Plant material and growth conditions: the Arabidopsis species used were Colombia ecotype. Seed culture conditions were 22°C, 16 h/d light, 8 h/d dark cycle, 60% relative humidity, and 2200 L× light intensity, and plants were grown in pots at 22°C, 12 h/d light, 12 h/d dark cycle, and 60% relative air humidity.

Genetic transformation of the sugarcane ScCaM gene into A. thaliana and screening of homozygous plants: the eukaryotic expression vector pBWA(V)HS-35S-ScCaM was constructed by introducing the ScCaM coding region into the overexpression vector pBWA(V)HS-35S using the in-fusion cloning method. The recombinant plasmid was validated and then transformed into Agrobacterium tumefaciens GV3101. After Arabidopsis thaliana had bolted, Agrobacterium containing the pBWA(V)HS-35S-ScCaM recombinant plasmid was titrated and used to infect wild-type Col-0 flowers. Next, the flowers were stored for 24 h in the dark, followed by a long period of irradiation (15 h light, 10,000–12,000 L×, 50%–60% relative humidity, and 21°C–24°C temperature) until pod maturity. T₃ generation homozygous strains of transgenic ScCaM Arabidopsis were finally obtained using hygromycin B screening and polymerase chain reaction (PCR) amplification assays.

Screening culture was performed by using Murashige and Skoog (MS) medium (Murashige and Skoog 4.4 g/L, agar 8 g/L, adjusted to pH5.8) containing 100 g/L of hygromycin B, and three seedlings were transplanted to one planting pot when they grew to the 3–4-leaf stage. On reaching the 5–8-leaf stage, RNA from plant leaf was extracted to detect whether the transgenic plants were successfully transformed to overexpress ScCaM. HYG-F/R. For performing RT-PCR, pBWA(V)HS-35S-ScCaM-F/R were used as primers, pBWA(V)HS-35S-ScCaM plasmid was used as a positive control, while water was used as a blank control. cDNA obtained following RNA reverse transcription was used as a template for PCR amplification and electrophoresis. The PCR procedure was as follows: pre-denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, denaturation at 55°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 10 min, and holding at 4°C. The primer sequences and RT-PCR system are shown in Suppl. Tables S1 and S2, respectively.

Seeds of T₃ generation transgenic overexpressing Arabidopsis thaliana and wild-type Col-0 Arabidopsis thaliana were sterilized using 2% sodium hypochlorite, and the seeds were grown on MS plates containing 0, 100, or 150 mM sodium chloride (NaCl) after sterilization and incubated continuously for 12 d, during which germination potential was observed, and germination rate was determined. A line graph of germination rate was plotted using Origin 8.0 software. Arabidopsis plants were grown for about 4 weeks, and those with uniform growth were selected and subjected to salinity stress. Specifically, 15 mL of 300 mM NaCl was poured on the Arabidopsis plants once every two days for 12 d. Wild-type Col-0 plants were used as controls, their phenotypes were observed, and photographs were acquired. At 12 d of treatment, Arabidopsis leaf RNA was extracted using Trizol and reversed transcribed into cDNA using the Novozymes HiScript® Q RT SuperMix for qPCR (+gDNA wiper) kit for further expression analysis of resistance-related genes.

Sterilized ScCaM-overexpressing T₃ generation Arabidopsis and wild-type Col-0 Arabidopsis seeds were spotted on MS medium plates containing 0, 200, or 300 mM mannitol and incubated continuously for 12 d. Germination potential was observed, and the germination rate was determined. A line graph of germination rate was plotted using Origin 8.0 software. When the Arabidopsis plants were grown for about 4 weeks, the plants with uniform growth were selected for drought stress treatment. Specifically, the conditions were natural drought (no watering) for 11 d and rehydration for 5 and 11 d, and wild-type Col-0 plants were used as the controls, during which their phenotypes were observed and photographs were acquired. At 11 d of treatment, Arabidopsis leaf RNA was extracted and reverse transcribed into cDNA for further expression analysis of resistance-related genes.

Quantitative reverse transcription-PCR (RT-qPCR) was used to analyze the expression of some Arabidopsis stress-related genes under salinity and drought stress, including genes related to reactive oxygen species (ROS) scavenging (AtSOD, AtCAT, and AtAPX) (Yang et al., 2015), ABA response (AtNCED3, AtP5CS, and AtRD29A) (Qiu et al., 2020), salinity stress (AtSOS3), and drought stress (AtCDPK1) (Yang et al., 2015), with Atactin2 as an internal reference gene (Zhu, 2002). Primer sequences are listed in Suppl. Table S1. The qRT-PCR steps were as follows: 50°C, 2 min; 95°C, 2 min; 40 cycles of 95°C, 10 min; 95°C, 15 s; 60°C, 1 min. Relative gene expression was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Significance (p < 0.05) was calculated using one-way ANOVA. Duncan’s new multiple range test was calculated using DPS 9.50 software, and data are expressed as the mean ± standard error (SE). Histograms were constructed using Origin 8.0 software.

