Overexpression of <i>IbSINA5</i> Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Shiyang Li; Xue-Ao Liu; Lizi Zhao; Huiqing Huang; Bei Li; Zhizhong Song; Meixia Liang; Hongxia Zhang; Limin Wang; Shenglin Zhou

doi:10.32604/phyton.2021.016314

Overexpression of IbSINA5 Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Shiyang Li1#, Xue-Ao Liu2#, Lizi Zhao2, Huiqing Huang3, Bei Li2, Zhizhong Song2, Meixia Liang2, Hongxia Zhang2,3, Limin Wang2,*and Shenglin Zhou1,*

1Shandong Technology and Business University, Yantai, 264005, China
2The Engineering Research Institute of Agriculture and Forestry, Ludong University, Yantai, 264025, China
3School of Life Sciences, Ludong University, Yantai, 264025, China
*Corresponding Authors: Limin Wang. Email: wanglimin9696@163.com; Shenglin Zhou. Email: zhoushenglin@163.com
Received: 20 February 2021; Accepted: 05 March 2021
#These authors contribute equally to this work

Sweet potato (Ipomoea batatas [L.] Lam) has been grown worldwide as a tuberous crop due to its food and commercial values, and resistance to adverse growth conditions [1–3]. However, owing to its tropical and subtropical origin, abiotic stresses such as low temperature and drought can severely affect the storage root yield of sweet potato, especially during the rapid root expansion period [4,5].

To increase the resistance of sweet potato to abiotic stresses, a number of stress related genes such as IbCBF3, IbMIPS1, IbNAC1, IbOR, IbTPS and IbLEA14 have been isolated, and transgenic sweet potato plants with improved tolerance to biotic and abiotic stresses have been generated [6–9]. Ectopic expression of rice OCI enhanced the resistance to stem nematode in transgenic sweet potato [10]. Constitutive expression of Arabidopsis AtNHX1, HDG11 or spinach BADH in sweet potato increased salt and cold resistance of transgenic plants [4,5,11]. Overexpression of IbMIPS1 or IbCBF3 increased the resistance to stem nematode, salt, drought and cold stress of transgenic sweet potato [12,13].

SINA protein is a subfamily of the tumor necrosis factor receptor-associated factor (TRAF) super family, which function in various developmental processes of animals and plants as ubiquitin ligases. They have been characterized by an N-terminal cysteine-rich really interesting new gene (RING) domain, two zinc-finger motifs and a conserved C-terminal coiled-coil domain responsible for substrate binding and homo- or hetero-dimerization [14–17]. In plants, SINA proteins were found to function in proteasome mediated protein ubiquitination [18]. In Arabidopsis, SINAT2 interacted with RELATED TO APETALA2 (AP2) 2 (AtRAP2.2), whereas SINAT5 worked in the degradation of NAC1 to regulate lateral root growth [19–21]. In rice, the SINA family protein OsDIS1 negatively regulated the drought tolerance in transgenic rice plants [22]. In alfalfa, ectopic expression of the dominant negative form of SINAT5 affected the growth and nodulation of transgenic plant [23]. In tomato, overexpression of SlSINA2 lead to altered chlorophyll level in the leaves of transgenic plants, whereas overexpression of SlSINA5 disturbed flower development [24].

Previously, we investigated the function of Arabidopsis SINA2 (AtSINA2), a SINA protein lacking RING domain, and found that it positively regulated the drought tolerance of Arabidopsis plants [25]. Here, we demonstrate for the first time that IbSINA5, a homolog of SINA2, functions as a positively regulator in the cold signaling pathway in sweet potato.

Sweet potato cultivars Xushu18 and Taizhong 6 were grown in greenhouse as described previously [13]. For cold, drought and salt stress treatments, three-week-old Taizhong 6 plants were kept at 4°C, or treated with 25% PEG6000 or 250 mM NaCl. The fourth fully expanded leaves counted from the tops of plants were collected at 3 h, 6 h, 12 h, 24 h and 48 h after the treatments for RNA isolation. Ten plants were used for each treatment. For IbSINA5 expression analysis, fibrous roots, storage roots, stems, young leaves, mature leaves and senescent leaves of ten-week-old Taizhong 6 sweet potato plants were used. Total RNA was extracted as described previously [26].

qRT-PCR was carried out using IbSINA5 primers (forward: 5’-ATGTATAAAATGGAGATTGAAAGC-3’; reverse: 5’-TTAAGTGCTACAAATATTCG-3’) as described previously [26]. The expression level of IbCBF3, IbCOR413, IbCOR314 and IbCOR27 were examined with the house keeping gene IbTubulin as an internal control [13].

