A novel sweetpotato bZIP transcription factor gene, IbbZIP1, is involved in salt and drought tolerance in transgenic Arabidopsis

Key message The overexpression of IbbZIP1 leads to a significant upregulation of abiotic-related genes, suggesting that IbbZIP1 gene confers salt and drought tolerance in transgenic Arabidopsis. Abstract Basic region/leucine zipper motif (bZIP) transcription factors regulate flower development, seed maturation, pathogen defense, and stress signaling in plants. Here, we cloned a novel bZIP transcription factor gene, named IbbZIP1, from sweetpotato [Ipomoea batatas (L.) Lam.] line HVB-3. The full length of IbbZIP1 exhibited transactivation activity in yeast. The expression of IbbZIP1 in sweetpotato was strongly induced by NaCl, PEG6000, and abscisic acid (ABA). Its overexpression in Arabidopsis significantly enhanced salt and drought tolerance. Under salt and drought stresses, the transgenic Arabidopsis plants showed significant upregulation of the genes involved in ABA and proline biosynthesis and reactive oxygen species scavenging system, significant increase of ABA and proline contents and superoxide dismutase activity and significant decrease of H2O2 content. These results demonstrate that the IbbZIP1 gene confers salt and drought tolerance in transgenic Arabidopsis. This study provides a novel bZIP gene for improving the tolerance of sweetpotato and other plants to abiotic stresses. Electronic supplementary material The online version of this article (10.1007/s00299-019-02441-x) contains supplementary material, which is available to authorized users.


Introduction
Salinity and drought seriously affect the productivity of agricultural crops in the world (Munns and Tester 2008;Yang et al. 2010;Zhao et al. 2013). Developing crops tolerant to salinity and drought is becoming important. Plants adapt to salinity and drought stresses by developing a variety of mechanisms, such as growth and development regulation, osmotic adjustment, detoxification, and ion homeostasis (Bohnert et al. 1995;Zhu 2001Zhu , 2002.
The basic region/leucine zipper motif (bZIP) proteins compose a large family of transcription factors in higher plants. The bZIP members have been reported to regulate flower development, seed maturation, pathogen defense and stress, light, hormone and sugar signaling pathways (Jakoby et al. 2002;Lindemose et al. 2013). Several bZIP genes have been found to confer the tolerance to abiotic stresses in some plant species. In Arabidopsis, AtABF3, AtbZIP24, and AtbZIP1 are positive regulators of plant tolerance to abiotic stresses (Kim et al. 2004;Yang et al. 2009;Sun et al. 2012). In rice, OsbZIP23, OsbZIP72, OsABF1/OsbZIP12, OsABF2/OsbZIP46, and OsbZIP71 increased the tolerance to abiotic stresses (Xiang et al. 2008;Lu et al. 2009; Communicated by Sang-Soo Kwak.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0029 9-019-02441 -x) contains supplementary material, which is available to authorized users. et al. 2010a, b;Liu et al. 2014a, b, c, d), but OsbZIP52 negatively regulates responses to cold and drought (Liu et al. 2012). GmbZIP44, GmbZIP62, and GmbZIP78 conferred salt and freezing tolerance in transgenic Arabidopsis (Liao et al. 2008). GmbZIP1 increased the tolerance to salinity, cold temperature and drought in transgenic Arabidopsis and improved drought tolerance in transgenic wheat (Gao et al. 2011a, b). ZmbZIP72 conferred salt and drought tolerance in transgenic Arabidopsis (Ying et al. 2012) and ZmABP9 enhanced salt and drought tolerance in transgenic cotton (Wang et al. 2017).
Sweetpotato is an important food crop worldwide (Zang et al. 2009). Its productivity is often limited by salinity and drought stresses (Liu et al. 2014a). Gene engineering is an alternative approach for improving abiotic stresses' tolerance in sweetpotato (Kim et al. 2012(Kim et al. , 2013(Kim et al. , 2014Liu et al. 2014aLiu et al. , b, c, 2015Wang et al. 2016a, b, c;Zhai et al. 2015;Li et al. 2017). To date, the bZIP family has not been reported in sweetpotato. Here, a novel bZIP transcription factor gene, named IbbZIP1, was isolated from sweetpotato and found to be involved in salt and drought tolerance in transgenic Arabidopsis.

