CIPK11: a calcineurin B-like protein-interacting protein kinase from Nitraria tangutorum, confers tolerance to salt and drought in Arabidopsis

Background The CIPKs are a group of plant-specific Ser/Thr protein kinases acting in response to calcium signaling, which plays an important role in the physiological and developmental adaptation of plants to adverse environments. However, the functions of halophyte-derived CIPKs are still poorly understood, that limits a potential application of CIPKs from halophytes for improving the tolerance of glycophytes to abiotic stresses. Results In this study, we characterized the NtCIPK11 gene from the halophyte Nitraria tangutorum and subsequently analyzed its role in salt and drought stress tolerance, using Arabidopsis as a transgenic model system. NtCIPK11 expression was upregulated in N. tangutorum root, stem and blade tissues after salt or drought treatment. Overexpressing NtCIPK11 in Arabidopsis improved seed germination on medium containing different levels of NaCl. Moreover, the transgenic plants grew more vigorously under salt stress and developed longer roots under salt or drought conditions than the WT plants. Furthermore, NtCIPK11 overexpression altered the transcription of genes encoding key enzymes involved in proline metabolism in Arabidopsis exposed to salinity, however, which genes showed a relatively weak expression in the transgenic Arabidopsis undergoing mannitol treatment, a situation that mimics drought stress. Besides, the proline significantly accumulated in NtCIPK11-overexpressing plants compared with WT under NaCl treatment, but that was not observed in the transgenic plants under drought stress caused by mannitol application. Conclusions We conclude that NtCIPK11 promotes plant growth and mitigates damage associated with salt stress by regulating the expression of genes controlling proline accumulation. These results extend our understanding on the function of halophyte-derived CIPK genes and suggest that NtCIPK11 can serve as a candidate gene for improving the salt and drought tolerance of glycophytes through genetic engineering. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-021-02878-x.


Background
Soil salinity and drought are critical environmental threats to plant development that limit plant growth by negatively affecting the availability, transportation, and partitioning of nutrients and water. These effects are threatening to decline crop productivity worldwide and increase the pace of soil desertification, further affecting the ecological balance [1]. Therefore, understanding halophyte plant tolerance to salt and drought stress is critical for sustaining agricultural productivity by breeding new stress-tolerant plants that may cope with abiotic stresses [2]. N. tangutorum belongs to the family Nitrariaceae Nitraria in Sapindales, which is widely distributed in northwestern China [3][4][5][6]. N. tangutorum is a desert halophyte adapted to severe drought and high salinity, and generally grows in arid or semiarid regions with high salinity [7,8]. Moreover, this species can efficiently alleviate the degree of soil salinity and fix moving sand, thus playing an important ecological role in environmental balance [8,9]. Previous studies have shown that Nitraria may adapt to abiotic stress conditions through increasing antioxidant enzyme activities, proline accumulation, level of soluble carbohydrates and reducing the intracellular Na + / K + ratio [7,[10][11][12][13]. However, the molecular mechanisms underlying the physiological adaptability of N. tangutorum to various stresses need further study [14][15][16].
To perceive salinity and drought stress, plants have evolved various stress sensors, signaling pathways, transcription factors and promoters to elicit the necessary responses by altering their metabolism, growth and/or development [17,18]. Ca 2+ acts as an ubiquitous messenger in various signal transduction networks to induce specific cellular responses, such as responses to signals of abiotic stress [19,20]. Previous studies have identified proteins able to sense Ca 2+ levels, including CaM, CDPK and CBL. CBLs function through interacting with CIPKs to activate specific targets and transduce signals [21,22]. CIPKs contain a highly conserved N-terminal kinase domain with a putative activation loop and a unique Cterminal regulatory region with a conserved NAF amino-acid motif that have been found to promote stress tolerance by regulating various physiological responses [23][24][25]. Overexpression of OsCIPK12 improved rice tolerance to cold, drought, and salt stress by inducing the accumulation of proline and soluble sugars [26]. CaCIPK6 from chickpea has been shown to mediate auxin transport to regulate the salt tolerance of tobacco seedlings [27]. Overexpression of BrCIPK1 enhanced abiotic stress tolerance by increasing proline biosynthesis in rice [28]. In addition, CIPKs may regulate the activity of the ROS scavengers POD, SOD and CAT to reduce the content of H 2 O 2 and MDA, and to improve stress tolerance [29,30] or they may control ion and water homeostasis to improve salt tolerance [31,32]. These findings have continuously revealed the importance of CIPKs in regulating physiological factors that may improve plant stress tolerance.
Here, we identified a novel member of the CIPK gene family from N. tangutorum, NtCIPK11, and describe its role in the molecular regulation of salt and drought tolerance. We found that NtCIPK11 was induced in root, stem and leaf tissues by 500 mM NaCl or 200 mM mannitol, with transcripts preferentially accumulating in leaves. To further explore how NtCIPK11 might function molecularly, we overexpressed it in Arabidopsis. The transgenic plants showed a higher germination rate and better growth than the WT plants after NaCl or mannitol treatment. In addition, we found that genes involved in glutamate-derived proline biosynthesis [33][34][35], were regulated in transgenic plants. Besides, the proline accumulation was found to be significantly higher in the transgenic plants than that of WT seedlings. On the contrary, the H 2 O 2 content showed a less level in NtCIPK11-overexpressing plants than WT. Our data show that NtCIPK11 is able to regulate the proline accumulation through mediating the expression of key genes of specific biochemical processes in Arabidopsis, thereby increasing tolerance of plants dealing with abiotic stresses.

