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Article

Functional Characterization of Tea Plant (Camellia sinensis L.) CsCBF2 Gene Involved in Multiple Abiotic Stress Response in Tobacco (Nicotiana tabacum L.)

by
Qiying Zhou
1,2,3,†,
Dongxiao Liu
1,2,3,†,
Yiwan Wei
1,2,3,
Ning Ma
1,2,3,
Ruijiao Zhang
1,2,3,
Zengya Zhang
1,2,3,
Changjun Jiang
1,2,3 and
Hongyu Yuan
1,2,3,*
1
Henan Key Laboratory of Tea Plant Biology, Xinyang Normal University, Xinyang 464000, China
2
Henan Engineering Research Center of Tea Deep-Processing, Xinyang Normal University, Xinyang 464000, China
3
Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2022, 8(9), 853; https://doi.org/10.3390/horticulturae8090853
Submission received: 29 July 2022 / Revised: 12 September 2022 / Accepted: 13 September 2022 / Published: 19 September 2022
(This article belongs to the Special Issue Advances in Tea Plant Biology and Tea Quality Regulation)
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Abstract

:
C-repeat binding factors/dehydration responsive element binding factors 1 (CBFs/DREB1s) are a small family of transcription factors that play important roles in plant resistance to various external stresses. However, functional characterization of tea plant (Camellia sinensis L.) CBF gene (CsCBF) was still seldom reported. Here, functional study of the cold-responsive CBF gene (CsCBF2) was done. Results showed that CsCBF2 had conserved AP2 DNA-binding domain and the typical PKK/RPAGRxKFxETRHP and DSAWR signature sequences of CBF/DREB1. Yeast one-hybrid and transcription activation assays revealed that the activation domain of CsCBF2 could activate the reporter gene expression, and the N terminal of CsCBF2 displayed an inhibitory effect. Although CsCBF2 was conserved to bind the C-repeat/dehydration-responsive element (CRT/DRE), intact CsCBF2 protein preferred the CRT cis element. Under normal growth conditions, CsCBF2-overexpressing tobacco plants (CsCBF2-OX) exhibited lighter green leaf color, growth retardation, and dwarfism. Smaller leaf of CsCBF2-OX was only seen in eight weeks after been sown in soil. Under cold, salinity, or drought stress, CsCBF2-OX displayed better growing with longer roots, heavier fresh weight, higher germination rate, and accumulated more proline and sugar contents, but lower electrolyte leakage. The results demonstrated that CsCBF2 enhanced plant tolerance to multiple abiotic stresses.

1. Introduction

Plants are sessile organisms that always encounter a series of abiotic stresses during their life span, especially cold, drought, and salt stresses. The adverse environmental stresses threaten plant growth and development, and limit plant geographical distribution, productivity, and quality as well [1,2,3]. To combat and survive adverse conditions, plant evolved to generate sophisticated physiological and morphological changes such as cell wall remodeling, membrane re-organization, osmolytes accumulation, anti-oxidation system activation, etc., which were realized by gene expression regulation [1,4,5]. Transcription factors function in gene expression regulation, and play crucial roles in plant stress tolerance [6,7,8].
CBFs (C-repeat binding factors), also known as dehydration responsive element binding factors (DREB1s), are a class of well-known transcription factors regulating resistant gene expression involved in cold, drought, and salt stresses [9,10,11,12,13]. CBFs/DREB1s can activate the stress-related gene expression by binding to the TGGCCGAC (CRT; C-repeat) or TACCGACAT (DRE; dehydration-responsive element) cis-acting element, which was known as CRT/DRE [2,14]. In Arabidopsis, DREB1 proteins activate an array of downstream CRT/DRE cis-element containing genes, which play important roles in enhancing plant cold, desiccation, and salinity tolerance [15]. Overexpression of CBF/DREB1 in Arabidopsis improved tolerance to drought, salinity, and freezing stresses [16]. Several DREB1s have been cloned from rice, overexpression of OsDREB1A, 1B, or 1F enhanced cold, drought, and salt tolerance in both Arabidopsis and rice plants [8].
DREB proteins are subfamily members of the AP2/ERF superfamily with a highly conserved AP2 DNA-binding domain, and are further divided into six groups, named A-1 to A-6, which may act different roles in plant stress response. The A-1 group (CBF/DREB1), including AtCBF1/DREB1B, AtCBF2/DREB1C, AtCBF3/DREB1A, has highly conserved AP2 DNA-binding domain and two typical signature sequences (PKK/RPAGRxKFxETRHP and DSAWR) [17]. The A-2 group (DREB2), including DREB2A and DREB2B, always had the conserved AP2 domain and CMIV-1 ([K/R]GKGGPxN) motif [18]. It was reported that the CBF/DREB1 group has a major function in cold stress response, while the DREB2 group functions in dehydration and salt stresses [12,13,19]. However, studies demonstrated that OsDREB2A was induced not only by drought and salinity, but also by low temperature [20]. Overexpression of EsDREB2B in tobacco enhanced plant resistance to multiple abiotic stresses, including salt, cold, heat, and osmotic stress [21]. Expression of Hemarthria compressa HcDREB2 gene was also induced by drought, salinity and cold [22]. Expression of the A-1 group EgCBF3 gene in oil palm was up-regulated by cold, as well as drought and salt stress [23]. Constitutive expression of A-1 group OsDREB1E and OsDREB1G could also increase drought tolerance of rice plant [8]. Overexpression of Chrysanthemum lavandulifolium ClCBF1 improved plant tolerance to salt and drought stress [24]. So, the regulating function of CBFs/DREB1s was complex, and there was functional cross between CBF/DREB1 and DREB2 subgroups in regulating plant abiotic stress.
Although CBFs/DREB1s are highly conserved in plants, significant differences exist in different CBF/DREB1 genes and different plant species [11,12,25]. First, CBF/DREB1 genes in the same species had functional divergence. In Arabidopsis, CBF1 and CBF3 are induced rapidly by cold treatment, while CBF2 is induced by drought or salt stress but not by cold [8,26]. Overexpression of rice OsDREB1D or OsDREB1G enhanced plant tolerance to cold stress, while overexpression of rice OsDREB1A, 1B or 1F enhanced cold, drought, and salt tolerance in both Arabidopsis and rice plants [8]. It was reported that Vitis CBF4 was more tolerant to freezing stress, while Vitis CBF1 was more tolerant to drought stress [27]. Second, the same family CBF genes in different plant species exhibited distinct functions. Overexpression of DREB1A/CBF3 in Arabidopsis improved tolerance to drought, salinity, and freezing stresses [16]. Lolium perenne LpCBF3 gene, an ortholog of OsDREB1A/CBF3 in rice plant, was induced by cold stress but not by drought or salinity [28]. In sweet potato, SwDREB1/IbCBF3 gene expression was induced by cold and drought stresses, but was little affected by salt stress [29]. Third, CBF genes could be regulated by each other. It was known that expression of AtCBF2, AtCBF1, and AtCBF3 was regulated interactively to control cold response in Arabidopsis, AtCBF2 was a negative regulator of AtCBF1 and AtCBF3 [2,30]. Overexpression of wheat DREB3 in barley led to up-regulation of seven HvCBF genes and down-regulation of three HvCBF genes. Activation of HvCBF9 and HvCBF14 genes could suppress the expression of HvCBF12 gene [31]. Fourth, different plant species have different CBF/DREB1 gene numbers. In Arabidopsis, Populus, Brassica, Betula, and Eucalyptus, there are more than six CBF/DREB1 genes in these plants [12]. In Vitis vinifera and Vitis riparia, four CBF genes have been identified [27]. It was estimated that at least 20 HvCBF genes existed in barley genome [31].
CBFs/DREB1s had been identified in various plant species, such as Arabidopsis (A. thaliana), rice (Oryza sativa), maize (Zea mays), grape (V. vinifera), poplar (Populus euphratica), apple (Malus domestica), and birch (Betula pendula), etc. [25,26,32]. Tea plant (C. sinensis) is an important economic crop for making beverage. The growth of tea plant is seriously affected by various environmental stresses, especially extreme low temperature and drought [3,33,34]. Genome-wide analysis suggested that six CsCBF family genes existed in tea plant genome [3]. Although function studies demonstrated that overexpression of tea plant CsCBF3 gene enhanced plant cold tolerance [35], overexpression of tea plant CsDREB gene enhanced salt and drought tolerance of Arabidopsis [33]. There were still very few studies on functional analysis of CsCBF genes. In our previous research, several cDNA clones including two tea plant CBF-coding genes, were identified by suppression subtractive hybridization (SSH) under cold stress treatment [36]. CsCBF1, one of the CBF-coding genes, was further analyzed and displayed an important role in transcription regulation and cold response [37]. Here, we reported the cloning of the other tea plant CBF gene (CsCBF2) isolated in our previous SSH analysis. In addition, the CRT/DRE cis-element binding and transcription activity of CsCBF2 was identified in yeast cells. CsCBF2-overexpressing transgenic tobacco plants were generated and function of CsCBF2 gene was analyzed. Our results revealed that CsCBF2 gene encodes a typical CBF/DREB1 protein. It displayed binding preference to the CRT cis element, and the N terminal of CsCBF2 had an inhibitory effect on its trans-activation activity. Overexpression of the CsCBF2 gene enhanced plant tolerance to multiple abiotic stresses.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Two-year old seedling cuttings of Xinyang “Quntizhong” tea plant (C. sinensis) were cultivated in a growth chamber under 24 °C, 70% relative humidity, 12 h photoperiod with 160 μM m−2 s−1 light intensity. The temperature of the growth chamber was adjusted to 4 °C after seven days, and then one to four leaves downward from the terminal bud were collected after 7 h. The leaves were frozen in liquid nitrogen quickly and used for RNA extraction.
Wild-type tobacco plants (Nicotiana tabacum L. cv. Petite Havana SR1) were grown in pots with soil and cultured in growth chamber under 25 °C, 16 h light/8 h dark, 70% humidity, 200 μM m−2 s−1 light intensity. Leaves of the wild-type tobacco plants were used for genetic transformation. The regenerated transformants were transferred to soil and cultured under the same conditions as those for wild-type.