Plant materials
Sweetpotato line HVB-3 was used to isolate the IbbZIP1 gene in this study. Its transcriptome sequencing was conducted by Li et al. (2017), from which one expressed sequence tag (EST) was obtained. Arabidopsis thaliana (Columbia-0, WT) was employed to analyze the function of IbbZIP1.

Cloning and analysis of IbbZIP1 and its promoter
Freshly-harvested storage roots of HVB-3 were used to extract total RNA. The first-strand cDNA was obtained using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China). A rapid amplification of cDNA ends (RACE) procedure was applied to amplify the fulllength cDNA of IbbZIP1 with specific primers (Supplementary Table S1). Genomic DNA extracted from in vitrogrown plants was used to amplify the genomic sequence of IbbZIP1 (Wang et al. 2016a). Its promoter was cloned with Universal GenomeWalker 2.0 Kit (TaKaRa, Dalian, China). The specific primers listed in Supplementary Table S1 were employed.

Expression analysis of IbbZIP1 in sweetpotato
Storage root, stem, and leaf tissues of HVB-3 grown for 100 days in a field were used to isolate total RNA. The expression of IbbZIP1 was analyzed by quantitative realtime PCR (qRT-PCR) with the specific primers of IbbZIP1 (Supplementary Table S1) and Ibactin (AY905538) as an internal control based on the method of Liu et al. (2014a). Comparative C T method was employed to quantify the gene expression (Schmittgen and Livak 2008). Furthermore, the HVB-3 plants cultured for 4 weeks on the basal Murashige and Skoog (MS) medium were transferred to liquid MS medium with H 2 O (control), 200 mM NaCl, 20% PEG6000 and 100 μM ABA, respectively, and sampled at 0, 2, 4, 6, 12, 24, and 48 h after treatment for analyzing the expression of IbbZIP1.

Transactivation assay of IbbZIP1 in yeast
Transactivation assay of the IbbZIP1 protein in yeast (Saccharomyces cerevisiae) was done as described by Jiang et al. (2014). Its encoding region obtained with specific primers (Supplementary Table S1) was integrated into the vector pGBKT7 (pBD). Expression vector pBD-IbbZIP1, pGAL4 (as positive vector) and pBD (as negative vector) were transferred into the yeast strain AH109, respectively. The transactivation activity was determined according to the method of Wang et al. (2016a).

Production of the transgenic Arabidopsis plants
The overexpression vector pC3301-121-IbbZIP1 was constructed by inserting 35S-IbbZIP1-NOS into pCAMBIA3301 at HindIII and EcoRI and then transferred into the Agrobacterium tumefaciens strain GV3101. The dipping flower method was applied to transform Arabidopsis (Clough and Bent 1998). The transgenic Arabidopsis plants were determined by histochemical GUS assay (Jefferson et al. 1987) and PCR analysis with 35S forward and IbbZIP1-specific reverse primers (Supplementary Table S1).

Assay for salt and drought tolerance
Transgenic Arabidopsis T 3 and WT seedlings were treated on MS medium containing 200 mM NaCl or 300 mM mannitol under 13 h day-light at 54 μM/m 2 /s and 22 °C. After 2 weeks, their root length and fresh weight were investigated. Furthermore, the T 3 and WT seedlings were planted for 2 weeks in pots with a soil, vermiculite, and humus mixture (1:1:1, v/v/v) and each pot was irrigated with a 33 mL of 300 mM NaCl solution once every 2 days for 2 weeks, or stressed by drought for 4 weeks and re-watered for 2 days. The contents of abscisic acid (ABA) and proline and the activity of superoxide dismutase (SOD) in the T 3 and WT plants treated in pots for 4 weeks under no stresses, 1 week under 300 mM NaCl stress or 2 weeks under drought stress were determined as described by Gao et al. (2011a)

Assay for ABA sensitivity
For ABA sensitivity assay, the transgenic Arabidopsis and WT seeds (50 seeds for each line) were sown on MS medium with 0, 0.5, and 1 μM ABA, respectively, under 13 h day-light at 54 μM/m 2 /s and 22 °C. After 1 week, their germination rate and cotyledon opening and greening rate were investigated.

