Effects of salt stress on seed germination and respiratory metabolism in different Flueggea suffruticosa genotypes

Plant materials

The mature fruits of F. suffruticosa from diverse genotypes were harvested from 30 provenances in the northeastern Hebei Province of China (Fig. 1). Detailed information on the provenance data from the original seed collection sites is shown in Table 1. Within several days of harvest, the seeds were abscised from the naturally dried fruit and collected in order of the location data for the salt tolerance screening test. The seeds of two F. suffruticosa genotypes (highly salt-sensitive in Jiaoshan and Shanhaiguan (P2) and highly salt-tolerant in Fengshan and Fengning (P27)) were chosen for further tests according to the results of salt tolerance of F. suffruticosa. All the chemicals used in this study were of analytical grade.

Experimental design

A salt tolerance screening test was established to ascertain the salt-tolerant ability of F. suffruticosa seeds according to the method described by Wu et al. (2019) and Fu et al. (2020), with some modifications. Seeds of different F. suffruticosa genotypes were sterilized with 1% sodium hypochlorite for 15 min, rinsed in distilled water five times, and placed in a Petri dish (nine cm diameter, two layers of filter paper on the bottom) containing 12 mL of NaCl solution with 0 (CK) and 200 mM, and germinated under dark conditions in a constant temperature incubator (Dongnan, Ningbo, China) at 25 ± 0.5 °C for 12 days. Each treatment contained three independent biological replicates. Seeds were considered to germinate when the seed coat was broken and the radicle was visible (Chen et al., 2020).

To evaluate the salt tolerance of F. suffruticosa at the germination stage, the germination rate, relative germination rate, germination potential, relative germination potential, germination index, relative germination index, relative salt harm rate, fresh weight, relative fresh weight, root length and relative root length were determined at 12 days after salt stress treatment (DAT). There were calculated using the formulae below (Deng et al., 2017; Sikder et al., 2020; Xu et al., 2021).

Germination rate = total germinated seeds/total seeds × 100%.

Relative germination rate = germination rate of salt treatment/germination rate of control × 100%.

Germination potential = number of germinated seeds in specified dates/ the tested seed number × 100%, germination potential was confirmed at the peak of sprouting.

Relative germination potential = germination potential of salt treatment/ germination potential of control × 100%.

Germination index = ΣGt/Dt, where Gt is expressed as the germination seed number in t days, and Dt refers to the day number of germination.

Relative germination index = germination index of salt treatment/germinationindex of control × 100%

Relative salt harm rate  = (germination rate of control − germination rate of salt treatment)/germination rate of control × 100%

Relative fresh weight = Fresh weight of salt treatment/Fresh weight of control × 100%

Relative root length  = Root length of salt treatment/Root length of control × 100%

Hierarchical cluster analysis was also used to evaluate salt tolerance based on the relative germination rate, relative germination potential, relative germination index, relative salt harm rate, relative fresh weight, and relative root length of the samples (Fig. 2). The salt tolerance was divided into four levels with a Euclidean distance of 6 between each level: highly salt tolerant (HST), salt tolerant (ST), moderately salt tolerant (MST), salt sensitive (SS). In addition, the F. suffruticosa inbred lines with a relative germination rate of less than 2% were classified as highly salt sensitive (HSS).

The seeds of two F. suffruticosa genotypes (HSS in Jiaoshan and Shanhaiguan (P2) and HST in Fengshan and Fengning (P27)) were chosen for the salt stress test as shown in Table 2. All the seeds were randomly divided into seven groups, each containing 30 uniform seeds. According to the results of a preliminary germination test, F. suffruticosa seeds would not germinate at NaCl concentrations greater than 240 mM. The experimental treatments were as follows: (1) no salt treatment (control, CK); (2) 40 mM NaCl treatment (S40); (3) 80 mM NaCl treatment (S80); (4) 120 mM NaCl treatment (S120); (5) 160 mM NaCl treatment (S160); (6) 200 mM NaCl treatment (S200); and (7) 240 mM NaCl treatment (S240). The F. suffruticosa seeds were sterilized with 1% sodium hypochlorite for 15 min. After being rinsed with distilled water five times, 30 seeds were sown in a Petri dish (nine cm diameter) with two layers of filter paper and cultured at 25 ± 0.5 °C for 12 days in an incubator in the dark. Three independent biological replicates were used for each treatment. The germination rate was measured at 12 DAT.

