Effect of Seed Soaking with Exogenous Proline on Seed Germination of Rice Under Salt Stress

Liu Hua-long, Sha Han-jing, Wang Jing-guo, Liu Yang, Zou De-tang, and Zhao Hong-wei

Rice Research Institute, Northeast Agricultural University, Harbin 150030, China

Effect of Seed Soaking with Exogenous Proline on Seed Germination of Rice Under Salt Stress

Liu Hua-long, Sha Han-jing, Wang Jing-guo, Liu Yang, Zou De-tang, and Zhao Hong-wei

Rice Research Institute, Northeast Agricultural University, Harbin 150030, China

To explore the germination mechanism of salt-stressed rice improved by exogenous proline, and provide a theoretical basis to rice direct sowing technology for salinized soil, the effects of soaking with proline on germination status, amylase activity and isoenzyme were studied in this paper. The results showed that germination status including germination energy (GE), germination rate (GR), relative germination energy (RGE) and relative germination rate (RGR) significantly decreased as the same as the activities of alpha-amylase, beta-amylase and the total amylase under salt stress. Soaking with exogenous proline improved the germination status of rice under salt stress. Moreover, GE and RGE of salt-stressed rice were improved with increasing of proline concentration at the range of 5-45 mmol ?L-1. Soaking with 15 mmol ?L-1and 30 mmol ?L-1proline significantly improved the amylase activities (e.g. alpha-amylase, beta-amylase and total amylase) of rice under salt stress. Salt stress inhibited the express of beta-amylase isoenzyme temporarily, but had few impacts on alpha-amylase isozyme. Soaking with 30 mmol ?L-1proline brightened District I and increased the width of 'i' brand in District II of alpha-amylase isoenzyme, but had few impacts on beta-amylase isoenzyme. In a word, soaking with proline could effectively alleviate the inhibitory effects of salt stress on seed germination.

exogenous proline, salt stress, rice, germination, amylase

Introduction

Rice is a moderately sensitive crop on salinity. Based on some published data, the salinity threshold for rice is around 3.0 dS ? m-1at 25℃, and a 1 ds ? m-1increase in salinity reduce yield by 12% (Maas, 1990). It is generally believed that most crops are more sensitive to salt during seed germination and seedling growth periods (Cuartero et al., 2006; Munns, 2002). Considering of the actual production, it is easy to damage the rice roots during seedling transplanting stage, and hard for seedlings to turn green in saline soil, which leads to the death of seedlings and eventually the reduction of yield. However, the direct sowing technology of rice, with fewer amounts of labor power and cost, can avoid the damage to seedlings. Because of fewer weeds surviving in salinity soil, the main problem of direct sowing technology is that germination state and seedling number are difficult to guarantee. Therefore, any measure to increase rice germination rate under salt stress plays a significant role in promoting rice direct sowing on saline soil.

Proline, as an organic osmoprotectant, largely accumulated in plants subjected to abiotic stresses. In addition to maintaining osmotic balance, proline is also able to stabilize subcellular structures, such as membranes and proteins, quench active oxygen,and protect cells against the adverse effects of salt stress (Ashraf and Foolad, 2007). In recent years, considerable amounts of literature have been published about exogenous proline improving the salt tolerance of crops, such as rice (Roy et al., 1993; Deivanai et al., 2011), sorghum (Nawaz et al., 2010), potato (Wang et al., 2005), soybean (He et al., 2000), canola (Athar et al., 2009), tobacco (Hoque et al., 2009) and melon (Kaya et al., 2007). Proline treatment in these studies mainly focused on adding proline to medium or foliar-spraying. However, there was little knowledge about the effect of soaking with proline. Thus, the effects of soaking with proline on germination status, amylase activity and isoenzyme were studied in this paper, so as to explore the germination mechanism of salt-stressed rice improved by exogenous proline, and provide a theoretical basis to rice direct sowing technology for salinized soil.

