2.1 Plant materials and growth conditions

Seeds of sweet sorghum, Sorghum bicolor (L.) Moench "KFJT-CK," were provided by the Institute of Modern Physics, Chinese Academy of Sciences (IMPCAS). Dry seeds of equal size without mold or lesions were selected. The carbon ions used for mutagenesis were generated by the Heavy Ion Research Facility in Lanzhou (HIRFL), IMPCAS. The energy of ¹²C⁶⁺-ions used was 100 MeV/u, with a ¹²C⁶⁺-ion irradiation dose rate of approximately 20 Gy/min. The dry seeds of sweet sorghum were irradiated by ¹²C⁶⁺-ion at doses of 0, 20, 40, 60, and 80 Gy. The KF1210-3 and KF1210-4 mutants were screened using an ¹²C⁶⁺-ion irradiation dose of 80 Gy provided by the accelerators at HIRFL and bred for further experiments.

Seeds of KF1210-3, KF1210-4, and KFJT-CK were grown in growth chambers at 22℃ and 70% relative humidity under continuous illumination at 5000 lux for 7 days. Three replicates of five seeds each were utilized for each treatment. NaCl concentrations of 0, 50, 100, 150, 200, 250, and 300 mM were used as salinity treatments. After 7 days, plant heights were measured. The plantlets were rinsed in deionized water to remove surface salts, and then the fresh tissue weight was measured. Thereafter, tissues were immediately dried at 108℃ for 1 h and 65℃ for 24 h, and the dry weight was measured.

2.2 Catalase analysis

Following the treatments with 0, 50, 100, 150, 200, 250, and 300 mM NaCl, root samples of sweet sorghum (100 mg each) were ground into a fine powder using liquid nitrogen in a pre-chilled mortar and pestle. Further grinding was performed in a solution of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 2% (w/v) polyvinyl polypyrrolidone (PVPP) for catalase (CAT) assays. The homogenates were centrifuged twice at 14000 × g for 15 min at 4℃. The final supernatants were used immediately for enzyme activity assays or stored at -20℃ for later use. Total CAT activity was determined using CAT kits (Keming Biotech Co., Ltd, Suzhou, China)

2.3 Real-time quantitative PCR analysis

The roots of sweet sorghum plants treated with 150 mM NaCl were harvested at 0, 2, 4, 8, 12, and 24 h. The expression levels of NHX, AKT1, and SOS1 genes were analyzed by real-time quantitative PCR. The roots were treated as follows. (1) Total RNA was extracted using an RNAprep pure plant kit (TianGen, Biotech Co., Ltd, Beijing, China). (2) RNA samples were quantified according to the absorbance at 260 nm, with the purity being evaluated according to the 260/280 nm ratio. (3) First-strand cDNA was synthesized using MMLV-reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). (4) The reverse-transcribed cDNA samples were used for real-time quantitative PCR, which was performed using a thermal cycler (QIAGEN, Dusseldorf, Germany). A specific fragment (158 bp) of SOS1 was amplified using the primer pair P1 and P2; the AKT1-specific fragment (111 bp) was amplified using primers P3 and P4; and the NHX-specific fragment (140 bp) was amplified using primers P5 and P6 (primer sequences are shown in Table 1). Experiments were performed at least three times. The Actin gene was also amplified for the purpose of RNA normalization. The specific primers for Actin (A1 and A2; Table 1) amplified a 170-bp fragment. SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA) was used for 20 μL PCR reactions, with amplification being performed under the following conditions: 95℃ for 30 s, followed by 40 cycles of 95℃ for 5 s, and 60℃ for 34 s. Each sample was amplified three times. The relative expression levels (RELs) of all the samples were calculated and analyzed (ABI PRISM 7500 sequence detection system). The REL of each sample was calculated using the following equation: REL = 2^-ddCt, where the Ct value of target genes and Actin in different samples were obtained after the quantitative real-time PCR reaction. In brief, the normalized Actin Ct value was subtracted from the Ct value of the gene of interest (target gene) to give the dCt value of the sample. The dCt value of the calibrator (the sample with the 0 h dCt value in our experiment) was subtracted from that of every other sample to yield the respective ddCt values. The two to the power -ddCt (2^-ddCt) values for each sample were used as the relative expression levels [31].

2.4 Statistical analysis

The results are presented as means with standard error of the means. Data analysis was performed with one-way analysis of variance (ANOVA) using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was applied to distinguish the difference between means at a significance level of P < 0.05.

3.1 Biomass production of KFJT-CK, KF1210-3, and KF1210-4

The effects of salt on the biomass production of sweet sorghum are shown in Fig. 1. Under control conditions, the KF1210-3 mutant had the highest plant height, fresh weight, and dry weight. These results revealed that the plant height and biomass of sweet sorghum can be affected by carbon beam irradiation. Moreover, the plant heights and fresh weights of KFJT-CK, KF1210-3, and KF1210-4 were reduced with an increase in salt concentrations, whereas the dry weights showed little variation with an increase in salinity. Among the KFJT-CK, KF1210-3, and KF1210-4 cultivars subjected to increasing salt concentrations in culture rooms, the KF1210-3 cultivar was characterized by the strongest resistance to salinity. These results indicate that carbon ion irradiation significantly altered the growth of sweet sorghum under both normal and saline conditions.

Fig. 1.Full-screen View

(Color online) Morphology (a), plant height (b), fresh weight (c), and dry weight (d) of 1-week-old sweet sorghum KFJT-CK, KF1210-3, and KF1210-4 treated with NaCl solutions of different concentrations for 7 days. Experiments were performed at least three times. Values are the mean ± SE (n = 3) and bars indicate SE.