The encoding cDNA of IbSINA5 was cloned from sweet potato cultivar Taizhong 6 using its forward and reverse primers based on the gene information from the database [27]. The amplified cDNA fragment was cloned to the pEASY-T5 Zero Cloning Vector (Transgen, China) after sequence confirmation.

To detect the subcellular localization of IbSINA5 protein, IbSINA5 was fused in frame to the N-terminal of GFP in the pBI221:GFP vector. The resultant pBI221:IbSINA5-GFP and pBI221:GFP was transiently expressed in onion epidermal cells, respectively, as described previously [28]. The location of GFP and IbSINA5-GFP in the onion cells was observed with a confocal laser scanning microscopy (Leica TCS SP2).

To construct the plant expression vector pCAMBIA-2301s2:IbSINA5, the full length cDNA of IbSNIA5 was cloned into the modified pCAMBIA-1301s2 vector, driven by two copies of cauliflower mosaic virus (CaMV) 35S promoter. The resultant construct pCAMBIA-2301s2:IbSINA5 was introduced into sweet potato cv. Xushu18 via Agrobacterium-mediated transformation as described previously [29].

For the molecular confirmation of transgenic plants, PCR and qRT-PCR were carried out to verify the integration and expression of IbSNIA5. To assess the expression levels of IbSNIA5, IbCBF3 and IbCOR genes, total RNAs extracted from the mature leaves (the fourth leaves counted from the tops of plants) of four-week-old wild type and transgenic plants before and after cold treatment were used for qRT-PCR analyses. All gene specific primer sequences were the same as used by Jin et al. [13].

For cold resistance analysis, four-week-old wild type and transgenic sweet potato plants grown in soil were kept at 4°C for 48 h and recovery at 25°C for 24 h. The phenotypes of plants before and after the treatments were photographed and the contents of proline, MDA and hydrogen peroxide (H2O2) in the mature leaves were measured as described previously [30,31].

To generate the reporter construct 35S::REN-ProIbSIN5::LUC, the promoter region (2001 bp) of IbSNIA5 was inserted into pGreenII0800. To generate the effecter vector pGreenII62-SK-IbCBF3, IbCBF3 were cloned into pGreenII62-SK. Sweet potato leaf protoplast preparation and transformation were carried out as described by Wang et al. [32]. Transient dual-luciferase assays in sweet potato leaf protoplast were performed as described previously [33,34].

To analyze the transcription function of IbCBF3, the transcriptional factor effector pGreenII62-SK-IbCBF3 was transfected into sweet potato leaf protoplast. The transient expressions of IbCBF3, IbSNIA5 and IbCOR genes in sweet potato leaf mesophyll protoplasts were examine by qRT-PCR as described previously [35].

ANOVA (one-way) was used to generate every P value. The variability was indicated with the standard deviation (SD). *, ** and *** indicate p-value < 0.05, < 0.01 and < 0.001, respectively. Data are shown as mean ± standard deviation (SD) from three biological replicates each.

Ubiquitination mediated protein degradation at different plant growth stages and in response to various abiotic stresses has been well studied [36,37]. The Arabidopsis SINAT5 (AtSINAT5) functioned as a RING type E3 ubiquitin ligase to control lateral root growth by degrading NAC1, whereas SINA2, a TRAF-like SINA family protein lacking RING domain, positively regulated the resistance to drought stress [19,25]. To explore the biological function of SINA family proteins in tuberous crops, we blast searched the sweet potato genome database using Arabidopsis SINA2 (https://www.ipomoea-genome.org/genome_ jbrowse.html). A total number of seven SINA2 homologues were identified. Similar to the previously reported SINA and some other TRAF-like family proteins, they all contain a conserved C-terminal TRAF-like domain [14–16]. However, among them, same as the Arabidopsis SINA2 and animal’s TRAF1 proteins, IbSINA5 also lacks the RING domain (Fig. 1). Therefore, IbSINA5 does not have E3 ligase activity required for its target protein degradation.

