15 s at a rate of 0.5 °C /s. Relative expression was calculated by the ∆∆Ct threshold method [24]. For heat and cold treatment, semi-quantitative RT-PCR was used to detect the expression pattern of BocMBF1c with Recombinant Taq DNA Polymerase TaKaRa Taq™ (Takara) and the same primers of qPCR. Semi-quantitative RT-PCR conditions: initial denaturation of 94 °C for 2 min, then 26 cycles for Tubulin8 and 30 cycles for BocMBF1c of 95 °C for 30 s, 56 °C for 15 s, 72 °C for 10 s. Three biological replicates with triple technical replicates were used for all samples.

4.6. Arabidopsis Transformation and Phenotypic Analysis of Transgenic Plants

The BocMBF1c coding sequence was fused with GUS and then expressed in Arabidopsis thaliana driven by the 35S CaMV promoter and then integrated into the pBI121 binary vector. The floral dipping method [25] was used for Arabidopsis transformation. Transgenic seeds were selected on a 0.5 MS agar plate with 50 mg/L kanamycin and 300 mg/L timentin and then transplanted to soil for setting the seeds under standard growth conditions. Three homozygous transgenic lines from independent transformation events were used for further study (Figure S3).

Five-day-old BocMBF1c-OE and Col-0 Arabidopsis seedlings were collected for GUS staining analysis after germination on MS plates [13]. Two-week-old seedlings grown on soil were harvested for measuring fresh weight. For thermal tolerance analysis, five-day-old seedlings grown on MS plates were treated at 46 °C for 2 h, and then these plates were put back into growth chambers under normal condition for another 2 days.