Effects of Artificial Aging on Physiological Characteristics of Rice Seeds with Different Dormancy Characteristics

Dongdong CAO Shanyu CHEN Yutao HUANG Yebo QIN Guanhai RUAN

Abstract［Objectives］ This study was conducted to study the Germination characteristics and secondary metabolism regulation of Scutellaria baicalensis Georgi under osmotic stress.

［Methods］ The activity of praline, soluble sugar and protein was detected with ultraviolet spectrophotometry. The activity of phenylalanine ammonialyase (PAL) and cinnamic acid 4hydroxylase (C4H) was determined with ultraviolet spectrophotometry. The secondary metabolites were detected by high performance liquid chromatography (HPLC).

［Results］ The germination percentage, germination potentiality and germination index decreased at first and increased subsequently along with the enhancement of PEG concentration. The praline content, the soluble sugar content and the soluble protein content in S. baicalensis seedlings increased along with the enhancement of PEG concentration and the differences from the CK were significant (P<0.05). The PAL and C4H in S. baicalensis seedlings decreased after osmotic stress and the differences from the CK were significant (P<0.05). The results indicate that, the scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylinA contents decreased after osmotic stress and the differences from the CK were significant (P<0.05).

［Conclusions］ The quality of the skullcaps could be improved by improving the activity of PAL and C4H.

Key wordsGermination characteristics; PAL; C4H; Secondary metabolites



Received: July 10, 2018Accepted: October 13, 2018

Supported by 2017 Traditional Chinese Medicine Public Health Service Specific Grant (CS［2017］66); Shandong Traditional Chinese Medicine Science and Technology Development Program (2017522).

Jinhua LIU (1979-), female, P. R. China, PhD, devoted to research about research on traditional Chinese medicine resources and quality control.

*Corresponding author. Email: jinhualiu1980@163.com.

Seed germination is affected by many factors, such as temperature, light, and soil water content. The availability of retrievable water in the soil is considered the most important factor for controlling seed germination［1-2］. This might be due to the fact that water influences the progress and kinetics of most biologically significant reactions.

Plants can respond and adapt to water stress spontaneously by altering their cellular metabolism and invoking various defense mechanisms［3］. Survival under this stressful condition depends on the plants ability to perceive the stimulus, generate and transmit the signals, and initiate various physiological and chemical changes［4-5］. The dried root of S. baicalensis (Labiatae family) is a kind of wellknown genuine medicinal material of Shandong Province［5-6］. Because of a sharp decline of wild resources, artificial cultivation must be performed to meet the increasing requirement as the science and technology develop gradually［7］. The regional plant of S. baicalensis is concentratedly distributed in mountainous and hilly areas. Water is one of the highly important ecological factors in plant growth at all times, especially for medicinal radix materials. So the understanding of seed germination physiology of a species can help evaluate safesite availability because seed dormancy/germination traits determine the number and timing of seedling emergence. However, few reports are available about the physiological properties response to water stress. As a result, in the present study, germination, osmotic adjustment, MDA, and the corresponding activity of catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) of S. baicalensis seeds subjected to water stress were studied. The present investigation also offers a reference opinion and a theoretical basis for improving the germination of S. baicalensis seeds and understanding the natural regeneration progress of S. baicalensis.

Materials and Methods

Materials

The seeds of S. baicalensis were identified by ZHANG Yongqing (Shandong University of Traditional Chinese Medicine). The seeds were selected and sterilized with 75% ethanol, washed for six times with distilled water, treated by osmotic stress with PEG6000 for 1 h and placed in a culture dish (10 cm) which was covered with three layers of filter paper. The PEG concentrations were 0, 1%, 5%, 10%, 15% and 20%, respectively. The seeds were put in the illumination box for germination. The number of germinated seeds was recorded every day. The germination percentage (6 d), vigor of germination (5 d) and germination index were determined according to FU Jiarui. The praline, soluble sugar, protein, PAL, C4H, CHS and the secondary metabolites were measured during cotyledon period.

Methods

Determination of activity of praline, soluble sugar and protein

The activity of praline, soluble sugar and protein was detected with ultraviolet spectrophotometry［8］.

PAL and C4H extraction and their activity determination

PAL and C4H extraction and their activity determination were carried out according to JIN et al.［9］.