Determination of water absorption

For water absorption, seeds were immersed in petri dishes containing 12.5 ml of the solution for each treatment, and successive weighing was carried out to measure the fresh weight gain every four hours until germination. The water on the seed epidermis should be blotted with absorbent paper before measurement. Then the seeds were placed in the treatment solution again after measurement. As the water content absorbed in each time is calculated as follows, wherein W_i and W_f are the initial and final weight of seeds at each time point, respectively (Sousa, Maia & Morais, 2016).

% of absorbed water = (W_f–W_i)/ W_i ×100%

Determination of soluble sugar content

The soluble sugar content was determined at different time points (3, 5, 7, 9, 11 DAT) using the method described by Chen et al. (2020) with slight modifications and included three independent biological replicates. Glucose (0.1 g) was weighed and diluted into 100 mL volumetric flasks to obtain a standard solution of 1 mg mL⁻¹. Then the standard solution was diluted to 5 concentration gradients, including 0 µg mL⁻¹, 10 µg mL⁻¹, 20 µg mL⁻¹, 30 µg mL⁻¹, 40 µg mL⁻¹ and 50 µg mL⁻¹. A total of 2 ml standard solutions and 4 ml acid-anthrone reagent were added to test tubes for 10 min. The reaction mixtures were determined by measuring the absorbance at 620 nm using a UV2600 spectrophotometer (Shimadzu, Kyoto, Japan) for making a standard curve. All germinated seeds were ground into powder after oven-drying at 85 °C for 4 h. A total of 0.1 g dried samples and 15 mL of 80% ethanol were added to test tubes and placed in boiling water for 30 min. The reaction mixture was transferred into a centrifuge tube and centrifuged at 800 g for 30 min, and the supernatant was transferred into test tubes. These steps were repeated twice, and the supernatants were combined. The supernatant (two mL) and sulfuric acid-anthrone reagent (four mL) were mixed. The soluble sugar contents were determined by measuring the absorbance of each sample at 620 nm using a UV2600 spectrophotometer (Shimadzu) and calculated using a standard curve.

Determination of glycolysis metabolizing enzyme activity

The activities of HK, PFK and PK were determined at different time points (3, 5, 7, 9, 11 DAT) using assay kits (HK-2-Y, PFK-2-Y and PK-2-Y, respectively; Suzhou Comin Biotechnology Co., Ltd., Suzhou, China). Three independent biological replicates were assessed. All germinated seeds were rapidly frozen in liquid nitrogen and stored at −80 °C until analysis.

NADPH is the product of HK-related reactions, which has characteristic absorption peak at 340 nm. While PFK and PK activities can be reflected by the determination of NADH decline rate at 340 nm. 0.1 g samples were ground in a mortar with one mL of corresponding extraction solution and transferred into EP tubes. The homogenate was centrifuged at 8,000 g 4 °C for 10 min, and the supernatant was collected. Related reagents were added to the above filtrate according to the manufacturer’s instructions. The reaction mixtures were measured at 340 nm with a UV2600 spectrophotometer (Shimadzu). The enzyme activity was calculated and expressed in nmol min⁻¹ g⁻¹ (fresh weight (FW)).

Determination of TCA cycle-related enzyme activity

The activities of PDH and CS were determined at different time points (3, 5, 7, 9, 11 DAT) using assay kits (BC0380, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; BC1060, Beijing Solarbio Science & Technology Co., Ltd.). Three independent biological replicates were assessed. All germinated seeds were rapidly frozen in liquid nitrogen and stored at −80 °C until analysis.

PDH catalyzes the dehydrogenation of PA, resulting in a reduction in absorbance at 605nm. 0.1 g samples were ground into a homogenate with one mL and 10 µL of corresponding reagents in a mortar. The homogenate was then collected and centrifuged at 11,000 g 4 °C for 10 min. Related reagents were added to the supernatant according to the manufacturer’s instructions. The reaction mixtures were measured at 605 nm with a UV2600 spectrophotometer (Shimadzu). The enzyme activity was calculated and expressed in nmol min⁻¹ g⁻¹ (FW).