N. tangutorum physiologically responded to salt treatment
As a halophyte with adaptability in a salt environment, N. tangutorum has been the focus of studies designed and implemented to investigate the mechanism of salt tolerance using biochemical methods [7,10,12] and molecular biology techniques [14,15,36]. To better understand the salt tolerance, we observed the growth morphology of N. tangutorum upon 400 mM NaCl treatment (Fig. 1). The seedlings watered with tap water showed unchanging growth state for 18 days (0 mM NaCl treated plants in Fig. 1a-h). However, the plants treated with 400 mM NaCl exhibited dynamic change in appearance. The bottom leaves gradually withered and turned yellow with treatment extension. After one week, the seedlings under salt stress conditions were significantly different from the untreated seedlings, especially the bottom leaves ( Fig. 1a-f). However, plants treated with salt for one week recovered when tap water was used for another 10 days and displayed more vigorous growth than the untreated plants. More new leaves appeared at the tip of the salt-treated seedlings (Fig. 1g, h and h'). To further study the physiological mechanism of salt tolerance, the activity of antioxidant enzymes POD, SOD and CAT, was tested in plants after 400 mM NaCl treatment ( Fig. 1i-k). The results showed that the activity of these antioxidant enzymes was differentially affected by salinity. The POD and SOD activity increased significantly at a 400 mM salinity level on the first day of treatment ( Fig. 1i and j). CAT did not positively respond to salt treatment in our experiment (Fig. 1k). Furthermore, we found that free proline, generally thought to have a positive role in plants responses to environmental stresses, such as drought and salinity [37,38], significantly accumulated in N. tangutorum after salt treatment (Fig. 1l). In addition, the MDA content, which indicates the integrity of the membrane [39], was slightly changed during the salt treatment (Fig. 1m). Thus, these data and 8 days (f); the appearance of the plants after the 8-day treatment as described above and 1-day re-watering (g) and 10-day re-watering (h and h′) with tap water; red arrowheads indicate withering leaves; red stars indicate new leaves; scale bar: 1 cm. i-m Effect of NaCl stress on biochemical parameters: activities of POD (i), SOD (j), and CAT (k), proline content (l), and MDA content (m) in the N. tangutorum leaves. The data represent means ± SD of three biological replicates; statistical analyses were performed with one-way ANOVA test with LSD multiple comparisons, '*' p < 0.05, '**' p < 0.01, '***' p < 0.001 taken together suggest that N. tangutorum significantly increased the activity of some antioxidant enzymes and the proline content to protect the cell membrane from being drastically affected by salinity stress under our experimental conditions.