Materials and Methods

Plant materials and experiment

Rice c.v. (Oryza sativa L.) Dongnong 425 was used in this experiment, and it was provided by Rice Research Institute of Northeast Agricultural University, Harbin, China. The cultivar Dongnong 425 was 98.5 cm in plant heignt and about 140 days in growing period. The active accumulated temperature above 10℃ in the whole growth season demand of the cultivar was 2 700℃. The experiment was laid out in Completely Random Design (CRD) with three replications for each treatment. Plump seeds were selected and sterilized with 10% sodium hypochlorite for 25 min, then washed 3 times with distilled water. The seeds were soaked with 0 (distilled water), 5, 15, 30, and 45 mmol ? L-1proline for two days at (30±1)℃. Then, 100 seeds from each proline-soaking treatment were placed on Petri dishes with 12 cm diameter on two layers of filter paper containing 25 mL 100 mmol ? L-1sodium chloride solution, compared with 25 mL distilled water control. To avoid artificial influence on seed germination and maintain the salt concentration, evaporated water was replenished at 8:00 a.m. and 20:00 p.m. respectively, each day according to the mark of water layer position on external Petri dishes, and solutions were renewed every three days. The dishes of each treatment were placed in dark incubator at (28±1)℃. After five days, the day/night temperature was (28±1)℃/(20±1)℃, and the photoperiod was 12 h/12 h, the light intensity was about 4 000 Lx. A seed was considered as germinated when the radicle length was half the length of seed. Germination was counted on the 3rd day and the 7th day after seed soaking, while the activity of amylase was determined on the 3rd day and the amylase isoenzyme of presoaking with 30 mmol ? L-1proline was assayed during the imbibitions and germination stage. All the treatments were as follows: soaking with distilled water+ non salt stress (CK); soaking with distilled water+100 mmol ? L-1NaCl (T0); soaking with 5 mmol ? L-1proline+ 100 mmol ? L-1NaCl (T1); soaking with 15 mmol ?L-1proline +100 mmol ? L-1NaCl (T2); soaking with 30 mmol ? L-1proline+100 mmol ? L-1NaCl (T3); soaking with 45 mmol ? L-1proline +100 mmol ? L-1NaCl (T4).

Determination of germination energy (GE), germination rate (GR) and their relative values

GE=germinated seed number within 3 days/total grains for test×100%.

GR=germinated seed number within 7 days/total grains for test×100%.

RGE=GE of treatment/ GE of control.

RGR=GR of treatment/ GR of control.

Assays of amylase activity and their isoenzyme determination

The activity of amylase was determined according to Gong (2000). While the amylase isoenzyme extraction and electrophoresis were according to He and Zhang (1999).

Statistical analysis

Results of all the data were shown as Mean±SD (n=3). One-way ANOVA (analysis of variance) and Duncan'smultiple range tests (DMRT) were used to evaluate the difference among treatment means at P＜0.05 with software SPSS v18.0.

Results

Effects of soaking with proline on seed germination of salt-stressed rice

Compared with control (CK), GE, GR and their relative values of salt stressed rice significantly decreased. While, presoaking with exogenous proline significantly alleviated the inhibition effects of salt stress on seed germination of rice c.v. Dongnong 425. In addition, exogenous proline also showed concentration effects on GE and RGE of salt stressed rice. With an increase of proline concentration, GE and relative germination energy (RGE) of rice under salt stress were significantly improved, but GR and relative germination rate (RGR) had no obvious changes among the concentrations of 15-45 mmol ? L-1. Moreover, germination states under salt stress and 45 mmol ? L-1proline treatment were similar as control (Table 1). The results indicated that presoaking with proline facilitated germination of salt-stressed rice.

Table 1 Effects of soaking with proline on seed germination of salt-stressed rice

Effect of proline on amylase activity in saltstressed rice

Salt stress significantly inhibited the activity of amylase during germination stage. Compared with control, the activities of amylases under salt stress, including α-amylase, β-amylase and total amylase, decreased by 10.1%, 13.9% and 13.2%, respectively. Soaking seed with 15 mmol ? L-1and 30 mmol ? L-1proline under salt stress significantly alleviated the decrease in the amylase activity, but 5 mmol ? L-1and 45 mmol ? L-1proline were not effective as the above concentrations (Fig. 1). Obviously, the activity of amylase improved by suitable concentration of proline under salt stress would accelerate the mobilization of storing substances, so as to provide adequate materials and energy for seed germination.

Effect of proline soaking on amylase isoenzyme in salt-stressed rice

To clarify how exogenous proline improved rice germination under salt stress, the amylase isoenzymes were analyzed by electrophoresis during germination stage. Fig. 2 showed that nine bands ('a-i') of amylase isoenzymes were detected during rice germination stage, and the relative mobility (Rm) were 9.4%, 12.3%, 17.2%, 24.9%, 29.9%, 38.9%, 41.8%, 47.9% and 52.3%, respectively. According to the report of He and Zhang (1999), the white and transparent band was α-amylase isoenzyme, and the orange, pink and red band was β-amylase isoenzyme. Based on this preliminary judgment, 'a-d'bands were β-amylase isoenzymes, while 'e-i' bands were α-amylase isoenzymes. At the initial stage of imbibitions (0-0.5 day of seed soaking), only β-amylase isoenzyme ('a' band) was detected. While α-amylase isoenzyme was observed after soaking seed for one day. The above results indicated that α-amylase was synthesized at the germination stage, but β-amylase enzyme existed in the mature seeds and played an important role at the early germination stage. Rm of β-amylase isoenzyme gradually increased under salt stress. Interestingly, 'd' band of β-amylase isoenzyme was not detected in the treatment with salinity on the 7th day of the salt stress, but observed two days later, indicating that the expression of β-amylase was temporarily inhibited by salt stress. β-amylase isoenzymes under control or salt stress conditions were less affected by soaking seed with proline, but α-amylase isoenzymes (District I, including bands 'e' and 'f') were brightened by exogenous proline on the 5th day of the salt stress. Similarly, the 'i' band of α-amylase isoenzymes in District II was broadened four days later (Fig. 2).