2.2. RACE Cloning of CsCBF2 Gene

The tea plant genome sequencing program had not yet been complete, and there was only partial ORF (open reading frame) sequence of CsCBF2 gene existed in the SSH identified cDNA clone when our study began. So, the full length cDNA of CsCBF2 gene was cloned by RACE (rapid amplification of cDNA ends) using the SMARTTM RACE cDNA kit (Clontech, Palo Alto, CA, USA). Total RNA of tea plant was isolated using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). 5′ and 3′ RACE ready cDNAs were prepared by using the total RNA of tea plant as template following the manufacture’s instruction, and the 10 × Universal Primer A Mix provided in the SMARTTM RACE cDNA kit and CsCBF2 gene specific primers (GSPs) were used for 5′ and 3′ RACE reactions. The GSPs were designed according to the partial CsCBF2 ORF sequence identified before using the web-based Primer 3.0 program (https://primer3.ut.ee/, accessed on 6 June 2014; Supplementary Table S1). PCR procedures for the 5′ and 3′ RACE reactions were as follows: 94.0 °C 5 min, followed by 30 cycles of 94.0 °C 30 sec, 55.0 °C (5′ RACE) or 50.0 °C (3′ RACE) 30 s, and 72.0 °C 1 min 30 s. The RACE reactions were completed on a PCR instrument (Bio-Rad, Hercules, CA, USA). The RACE products were characterized and sequenced, and were assembled with the partial CsCBF2 gene sequence inserted in the SSH-identified cDNA clone. The full-length cDNA of CsCBF2 was confirmed by long distance PCR (LdPCR) with specific primers (Table S1) following the manufacture’s instruction. The ORF of CsCBF2 was predicted by ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 1 July 2014). Molecular weight and isoelectric point of CsCBF2 were analyzed by the ExPASy online tool (https://www.expasy.org/, accessed on 15 July 2014).

2.3. Sequence Analysis and Phylogenetic Tree Building

Conserved protein domain of CsCBF2 was analyzed by the online CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 8 April 2021) and SMART (http://smart.embl-heidelberg.de/, accessed on 8 April 2021) tools. Amino acid sequence alignment of CsCBF2 with the homologous proteins from other plants was done by the BioEdit software (version 7.2.6.1, Tom Hall, Raleigh, NC, USA). The phylogenetic tree of CsCBF2 was constructed with 1000 bootstrap trials by the MEGA-X software (version 10.2.2, Sudhir Kumar, Tokyo, Japan) with Neighbor Joining method.

2.4. Yeast One-Hybrid and Transcription Activation Assays

For transcription activation analysis, CsCBF2 ORF sequence and CsCBF2 gene fragment coding the transcription activation domain (CsCBF2AD) were respectively PCR amplified from the full-length cDNA of tea plant by KOD_Plus-Neo DNA polymerase (Toyobo, Osaka, Japan) with the specific primers (Table S1) containing EcoRI and PstI restriction enzyme sites. Then, the amplified fragments were respectively cloned into the pGBKT7 yeast expression vector and transformed into AH109 yeast cells. The transactivation activities of CsCBF2 and CsCBF2AD were first selected on SD/-Trp plates, then confirmed by transformed yeast cells growth on the SD/-Trp/-His/-Ade plates.
For the specific binding analysis of CsCBF2 to the CRT/DRE cis-acting element, yeast one-hybrid assay was designed. The 4 × TGGCCGAC (CRT) and 4 × TACCGACAT (DRE) core sequences of CRT/DRE and their complementary sequences, with sticky end of EcoRI and SacI restriction site respectively on each side (Table S1), were synthesized by Genscript. Then, the synthesized core sequences of CRT/DRE and their complementary sequences with the sticky ends were respectively annealed to form short dsDNA fragments with EcoRI and SacI sticky ends. To check the binding specificity of CsCBF2 to the CRT/DRE sequences, the mutated 4 × CRT/DRE core sequences (4 × TGGCTTTC and 4 × TACTTTCAT) and their complementary sequences, with sticky end of EcoRI and SacI restriction site respectively on each side, were also synthesized and annealed to form mutated 4 × CRT/DRE short dsDNA fragments. Both the normal and the mutated 4 × CRT/DRE short dsDNA fragments were cloned into the pHIS2.1 through the EcoRI and SacI cloning sites. The ORF sequence and nucleotide sequence coding the AP2 binding domain of CsCBF2 (CsCBF2BD) were respectively PCR amplified from the full-length cDNA of tea plant by KOD_Plus-Neo DNA polymerase with the specific primers (Table S1) containing EcoRI and BamHI restriction enzyme sites. Then, the amplified fragments were cloned into the pGADT7-Rec2 yeast expression vector. CsCBF2-pGADT7-Rec2 or CsCBF2BD-pGADT7-Rec2 vector was transformed into Y187 yeast cells with the normal or mutated 4 × CRT/DRE-pHIS2.1 vector. The yeast one-hybrid assay was done as the protocol supplied by Matchmaker™ One-Hybrid Library Construction & Screening Kit (Clontech, Palo Alto, CA, USA).

2.5. Agrobacterium-Mediated Transformation of Tobacco Plants

For genetic transformation, ORF sequence of the CsCBF2 gene was amplified by KOD_Plus-Neo DNA polymerase with the specific primers containing BamHI and SacI restriction enzyme sites, with the CsCBF2-pGADT7-Rec2 plasmid as template. The amplified CsCBF2 gene was purified, and then cloned into the pCAMBIA1300 binary vector by the BamHI and SacI cloning sites after the cauliflower mosaic virus (CaMV) 35S promoter. The recombinant vector 35S:pCAMBIA1300-CsCBF2 was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation and used for tobacco transformation by leaf disc transformation method [38]. Regenerated CsCBF2-overexpressing transformants were selected on 1/2 MS medium containing 250 mg L−1 carbenicillin and 50 mg L−1 hygromycin B. The specific primers used for CsCBF2 gene cloning were listed in Table S1.