Expression analysis of the related genes
The T 3 and WT plants potted for 4 weeks without stress, 1 week stressed with 300 mM NaCl or 2 weeks stressed by drought were employed to analyze the expression of the genes involved in ABA and proline biosynthesis and reactive oxygen species (ROS) scavenging system using qRT-PCR protocols of Liu et al. (2014a). The specific primers of Atactin (internal control, NM112764) and the related genes were listed in Supplementary Table S1.

Statistical analysis
All experiments were performed with three biological replicates. Data presented as the mean ± SE were analyzed by Student's t test (two-tailed analysis) at P < 0.05 (*) and P < 0.01 (**).

Cloning and sequence analysis of IbbZIP1 and its promoter
The IbbZIP1 gene was cloned from sweetpotato line HVB-3. The cDNA sequence was 1757 bp in length and contained an 1080 bp ORF encoding a 359-aa polypeptide with a molecular weight of 41.26 kDa and a predicted pI of 7.89. The IbbZIP1 protein was subjected to phylogenetic analysis together with 44 Arabidopsis bZIP proteins belonging to 10 groups (Jakoby et al. 2002). The results revealed that IbbZIP1 belonged to group E of the bZIP family and had a close relationship with At1g58110 (Fig. 1). IbbZIP1 contained one bZIP domain (Fig. 2a). The 1946 bp genomic DNA of IbbZIP1 contained 4 exons and 3 introns (Fig. 2b). Its promoter region (~ 2056 bp) had the stress-responsive cis-acting regulatory elements, such as HSE, MBS, TCA, GARE, TGA, ERE, and ABRE (Supplementary Fig. S1; Supplementary Table S2). Fig. 1 Phylogenetic analysis of IbbZIP1 with 44 Arabidopsis bZIP proteins. Ten groups of bZIP proteins were defined based on the classification of Arabidopsis bZIP proteins (Jakoby et al. 2002) 1 3

Expression of IbbZIP1 in sweetpotato
The IbbZIP1 gene exhibited significantly higher expression level in the leaves of HVB-3 than in the stems and storage roots (Fig. 3a). Its expression in the in vitro-grown plants of HVB-3 was strongly induced by NaCl, PEG6000 and ABA, and peaked (6.79-fold) at 12 h of 200 mM NaCl treatment, (6.58-fold) at 24 h of 20% PEG6000 treatment and (7.51fold) at 24 h of 100 μM ABA treatment (Fig. 3b). These results showed that IbbZIP1 might be involved in the tolerance to salt and drought in sweetpotato.

Transactivation activity of IbbZIP1 in yeast
The yeast one-hybrid system was applied to identify a possible transactivation activity of the IbbZIP1 protein. The yeast cells harboring pGAL4 and pGBKT7-IbbZIP1 grew well on SD plate without tryptophan and histidine and exhibited β-galactosidase activity, but the cells bearing pBD failed to grow (Fig. 4). These results demonstrated that IbbZIP1 had transactivation activity in yeast and has potential ability to activate the transcription of related genes by directly binding to cis-acting elements in their promoter region or interacting with other proteins.

Production of the transgenic Arabidopsis plants
Lots of putatively transgenic Arabidopsis seeds were obtained by the dipping flower method. The randomly sampled seeds were sown on MS medium with 12.5 mg/L phosphinothricin (PPT) and produced plants. GUS assay and PCR analysis confirmed that 8 of the randomly sampled 60 plants were transgenic plants, named L1, L2, …, L8, respectively ( Supplementary Fig. S2), from which T 3 were generated. The expression of IbbZIP1 in the leaves of the T 3 and WT plants was analyzed by qRT-PCR and 4 of them (L1, L3, L6 and L8) exhibited significantly higher expression level compared with other plants (Supplementary Fig. S2).

