2.1. N. tangutorum physiologically responded 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 (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 top 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. POD and SOD activity increased significantly at a 400 mM salinity level on the first day of treatment (Fig. 1I and J). However, CAT did not positively respond to salt treatment in our experiment (Fig. 1K). However, free proline, generally thought to have a positive role in plants responses to environmental stresses, such as drought and salinity [37, 38], was observed to significantly accumulate 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 taken together suggested that N. tangutorum significantly increased the activity of some antioxidant enzymes and increased to proline content to protect the cell membrane from being drastically affected by salinity stress under our experimental conditions.
2.2. NtCIPK11 identification and bioinformatics analysis
The large number of genes showing responses to stresses have been 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).
2.3. NtCIPK11 in N. tangutorum positively responds 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 the roots, 17-fold in the stems and up to 118-fold in the leaves compared to the expression level in the untreated roots. This finding shows that NtCIPK11 transcripts accumulate preferentially in leaf tissues after salt treatment (Fig. 4A).
2.4. 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 and a higher number of leaves and roots than the WT plants, with the difference particularly large between the plants treated with 150 mM NaCl-treated medium (Fig. 5). Therefore, we concluded that NtCIPK11 overexpression significantly promoted the seed germination and induced the salt tolerance of Arabidopsis.
2.5. Overexpression of NtCIPK11 altered the transcription patterns of genes involved in proline metabolism
In plants, proline has been reported to accumulate after exposure to various stresses including salt, drought and cold [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). These results showed that NtCIPK11 overexpression affected the expression of proline-related genes.
2.6. 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 hours 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.
2.7. 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 the 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 post-germination (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.
2.8. Overexpression of NtCIPK11 promoted Arabidopsis root growth under drought stress
To further study the function of NtCIPK11 during drought treatment, we observed plant growth on medium containing different concentrations of mannitol for 20 days. 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 NtCIPK11-overexpressing 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 positive effect on proline accumulation. Nevertheless, the proline synthesis genes exhibited a different expression pattern compared to the genes in Arabidopsis under salt treatment in Arabidopsis (Fig. 9B-D). These results suggest that NtCIPK11 is involved in drought and salt stress signaling by influencing the expression of proline metabolism regulators but to different degrees.