2.6. Molecular Characterization of CsCBF2-Overexpressing Transgenic Tobacco Plants

DNA was extracted from leaves of the regenerated CsCBF2 transgenic plants using the CTAB method [39]. Then, PCR amplification was done using the CsCBF2 gene primers for tobacco transformation vector building, and 2 × Power Taq PCR MasterMix (Bioteke, Beijing, China). The amplicon of CsCBF2 was used to identify the positive CsCBF2 transgenic plants by electrophoresis detection.
To identify the positive CsCBF2-overexpressing tobacco plants (CsCBF2-OX) on transcriptional level, reverse transcription PCR (RT-PCR) was employed. Total RNA was isolated from CsCBF2-OX with TRIzol reagent (Takara, Osaka, Japan). Reverse transcription of total RNA into cDNA was done as we did before [40]. Then, RT-PCR was done by using the CsCBF2 gene primers for tobacco transformation vector building, 2 × Power Taq PCR MasterMix (Bioteke, Beijing, China), and the cDNA from reverse transcription. Size and intensity of the CsCBF2 amplicon were used to confirm the overexpression of CsCBF2 by electrophoresis detection. Tobacco Actin (Ntactin) gene was amplified and used as equal cDNA loading control. The primers used for Ntactin amplification are listed in Table S1.
The DNA amplification result and expression level of CsCBF2 gene in wild type tobacco plant (WT) was respectively used as control for DNA and expression analysis of CsCBF2-OX.
For Western blot analysis, proteins were extracted from leaves of CsCBF2-OX according to Zhou at al. [40]. A total of 15 μg proteins of each sample were fractionated on 12% (w/v) SDS–PAGE. Proteins were electroblotted onto nitrocellulose membranes after electrophoresis. Overexpressed CsCBF2 protein was detected by using the CsCBF2 antibody (1:800) from immunized mouse. The antibody was prepared by expressing the CsCBF2 coding sequence carried by the pCzn1 vector through NdeI and XbaI restriction enzyme sites in Escherichia coli (ArcticExpress, DE3). The CsCBF2 recombinant protein was induced by 0.5 M IPTG (isopropyl-β-D- thiogalactopyranoside) under 37 °C and purified on the sepharose CL-6B affinity chromatography column following the manufacturer’s instructions. The purified CsCBF2 recombinant protein was detected by SDS-PAGE, and its concentration was measured by Bradford method [41]. The purified CsCBF2 recombinant protein was then injected into a mouse. The mouse serum was used as the primary antibody for Western blot analysis of CsCBF2. Tobacco Actin (Ntactin) protein was used as the control for equal protein loading. The mouse sourced-Ntactin antibody was bought from a company (Abmart, Shanghai, China). The HRP-conjugated goat anti-mouse IgG (Abcam, Shanghai, China) was used as the secondary antibody. The protein blotted membranes were incubated with the chemiluminescent HRP substrate, the protein signals were detected by ChemiDoc MP Imaging System with a CCD camera (Bio-Rad, Hercules, CA, USA).
The experiments of DNA amplification, RT-PCR, and Western blot were repeated three times.

2.7. Low Temperature Tolerance Assays of CsCBF2-Overexpressing Transgenic Plants

Surface-sterilized seeds of T2 generation CsCBF2-OX and WT were sown on 1/2 MS culture medium for 16 days. The plants were transferred to the newly prepared MS culture medium growing for 5 days, and then they were used for cold and freezing treatments.
For cold stress treatment, the CsCBF2-OX and WT were transferred to the growth chamber under 4 °C, 16 h light/8 h dark, 70% humidity, 200 μM m−2 s−1 light intensity, for 16 days. CsCBF2-OX and WT kept in growth chamber under 25 °C, 16 h light/8 h dark, 70% humidity, 200 μM m−2 s−1 light intensity were used as controls. After the treatment, the cold-treated plants were transferred back to the normal growth chamber under 25 °C, 16 h light/8 h dark, 70% humidity, 200 μM m−2 s−1 light intensity for growing for 5 days. Then, root length and fresh weight of the treated plants and the control plants were respectively measured.
For freezing stress treatment, the plants were transferred to soil and cultivated in normal growth conditions for 21 days under 25 °C. The plants were moved to the growth chamber under 0 °C for 5 days, and then under −2 °C for 16 h. At last, the plants were transferred back to 25 °C for 5 days recovery. For all the treatments, the photoperiod was 16 h light/8 h dark. Plants before and after the freezing treatment were respectively photographed. Survival rates of CsCBF2-OX and WT were respectively calculated after 5 days’ recovery.

2.8. Salt and Drought Tolerance Assays of CsCBF2-Overexpressing Transgenic Plants

Seeds of T2 generation CsCBF2-OX and WT were surface-sterilized in 10% sodium hypochlorite for 5 min and rinsed five times with sterile distilled water. The seeds were kept in autoclaved tube with small amount of sterile water at 25 °C under dark for 4 days. For salt or drought stress treatment, the sterilized seeds of CsCBF2-OX were sown on 1/2 MS culture medium containing 20 mg L−1 hygromycin B and 150 mM NaCl or 400 mM mannitol, respectively. The sterilized seeds of WT were sown on 1/2 MS culture medium containing 150 mM NaCl or 400 mM mannitol, respectively, without hygromycin B. The seeds germination number and germination rate of both CsCBF2-OX and WT were recorded daily.
For root length and fresh weight comparative analysis, seeds of T2 generation CsCBF2-OX and WT were surface-sterilized and sown on 1/2 MS culture medium for 16 days. Then, seedlings of WT and CsCBF2-OX were transferred to 1/2 MS culture medium containing 200 mM NaCl and 200 mM mannitol for 20 days, respectively. As controls, surface-sterilized seeds of T2 generation CsCBF2-OX and WT were sown on 1/2 MS culture medium without NaCl or mannitol.
The cultivation plates were placed in growth chamber under 25 °C, 16 h light/8 h dark, 70% humidity, 200 μM m−2 s−1 light intensity conditions.

2.9. Cell Membrane Stability Assay

For cell membrane stability test, the CsCBF2-OX and WT were planted first as we did before the freezing treatment. After the plants were transferred to soil and cultivated in growth chamber with normal growth conditions for 21 days, both the CsCBF2-OX and WT were transferred to growth chamber under −6 °C for 2 h. Leaves from the same position of the CsCBF2-OX and WT were respectively collected before and after the freezing treatment, washed clean with tap water and rinsed thoroughly with double-distilled water. Leaves were cut into small pieces of the same size after drained with sterilized filter paper. About 0.5 g leaf pieces of CsCBF2-OX and WT were immersed in 20 mL double-distilled water in Erlenmeyer flasks, and vacuumed for 30 min at room temperature. The Erlenmeyer flasks were transferred to shaking tables at 170 rpm, 25 °C for 1.5 h, and then allowed to stand for 20 min. Relative electrical conductivity (S1) of solution in each of the Erlenmeyer flask was measured by a conductivity meter (SuoShen, Shanghai, China). Then, the solutions were boiled for 15 min, cooled to room temperature, and the relative electrical conductivity (S2) of each solution was measured. The relative electrical conductivity (S0) of the double-distilled water was also measured before the leaf pieces were immersed in. The electrolyte leakage was calculated by (S1 − S0)/(S2 − S0) × 100.

2.10. Soluble Sugar and Proline Contents Analysis

The CsCBF2-OX and WT were planted first as we did before the cold stress treatment. After the plants were transferred to soil and cultivated in normal growth conditions under 25 °C for 21 days, both the CsCBF2-OX and WT were transferred to the growth chamber under 4 °C for 8 h. Leaves from the same position of CsCBF2-OX and WT were collected. About 0.5 g collected leaf samples were ground into powder, and extracted with distilled water and sulphosalicylic acid respectively. The extracted products were used for soluble sugar and proline contents analysis by anthrone-sulfuric acid and acid ninhydrin method, respectively [42].

3. Results

3.1. Isolation and Characterization of CsCBF2 Gene

Full-length cDNA of CsCBF2 gene was acquired by RACE clone as described in Materials and Methods. Results showed that about 600 bp bands were produced by both 5′ RACE and 3′ RACE (Figure 1A). After sequencing and assembly, result suggested that the full-length cDNA of CsCBF2 gene was 1162 bp (GenBank accession no. KC702795). The long distance PCR of CsCBF2 gene resulted in a 1062 bp band (Figure 1A), and the sequencing result was 100% identical to the assembled full-length cDNA of RACE cloning. The full-length cDNA of CsCBF2 contains 193 bp 5′ untranslated region, 720 bp ORF, and 249 bp 3′ untranslated region. CsCBF2 encoded 239 amino acid residues with predicted molecular mass of 26.44 kDa and pI value of 5.22. Protein domain analysis showed that CsCBF2 had conserved AP2 domain (Supplementary Figure S1; Figure 1B). In addition, protein sequence alignment showed that two typical signature sequences PKK/RPAGRxKFxETRHP and DSAWR were respectively laid before and after the AP2 domain of CsCBF2 but not found in AtDREB2A and AtDREB2B (Figure 1B). The transcription activation domain of CsCBF2 was more similar to CBF/DREB1 proteins than to AtDREB2 proteins (Figure 1B). Phylogenetic analysis was done by using CsCBF1, 2, 3, and AtCBF1, 2, 3, and other CBF proteins from Actinidia deliciosa, Nyssa sinensis, Camptotheca acuminate, P. euphratica, Ipomoea batatas, Lycopersicon esculentum, M. domestica, V. vinifera, Hordeum vulgare, Deschampsia Antarctica, Malus baccata, B. pendula, Eucalyptus gunnii, Prunus avium, Triticum aestivum, and O. sativa. Result showed that CsCBF2 was clustered with CsCBF1, and was closely related to homologues from A. deliciosa, N.sinensis, C. acuminate, and was on the same branch with LeCBF1 and IbCBF3 (Figure 1C).