Enhanced salt and drought tolerance of transgenic Arabidopsis
Four transgenic lines (L1, L3, L6, and L8) and WT seedlings were cultured on MS medium with 200 mM NaCl, 300 mM mannitol or without stresses for 2 weeks, respectively. The transgenic and WT plants exhibited no differences in growth without stresses, but the transgenic plants showed significantly better growth and increased physical size than WT under NaCl and mannitol stresses (Fig. 5).
Two-week-old transgenic and WT plants grown in pots were stressed by 300 mM NaCl and drought, respectively. The transgenic plants and WT showed no differences in growth without stresses (Fig. 6a). Under salt and drought stresses, the transgenic plants exhibited good growth, increased ABA and proline contents, enhanced SOD activity and decreased H 2 O 2 content, while WT almost died (Fig. 6a, b). These results indicated that L1, L3, L6, and L8 had significantly enhanced salt and drought tolerance compared with WT.

Increased ABA sensitivity of transgenic Arabidopsis
Seed germination in response to ABA was tested to determine whether IbbZIP1 is involved in the ABA signaling pathway. No obvious differences in germination rate and cotyledon opening and greening rate were observed between the transgenic lines (L1, L3, L6, and L8) and WT under normal condition (Fig. 7). With exposure to different concentrations of ABA, both germination rate and cotyledon opening and greening rate of the transgenic and WT seeds declined, but the germination of L1, L3, L6, and L8 seeds were more sensitive to ABA-elicited inhibition, indicating that this gene might be involved in the ABA signaling pathway (Fig. 7).

Expression of the stress-responsive genes in the transgenic Arabidopsis plants
The genes encoding ABA biosynthetic 9-cis-epoxycarotenoid dioxygenase (NCED) and xanthoxin dehydrogenase (ABA2) and proline biosynthetic pyrroline-5-carboxylate synthase (P5CS) showed significantly higher expression levels in the transgenic plants in comparison with WT under salt and drought stresses (Fig. 8). The ROS scavenging genes encoding SOD, glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were found to show a systematic upregulation in the transgenic plants under salt and drought stresses (Fig. 8).