3.2 Catalase activity

The effects of NaCl on the CAT activities of KFJT-CK, KF1210-3, and KF1210-4 are shown in Fig. 2. The CAT activity of the sweet sorghums was observed to increase with an increase in the concentrations of NaCl from 0 to 150 mM (P < 0.05). At a concentration of 150 mM NaCl, the CAT activity in KFJT-CK was the highest. However, whereas at a concentrations of 200 mM NaCl, the CAT activity in KF1210-3 continued to increase, that in KFJT-CK and KF1210-4 showed a decrease. An increase in the antioxidant activities of sweet sorghum could be a response to cellular damage induced by salinity, and may thus represent a defense mechanism against the generation of NaCl-induced O₂^.- and H₂ O₂. CAT can eliminate H₂ O₂ via the direct formation of H₂ O and O₂ [32]. Our results indicate that among the three sorghum cultivars examined, KF1210-3 has the highest dismutation capacity in response to an increase in salt concentration. It is notable that the CAT is a relatively effective antioxidant enzyme in preventing cellular damage, which is related to an enhanced tolerance to salt stress [33]. Accordingly, CAT analysis represents an effective approach for selecting salt-tolerant cultivars of sweet sorghum generated by ¹²C⁶⁺-ion irradiation.

Fig. 2.Full-screen View

(Color online) Catalase (CAT) activities of sorghum cultivars KFJT-CK, KFJT-1210-3, and KFJT-1210-4 under stress induced by salt different concentrations. Experiments were performed at least three times. Values are the mean ± SE (n = 3) and bars indicate the SE. Means with the same letter for each cultivar are not significantly different according to Duncan’s test at P < 0.05.

3.3 Expressions of NHX, AKT1 and SOS1 in the roots of sweet sorghum under saline conditions

NHX, which is located in the tonoplast, plays a critical role in minimizing the concentration of cytoplasmic Na⁺ through sequestration of Na⁺ into the vacuoles of many plants [21]. Previous results have shown that overexpression of NHX enhances salt tolerance by increasing the accumulation of Na⁺ in plants [23]. However, none of the previous studies have addressed the role of the NHX protein in relation to the regulation of Na⁺ concentration. In the present study, the expression levels of NHX in the root of KFJT-CK, KF1210-3, and KF1210-4 sorghum were determined by real-time quantitative PCR at 0, 2, 4, 8, 12, and 24 h after the samples had been treated with 150 mM NaCl. At 8 h after treatment, the expression level of NHX in the roots KF1210-4 and KF1210-3 was 7% and 73% higher, respectively, than in the roots of the control (KFJT-CK) (Fig. 3a). In response to treatment with 150 mM NaCl, the expression of NHX in the roots of KF1210-3 was significantly higher than that in KFJT-CK roots. These results indicate that expression levels of NHX in the roots of the mutant KF1210-3 were upregulated under saline conditions. The experimental results also demonstrated that NHX is crucial for the control of cytoplasmic Na⁺homeostasis and maintenance of salt accumulation in the vacuoles of sweet sorghum under salt stress.

Fig. 3.Full-screen View

(Color online) Time courses of NHK, AKT1, and SOS1 expressions in the roots of sweet sorghum treated with 150 mM NaCl. Experiments were performed at least three times. Values are the mean ± SE (n = 3) and bars indicate the SE.

It is well known that AKT1 plays a crucial role in controlling the homeostasis between Na⁺ and K⁺in plants. AKT1 can unload Na⁺ from the xylem to the xylem parenchyma cells (XPCs) [34]. In the present study, we demonstrated that 150 mM NaCl significantly decreased the expression level of AKT1 in the roots of KFJT-CK, KF1210-3, and KF1210-4 (Fig. 3b). In this regard, Zhang et al. found that a reduction of AKT1 in roots could limit the entry of Na⁺ into plants [5,6], which implies that the main role of AKT1 is the mediation of K⁺ and Na⁺ under salt stress.

SOS1 has been proposed to be an important transporter for the spatial distribution of Na⁺, and could therefore be a plasma membrane Na⁺/H⁺ antiporter [35]. It has been suggested that SOS1 plays a necessary role in protecting K⁺ uptake mediated by AKT1. In the present study, we found that NaCl at a concentration of 150 mM significantly increased the expression level of SOS1 by 423% in KF1210-3 but only by 338% in the roots of KFJT-CK at 12 h after treatment (Fig. 3c). These results indicate that SOS1 may trigger a significant regulation in the roots of sweet sorghum under saline conditions. SOS1 could load Na⁺ into the xylem, thereby controlling delivery to the shoot and storage in leaf mesophyll cells. We found that downregulated expression of AKT1 in roots may also be involved in a decrease in K⁺ accumulation in root cells. Furthermore, elevated cytoplasmic Na⁺ levels could influence K⁺ uptake under conditions of salt stress.

Under salt stress, both AKT1 and SOS1 play important roles in the unloading and exclusion of Na⁺ to maintain a low level of Na⁺in plants. AKT1 retrieves Na⁺ from the xylem under conditions where absorption and accumulation of Na⁺ could induce damage in plants. Furthermore, NHX could minimize the concentration of cytoplasmic Na⁺ by the sequestration of Na⁺ in vacuoles, thereby enhancing the salt tolerance of sweet sorghum. This process would not only contribute to limiting the rapid accumulation of Na⁺ in the root but also alleviate the damage caused by high concentrations of Na⁺. On the basis of our results, we propose a model in which the cooperation of NHX, AKT1, and SOS1 facilitates Na⁺homeostasisin sweet sorghum under conditions of saline stress.