3.2. CRT/DRE-Binding and Transactivation Assay of CsCBF2

To confirm that CsCBF2 was a CBF/DREB1 protein of AP2 superfamily, the transactivation activity and the specific binding to CRT/DRE cis-element were examined in yeast cells. To examine the transactivation activity, CsCBF2 ORF sequence and nucleotide sequence coding the transcription activation domain of CsCBF2 (CsCBF2AD) were respectively cloned into pGBKT7 vector and subsequently transformed into AH109 yeast cells. The yeast cells harboring pGBKT7-CsCBF2 and pGBKT7-CsCBF2AD grew well on SD/-Trp plate, but only yeasts harboring pGBKT7-CsCBF2AD could grow on SD/-Trp/-His/-Ade plate (Figure 2A–D). To examine the specific binding of CsCBF2 to CRT/DRE, yeast one-hybrid assay was employed. Four-CRT/DRE tandem core elements (4 × CRT/DRE; 4 × TGGCCGAC, 4 × CRT; 4 × TACCGACAT, 4 × DRE) and the mutated 4 × CRT/DRE core elements (4 × mCRT/DRE; 4 × TGGCTTTC, 4 × mCRT; 4 × TACTTTCAT, 4 × mDRE) were respectively cloned into the pHIS2.1 vector. CsCBF2 ORF sequence and nucleotide sequence coding the AP2 domain of CsCBF2 (CsCBF2BD) were cloned into pGADT7-Rec2 vector containing the GAL4 DNA activation domain, respectively. According to the instruction of Matchmaker™ One-Hybrid Library Construction & Screening Kit (Clontech, Palo Alto, CA, USA), the optimum 3-amino-1, 2, 4-triazole (3-AT) concentration for suppressing the basal expression of HIS3 reporter gene in the pHIS2.1 vector was done first. It displayed that 25 mM 3-AT had an obvious inhibition to the basal expression of HIS3 reporter gene (Figure 2E,F). Then, the pGADT7-Rec2-CsCBF2 or the pGADT7-Rec2-CsCBF2BD plasmid was transformed into Y187 yeast cells together with pHIS2.1-4 × CRT/DRE, or pHIS2.1-4 × mCRT/DRE vector, and selected on SD/-Trp/-His/-Leu plate with 25 mM 3-AT. Results showed that cells harboring pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × CRT or pHIS2.1-4 × DRE plasmids, and cells harboring pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × CRT plasmids grew well on SD/-Trp/-His/-Leu plated with 25 mM 3-AT. However, yeast cells harboring pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mCRT/DRE, those harboring pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mCRT/DRE, or those harboring pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × DRE displayed obvious growth inhibition on SD/-Trp/-His/-Leu plated with 25 mM 3-AT (Figure 2G).

3.3. Generation and Molecular Identification of CsCBF2 Transgenic Plants

To reveal biological function of CsCBF2 gene, 35S:CsCBF2-pCAMBIA1300 overexpression recombinant vector was built and transformed into tobacco plants by agrobacteria-mediated transformation (Figure 3A). In total, 21 independent T0 generation CsCBF2-overexpressing transgenic lines were acquired. About 720 bp target bands were identified in 17 of the 21 independent CsCBF2 transgenic lines (Figure 3B). Seeds of the positive T1 generation CsCBF2-overexpressing tobacco plants (CsCBF2-OX) were surface-sterilized and selected on 1/2 MS culture medium containing hygromycin B for 20 days. Then, leaf samples were respectively collected from nine hygromycin resistant transgenic plants, which belonged to three independent T0 generation CsCBF2-OX L5, L15, and L17. Total RNAs were isolated from the leaf samples, and RT-PCR amplification showed that all of the identified CsCBF2-OX had the 720 bp target bands, while WT had no amplification of CsCBF2 (Figure 3C). Three T2 generation CsCBF2 transgenic plants, which were from the L5, L15, and L17 independent T0 generation transgenic lines, were randomly selected for Western blot analysis. Result showed that CsCBF2 protein was overexpressed in the transformed tobacco plants, and the WT had no CsCBF2 protein expression (Figure 3D). These results confirmed that CsCBF2 gene was successfully transformed and overexpressed in tobacco plants.

3.4. Overexpression of CsCBF2 Gene Leads to Phenotypic Alterations of Tobacco Plant

Under normal growth conditions, the CsCBF2-OX displayed different phenotype compared with WT. It showed that the CsCBF2-OX grew much slower than WT after two-week of sowing in soil. The WT had four leaves, but the CsCBF2-OX displayed two to four leaves with much smaller leaf area. The plant height of CsCBF2-OX was shorter than WT (Figure 4). Four-week later, the WT grew faster and the leaf area was obviously increased, while leaf area of the CsCBF2-OX was still much smaller than that of WT (Figure 4). After six-week of sowing in soil, growth retardation was still seen in CsCBF2-OX. Leaves on the same position were still smaller in CsCBF2-OX than those in WT, and WT were taller than CsCBF2-OX. Moreover, it showed obviously that leaves of WT were dark green while those of CsCBF2-OX were light green (Figure 4). After eight-week of sowing in soil, the leaf area and plant height was not significantly different between CsCBF2-OX and WT, but the leaf color of WT was still darker than that of CsCBF2-OX (Figure 4).

3.5. Overexpression of CsCBF2 Confers Cold Tolerance in Tobacco Plant

Under the normal control conditions for 20 days, both CsCBF2-OX and WT grew well except that the CsCBF2-OX displayed growth retardation with smaller leaf area (Figure 5A). The average fresh weight of WT was 1.56 g, while that of CsCBF2-OX L5, L15, and L17 was 1.42, 1.41, and 1.44 g, respectively, which was significantly lighter than that of WT (Figure 5B). The average root length of WT, L5, L15 and L17 plants was respectively 8.96, 6.91, 6.58, and 8.02 cm. Although the root length of CsCBF2-OX was slightly shorter than that of WT, there was no significant difference between WT and CsCBF2-OX (Figure 5C). Under the cold stress conditions for 20 days, although growth of both WT and CsCBF2-OX was inhibited, CsCBF2-OX were more resistant to the cold stress than WT (Figure 5D). WT showed obvious leaf-chlorosis after the cold treatment, and some of them had all the leaves wilted. However, the leaf-chlorosis symptom was rarely seen on the CsCBF2-OX. After growing 20 days under the cold stress conditions, CsCBF2-OX grew much bigger than WT, with bigger leaf size and longer root length (Figure 5D). The average fresh weight of CsCBF2-OX was much heavier than that of WT. The average fresh weight of WT, L5, L15, and L17 plants was respectively 0.083, 0.138, 0.133, and 0.132 g (Figure 5B). Root length of CsCBF2-OX was significantly longer than that of WT after the cold treatment. The average root length of WT was 1.69 cm, while that of CsCBF2-OX L5, L15, and L17 was respectively 3.61, 2.94, and 2.74 cm (Figure 5C). The root length after the cold treatment to that under the normal growth conditions (root length ratio) of CsCBF2-OX was bigger than that of WT (Figure 5E).
Physiological changes of CsCBF2-OX after cold treatment were also investigated. Under normal growth conditions, results showed that soluble sugar content had little difference between WT and CsCBF2-OX, but the proline content in CsCBF2-OX L15 and L17 was significantly higher than that in WT (Figure 5F,G). After 8 h of the cold treatment, both the proline and soluble sugar contents were significantly higher in CsCBF2-OX than those in WT. The proline content was respectively 0.184, 0.286, 0.291, and 0.278 μg g−1 FW in WT and the CsCBF2-OX L5, L15, and L17, it was respectively 1.55, 1.58, and 1.51 folds in L5, L15, and L17 of that in WT (Figure 5F). The soluble sugar content was respectively 3.48, 4.58, 4.26, 4.46 μg g−1 FW in WT and the CsCBF2-OX L5, L15, L17, the content was respectively 1.32, 1.22, and 1.28 folds in L5, L15 and L17 of that in WT (Figure 5G).