Discussion
Several bZIP transcription factor genes have been cloned from Arabidopsis, rice, soybean, maize etc., and found to play roles in plant stress-responsive and hormone signal transduction (Sun et al. 2012;Liu et al. 2014a;Gao et al. 2011a;Wang et al. 2017). However, there is no report on the bZIP transcription factors in sweetpotato. This study reported, for the first time, the cloning and characterization of a novel transcription factor gene, IbbZIP1, from sweetpotato. The IbbZIP1 protein contains a typical bZIP domain which is essential for bZIP transcription factor, belongs to E group of bZIP family and has a close relationship with At1g58110 (Figs. 1, 2). To date, the function of the At1g58110 gene is still unknown. We found that the promoter region of IbbZIP1 had the stress-responsive cisacting regulatory elements (Yamaguchi-Shinozaki et al. 2005, 2006Xiang et al. 2008). The IbbZIP1 expression was induced by NaCl, PEG, and ABA (Figs. S1, 4; Supplementary Table S2) and its overexpression increased salt and drought tolerance in transgenic Arabidopsis (Figs. 6, 7).
The bZIP transcription factors regulate the ABA-mediated abiotic stresses signaling pathways in plants (Mehrotra et al. 2014). In rice, overexpression of OsABI5 and OsbZIP23 upregulated the ABA biosynthetic gene ABA2 Xiang et al. 2008). The ABA biosynthetic gene NCED2 was upregulated in the ZmABP9-overexpressing cotton plants (Wang et al. 2017). ABA regulates the expression of ABA dependent stress-responsive genes and higher levels of ABA can enhance salt and drought tolerance in Arabidopsis (Tuteja 2007;Liu et al. 2013;Wang et al. 2016a). In the present study, increased ABA sensitivity of transgenic Arabidopsis to exogenous ABA showed that IbbZIP1 might be involved in the ABA signaling pathway Fig. 6 Responses of the transgenic Arabidopsis plants and WT grown in pots under salt and drought stresses. a Phenotypes of transgenic vs. WT plants grown for 6 weeks under normal condition, 2 weeks under 300 mM NaCl stress and 4 weeks under drought stress and then 2 days after re-watering, respectively. b ABA, proline, and H 2 O 2 contents and SOD activity in the transgenic plants and WT grown for 4 weeks under normal condition, 1 weeks under 300 mM NaCl and 2 weeks under drought stress. Data are presented as mean ± SE (n = 3) (Fig. 7). The IbbZIP1-overexpressing Arabidopsis plants exhibited significant upregulation of NCED and ABA2 and significant increase of ABA content under salt and drought stresses (Figs. 7,8). It is suggested that overexpression of IbbZIP1 confers tolerance to salt and drought due to the increased expression level of the ABA biosynthetic genes, which increases the production of ABA as a signaling molecule and further the expression of stress-responsive genes.
It has been reported that the high level of ABA in rice increases the transcript level of OsP5CS1, which lead to accumulation of proline under abiotic stresses (Sripinyowanich et al. 2013). It has been shown that proline accumulation resulted in the enhanced salt and drought tolerance in several plant species (Szabados and Savouré 2010;Krasensky and Jonak 2012;Zhang et al. 2012;Liu et al. 2015). Overexpression of GmbZIP62 and GmbZIP78 upregulated the genes involved in ABA signaling pathways and GmP5CS1, resulting the increased proline content and the enhanced salt and freezing tolerance in transgenic Arabidopsis (Liao et al. 2008). In this study, the IbbZIP1-overexpressing Arabidopsis plants exhibited the increased P5CS expression level and proline content under salt and drought stresses (Figs. 7, 8). These results suggested that more proline might be accumulated because of the increased ABA level, which leads to the enhanced salt and drought tolerance.
In plants, overproduction of ROS often occurs under salinity and drought stresses, which leads to oxidative damage. ROS can be detoxified by activating ROS scavenging enzymes (Gill and Tuteja 2010). The increased proline level led to upregulation of the ROS scavenging genes, which resulted in the enhanced tolerance to salt and drought in transgenic sweetpotato (Liu et al. 2014b, Fig. 7 Responses of the transgenic Arabidopsis seeds and WT sown on MS medium with 0, 0.5, and 1 μM ABA for 1 week. a Growth vigor of the transgenic and WT seedlings. b Germination rates of the transgenic Arabidopsis seeds and WT. c Cotyledon opening and greening rates of the transgenic Arabidopsis seeds and WT Liu et al. 2015;Wang et al. 2016b;Zhai et al. 2015). We found that under salt and drought stresses, the ROS scavenging genes (SOD, GPX, CAT , APX, and DHAR) were systematically upregulated in the IbbZIP1-overexpressing Arabidopsis plants (Fig. 8). Thus, more accumulation of proline in the IbbZIP1-overexpressing Arabidopsis plants might upregulate the ROS scavenging genes and further stimulate the ROS scavenging system, which results in the enhanced salt and drought tolerance.
In conclusion, a novel sweetpotato bZIP transcription factor gene, IbbZIP1, has been successfully isolated. The IbbZIP1 gene is involved in salt and drought tolerance in transgenic Arabidopsis. Its overexpression might upregulate the genes involved in ABA and proline biosynthesis and ROS scavenging system and increase the ABA and proline contents, which leads to enhanced salt and drought tolerance. This study provides a novel bZIP gene for improving the tolerance of sweetpotato and other plants to abiotic stresses.
Author contribution statement Conceived and designed the experiments: QCL CK. Performed the experiments: CK. Analyzed the data: CK. Contributed reagents/materials/analysis tools: QCL HZ SZH NZ. Wrote the paper: QCL CK.
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