NtCIPK11 identification and bioinformatics analysis
A large number of plant genes that show a response to different stresses have been previously identified as potential resources for genetic engineering. However, most of these candidate genes were isolated from glycophytes, which possess a relatively poor ability to tolerate environmental stresses [40]. Thus the molecular information from halophytes that can be used to analyze the mechanisms of stress tolerance is limited. As a consequence, N. tangutorum was selected for functional gene exploration in our study. We used 5′ and 3′ RACE to determine the complete cDNA nucleotide sequence of the novel gene and found that it is 1677 bp in length, with a 236 bp 5'UTR and a 127 bp 3'UTR. The coding region is 1314 bp long and encodes a 438 amino acid polypeptide with a calculated molecular mass of 49.4126 kDa. BLASTP searches and multiple alignment analyses showed that the deduced protein sequence of this clone displayed a high identity with CIPK orthologs in other species (Fig. 2a). The protein sequence showed 73.48% identity with Hevea brasiliensis CIPK11 (XP_021639925.1), 72.62% identity with CIPK11 (XP_006431996.1) of Citrus clementina and 67.34% identity with AtCIPK11 (AAK16686.1) of Arabidopsis thaliana (Fig. 2a). Similar to its homologues, this deduced protein possesses an N-terminal serine/threonine protein kinase domain (26-279 aa) with an ATP-binding site, an active site and a C-terminal regulatory domain (310-369 aa) with a CBL-interacting NAF/FISL module (Fig. 2a), motifs that are highly conserved in the CIPK family. A hydrophobicity blot and transmembrane domain prediction indicated that the most hydrophobic segment of NtCIPK11 is located between amino acid residues 210 and 221 ( Fig. 2b and c). In addition, a phylogenetic analysis of the N. tangutorum CIPK protein and 26 Arabidopsis thaliana CIPK proteins showed that the novel halophyte CIPK clusters as a sister branch of AtCIPK11 to the intron-free subgroup [41]; hence we referred to it as N. tangutorum CIPK11 (NtCIPK11) (Fig. 3).

NtCIPK11 in N. tangutorum positively responded to salt treatment
To study whether NtCIPK11 expression is regulated by salt in Nitraria, we treated seedlings with 500 mM NaCl for a duration of two hours. The qPCR expression profiling showed that untreated NtCIPK11 was expressed in the roots, stems and leaves, with the latter two tissues expressing 1.4-and 1.8-fold higher levels than the roots (Fig. 4a). After treatment with 500 mM NaCl, we found that the NtCIPK11 transcript level increased 7-fold in roots, 17-fold in stems and up to 118-fold in leaves compared to the expression level in untreated roots. This finding shows that NtCIPK11 transcripts accumulate preferentially in leaf tissues after salt treatment (Fig. 4a).

NtCIPK11 overexpression led to improved salt resistance in Arabidopsis
To investigate how NtCIPK11 acts molecularly, we cloned and overexpressed the gene in Arabidopsis. The seeds of transgenic Arabidopsis plants showed a 95.66% germination rate on average, close to that of WT seeds (96.05%) on ½ MS medium without salt; however, the NtCIPK11-transformed seeds showed 88% or 57% germination rates, respectively, after 5 days of 100 mM NaCl or 150 mM NaCl treatment, approximately twice as high as the WT germination rates of 45% and 25% respectively under the same salt conditions ( Fig. 4b and c). After 20 days, the NtCIPK11-overexpressing plants showed longer roots (Fig. 5b) and a higher number of leaves ( Fig. 5a and c) and roots ( Fig. 5d) than the WT plants, with the difference particularly large between the plants treated with 150 mM NaCl-treated medium. Therefore, we concluded that NtCIPK11 overexpression significantly promoted the seed germination and induced the salt tolerance of Arabidopsis.