Figure 1: Sequence alignment and phylogenetic analyses of SINAs from different plant species. Comparison of Ipomoea batatas IbSINA5 with Arabidopsis thaliana AtSINAT1 (NP_181729.1), AtSINA2 (NP_187978.1), Solanum lycopersicum SlSINA1 (XP_004228679.1), and Populus trichocarpa PtSINA2 (XP_002299212.1), and phylogenetic tree of IbSINA5 homologs in different plants are shown. The gene and amino acid sequences of IbSINA1 (g919), IbSINA2 (g4297), IbSINA3 (g8848), IbSINA4 (g17091), IbSINA5 (g20660), IbSINA6 (g30553) and IbSINA7 (g58368) are extracted from the sweet potato genome database (https://www.ipomoea-genome.org/genome_ jbrowse.html). Based on their conservation levels, residues in the sequence alignment analyses are respectively highlighted in black, dark gray and light gray. The bar in phylogenetic tree represents the number of amino acid substitutions per site. Asterisk and plus signs represent the RING domain and zinc-finger motif, respectively

To dissect the function of IbSNIA5 in the resistance to abiotic stresses in sweet potato, its expression levels in wild type Taizhong 6 plants were examined by qRT-PCR. IbSNIA5 was ubiquitously expressed in all the tested tissues and organs including fibrous roots, storage roots, stems, young leaves, mature leaves and senescent leaves, with a predominant expression in fibrous roots (Fig. 2A). Previously, we reported that SINA2 was strongly induced by ABA and drought [25]. We examined the expression levels of IbSINA5 in three-week-old Taizhong 6 plants after low temperature (4°C), 25% PEG6000 or 250 mM NaCl treatments by qRT-PCR. We found that IbSNIA5 was remarkably induced by cold, drought and salt stress, indicating its possible roles in plant response to various abiotic stresses (Figs. 2B–2D).

Figure 2: IbSINA5 expression and IbSINA5 subcellular localization. (A) Expression pattern of IbSINA5. The transcription levels of IbSINA5 in fibrous roots, storage roots, stems, young leaves, mature leaves and senescent leaves of ten-week-old sweet potato plants were shown. (B–D) Expression of IbSINA5in response to different abiotic stress treatments. Three-week-old sweet potato plants grown in greenhouse were kept at 4°C, or treated with 25% PEG6000 or 250 mM NaCl. The fourth fully expanded leaves counted from the tops of plants were collected at 3, 6, 12, 24 and 48 h after the treatments for RNA isolation. The transcription levels of IbSINA5 were measured by qRT-PCR and normalized to IbTubulin expression. Values are means and standard deviations of three biological replicates. * and ** indicate p-value < 0.05 and < 0.01, respectively. (E) IbSINA5 is mainly localized in nuclei and cytoplasm. The location of GFP and IbSINA5-GFP in the onion epidermal cells was observed with a confocal laser scanning microscopy (Leica TCS SP2). Scale bars = 100 μm

The subcellular localization of a certain protein may reflect its possible functions in the relevant biological processes it participates. To determine the subcellular localization of IbSNIA5, pBI221:IbSINA5-GFP and pBI221:GFP was transiently expressed in onion epidermal cells. IbSINA5-GFP fusion protein was mainly localized in nuclei and cytoplasm (Fig. 2E). Similar subcellular localization was also observed with SINA2-YFP fusion protein in Arabidopsis protoplasts [25]. The nuclei and cytoplasm localization of IbSINA5 implies that it may have a regulatory role in the expression of its upstream transcriptional factors in nuclei and its downstream functional genes in cytoplasm.