3.6. Freezing Tolerance of CsCBF2-Overexpressing Transgenic Tobacco Plants

To test the function of CsCBF2 in freezing tolerance, CsCBF2-OX and WT were first treated under 0 °C for 5 days, and then transferred to −2 °C for 16 h. Before the freezing treatment, results showed that CsCBF2-OX and WT grew well, except that CsCBF2-OX had relative smaller size and lighter leaf color than WT (Figure 6A). After being treated under 0 °C for 5 days, CsCBF2-OX still grew well, while WT showed obvious dehydration (Figure 6B). After the continued treatment under −2 °C for 16 h, only a small part of CsCBF2-OX showed dehydrated phenotype, but almost all of WT showed heavy dehydration and leaf collapse (Figure 6C). After the treated plants were transferred from −2 °C to 25 °C conditions for 5 days’ recovery, almost all of the WT could not keep alive and died finally; however, most of the CsCBF2-OX were still alive (Figure 6D). Statistical analysis showed that the survival rate of CsCBF2-OX was much higher than that of WT. The survival rate of WT and CsCBF2-OX L5, L15, and L17 was respectively 28%, 81%, 78%, and 64%, after the freezing treatment (Figure 6E).
To investigate the effect of CsCBF2 on cell membrane stability under freezing stress, the relative electrical conductivity was measured. The relative electrical conductivity showed no difference between WT and CsCBF2-OX before freezing treatment. After the freezing treatment, the relative electrical conductivity increased in both WT and CsCBF2-OX. However, the relative electrical conductivity of CsCBF2-OX was significantly lower than that of WT (Figure 6F). The result suggested that CsCBF2-OX could alleviate cellular damage by lowering the electrolyte leakage under freezing stress.

3.7. CsCBF2 Transgenic Plants Exhibit Increased Tolerance to Drought and Salt Stress

To assess the function of CsCBF2 in drought and salt stress, seed germination rates of CsCBF2-OX and WT was analyzed first. Under normal conditions without stress, the germination rate showed no significant difference between CsCBF2-OX and WT (Figure 7A). Under salt stress with 150 mM NaCl, it showed no obvious difference between CsCBF2- OX and WT until 8 days of the stress treatment. On the 9th day of the salt stress, seed germination rate was much lower in WT than that in CsCBF2-OX. It was 46%, 74%, 72%, and 64%, respectively, in WT and CsCBF2-OX L5, L15, and L17. On the 10th day of the salt stress, seed germination rate of WT and CsCBF2-OX reached the maximum value, it was respectively 68%, 86%, 92%, and 88% in WT and the CsCBF2-OX L5, L15, L17. The germination rate of WT and CsCBF2-OX showed no change after even twelve days of salt stress (Figure 7B). Under 0.4 M mannitol-induced drought stress, significant lower seed germination rate was seen in WT after 5 days of the treatment. Seed germination rate of WT and CsCBF2-OX L5, L15, and L17 was 2%, 14%, 16%, and 10%, respectively. After 10 days of the treatment, seed germination rate of WT and CsCBF2-OX plants L5, L15, and L17 was respectively 82%, 92%, 94%, and 94% (Figure 7C).
Then, root length and fresh weight of WT and CsCBF2-OX were measured under normal and stress conditions. After growing on MS medium for 20 days under normal conditions, root lengths of WT and CsCBF2-OX had no significant difference (Figure 7D,F); but the fresh weight of WT was significantly heavier than that of CsCBF2-OX, which was associated with the smaller leaf size of CsCBF2-OX (Figure 7D,E). After 20 days under salt stress, root length of CsCBF2-OX was significantly longer than that of WT, and fresh weight of CsCBF2-OX was also significantly heavier than that of WT (Figure 7D–F). The root length after the salt stress treatment to that under the normal growth conditions (root length ratio) of CsCBF2-OX was much bigger than that of WT (Figure 7G). After 20 days under mannitol simulated drought stress, although the root length of CsCBF2-OX was similar to that of WT (Figure 7D,F) and the root length after the mannitol treatment to that under the normal growth conditions (root length ratio) of CsCBF2-OX showed no significant difference to that of WT (Figure 7G), the fresh weight of CsCBF2-OX was significantly heavier than that of WT (Figure 7E).