Overexpression of NtCIPK11 altered the transcription pattern of genes involved in proline metabolism and accumulation
In plants, proline has been reported to accumulate after exposure to various stresses, including salt, drought and cold stress [42]. As shown in previous research, CIPK overexpression promoted proline accumulation and improved the tolerance of plants exposed to cold and drought stress [43]. To determine the potential mechanism of how ectopic expression of NtCIPK11 increases salt tolerance, four key genes of proline metabolism, P5CS1, P5CS2, P5CR [34] and ProDH1 [35], in WT and transgenic plants were measured via qPCR. As shown in Fig. 6, the genes related to proline synthesis had significantly higher expression levels in the NtCIPK11-overexpressing plants than they did in the WT plants under the salt stress conditions (Fig. 6a-c). However, ProDH1, which regulates proline catabolism, had a lower expression level in the transgenic plants than in the WT plants (Fig. 6d). Importantly, the proline content was significantly higher in the transgenic seedlings than that of in the WT plants under 100 mM NaCl treatment (Fig. 6e). Besides, H 2 O 2 staining was observed as light brown in the root of transgenic plants especially in the OX-1 seedlings, but dark brown in WT plants under 100 mM NaCl treatment (Fig. S1). These results showed that NtCIPK11 overexpression affected the expression of proline metabolism-related genes and proline accumulation, which might mediate the reduction of ROS production to mitigate the damage in plants exposed to salt stress.
NtCIPK11 positively responded to drought treatment in N. tangutorum To investigate the function of NtCIPK11 in drought tolerance, we simulated drought stress by treating plants with 200 mM mannitol for 2 h and observed how NtCIPK11 expression changed. We found that NtCIPK11 transcript levels increased dramatically after mannitol treatment, but to a slightly lesser extent than they did upon salt treatment, increasing 15-, 20-and 38-fold in root, stem and leaf tissues, respectively (Fig. 7a). Taken together, these results show that in response to at least two kinds of abiotic stresses, salt and drought stress, NtCIPK11 expression is increased.

Overexpression of NtCIPK11 enhanced the development of Arabidopsis seedlings under drought stress
To study how NtCIPK11 affects the drought stress response, seeds of transgenic Arabidopsis and those of WT plants were sown on ½ MS-agar plates containing various concentrations of mannitol. Compared to the seedlings exposed to the salt treatment, the seed germination of both WT and transgenic plants was not affected by the mannitol treatment (Fig. 7c). However, we found that WT seedlings developed more slowly than those of the transgenic plants, as indicated by the percentage of seedlings that formed two cotyledons 4 days postgermination ( Fig. 7b and c). Adding mannitol to the ½ MS medium caused a high number of WT seeds to undergo arrested development, with 31%, 20% and 5% of the seedlings reaching the two-cotyledon stage at concentrations of 100 mM, 150 mM and 200 mM mannitol, respectively (Fig. 7b). In contrast, as many as 91%, 80% and 70% of the NtCIPK11-transformed seeds developed two cotyledons (Fig. 7b). Therefore, these results showed that NtCIPK11 can promote seedling development under drought stress conditions at an early stage of plant growth.

Overexpression of NtCIPK11 promoted Arabidopsis root growth under drought stress
To further study the function of NtCIPK11 during drought treatment, we observed plant growth for 20 days on medium containing different concentrations of mannitol. The NtCIPK11-overexpressing plants showed better growth than the WT plants after mannitol treatment (Fig. 8a). The transgenic lines developed a longer primary root than the WT line, especially after treatment with 150 mM or 200 mM mannitol ( Fig. 8a and b). To determine whether NtCIPK11 functions like its orthologs to regulate the expression of genes related to proline-mediated drought tolerance, the transcripts of four genes (ProDH1, P5CS1, P5CS2, and P5CR) were measured by qPCR, and the results were compared to the transcription patterns of the WT and NtCIPK11overexpressing plants. We found that ProDH1 transcription in the transgenic plants was lower than it was in the WT plants after mannitol treatment, which indicates a  (Fig. 9a). Nevertheless, the proline synthesis genes exhibited a different expression pattern compared to the genes under salt treatment in Arabidopsis (Fig. 9b-d). At the same time, we observed that the proline content was increased in both WT and transgenic plants under mannitol treatment (Fig. 9e). The proline content of transgenic seedlings was higher than that in the WT. However, that result did not show a significant difference between WT and NtCIPK11-overexpressing seedlings (Fig. 9e). These results suggest that NtCIPK11 is involved in drought and salt stress signaling by influencing the expression of Fig. 4 NtCIPK11 responded to salt stress in N. tangutorum and Arabidopsis. a NtCIPK11 transcription increased after 500 mM NaCl treatment of N. tangutorum. b Germination rate of WT and NtCIPK11 overexpressing seeds. c Growth of the seeds germinated on medium with different salt contents. Three biological replicates and three technical replicates were conducted. The data represent the means ± SD from three biological replicates. '**' p < 0.01 proline metabolism regulators and proline accumulation but to different degrees.