PAL extraction and its activity determination

0.2 g of S. baicalensis seedling was ground to a fine power with a mortar and pestle using liquid N2. The powder was then extracted with 5 ml of 0.1 mol/L borax buffer (pH=8.8) containing 0.02 g pvp, 5 mmol/L βmercaptoethanol and 1 mmol/L EDTA, and then centrifuged at 10 000 r/min for 15 min. The supernatant was used to measure the activity of PAL. The assay mixture consisted of 2 ml of borax buffer (0.1 mol/L, pH= 8.8) and crude enzyme (100 μl). The reaction was initiated by the addition of Lphenylalanine (800 μl). Tubes were incubated at 30 ℃ for 30 min, and the reaction was stopped by the addition of 6 mmol/L HCl (200 μl). The yield of cinnamic acid was estimated at 290 nm. Triplicate assays were performed for each extract.

C4H extraction and its activity determination

0.2 g of S. baicalensis seedlings were ground to a fine power with a mortar and pestle using liquid N2. The powder was then extracted with 3 ml of 0.05 mol/L TrisHCl buffer (pH=8.9) containing 15 mmol/L βmercaptoethanol, 4 mmol/L MgCl2, 5 mmol/L Vc, 10 μmol/L Leupetin, 1 mmol/L PMSF, 0.15% PVP (m/V), 10% Glycerol and then centrifuged at 10 000 r/min for 20 min, and the supernatant was used to measure the activity of C4H. The assay mixture consisted of 2.2 ml buffer containing 2 μmol/L transcinnamic acid, 50 mmol/L TrisHCl (pH=8.9), 2 μmol/L NADPNa2, 5 μmol/L G6PNa2 and crude enzyme (100 μl). Tubes were incubated at 25 ℃ for 30 min with dscillator, and the reaction was stopped by the addition of 6 mmol/L HCl (100 μl). The yield of cinnamic acid was estimated at 340 nm. Triplicate assays were performed for each extract.

Determination of the secondary metabolites

All HPLC measurements were performed with an Agilent 1100 series HPLCDAD system. The mobile phase consisted of MEOH, H2O and H3PO4. The mobile phase gradient program was shown in Table 1. The flow rate was kept at 1.0 ml/min, and the detection wavelength was set at 276 nm.

Table 1Mobile phase gradient program

Time∥min

Mobile phases

MEOHH2O2% H3PO4

0405010

10504010

20505010

30603010

40801010

50801010

Linearity investigation

The blended reference substance solutions were prepared by dissolving 6.1, 3.0, 2.1, 1.1, 10.3, 1.5, 2.9 mg of scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylina reference substance, respectively. After accurately weighed, they were put into a 100 ml volumetric flask with methanol, respectively. The series standard solutions were injected into the HPLC under the chromatographic conditions. The representative linear equations for scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylina were Y=90.464x-182.57 (R=0.999 1), Y=193.59x-311.68 (R=0.999 2), Y=86.437x-37.023 (R=0.999 2), Y=156.23x-207.14 (R=0.999 1), Y=49.077x-15.829 (R=0.999 3) and Y=1 203.84x-110.16 (R=0.999 2), respectively.

Solution preparation

The tested samples were pulverized into powder and passed through a 40mesh sieve．Each sample powder (0.5 g) was weighted accurately and extracted by ultrasonator with 20 ml of ethanolwater (7∶3) for 60 min. The extract was filtered finally.

Precision test

The solution for test was made according to the solution preparation method, injected for five times repeatedly to record the peak areas of the scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylina content. The RSD value of them were 0.23%, 0.32%, 0.41%, 0.29%, 0.82% and 0.61%, respectively. The results showed that the solution had good precision.

Reproducibility test

0.5 g of S. baicalensis seedling was taken precisely for five times. The solution for test was made according to the solution preparation method, and injected to record the peak areas of the scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylina. The RSD values of them were 0.43%, 0.22%, 0.51%, 0.69%, 0.32% and 0.71%, respectively. The results showed that the solution had good reproducibility.

Inspection of the stability of solutions

The stability of the assayed sample solution was monitored by measuring the peak areas. Each assayed sample solution was determined at 0, 2, 4, 6, 12, 18, 24, 48, 72 h, respectively. The results indicated that the assayed sample solutions were stable, and their RSD values were 0.86%, 0.57%, 1.02%, 0.93%, 1.12% and 0.95%, respectively.