We constructed the prokaryotic expression vector ScCaM-pGEX-4T-1 and determined the optimal conditions for ScCaM expression in E. coli cells as 16°C, 20 h, and induced by 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) (Su et al., 2020b). In this study, we first tested whether the optimal temperature and IPTG concentration could induce the expression of ScCaM at the protein level. Following the successful induction of ScCaM-pGEX-4T-1, recombinant E. coli cells were subjected to salinity and drought stress experiments to analyze the tolerance of ScCaM protein to salinity and drought in E. coli cells. The procedure was as follows: pGEX-4T-1 and pGEX-4T-1-ScCaM were transformed in prokaryotic expression strain E. coli BL21 (DE3), and single colonies of recombinant bacteria were cultured in ampicillin (70 µg/mL)-containing LB broth. The cells were incubated at 37°C with shaking at 200 rpm until OD₆₀₀ = 0.6. Subsequently, 0.5 mM IPTG (to analyze the expression of TCGT transcript) was added, and the cells were incubated at 16°C in a 200 rpm shaking incubator for induction. The bacterial suspensions were collected at intervals of 0, 2, 8, and 20 h. The bacteria were then collected via centrifugation at 10,000 g for 10 min at 26°C, mixed with 6× protein loading buffer, boiled at 100°C for 5 min, and centrifuged again at 10,000 g for 5 min at room temperature. Next, 10 µL of supernatant was resolved by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, followed by coomassie brilliant blue staining and photograph acquisition. The above bacterial broth collected after 20 h of IPTG induction was diluted to the same concentration using LB broth, i.e., OD₆₀₀ = 0.7, following which the broth was diluted to 10⁻³ and 10⁻⁴ and used as a stock solution. The diluted broth was spotted onto LB agar plates containing 70 µg/mL ampicillin, incubated overnight at 37°C, and photographed for retention.

As shown in Suppl. Fig. S1, T₃ generation ScCaM-overexpressing Arabidopsis seeds were essentially homozygous by screening cultures on MS medium plates exhibiting hygromycin resistance.

Fig. 1 shows the amplification products of HYG and ScCaM genes in Arabidopsis plants. As shown in Figs. 1A and 1C, a single band of approximately 750 bp in size was amplified when the overexpression plasmid was used as a template and generally matched the length of the vector HYG tag. A single band consistent with the overexpression plasmid was identified in samples 4 to 9, and no band was identified in samples 1 to 3. As shown in Figs. 1B and 1D, a single band was amplified when the overexpression plasmid was used as a template, which was approximately 450 bp in size and generally matched the length of the ScCaM open reading frame. The results indicate that pBWA(V)HS-35S-ScCaM was successfully expressed in the Arabidopsis transgenic strain, and the selected Arabidopsis plants could be used for the next step of analysis for salinity and drought tolerance.

Selected transgenic ScCaM T₃ generation lines, T₃-1 and T₃-5, and wild-type Col-0 Arabidopsis seeds were grown on MS blank medium plates and MS medium plates containing 100 mM NaCl, 150 mM NaCl, 200 mM mannitol, and 300 mM mannitol, respectively. The germination rate and growth after 12 days of continuous incubation are shown in Fig. 2, according to which both transgenic lines T₃-1 and T₃-5 were able to germinate on MS medium plates. The germination rate of Col-0 seeds was 95.24%, and all germinated plants were green, suggesting that they were able to grow normally (Figs. 2A and 2B). The germination rates differed in T₃-1, T₃-5, and wild-type Col-0 seeds on MS medium plates containing different concentrations of NaCl and mannitol, but the differences were generally small (Figs. 2C–2F). Thus, overexpression of the ScCaM gene did not affect the tolerance of transgenic Arabidopsis to salinity and drought stresses during seed germination.

To investigate the salinity and drought tolerance of transgenic ScCaM Arabidopsis plants, we treated the plants with 300 mM NaCl solution in water and performed natural drought treatments. As shown in Fig. 3A, there was no significant difference in the growth of Col-0, T₃-1, and T₃-5 Arabidopsis plants before salinity stress, and after 7 days and 12 days of salinity stress, chlorosis was observed in the leaves of Col-0, T₃-1, and T₃-5 plants; however, it was more severe in the leaves of T₃-1 and T₃-5 plants than in the leaves of Col-0 plants. As shown in Fig. 3B, there was no significant difference in the growth of Col-0, T₃-1, and T₃-5 Arabidopsis plants before drought treatment, and after 11 days of drought treatment, chlorosis was observed in the leaves of Col-0 and ScCaM-transformed T₃-1 and T₃-5 plants; however, chlorosis was more severe in the leaves of T₃-1 and T₃-5 plants than in the leaves of Col-0 plants. Thus, the transgenic ScCaM Arabidopsis plants were less tolerant to salinity and drought stress than the Col-0 plants.