Discussion
Salt and drought stress are major environmental factors that threaten agricultural productivity and ecological balance. As a result of natural selection and adaptation to a stressful environment, halophytes have evolved specific and diverse regulatory mechanisms for high stress tolerance, that lead to a significant plasticity in environmental adaptation [44,45]. Thus, the basic machinery of halophytes for adaptation to harsh stresses deserves further research. In addition, understanding the genetics of halophyte responses to a variety of stress conditions is critical for developing transgenic treatment strategies [46][47][48].
In our study, we reached three major conclusions on how the NtCIPK11 gene isolated from N. tangutorum increases the salt tolerance of Arabidopsis. First, the overexpression of NtCIPK11 in Arabidopsis resulted in a significantly higher seed germination rate after NaCl treatment (Fig. 4b and c). Second, the transgenic plants grew better than the WT plants during salt treatment (Fig. 5). Third, the NtCIPK11 overexpression caused a The data represent means ± SD from three biological replicates, and the statistics analyses were performed with one-way ANOVA test, '*' p < 0.05, '**' p < 0.01 higher proline accumulation than WT plants under salt stress (Fig. 6). These results revealed the function of this novel gene from the halophyte on salt tolerance were very consistent with the findings of previous studies [49]. The mechanism for salt tolerance induction through CIPKs have been previously identified: CIPKs mediate the expression of genes encoding various transporters important for ion homeostasis [49][50][51], increase the amount of antioxidant metabolites [52], or promote the accumulation of compatible osmolytes such as soluble sugars and proline [53,54] under salt stress conditions. Moreover, the previous report discussed the active response of N. tangutorum P5CS to salt stress [55], which helps to explain the proline accumulation in N. tangutorum we observed in our study. Therefore, we hypothesized that NtCIPK11 functions through proline accumulation to protect plants under high salinity conditions. As a result, the key genes regulating proline levels were found to be differentially expressed in NtCIPK11-overexpressing plants and WT plants under salt stress. The genes modulating proline synthesis were upregulated (Fig. 6a-c); in contrast, gene regulating , and P5CR (c). d Expression level of the proline catabolism gene ProDH1. e Proline content in one-week-old WT and transgenic seedlings germination on the medium containing 0 mM NaCl or 100 mM NaCl. The data represents means ± SD of three replicates and the statistical analyses were performed with one-way ANOVA test, '*' p < 0.05, '**' p < 0.01 proline catabolism was downregulated (Fig. 6d), which would improve proline content in theory. The increased proline content further supported the differential expression of genes related to proline metabolism under salt treatment.
The importance of this study is shown by the finding that the transcription levels of specific genes regulating proline content in transgenic plants were significantly different from those in WT plants when NaCl was applied. However, we found that P5CS1 and P5CS2 significantly upregulated under salt stress were downregulated in transgenic plant OX-1 and upregulated in OX-2 under the normal condition (without NaCl treatment). Moreover, we also observed a less proline content of OX-1 than that of WT plants under control condition, that further supported the low expression of genes involved in proline synthesis in transgenic seedlings. The possible reason for this result was supposed to be related with the identity of CBL-CIPK complex. CBL sensor proteins need to bind Ca 2+ and activate the downstream targets CIPKs, thereby regulating the specific biochemical processes [56]. In other words, a relatively low On the contrary, abiotic stresses inducing a high concentration of Ca 2+ signal requires a high activity of CBL-CIPK signal network to activate downstream target genes for responses to stresses. The particular genes involved in abiotic stresses would function as the regulatory element in Ca 2+ transduction. In our study, the overexpression of NtCIPK11 regulated the differential expression of genes related to proline metabolism under salt stress. Moreover, decrease of ROS in the transgenic Arabidopsis further explained the positive function of CIPK11 from N. tangutorum on plants dealing with salt stress (Fig. S1). Our investigation shared partial points with the research of CIPKs from rice [43]. Ectopic expression of rice OsCIPK03 and OsCIPK12 led to a significant accumulation of proline under cold and drought stress conditions [43]. Thus, we suggest that our halophyte-derived NtCIPK11 enhances salt tolerance by inducing gene expression to enhance the proline accumulation in plants exposed to salt stress. Proline has been proposed to act as a compatible osmolyte [57,58], a reactive oxygen species scavenger [59], and a protectant of macromolecules such as enzymes and cellular structures [42,60], thus affecting plant adaptability to stress. Salt-induced NtCIPK11 regulation of genes associated with proline accumulation led to our speculation about the capacity of this gene to cope with drought stress. Surprisingly, the drought conditions simulated by 100 mM, 150 mM, and 200 mM mannitol application did not affect seed germination but limited seedling development (Fig. 7c). Correspondingly, proline synthetases were not upregulated in transgenic plants (Fig. 9b-d). However, the transcript level of the enzyme leading to proline degradation was higher in the WT seedlings than in the NtCIPK11-overexpressing seedlings (Fig. 9a). These results seem to indicate that NtCIPK11 has no dramatic effect on the proline level under drought stress. The possible reason for this outcome might be attributed to the different strategies of plants in adapting to salinity and drought. Although stress regulators have multiple functions, they have a low probability of showing the same capacity in response to different stresses. For example, Arabidopsis CIPK11 has been reported as a negative regulator of the drought The data represent the means ± SD; three technical and biological replicates were conducted; one-way ANOVA test was used for the p-value calculations, '*' p < 0.05, '**' p < 0.01, '***' p < 0.001 stress response by controlling the expression of the transcription factor Di19-3, a gene reported to be involved in the abiotic stress response [61]. NtCIPK11 overexpression led to the advanced development of seedlings after germination under drought stress caused by mannitol treatment (Fig. 8). Thus, we suggest that NtCIPK11 can promote drought tolerance but the mechanism may not involve proline accumulation under drought conditions.