Fig. 1 Effects of seed soaking with proline on activities of α-(A), β-(B), and total amylase (C) in salt-stressed rice

Fig. 2 Effects of soaking seed with 30 mmol ? L-1proline on amylase isoenzyme expression in salt-stressed rice

Discussion

Germination stage is the initial stage of plant development, and plays an important role in life cycle. It is generally believed that rice is sensitive to salinity at the germination stage. The reason that salt stress inhibit seed germination is mainly due to imbalance in water uptake, destruction of cell membrane and reduction of enzyme activity, which limits the hydrolysis of food reserves and immobilizes the translocation of food reserve from storage tissue to develop embryo axis (Guo et al., 2004; Ke et al., 2002; Qi et al., 2007; Deivanai et al., 2011). Han et al. (1998) confirmed thatsalt could inhibit the germination of imbibed seeds. It was also found in the present study that GE, GR, RGE and RGR of imbibed seeds decreased significantly under salt stress (Table 1).

Proline is a kind of soluble osmolyte, which is uncharged under neutral pH and highly soluble in water. In addition, proline with high concentrations could not affect the interaction between solvent molecules. Numerous studies have demonstrated that exogenous proline could improve the germination rates of different crops under salt stress, such as sorghum (Nawaz et al., 2010), canola (Athar et al., 2009), and rice (Deivanai et al., 2011). Our work also confirmed that exogenous proline could effectively improve seed germination status under salt stress. Water absorption of the imbibition has three phases. In phase I, there is rapid water absorption dependent on physical process. Phase II shows stagnation of water absorption since the seed is saturated. Phase III is associated with another water uptake where radical emergence and elongation occurs (Li, 2006). In our present study, phase I was approximately completed after soaking with water or proline. Thus, GE and RGE improved by soaking with proline under salt stress were mainly due to increase of intracellular osmolytes and alleviation of water uptake imbalance in phases II and III.

The mobilization of food reserve from storage tissue to develop embryo axis depended on the activity of amylase (including α-amylase and β-amylase) until the seedling became photosynthetically self sufficient (Nandi et al., 1995). Starch was directly hydrolyzed to dextrin by α-amylase, and then turned to glucose and maltose by β-amylase, de-branching enzyme and α-glucosidase. Thus, α-amylase is often regarded as a key enzyme in seed germination (Lovegrove et al., 2000). Extensive literature indicates that α-amylase is synthesized de novo in the aleurone layer and the scutellar epithelium of germination seeds (Okamot and Akazawa, 1979; Higgins et al., 1982). However, whether β-amylase presented in germinating rice seed is still controversial (Nandi et al., 1995; Yamaguchi, et al., 1998). It is reported that β-amylase was formed during the development and maturation of cereal crops (except rice), accumulated and stored in dry seeds in the form of inactive polymer and gradually released and activated in germination stage (Yamaguchi et al., 1998). While Nandi et al. (1995) found that β-amylase was present in dry seeds of rice, and could be regarded as reliable indicator of germination ability of rice seed stocks and of their vigour during rice seed germination. Studies on the β-amylase in Triticeae tribe members, such as barley, wheat and rye, revealed that a proteolytic mechanism activates the pre-existing β-amylase molecules in the starchy endosperm during seed germination (Wang et al., 2004). Under the action of hydrolytic enzyme, the molecular weight of β-amylase present in dry seeds of barley in the form of granule-bound starch reduced from 64 ku to 59 ku (Sopanen et al., 1989; Loreti et al., 1998). Our results showed that α-amylase was synthesized in germination stage, while β-amylase existed in dry rice seeds. Moreover, Rm of β-amylase isoenzyme increased with the days after salt stress went on, confirming that β-amylase in rice as other cereals also had a gradual hydrolysis process.

Generally, salt stress could inhibit amylase activity during seed germination, which limits the hydrolysis of stored substances. Through the gel electrophoresis of amylase isoenzyme in this experiment, it was found that salt stress could temporarily inhibit the expression of β-amylase isoenzyme ('d' band), but had no effect on α-amylase isoenzyme. Soaking with suitable concentration of proline could effectively improve amylase activities under salt stress, thus accelerated the mobilization of storing substances and provided adequate material and energy for seed germination.

Conclusions

Soaking with proline relieved the inhibition effect of salt stress on rice germination. Maybe it was because soaking with proline could improve the internal osmotic potential of rice seeds and enable them to maintain adequate water supply under salt stress.Thus, the activity of the amylase in the seed kept as normal and abundant materials and energy required for germination could be provided.

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S511

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1006-8104(2014)-03-0001-06

Received 12 August 2013

Supported by the Project of Twelfth Five-year Plan for Sci & Tech Research of China in Rural Areas (2011BAD35B02-01); Program of Sci & Tech Research of China (2011BAD16B11)

Liu Hua-long (1978-), male, assistant researcher, Ph. D, engaged in the research of rice breeding and high-yield cultivation. E-mail: nkxhahanjing@163.com