4. Discussion

CBFs/DREB1s are subgroup of the AP2/ERF superfamily with typical PKK/RPAGRxKFxETRHP and DSAWR signature sequences respectively laid on each side of the AP2 DNA binding domain [13,17]. It has reported that CBFs/DREB1s played important role in plant abiotic stress resistance [8,11,43]. However, function of CBFs/DREB1s was still poorly characterized in tea plant. Accordingly, cloning and functional characterization of the cold responsive CsCBF gene (CsCBF2) identified in our previous study was done. The results demonstrated that CsCBF2 encoded a protein with 239 aa, which had similar size to the formerly cloned CBFs/DREB1s-coding products in tea plant (Figure 1A) [3,33,37]. Sequence analysis showed that CsCBF2 had the conserved AP2 domain, which was more similar to the CBF/DREB1 group than to the DREB2 group. The typical PKK/RPAGRxKFxETRHP and DSAWR signature sequences of CBF/DREB1 proteins were also found in the CsCBF2 protein (Figure 1B). Phylogenetic analysis showed that CsCBF2 was tightly clustered with CsCBF1 and AdCBF, and other CBF proteins from woody plants (Figure 1C). It is consistent with the fact that tea plant is a woody plant and has a very close phylogenetic relationship with A. deliciosa on the whole genome evolution analysis [44]. Taken together, these results revealed that CsCBF2 was a typical CBF/DREB1 gene in tea plant.
CBFs/DREB1s, as transcription factors, could specifically bind to the CRT/DRE cis-element in the target gene promoter and regulate its expression [11,17,26]. Although the cis-element CRT and DRE share conserved core nucleotide sequence CCGAC, the intact sequence of CRT cis-element is TGGCCGAC and that of DRE is TACCGACAT [14]. By yeast one-hybrid assay, our results demonstrated that CsCBF2 binding domain (CsCBF2BD) could bind to the CRT/DRE cis-element to activate the reporter gene expression and keep the yeast cells alive on the selective medium (Figure 2G). But intact CsCBF2 could only bind to the CRT element, not to the DRE element. Neither CsCBF2BD nor intact CsCBF2 could bind to the mutated CRT/DRE element (Figure 2G). These results suggested that CsCBF2 specifically binds to the conserved CRT/DRE cis-element. The intact CsCBF2 could not bind to the DRE element, indicating that CsCBF2 had a preferred binding to the CRT element. This preference of CBFs/DREB1s to CRT is also reported in many other studies [6,25]. Moreover, except the CsCBF2BD, other part of CsCBF2 had an inhibitory effect on its binding to DRE, which might be another possible reason. Analysis of the transactivation activity of CsCBF2 demonstrated that the CsCBF2AD could activate the reporter gene expression in the AH109 yeast cells and made them alive on the selective medium (Figure 2C). However, the intact CsCBF2 protein suppressed the growth of AH109 yeast cells on the selective media (Figure 2D). The result suggested that the N terminal of CsCBF2 protein had an inhibitory effect, transcription activation activity of CsCBF2 may need other proteins or modifications to release the inhibition. Reports had demonstrated that transcription factors could realize fine gene expression regulation by using their inhibitory domain or post-translation modification [7,45]. Sakuma et al. [46] found that an inhibitory domain existed in AtDREB2A, and modification of the inhibitory domain occurred under stress conditions. Research also revealed that CBF/DREB1 proteins may employ the feedback mechanism to suppress their own transcription, and AtCBF2 could suppress the expression of AtCBF1 and AtCBF3 [3]. In tea plant, Wang et al. [33] observed that CsDREB had no transcriptional activity in yeast but the reason was not further studied. Although the authors speculated that a post-translational modification was required to activate CsDREB, existing inhibitory domain in CsDREB might be another possible reason. Our previous study had found that the N terminal of CsCBF1 protein had an inhibitory effect on its transactivation activity [37]. So, function of CsCBF2 protein is finely regulated, and the regulatory mechanism needs further study.
Constitutively expression of CBF/DREB1 gene always affects plant growth and development, such as growth retardation, dwarfism, and flowering delay [1,12,47]. Our study showed that the leaf color of CsCBF2-OX was lighter than that of WT, CsCBF2-OX grew slower, with smaller leaf and shorter plant height than WT until eight-week after sowing (Figure 4). The results were very similar to those reported in other plants overexpressing CBF/DREB1 gene, except that the leaf color was often reported darker green in the transgenic plants. Research has demonstrated that GA regulated accumulation of DELLA proteins or ABA down-regulated phosphatase signal pathway was important factors for the growth inhibition of CBF/DREB1-overexpressing transgenic plants [12,30,48]. However, growth inhibition of CsCBF2-OX disappeared, and there was no obvious difference between CsCBF2-OX and WT except the leaf color was still lighter in the transgenic plants when the phenotype was recorded on the eight-week after the seeds sowing (Figure 4). The result was unexpectedly different to the growth inhibition phenotype displayed by CBF/DREB1-overexpressing plants. It suggested that growth inhibition displayed by CBF/DREB1-overexpressing plants might be related to plant growth stage. Report demonstrated that overexpression of zoysiagrass CBF/DREB1 gene (ZjDREB1.4) in Arabidopsis enhanced temperature stress tolerance without growth inhibition but only a few days delay in bolting. Overexpression of OsDREB1B and OsDREB1D showed no obvious retardation at seedling stage [15]. Interestingly, overexpression of OsDREB1B in rice inhibited plant growth, while OsDREB1B-overexpressing transgenic tobacco plants displayed neither growth inhibition nor visible phenotypic alterations [11]. It seems that tobacco plant has a specific regulating mechanism to remove the phenotypic alterations caused by overexpressing of CBF/DREB1. However, it was reported that rice plants overexpressing AtDREB1A/CBF3 exhibited no visible phenotypic alterations [49]. So, the growth abnormalities observed in CBF/DREB1 constitutively expressed transgenic plants under non-stress conditions is not a common phenomenon. Many factors including growth stage of transgenic plant, transgenic receptor, source of CBF/DREB1 genes and the positional effects, etc., may affect the phenotype of CBF/DREB1-overexpressing transgenic plants. Using tissue-specific promoter or inducible promoter to control the spatiotemporal expression or the expression level of CBF/DREB1 gene would be an effective way to eliminate the growth inhibition without affecting the stress resistance function. Indeed, research demonstrated that modulating AtDREB1C expression by using drought-inducible promoter displayed no phenotypic alterations, but enhanced drought tolerance of the transgenic plants [1].
In Arabidopsis, studies reported that CBF/DREB1 primarily functioned in cold stress, while DREB2 was induced by salinity and drought but not by cold [8,12,26]. However, studies demonstrated that the CBF/DREB1 homologs in many other plants were regulated not only by cold, but also by drought and salinity [12,13,27]. Ectopic expression of CBF/DREB1 or DREB2 gene also showed enhanced plant resistance to cold, drought, and salinity [1,50]. Our study revealed that overexpression of CsCBF2 gene enhanced plant resistance to multiple stresses, such as cold (low temperature and freezing), salinity, and high concentration of mannitol simulated drought stress (Figure 5, Figure 6 and Figure 7). Under cold stress (low temperature and freezing), CsCBF2-OX displayed better growth than WT. While growth of WT was retarded, and most of them died after the freezing treatment (Figure 5A–E and Figure 6A–E). Under low nonfreezing temperatures, CBF gene expression was induced followed by activation of downstream regulated genes (CBF regulon), which could further induce plant freezing tolerance [15,47]. It was reported that cold acclimation was required for CBF-mediated freezing resistance [30]. Our research showed that CsCBF2-OX was more resistant to freezing stress without cold acclimation (Figure 6A–D). This may be caused by the overexpression of CsCBF2 in transgenic plants, and the CBF regulons were always activated in CsCBF2-OX. In fact, there were many studies that demonstrated that overexpression of CBF/DREB1 gene induced expression of CBF regulons under normal conditions, and enhanced plant freezing resistance without cold acclimation [12,29,51]. Further study showed that proline and soluble sugar contents were much higher in CsCBF2-OX than those in WT (Figure 5F,G). Several studies demonstrated that overexpression of DREB gene induced proline and sugar accumulation under cold stress [21,52]. Reports also suggested that high level of free proline and soluble sugars within cells would protect membranes at freezing temperatures [15,52]. Although the damage degree of cell membrane was not measured under low temperature (4 °C) stress, our study demonstrated that the electrolyte leakage value of WT was much bigger than that of CsCBF2-OX under freezing (Figure 6). These results suggested that accumulation of proline and soluble sugars to suppress membrane damage would be an important resistance mechanism of CsCBF2 gene to cold stress. Root length, fresh weight, and germination rate are important indicators reflecting plant resistance to various abiotic stresses, and research demonstrated that CBF/DREB1-overexpressing plants always had longer roots, heavier fresh weight, and higher seed germination rate than WT [12,29,53]. Under cold and salt stresses, CsCBF2-OX had longer roots and heavier fresh weight than WT (Figure 5 and Figure 7), indicating that overexpression of CsCBF2 enhanced plant resistance to cold and salinity. Seed germination rate, root length, and fresh weight were also measured in CsCBF2-OX and WT under different mannitol concentrations simulated drought stress. Results showed that seed germination rate of CsCBF2-OX were higher than that of WT under relative higher mannitol concentration (0.4 M; Figure 7C). Under lower mannitol concentration (0.2 M), the fresh weight of CsCBF2-OX was heavier than that of WT, but the root length had no significant difference between CsCBF2-OX and WT (Figure 7E–G). The result suggested that the mannitol treatment had no effect on root elongation, but growth and substance accumulation in WT was inhibited when compared with CsCBF2-OX. There were some reports that showed overexpression of DREB gene had no obvious function on root elongation, but enhanced fresh weight of DREB transgenic plants under stress conditions [21,54,55].

5. Conclusions

CsCBF2, a cold-responsive CBF gene isolated by suppression subtractive hybridization (SSH) in our previous study, was cloned by RACE technology. It showed that the full-length cDNA of CsCBF2 was 1162 bp, including 193 bp 5′ untranslated region, 720 bp ORF, and 249 bp 3′untranslated region. Sequence analysis and transcription activity assays demonstrated that CsCBF2 was a typical CBF/DREB1 transcription factor of the AP2/ERF superfamily. CsCBF2 without the activation domain (CsCBF2BD) showed specific binding to the conserved CRT/DRE cis element, but the intact CsCBF2 protein only bound to the CRT cis element. Transcription activation assays revealed that N terminal of CsCBF2 had an inhibitory effect. Overexpression of CsCBF2 in tobacco plants enhanced resistance to multiple abiotic stresses, such as cold, salinity, and drought. Accumulation of more proline and sugar contents, along with lower electrolyte leakage was related to the abiotic stress resistance of CsCBF2-OX. The CsCBF2-OX displayed light green leaf color, growth retardation, and dwarfism. Except the light green leaf color, other phenotypic alterations of CsCBF2-OX disappeared from the eighth week after sowing. Overall, our results suggest that CsCBF2 is an important resistance gene for plant breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8090853/s1. Figure S1: Conserved protein domain analysis of CsCBF2 by the online CD-Search (A) and SMART (B) tools. Table S1: Gene primers used in this study. Table S2: The accession numbers of the CBF/DREB1 proteins used for CsCBF2 phylogenetic analysis.