Figure 3: Molecular confirmation of IbSINA5 transgenic plants. (A) Schematic map of pCAMBIA1301s2-IbSINA5 construct. Expression of IbSINA5 was driven by two copies of the cauliflower mosaic virus 35S promoter. (B) PCR analysis of IbSINA5 integration in different transgenic lines. M, molecular marker; WT, wild type; L1-7, different transgenic lines; P, plasmid. (C) qRT-PCR analyses of IbSINA5 in wild type and different transgenic lines. Gene expression level in WT was set to 1. Values are means and standard deviations of three biological replicates. ** and *** indicate p-value < 0.01 and < 0.001, respectively

Base on our previous study that overexpression of SINA2 significantly augmented the resistance to drought stress in Arabidopsis plants, and the observation that IbSINA5 was dramatically induced by cold and drought stresses, we speculated that overexpression of IbSINA5 in sweet potato may also promote the cold resistance [25]. To verify this hypothesis, the plant expression construct pCAMBIA-2301s2:IbSINA5 containing the coding sequence of IbSINA5 was introduced into the genome of Xushu18 (Fig. 3A). A total number of 34 independent hygromycin resistant lines were obtained, and seven of them were randomly selected. PCR and qRT-PCR analyses confirmed the integration of IbSINA5 in the sweet potato genome, and the overexpression of IbSINA5 in all the tested transgenic lines (Figs. 3B and 3C).

To determine whether overexpression of IbSINA5 would improve the resistance of transgenic sweet potato plants to cold stress, we compared their growth phenotypes with wild type plants. Three transgenic lines with different IbSINA5 overexpression levels (L2, L5 and L7) were selected. Four-week-old wild type and transgenic plants grown in greenhouse were kept at 4°C for 48 h, then recovered at 25°C for 24 h. All transgenic plants showed less cold damage and successfully recovered after they were transferred back to room temperature, whereas wild type plants exhibited more severe cold damage and failed to recover (Fig. 4A). The damage extents were consistent with IbSINA5 overexpression levels, as indicated by the more severe damage in transgenic line L5, which showed relatively lower IbSINA5 overexpression level, than in transgenic lines L2 and L7, which showed relatively higher IbSINA5 overexpression levels (Fig. 4B).

Figure 4: Cold tolerance and gene expression analyses of wild type and transgenic plants. (A) Phenotypes of sweet potato plants before and after cold stress treatment. Four-week-old wild type and transgenic were treated at 4°C for 48 h and recovered at 25°C for 24 h. Cold damage in the leaves of wild type plants was significantly severe than that of transgenic plants after 48 h cold treatment and 24 h recovery. Ten plants of wild type and each transgenic line were used for the treatment. Photos were the representatives from three replicates. (B) qRT-PCR analyses. The transcription levels of IbSINA5 in the leaves of wild type and transgenic lines L2, L5 and L7 before cold treatments were measured by qRT-PCR. (C) IbCOR gene expression analyses. The transcription levels of IbCOR413, IbCOR314 and IbCOR27 in the leaves of four-week-old wild type and transgenic plants were examined by qRT-PCR. The transcript levels of these genes were normalized to IbTubulin expression. (D–F) Proline, MDA and H2O2 content analyses in the leaves of wild type and transgenic plants before and after the cold treatments. Values are means and standard deviations of three biological replicates. *, ** and *** indicate p-value < 0.05, < 0.01 and < 0.001, respectively

3.5 IbSINA5 Increases IbCOR Gene Expression and Oxidative Stress Tolerance in Transgenic Plants

It is well known that COR genes play a crucial role in plant response to cold stress [38]. We examined the expressions of IbCOR413, IbCOR314 and IbCOR27 in wild type and transgenic plants. As we have expected, the expression levels of IbCOR genes in transgenic plants were significantly higher than in wild type plants (Fig. 4C). Previous study has showed that overexpression of IbCBF3 conferred improved tolerance to low temperature and drought stress on transgenic sweet potato, leading to decreased accumulation of MDA and H2O2 [13]. Similar results were also observed in transgenic sweet potato plants overexpressing IbSINA5. Under normal growth condition, no significant difference was observed. However, under cold stress condition, a higher proline, and lower MDA and H2O2 content was detected in transgenic plants (Figs. 4D–4F). The increased IbCOR gene expression and antioxidant ability might help explain the improved cold resistance in IbSINA5 transgenic plants.