Conclusion
In summary, we identified a stress-responsive gene NtCIPK11 from halophyte N. tangutorum. Ectopic expression of NtCIPK11 promoted the seed germination and seedling growth of Arabidopsis upon salt treatment. Moreover, the overexpression of NtCIPK11 caused a different transcription of genes related to proline metabolism and a relatively high proline accumulation in the seedlings treated with NaCl. Although the transcription patterns of the proline synthesis genes were differentially regulated under mannitol treatment compared to the genes in seedlings under salt stress, NtCIPK11 overexpression still induced a higher tolerance of the seedlings to drought stress. Hence, it is concluded that NtCIPK11 is a novel halophyte gene that plays positive roles in plant responses to salt and drought. The novel halophyte-derived gene identified may be used as a candidate gene in molecular breeding of commercial plants to obtain better stress tolerance.

Plant culture and treatment N. tangutorum
Seeds of N. tangutorum were harvested from the Experimental Center for Desert Forestry of Chinese Academy of Forestry at Dengkou Inner Mongolia in China. The research institution providing the seeds of N. tangutorum has a cooperative relationship with Nanjing Forestry University. The author Jingbo Zhang, a professional researcher on genus Nitraria, undertook the formal identification of N. tangutorum, who harvested the seeds totally complying with the institutional guidelines. We were unfortunately unable to find a voucher specimen of N. tangutorum deposited in any publicly available herbarium.
For successful germination, N. tangutorum seeds were kept in sand with a relative water content of 7% at 4°C for eight weeks. The seeds germinated in pots containing a mixture of soil and sand (1:1) in a chamber with 55% to 60% relative humidity, 26°C~28°C, and a 16-h light/8-h dark light regime. Two-month-old seedlings has been used for biochemical parameter assays and qPCR analysis.

Arabidopsis thaliana
The seeds of Arabidopsis thaliana (Columbia ecotype) wild type used in this study were kindly provided by Prof. Thomas Laux (Signalling Research Centres BIOSS and CIBSS, Faculty of Biology, University of Freiburg, Germany). Transgenic Arabidopsis plants were obtained using the floral dip method [62]. To generate seeds for phenotypic analyses, NtCIPK11 overexpressing plants were screened until homozygous seeds were obtained. Each experiment was performed in triplicate, with at least 120 seeds of each genotype. Arabidopsis seeds were surface sterilized and sown on ½ MS containing different concentrations of NaCl or mannitol, and then cultured in a growth chamber at 23°C using a 16-h-light/8-hdark cycle. Four days post-germination, the germination rate and seedling development of the plants were observed; subsequently, the growth state was analyzed 20 days post-germination. Arabidopsis seedlings at 20 days post germination were immediately frozen in liquid nitrogen and stored at − 80°C for qPCR detection.