Author Contributions

Conceptualization, H.Y. and D.L.; project administration, D.L., Y.W. and N.M.; formal analysis, Q.Z. and D.L.; data curation, Y.W. and N.M.; methodology, Q.Z. and C.J.; visualization, Q.Z. and Z.Z.; investigation, D.L., N.M. and Y.W.; supervision, H.Y. and C.J.; funding acquisition, Q.Z. and H.Y.; software, Q.Z. and R.Z.; validation, R.Z. and Z.Z.; writing—original draft, Q.Z. and D.L.; writing—review and editing, H.Y. and C.J. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the National Key Research and Development Program of China (2021YFD1601100), the National Natural Science Foundation of China (No. U1404319), Scientific and Technological Research Projects of Henan Province (202102110230, 212102110399, 222102110238), the Training Program for Young Backbone Teachers in Colleges and Universities of Henan Province (2021GGJS100), Key Scientific Research Projects of Universities in Henan Province (22A210023), Nanhu Scholars Program for Young Scholars of XYNU (2016060), and National Training Programs of Innovation and Entrepreneurship for Undergraduates (202110477011).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available within the article and the supplementary materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. RACE clone, sequence alignment and phylogenetic analysis of CsCBF2. (A) Electrophoresis detection of RACE cloning product. M: DNA Marker; 5′ RACE: 5′ RACE cloning product of CsCBF2 gene; 3′ RACE: 3′ RACE cloning product of CsCBF2 gene; LdPCR: long distance PCR product of CsCBF2 gene. (B) Sequence alignment of CsCBF2 with DREB proteins of Arabidopsis. The two signature sequences of CBF/DREB1 proteins are indicated by triangle. The box encloses the AP2 DNA binding domain. The arrow indicates the carboxyl-terminal acidic activation region. Black shading indicates identical amino acid residues, grey shading indicates amino acids with similar charge or hydrophobicity. CsCBF1 (ACB20695), CsCBF3 (ACF71456), C. sinensis; AtCBF1 (ACI15576), AtCBF2 (ABV27114), AtCBF3 (ABV27138), AtDREB2A (O82132), AtDREB2B (O82133), A. thaliana. (C) Phylogenetic analysis of CsCBF2 protein. The phylogenetic tree was constructed with 1000 bootstrap trials using MEGA-X software with Neighbor Joining method. CBF proteins from C. sinensis (CsCBF, red circle), A. thaliana (AtCBF, red triangle), B. pendula (BpCBF, red solid diamond), P. euphratica (PopCBF, red hollow diamond), M. domestica (MdCBF, green solid square), A. deliciosa (AdCBF, orange hollow circle), N. sinensis (NsCBF, blueviolet hollow circle), C. acuminate (CaCBF, blue hollow circle), L. esculentum (LeCBF, darkblue solid square), I. batatas (IbCBF, orange solid square), E. gunnii (EgCBF, brown solid diamond), P. avium (PaCBF, copper triangle), M. baccata (MbCBF, green triangle), V. vinifera (VvCBF, red solid square), D. Antarctica (DaCBF, blueviolet hollow square), H. vulgare (HvCBF, plum triangle), O. sativa (OsDREB, blue solid diamond), T. aestivum (TaCBF, blueviolet solid square) were used for the phylogenetic tree construction. The accession numbers of the CBF/DREB1 proteins are shown in Table S2.
Figure 1. RACE clone, sequence alignment and phylogenetic analysis of CsCBF2. (A) Electrophoresis detection of RACE cloning product. M: DNA Marker; 5′ RACE: 5′ RACE cloning product of CsCBF2 gene; 3′ RACE: 3′ RACE cloning product of CsCBF2 gene; LdPCR: long distance PCR product of CsCBF2 gene. (B) Sequence alignment of CsCBF2 with DREB proteins of Arabidopsis. The two signature sequences of CBF/DREB1 proteins are indicated by triangle. The box encloses the AP2 DNA binding domain. The arrow indicates the carboxyl-terminal acidic activation region. Black shading indicates identical amino acid residues, grey shading indicates amino acids with similar charge or hydrophobicity. CsCBF1 (ACB20695), CsCBF3 (ACF71456), C. sinensis; AtCBF1 (ACI15576), AtCBF2 (ABV27114), AtCBF3 (ABV27138), AtDREB2A (O82132), AtDREB2B (O82133), A. thaliana. (C) Phylogenetic analysis of CsCBF2 protein. The phylogenetic tree was constructed with 1000 bootstrap trials using MEGA-X software with Neighbor Joining method. CBF proteins from C. sinensis (CsCBF, red circle), A. thaliana (AtCBF, red triangle), B. pendula (BpCBF, red solid diamond), P. euphratica (PopCBF, red hollow diamond), M. domestica (MdCBF, green solid square), A. deliciosa (AdCBF, orange hollow circle), N. sinensis (NsCBF, blueviolet hollow circle), C. acuminate (CaCBF, blue hollow circle), L. esculentum (LeCBF, darkblue solid square), I. batatas (IbCBF, orange solid square), E. gunnii (EgCBF, brown solid diamond), P. avium (PaCBF, copper triangle), M. baccata (MbCBF, green triangle), V. vinifera (VvCBF, red solid square), D. Antarctica (DaCBF, blueviolet hollow square), H. vulgare (HvCBF, plum triangle), O. sativa (OsDREB, blue solid diamond), T. aestivum (TaCBF, blueviolet solid square) were used for the phylogenetic tree construction. The accession numbers of the CBF/DREB1 proteins are shown in Table S2.
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Figure 2. Transcription activation and the CRT/DRE binding activity analysis. (A,C) respectively exhibited the growth of yeast cells harboring the pGBKT7-CsCBF2AD recombinant plasmid on SD/-Trp and SD/-Trp/-His/-Ade plate. (B,D) respectively exhibiting the growth of yeast cells harboring the pGBKT7-CsCBF2 recombinant plasmid on SD/-Trp and SD/-Trp/-His/-Ade plate. (E,F) respectively display the growth of yeast cells harboring the pGADT7-Rec2 and pHIS2.1 plasmids on SD/-Trp/-His/-Leu plated with no 3-amino-1, 2, 4-triazole (3-AT) and 25 mM 3-AT. (G) The growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × CRT/DRE, pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mCRT/DRE, pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × CRT/DRE, pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mCRT/DRE on SD/-Trp/-His/-Leu plated with 25 mM 3-AT. 1-1, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mDRE; 1-2, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × DRE; 1-3, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mCRT; 1-4, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × CRT; 1-5, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1; 2-1, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mDRE; 2-2, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × DRE; 2-3, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mCRT; 2-4, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × CRT; 2-5, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1.
Figure 2. Transcription activation and the CRT/DRE binding activity analysis. (A,C) respectively exhibited the growth of yeast cells harboring the pGBKT7-CsCBF2AD recombinant plasmid on SD/-Trp and SD/-Trp/-His/-Ade plate. (B,D) respectively exhibiting the growth of yeast cells harboring the pGBKT7-CsCBF2 recombinant plasmid on SD/-Trp and SD/-Trp/-His/-Ade plate. (E,F) respectively display the growth of yeast cells harboring the pGADT7-Rec2 and pHIS2.1 plasmids on SD/-Trp/-His/-Leu plated with no 3-amino-1, 2, 4-triazole (3-AT) and 25 mM 3-AT. (G) The growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × CRT/DRE, pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mCRT/DRE, pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × CRT/DRE, pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mCRT/DRE on SD/-Trp/-His/-Leu plated with 25 mM 3-AT. 1-1, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mDRE; 1-2, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × DRE; 1-3, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × mCRT; 1-4, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1-4 × CRT; 1-5, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2 and pHIS2.1; 2-1, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mDRE; 2-2, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × DRE; 2-3, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × mCRT; 2-4, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1-4 × CRT; 2-5, the growth of yeast cells harboring the pGADT7-Rec2-CsCBF2BD and pHIS2.1.
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Figure 3. Identification and validation of CsCBF2-overexpressing transgenic tobacco plants. (A) Constructs used to overexpress CsCBF2 in tobacco. 35S-Pro, 35S cauliflower mosaic virus 35 S promoter; NOS-T, 3′ terminator region of the nopaline synthase gene; Hyg, hygromycin phosphotransferase-coding gene; polyA, cauliflower mosaic virus polyA signal; RB, right border; LB, left border. (B) PCR analysis of CsCBF2 open reading frame (ORF) sequence in T0 transgenic lines. (C) RT-PCR identification of CsCBF2 gene transcript in the T2 transgenic plants from three different T0 transgenic lines L5, L15 and L17. The tobacco housekeeping gene Ntactin was used as equal loading control. M: DNA marker. WT, wild type tobacco plant. (D) Western blot analysis of CsCBF2 protein expression in T2 transformants L5, L15, and L17 using the antibody against CsCBF2. Expression of Ntactin in T2 transformants L5, L15, and L17 was detected by Ntactin antibody and used as equal loading control of the total protein. WT, wild type tobacco plant.
Figure 3. Identification and validation of CsCBF2-overexpressing transgenic tobacco plants. (A) Constructs used to overexpress CsCBF2 in tobacco. 35S-Pro, 35S cauliflower mosaic virus 35 S promoter; NOS-T, 3′ terminator region of the nopaline synthase gene; Hyg, hygromycin phosphotransferase-coding gene; polyA, cauliflower mosaic virus polyA signal; RB, right border; LB, left border. (B) PCR analysis of CsCBF2 open reading frame (ORF) sequence in T0 transgenic lines. (C) RT-PCR identification of CsCBF2 gene transcript in the T2 transgenic plants from three different T0 transgenic lines L5, L15 and L17. The tobacco housekeeping gene Ntactin was used as equal loading control. M: DNA marker. WT, wild type tobacco plant. (D) Western blot analysis of CsCBF2 protein expression in T2 transformants L5, L15, and L17 using the antibody against CsCBF2. Expression of Ntactin in T2 transformants L5, L15, and L17 was detected by Ntactin antibody and used as equal loading control of the total protein. WT, wild type tobacco plant.
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Figure 4. Phenotype of CsCBF2-overexpressing transgenic tobacco plants after different periods of sowing under normal growth conditions.
Figure 4. Phenotype of CsCBF2-overexpressing transgenic tobacco plants after different periods of sowing under normal growth conditions.
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Figure 5. Phenotypic and physiological responses of CsCBF2-overexpressing transgenic plants under cold stress conditions. Data are means ± standard deviation (SD) of three independent experiments. Significant differences between WT and transgenic plants under control and cold stress conditions are indicated by * p < 0.05, ** p < 0.01; Student’s t-test. (A,D) exhibit the phenotypes of CsCBF2-overexpressing tobacco plants (CsCBF2-OX) and wild type tobacco plant (WT) growing on 1/2 MS culture medium for 20 days, respectively, under normal growth conditions (control) and cold stress conditions (4 °C). (B) The fresh weight of CsCBF2-OX L5, L15, L17, and WT under normal (control) and cold stress (4 °C) conditions, after growing on 1/2 MS culture medium for 20 days. (C) The root length of CsCBF2-OX L5, L15, L17, and WT under normal (control) and cold stress (4 °C) conditions, after growing on 1/2 MS culture medium for 20 days. (E) The root length ratio of CsCBF2-OX L5, L15, L17, and WT. The root length ratio is the root length of CsCBF2-OX L5, L15, L17, and WT under cold stress conditions (4 °C) correspondingly to that under normal growth conditions (control). (F,G) respectively represents the proline and soluble sugar content in CsCBF2-OX L5, L15, L17, and WT under normal growth conditions (control) and cold stress (4 °C).
Figure 5. Phenotypic and physiological responses of CsCBF2-overexpressing transgenic plants under cold stress conditions. Data are means ± standard deviation (SD) of three independent experiments. Significant differences between WT and transgenic plants under control and cold stress conditions are indicated by * p < 0.05, ** p < 0.01; Student’s t-test. (A,D) exhibit the phenotypes of CsCBF2-overexpressing tobacco plants (CsCBF2-OX) and wild type tobacco plant (WT) growing on 1/2 MS culture medium for 20 days, respectively, under normal growth conditions (control) and cold stress conditions (4 °C). (B) The fresh weight of CsCBF2-OX L5, L15, L17, and WT under normal (control) and cold stress (4 °C) conditions, after growing on 1/2 MS culture medium for 20 days. (C) The root length of CsCBF2-OX L5, L15, L17, and WT under normal (control) and cold stress (4 °C) conditions, after growing on 1/2 MS culture medium for 20 days. (E) The root length ratio of CsCBF2-OX L5, L15, L17, and WT. The root length ratio is the root length of CsCBF2-OX L5, L15, L17, and WT under cold stress conditions (4 °C) correspondingly to that under normal growth conditions (control). (F,G) respectively represents the proline and soluble sugar content in CsCBF2-OX L5, L15, L17, and WT under normal growth conditions (control) and cold stress (4 °C).
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Figure 6. Phenotypic and electrolyte leakage analysis of CsCBF2-overexpressing transgenic plants under freezing stress conditions. Significant differences between WT and transgenic plants under control and freezing conditions are indicated by * p < 0.05, ** p < 0.01; Student’s t-test. (A–D) respectively displays the phenotype of WT and CsCBF2-OX L5, L15, L17 before freezing treatment, after 0 °C freezing treatment for 5 days, after −2 °C freezing treatment for 5 days, and after transferred back to normal growth conditions and restored growing for 5 days. (E) The survival rates of WT and CsCBF2-OX L5, L15, L17 after transferred back to normal growth conditions and restored growing for 5 days. (F) The electrolyte leakage analysis of WT and CsCBF2-OX L5, L15, L17 under normal growth conditions (control) and after −6 °C freezing treatment for 2 h.
Figure 6. Phenotypic and electrolyte leakage analysis of CsCBF2-overexpressing transgenic plants under freezing stress conditions. Significant differences between WT and transgenic plants under control and freezing conditions are indicated by * p < 0.05, ** p < 0.01; Student’s t-test. (A–D) respectively displays the phenotype of WT and CsCBF2-OX L5, L15, L17 before freezing treatment, after 0 °C freezing treatment for 5 days, after −2 °C freezing treatment for 5 days, and after transferred back to normal growth conditions and restored growing for 5 days. (E) The survival rates of WT and CsCBF2-OX L5, L15, L17 after transferred back to normal growth conditions and restored growing for 5 days. (F) The electrolyte leakage analysis of WT and CsCBF2-OX L5, L15, L17 under normal growth conditions (control) and after −6 °C freezing treatment for 2 h.
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Figure 7. Phenotypic and physiological responses of CsCBF2-overexpressing transgenic plants under salt and drought stress conditions. Data are means ±standard deviation (SD) of three independent experiments. Significant differences between WT and transgenic plants under control and stress conditions are indicated by * p < 0.05, ** p < 0.01; Student’s t-test. (A–C) respectively show the germination rate of WT and CsCBF2-OX L5, L15, L17 growing on 1/2 MS culture medium, on 1/2 MS culture medium with 150 mM NaCl, on 1/2 MS culture medium with 400 mM mannitol. (D) The phenotype of WT and CsCBF2-OX growing on 1/2 MS culture medium, on 1/2 MS culture medium with 200 mM NaCl, on 1/2 MS culture medium with 200 mM mannitol. (E) The fresh weight of WT and CsCBF2-OX L5, L15, L17 under normal conditions (control), salt stress, and high concentration of mannitol simulated drought stress for 20 days. (F) Root length of WT and CsCBF2-OX L5, L15, L17 under normal conditions (control), salt stress, and high concentration of mannitol simulated drought stress for 20 days. (G) The root length ratio of WT and CsCBF2-OX L5, L15, L17. The root length ratio is the root length of CsCBF2-OX L5, L15, L17, and WT respectively under salt and mannitol-simulated drought stress correspondingly to that under normal growth conditions (control).
Figure 7. Phenotypic and physiological responses of CsCBF2-overexpressing transgenic plants under salt and drought stress conditions. Data are means ±standard deviation (SD) of three independent experiments. Significant differences between WT and transgenic plants under control and stress conditions are indicated by * p < 0.05, ** p < 0.01; Student’s t-test. (A–C) respectively show the germination rate of WT and CsCBF2-OX L5, L15, L17 growing on 1/2 MS culture medium, on 1/2 MS culture medium with 150 mM NaCl, on 1/2 MS culture medium with 400 mM mannitol. (D) The phenotype of WT and CsCBF2-OX growing on 1/2 MS culture medium, on 1/2 MS culture medium with 200 mM NaCl, on 1/2 MS culture medium with 200 mM mannitol. (E) The fresh weight of WT and CsCBF2-OX L5, L15, L17 under normal conditions (control), salt stress, and high concentration of mannitol simulated drought stress for 20 days. (F) Root length of WT and CsCBF2-OX L5, L15, L17 under normal conditions (control), salt stress, and high concentration of mannitol simulated drought stress for 20 days. (G) The root length ratio of WT and CsCBF2-OX L5, L15, L17. The root length ratio is the root length of CsCBF2-OX L5, L15, L17, and WT respectively under salt and mannitol-simulated drought stress correspondingly to that under normal growth conditions (control).
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Zhou, Q.; Liu, D.; Wei, Y.; Ma, N.; Zhang, R.; Zhang, Z.; Jiang, C.; Yuan, H. Functional Characterization of Tea Plant (Camellia sinensis L.) CsCBF2 Gene Involved in Multiple Abiotic Stress Response in Tobacco (Nicotiana tabacum L.). Horticulturae 2022, 8, 853. https://doi.org/10.3390/horticulturae8090853