Previous work has demonstrated that IbCOR genes were up-regulated by IbCBF3 [13]. We performed PlantCARE analysis with the promoter sequence of IbSINA5, and a dehydration-responsive element-binding/C-repeat-binding factor (DREB/CBF) binding (DRE core) element was identified (Fig. 5). To explore whether IbSINA5 expression was regulated by CBFs in sweet potato, dual-luciferase assays were carried out. We found that IbCBF3 significantly activated the promoter of IbSINA5, implying that IbSINA5 expression could be regulated by IbCBF3 (Figs. 6A and 6B). We then transiently expressed IbCBF3 in the leaf protoplasts of wild type Xushu 18 plants. We observed that transient expression of IbCBF3 increased the transcription of IbSINA5, IbCOR413, IbCOR314 and IbCOR27 (Figs. 6C and 6D).

Figure 5: Plant CARE analysis. The Cis-acting elements in the promoter region of IbSINA5 (2001 bp) were analyzed. The predicted CBF binding DRE core element was marked in red

Taken together, our results suggests that overexpression of IbSINA5 up-regulates IbCOR gene expression and enhances cold resistance in transgenic sweet potato plants. IbCBF3 could be an efficient activator of IbSINA5 to regulate the expression of IbCOR genes, possible in a CBF-SINA-COR module.

Figure 6: IbCBF3 positively regulates the expression of IbSINA5 and IbCOR genes. (A) Schematic diagrams of the effector and reporter constructs. (B) IbCBF3 activates the promoter of IbSINA5. The control vector or pGreenII62-SK-IbCBF3 were co-expressed with 35S::REN-ProIbSIN5::LUC in the leaf protoplasts of wild type sweet potato plants. LUC/REN ratios indicate the promoter activity of IbSINA5 activated by IbCBF3. Control, protoplasts co-transfected with pGreenII62-SK and 35S::REN-ProIbSIN5::LUC; IbCBF3, protoplasts co-transfected with pGreenII62-SK-IbCBF3 and 35S::REN-ProIbSIN5::LUC. (C) Expression of IbCBF3 in the transfected sweet potato mesophyll protoplasts. (D) Expression levels of IbSINA5 and IbCOR genes in the transfected protoplasts. pGreenII62-SK-IbCBF3 was transfected into the sweet potato mesophyll protoplasts. pGreenII62-SK was used as a negative control. Total RNA was isolated from the transfected protoplasts for qRT-PCR analyses. The expression of gene in control was set to 1. Control, protoplasts transfected with pGreenII62-SK; IbCBF3, protoplasts transfected with pGreenII62-SK-IbCBF3. Values are means and standard deviations of three biological replicates. ** indicates p-value < 0.01

Acknowledgement: We thank Prof. Jun-jie Han (Yantai Academy of Agricultural Sciences, Yantai, Shandong Province, China) for providing the sweet potato cultivars Xushu18 and Taizhong6.

Authors Contribution: LMW, SLZ and HXZ designed the experiments; SYL, XAL, LZZ, HQH and BL performed the experiments; ZZS and LMW analyzed the data; SYL and HXZ wrote the manuscript. All authors have read and approved the final manuscript.

Funding Statement: This work was jointly supported by the following grants: Agricultural Variety Improvement Project of Shandong Province [Grant Nos. 2019LZGC009, 2019LZGC010, 2020LZGC007]; the National Key R&D Program of China [Grant Nos. 2018YFD1000500, 2019YFD1000500]; the National Natural Science Foundation of China [Grant Nos. 31870576, 31901572, 32071733]; the Natural Science Foundation of Shandong Province [Grant Nos. ZR2018PH041, ZR2019PC015, ZR2020MC138]; the Modern Agricultural Industry Technology System Innovation Team of Shandong Province of China [Grant No. SDAIT-02-05]; the Key R&D Program of Shandong Province of China [2019GSF108154]; the Science and Technology Development Project in Yantai [Grant No. 2018XSCC041].

Conflicts of Interest: We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

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