Biochemical parameter assay
Two-month-old N. tangutorum seedlings were watered with 400 mM NaCl for morphological observation and biochemical parameter assays. The analyses of enzyme activity, proline content and MDA content were conducted following the methods of Janmohammadi et al.  [64]. One-week-old Arabidopsis seedlings were harvested for the measurement of proline content. Three technical and biological replicates were performed for each biochemical parameter test. One-way ANOVA test combined with LSD multiple comparisons was used for statistical analysis.

NtCIPK11 gene cloning
Total RNA was extracted from the leaves of N. tangutorum seedlings using a total RNA purification kit (Norgen, Thorold, ON, Canada), followed by removal of genomic DNA contaminant using DNase I (TaKaRa, Japan). Ultraviolet spectrophotometry was used to quantify the total RNA concentration and gel electrophoresis was used to evaluate its integrity. Double-stranded cDNA was synthesized by reverse transcriptase according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). Degenerate primers for NtCIPK11 fragment isolation were designed based on the poplar CIPK homeodomain. Primers used for NtCIPK11 fragment isolation are listed in Supplementary  Table 1. The full length of NtCIPK11 sequence was cloned by 5′ and 3′ RACE using the primers listed in Supplementary Table 2, as indicated in the SMARTerTM RACE cDNA amplification kit user manual (BD Bioscience Clontech, USA). The complete coding sequence of NtCIPK11 was obtained from cDNA based on the assembled RACE sequences, using the primers listed in Supplementary Table 3.

NtCIPK11 sequence analysis
NtCIPK11 orthologues from other species were searched with NCBI BLASTP. The molecular mass of NtCIPK11 was predicted by the online software package ExPASy (https://web.expasy.org/cgi-bin/protparam/). Multiple sequence alignments of NtCIPK11 and its orthologs were performed using DNAMAN 6.0. The feature motifs and domains in NtCIPK11 were predicted using Inter-ProScan online software (http://www.ebi.ac.uk/ InterProScan/). The accession numbers of the sequences and species used for the alignment are listed in Supplementary Table 4. Hydrophobic analysis and transmembrane domain prediction of the NtCIPK11 protein were conducted using the ProtScale tool (http://ca.expasy.org/ tools/protscale.html) and the TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Phylogenetic analysis was performed with amino acid sequences of NtCIPK11 and 26 CIPKs from Arabidopsis using Mega 6 by the NJ method with 1000 bootstrap replications and the JTT model. The accession numbers of the sequences used for the phylogenetic tree are listed in Supplementary Table 5.

Quantitative real-time PCR analyses
To confirm the response of NtCIPK11 to salt and drought stress, qPCR was performed using total RNA from the root, stem and leaf tissues of two-month-old N.tangutorum seedlings treated with 500 mM NaCl or 200 mM mannitol for 2 h. NtCIPK11-overexpressing and WT Arabidopsis germinated on medium with 100 mM NaCl and 200 mM mannitol were used for the transcription analysis of proline-related genes. Total RNA was reverse transcribed as mentioned previously. qPCR was carried out using a SYBR-Green PCR Master Mix on a LightCycler®480 real-time PCR detection system (Roche, Basel, Switzerland) according the manufacturer's instructions. The expression levels of the target genes were normalized by the transcription of the housekeeping gene actin in Nitraria [65] and UBQ10 in Arabidopsis [66]. Three technical replicates for each experiment was performed in three biological replicates. The primers used for the qPCR analyses were designed with Primer3 http://frodo.wi.mit.edu/). The sequences of the specific primers for each gene are listed in Supplementary  Table 6.

Detection of H 2 O 2 accumulation
One-week-old Arabidopsis seedlings have been used for H 2 O 2 accumulation analysis. Eighteen plants from each line (WT and two transgenic lines) were immersed with DAB (Sigma-Aldrich, catalog number: D12384) staining solution for the detection of H 2 O 2 [67]. Morphology of seedlings staining for four hours was imaged using a Leica M165FC microscope.