CS-related reactions cause the transfer of DTNB into TNB, which has characteristic absorbance at 412 nm. 0.1 g samples were ground into a homogenate with one mL and 10 µL of corresponding reagents in a mortar. The homogenate was centrifuged at 600 g 4 °C for 10min. The supernatant was then collected and centrifuged at 11,000 g 4 °C for 10min. Related reagents were added to the supernatant according to the manufacturer’s instructions. The reaction mixtures were measured at 412 nm with a UV2600 spectrophotometer (Shimadzu). The enzyme activity was calculated and expressed in nmol min⁻¹ g⁻¹ (FW).

Determination of G6PDH activity

The activity of G6PDH was determined at different time points (3, 5, 7, 9, 11 DAT) using a glucose-6-phosphate dehydrogenase activity assay kit (QYS-231047, Qiyi Biological Technology (Shanghai) Co., Ltd., Shanghai, China). Three independent biological replicates were assessed. All germinated seeds were rapidly frozen in liquid nitrogen and stored at −80 °C until analysis.

G6PDH activity can be reflected by the determination of NADPH increase rate at 340 nm because it catalyzes the conversion of NADP⁺ to NADPH. 0.05 g samples were ground in a mortar with one mL of the corresponding extraction solution and transferred into EP tubes. The homogenate was centrifuged at 8,000 g 4 °C for 10 min, and the supernatant was collected. Related reagents were added to the above filtrate according to the manufacturer’s instructions. The reaction mixtures were measured at 340 nm with a UV2600 spectrophotometer (Shimadzu). The enzyme activity was calculated and expressed in nmol min⁻¹ g⁻¹ (FW).

Determination of the contents of PA and CA

The contents of PA and CA were measured at different time points (3, 5, 7, 9, 11 DAT) using pyruvic acid and citric acid assay kits (BC2200 and BC2150, respectively; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer’s instructions. Three independent biological replicates were assessed. All germinated seeds were rapidly frozen in liquid nitrogen and stored at −80 °C until analysis. The reaction mixtures were measured at 520 nm for PA and 545 nm for CA with a UV2600 spectrophotometer (Shimadzu). The PA content was calculated using a standard curve and expressed in µg g⁻¹ (FW), while the CA content was calculated and expressed in µmol g⁻¹ (FW).

Statistical analysis

Microsoft Excel 2013 (Redmond, WA, USA) was used for data processing. SPSS 21.0 (IBM, Inc., Armonk, NY, USA) was used to analyze variance (ANOVA). Differences among the treatment means were assessed using a least significant difference (LSD) test at a p < 0.05 threshold.

Effect of salt stress on the germination rate

As shown in Fig. 3, the germination rate in P2 and P27 decreased as the NaCl concentration increased, reaching its minimum at 240 mM NaCl. Under normal conditions (CK), the seed germination rate reached 84.44% and 85.56% in P2 and P27, respectively. In comparison, the germination rate was only about 1.11% and 5.56% under 240 mM NaCl treatment, indicating that NaCl indeed inhibited seed germination. Compared with the CK treatment, the germination rate in P2 was reduced by approximately 9.21%, 28.95%, 56.58%, 64.47%, 96.05% and 98.68%, respectively, at the 40, 80, 120, 160, 200, and 240 mM NaCl treatments. While the germination rate in P27 was reduced by approximately 0.00%, 7.79%, 25.97%, 35.06%, 88.31% and 93.51%, respectively, at the 40, 80, 120, 160, 200, and 240 mM NaCl treatments.

Effect of salt stress on the water absorption

There are three phases in germination: phase I, phase II, phase III, which represent imbibition and imbibitional damage, the lag phase and the completion of germination, respectively (Bewley et al., 2013). For the water absorption of the seeds P2 and P27, the physiological phase I imbibition of the CK treatment occurred in about 24 h with a noticeable increase to stabilize water absorption, and phase II was extended to about 60 h. The phase III of the CK treatment occurred in about 108 and 96 h in P2 and P27, respectively, with increases in absorption to germination. Seed germination was delayed by salt stress. For seeds under salt stress, is that phase I went up to approximately 36 and 28 h in P2 and P27 at 240 mM NaCl, respectively, when there was a more significant increase in water absorption since phase II was extended up to 120 and 108 h. The phase III occurred in about 264 and 228 h in P2 and P27 at the highest salinity level, respectively, until germination (Table 3).