AMA Style

Zhou Q, Liu D, Wei Y, Ma N, Zhang R, Zhang Z, Jiang C, Yuan H. Functional Characterization of Tea Plant (Camellia sinensis L.) CsCBF2 Gene Involved in Multiple Abiotic Stress Response in Tobacco (Nicotiana tabacum L.). Horticulturae. 2022; 8(9):853. https://doi.org/10.3390/horticulturae8090853

Chicago/Turabian Style

Zhou, Qiying, Dongxiao Liu, Yiwan Wei, Ning Ma, Ruijiao Zhang, Zengya Zhang, Changjun Jiang, and Hongyu Yuan. 2022. "Functional Characterization of Tea Plant (Camellia sinensis L.) CsCBF2 Gene Involved in Multiple Abiotic Stress Response in Tobacco (Nicotiana tabacum L.)" Horticulturae 8, no. 9: 853. https://doi.org/10.3390/horticulturae8090853

APA Style

Zhou, Q., Liu, D., Wei, Y., Ma, N., Zhang, R., Zhang, Z., Jiang, C., & Yuan, H. (2022). Functional Characterization of Tea Plant (Camellia sinensis L.) CsCBF2 Gene Involved in Multiple Abiotic Stress Response in Tobacco (Nicotiana tabacum L.). Horticulturae, 8(9), 853. https://doi.org/10.3390/horticulturae8090853

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