We analyzed the expression of relevant stress resistance genes to further clarify the molecular mechanisms underlying drought and salinity tolerance in ScCaM overexpressing transgenic Arabidopsis.

As shown in Fig. 4A, the expression of AtNCED3, AtP5CS, and AtRD29A was significantly downregulated in ScCaM overexpressing transgenic Arabidopsis T₃-1 and T₃-5 lines after 12 d of salinity stress, which was lower than that of the wild type, respectively. However, the expression of the ROS scavenger gene, AtSOD, and salinity stress response-related gene, AtSOS3, was significantly upregulated in the T₃-1 line, which was 4.28-and 2.04-fold higher than that of the control, respectively. Interestingly, the expression of this gene was not significantly different in the T₃-5 line compared with the control, and the expression of AtCAT, AtAPX, and AtCDPK1 was not significantly different in Col-0, T₃-1, and T₃-5 plants.

As shown in Fig. 4B, after 12 d of drought stress, the expression of AtP5CS and AtRD29A was significantly downregulated in both T₃-1 and T₃-5 plants, which was lower than that of the control, respectively. The ROS scavenging enzyme-coding genes, AtSOD and AtCAT, were significantly upregulated in both the T₃-1 and T₃-5 lines, which were higher than that of the wild type, respectively. The ABA pathway-related gene, AtNCED3, was significantly upregulated in the T₃-1 line, which was 5.11-fold higher than that of the wild type, and the expression of this gene was not significantly different in the T₃-5 line compared with the wild type. The expression of AtAPX, AtSOS3, and AtCDPK1 was not significantly different in Col-0, T₃-1, and T₃ -5 plants.

Overall, the expression of AtNCED3, AtP5CS, and AtRD29A was significantly downregulated, and the expression of AtSOD and AtSOS3 was significantly upregulated in the transgenic lines under salinity stress. However, the expression of AtP5CS and AtRD29A was significantly downregulated, and the expression of AtSOD, AtCAT, and AtNCED3 was significantly upregulated in the transgenic lines under drought stress. Notably, the expression of AtP5CS and AtRD29A was suppressed in both T₃-1 and T₃-5 lines under salinity and drought stresses, and it is speculated that ScCaM attenuated the tolerance of the transgenic plants to salinity and drought mainly by reducing the expression of AtP5CS and AtRD29A genes.

To find the role of the ScCaM gene in salinity and drought resistance in prokaryotes, ScCaM protein expression was induced as previously described (Liu et al., 2021), and stress experiments in LB plates with different concentrations of NaCl and mannitol were performed. As shown in Fig. 5, the accumulation of recombinant protein was observed after 8 h and 20 h induction by 0.5 mM IPTG. Based on EXPASY ProtParam, the estimated molecular weight of ScCaM protein was 16.83 kDa, and the molecular weight of GST protein was 26 kDa; the target protein of GST-ScCaM of about 43 kDa molecular weight was induced and was used for further plate stress experiments.

Plate stress experiments for the prokaryotic expression of the recombinant bacterium BL21 with pGEX-4T-1-ScCaM are shown in Fig. 6. According to Fig. 6A, after 12 h of incubation at 37°C, there was no significant difference in the growth of pGEX-4T-1-ScCaM and pGEX-4T-1 cells on LB plates containing 70 μg/mL ampicillin. Besides, pGEX-4T-1-ScCaM and pGEX-4T-1 cells did not differ in growth on 100 mM NaCl and 500 mM NaCl stress plates, i.e., both could grow at 100 mM NaCl with no significant difference in growth, whereas neither could grow on 500 mM NaCl stress plates. However, the growth of both pGEX-4T-1 and pGEX-4T-1-ScCaM cells was inhibited on 300 mM NaCl stress plates, with the growth status of pGEX-4T-1-ScCaM being significantly weaker than that of pGEX-4T-1. It is thus hypothesized that ScCaM attenuated the tolerance of E. coli to NaCl stress.

Fig. 6B showed that the growth of pGEX-4T-1-ScCaM and pGEX-4T-1 cells on LB plates with 70 μg/mL ampicillin did not differ much after 12 h of incubation at 37°C. Non-recombinant pGEX-4T-1 could grow on 500, 750, and 1000 mM mannitol stress plates. In contrast, pGEX-4T-1-ScCaM cells only grew on 500 mM mannitol stress plates, and there was no colony on 750 mM mannitol or 1000 mM mannitol stress plates; besides, the growth of pGEX-4T-1-ScCaM cells on 500 mM mannitol stress plates was weaker than that of pGEX-4T-1 cells. Thus, it was hypothesized that ScCaM also attenuated the tolerance of E. coli to mannitol stress.