Different changes in relative absorption value were detected in P2 and P27 under salt stress. By comparing the relative absorption of distilled water (CK) with the other treatments in P2, the relative absorption values of phase II under salt stress were lower than CK except for the seeds that were exposed to 80 mM NaCl. The relative absorption values of phase II in P2 was decreased by approximately 1.05%, 2.12%, 0.46%, 1.98%, and 8.36%, respectively, at the 40, 120, 160, 200, and 240 mM NaCl treatments. In contrast, the relative absorption values of phase II with salt treatments were higher in P27, which increased by 4.90%, 10.51%, 9.04%, 29.47%, 10.62%, and 3.61%, respectively, compared with the CK treatment. However, the relative absorption values of phase III in P2 and P27 under salt stress were higher than those with CK treatments.

Effect of salt stress on the soluble sugar content during seed germination

As shown in Fig. 4A, as the salt concentrations increased, salt stress overall increased the soluble sugar content in P2. A significant increase was observed at 5, 7, and 9 DAT. Compared with the CK treatment, the soluble sugar content in P2 increased by approximately 35.37%, 44.85%, 76.56%, 38.65%, and 17.65% at the highest level (240 mM) at 3, 5, 7, 9, and 11 DAT, respectively. In P27, the content of soluble sugar increased first and then decreased at 5, 7, and 11 DAT, and continued to increase at 3 and 9 DAT as the salt concentrations increased (Fig. 4B). Compared with the CK treatment, the soluble sugar content in the NaCl seeds of P27 was significantly higher. As the time of salt stress continued, the soluble sugar content in P27 increased by approximately 56.70%, 77.33%, 56.35%, 42.93%, and 75.14% at the highest level (240 mM) at 3, 5, 7, 9, and 11 DAT, respectively, compared with the CK treatment.

Effect of salt stress on the glycolysis metabolizing enzyme activity during seed germination

With increasing salt concentrations, the HK activity in P2 reached its maximum value at 160 mM NaCl, then reduced at 3 and 5 DAT. An increase in the concentration of salt treatment was accompanied by a significant increase in the HK activity in P2 at 7, 9, and 11 DAT (Fig. 5A). As stress time went on, a decrease in HK activity of P2 was observed in CK treatment. Compared with the CK treatment, the HK activity of P2 increased by approximately 81.57%, 181.66%, and 133.30% at the 240 mM NaCl concentration at 7, 9, and 11 DAT, respectively. With the increase in salt concentrations, a significant increase in the HK activity of P27 was observed at 3, 5, 7, and 9 DAT, which was observed at higher levels (80, 120, 160, 200, and 240 mM) of salinity at 11 DAT (Fig. 5B). At 3, 5, 7, and 9 DAT, the HK activity of P27 reached its maximum at 200 mM NaCl, with increases of 108.33%, 222.54%, 167.06%, and 290.47%, respectively, compared with their respective control.

PFK activity increased first and then decreased in the seeds P2 and P27 after salt treatment (Figs. 5C, 5D). With the increase in salt concentrations, the PFK activity of P2 increased the most significantly following treatment with 120 mM NaCl at 3, 7, 9, and 11 DAT, which increased by 36.95%, 40.07%, 20.35%, and 11.25%, respectively, compared with the CK treatment. In P27, the activity of PFK at all the levels of NaCl was higher than that with the CK treatment in general. A more significant increase occurred following exposure to 40 and 80 mM NaCl at 3 and 5 DAT. Compared with the CK treatment, PFK activity increased significantly at 7, 9, and 11 DAT under salt stress.

A similar response was observed in the activity of PK in P2 and P27 seeds under salt stress (Figs. 5E, 5F). Simultaneously, the PK activity increased first and then decreased under different concentrations of salt stress. The PK activity increased over time and reached its maximum on 11 DAT. Compared with the CK treatment, the PK activity with the salt treatment was simultaneously higher, and it increased to higher levels in P27 than in P2. At 11 d, the PK activity of P2 under the salt stress treatment increased by only 12.01%, 13.28%, 16.94%, 10.64%, 2.71%, and 0.92%, respectively, compared with the CK treatment. However, compared with the CK treatment, the PK activity of P27 increased by approximately 68.61%, 164.29%, 227.61%, 147.12%, 132.14%, and 82.01%, respectively, which was much higher than that of P2.

Effect of salt stress on the TCA cycle-related enzyme activity during seed germination

The PDH activity in the seeds of P2 increased first and then decreased after salt treatment. At 9 DAT, the activity of PDH in P2 was slightly lower at 200 and 240 mM NaCl concentrations than that with the CK treatment. Compared with the CK treatment, the PDH activity of P2 significantly decreased by approximately 25.84%, 33.59%, and 30.43% at the 160, 200, and 240 mM NaCl concentrations, respectively, at 11 DAT (Fig. 6A). In P27, the PDH activity was significantly increased at 5, 7, 9, and 11 DAT after salt treatment except for the seeds that were exposed to 40 mM NaCl at 11 DAT (Fig. 6B).

With the increase in salt concentrations, different changes in CS activity in P2 and P27 were apparent over time. At 3, 5, and 7 DAT, the activity of CS in P2 increased first and then decreased under salt stress. At the same time, a decrease was observed at 9, and 11 DAT (Fig. 6C). At 7 and 9 DAT, the CS activity under the CK treatment was significantly higher than those under the 120, 160, 200, and 240 mM NaCl treatments. As the salt stress continued, the CS activity of P27 increased first and then decreased at 3, 5, and 7 DAT. Salt stress treatment significantly increased CS activity at 9 and 11 DAT. Compared with each CK treatment, the activity of CS in P27 increased by approximately 27.00%, 52.56%, 57.04%, 48.57%, 61.69%, and 62.51% at 9 DAT and increased by approximately 15.40%, 29.20%, 23.85%, 35.00%, 66.71% and 75.00% at 11 DAT as the salt stress time continued (Fig. 6D).

Effect of salt stress on the G6PDH activity during seed germination

Different changes of G6PDH activity were detected in P2 and P27 under salinity stress. With increasing salt concentrations, the NaCl treatment in P2 caused a general decrease in the activity of G6PDH (Fig. 7A). The G6PDH activity in seeds with the CK treatment was significantly higher compared with those treated with higher levels (120, 160, 200, and 240 mM) of NaCl (Fig. 7A). The activity of G6PDH in P27 increased first and then decreased with the increase in the salt concentrations (Fig. 7B). The G6PDH activity of P27 under 120, 160, and 200 mM of NaCl stress was significantly higher than that with the corresponding CK treatment at 3, 7, 9, and 11 DAT.

Effect of salt stress on the PA and CA contents during seed germination

The PA content in P2 and P27 showed similar trends under salt stress. The PA content was increased first, then decreased at 3, 5, and 7 DAT, and continued to increase at 9 and 11 DAT. In P2, no significant change in PA content was detected under the NaCl treatment at 5 DAT. At 7, 9, and 11 DAT, a noticeable change appeared at higher levels (160, 200, and 240 mM) of NaCl (Fig. 8A). With the increase in salt concentrations, the PA content in seeds of P27 increased significantly first and then decreased at 3 and 5 DAT, and an evident increase was observed under the 80, 120, 160, 200, and 240 mM NaCl treatments at 7 and 9 DAT (Fig. 8B). The PA content under each salt stress concentration was higher than the corresponding CK treatment in general.

The effect of salinity on the CA content in P2 and P27 is shown in Figs. 8C, 8D. The CA content of P2 increased first and then decreased with the increase in salt concentration at 3 and 5 DAT. At 7, 9, and 11 DAT, a more significant decrease was observed with the increase in salt concentration. Overall, the content of CA was lower in the seeds exposed to 160, 200, and 240 mM NaCl concentrations compared with the CK treatment. In P27, the content of CA increased first and then decreased under salt stress at 3 DAT, which was significantly higher at the 40, 80, 120, 160, and 200 mM NaCl treatments on the day before germination compared with the CK treatment. The CA content increased at 5, 7, 9, and 11 DAT with the increased salt concentrations. No significant change in the CA content of P27 was apparent at 7, 9, and 11 DAT, while the CA content under salt stress was slightly higher than that with the CK treatment.