Recovery experiment

0.5 g of S. baicalensis seedling was taken precisely for five times. Proper amounts of scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylina were added, respectively. The solution for test was made according to the solution preparation method, and the content was determined. The average recovery rates (n=5) were 101.22%, 99.87%, 102.45%, 103.21%, 98.82%, 100.67%, respectively, and the RSD values were 0.63%, 0.93%, 0.89%, 1.29%, 1.02% and 0.91%, respectively. The results showed that the solution had good recovery rate.

Statistical analysis

Each experiment was repeated for three times at least. Values were expressed as means±s.e. Data were analyzed by Statistical Analysis System (version 8.0, SAS Institute Inc., NC, USA).

Results

Effect of osmotic stress with PEG on seed germination

Table 2 showed that after osmotic stress with 0%, 1%, 5%, 10%, 15% and 20% PEG, The germination percentage, germination potentiality and germination index decreased at first and increased subsequently along with the enhancement of PEG concentration, indicating that 15% and 20% PEG promoted the germination of the S. baicalensis seeds. The germination percentage, germination potentiality and germination index reduced obviously and had significant differences from the CK (P<0.05) after osmotic stress with 1%, 5%, and 10% PEG. Under the stress with 1% PEG, the germination percentage, germination potentiality and germination index reduced slowly, but under the stress with 5% and 10% PEG, the germination percentage, germination potentiality and germination index reduced more. Under the stress with 15% and 20% PEG, the germination percentage, germination potentiality and germination index increased to 52.07%, 49.67%, 35.60% and 54.67%, 51.21%, 38.40%, respectively.

Table 2Effects of osmotic stress with PEG on the germination of the seeds

PEG∥%Germination percentage∥%Germination potentiality∥%Germination index

0(CK)49.33±1.5348.67±0.5833.21±0.79

147.33±0.5845.00±1.0030.03±0.72

540.33±1.5339.67±0.5828.21±0.79

1034.00±1.0033.00±0.8926.41±0.56

1552.07±0.4849.77±0.6135.60±0.29

2054.79±0.3551.21±0.3238.40±0.32

Jinhua LIU et al. Germination Characteristics and Secondary Metabolism Regulation of Scutellaria baicalensis Georgi Under Osmotic Stress with PEG

Effect of osmotic stress with PEG on proline content in S. baicalensis seedlings

Fig. 1 showed that under osmotic stress with PEG, a great deal of proline was accumulated in S. baicalensis seedlings. The proline content increased along with the enhancement of PEG concentrate and the difference from the CK was significant (P<0.05).



Fig. 1Effects of osmotic stress with PEG on the proline content in the S. baicalensis seedlings

Effect of osmotic stress with PEG on soluble sugar content in S. baicalensis seedlings

Fig. 2 showed that the soluble sugar content in the seedlings of S. baicalensis seedlings increased along with the enhancement of PEG concentration and the difference from CK was significant (P<0.05).



Fig. 2Effects of osmotic stress with PEG on the soluble sugar content in the S. baicalensis seedlings

Effect of osmotic stress with PEG on soluble protein content in S. baicalensis seedlings

Fig. 3 showed that the soluble protein content in S. baicalensis seedlings increased after osmotic stress with 5%, 10%, 15%, and 20% PEG and the differences from the CK were significant (P<0.05). Under the stresses with 20% PEG, the soluble protein content reached its maximum value.



Fig. 3Effects of osmotic stress with PEG on the soluble protein content in the S. baicalensis seedlings

Effects of osmotic stress with PEG on PAL and C4H in S. baicalensis seedlings

Fig. 4 showed that the PAL and C4H in S. baicalensis seedlings decreased after osmotic stress and the differences from the CK were significant (P<0.05). It was shown that PAL and C4H were generally inhibited in seeds exposed to osmotic stress with PEG.



Fig. 4Effects of osmotic stress with PEG on the PAL and C4H in the S. baicalensis seedlings

Effects of osmotic stress with PEG on the secondary metabolites in the S. baicalensis seedlings

The changes of secondary metabolites in S. baicalensis seedlings exposed to different osmotic stress are shown in Fig. 5, Fig. 6 and Fig. 7. The results indicate that the scutellarin, baicalin, wogonoside, baicalein, wogonin and oroxylinA contents decreased after osmotic stress and the differences from CK were significant (P<0.05).



A: Scutellarin; B: baicalin; C: wogonoside; D: baicalein; E: wogonin; F: oroxylina

Fig. 5HPLC chromatograms of mixed reference substances



Fig. 6HPLC chromatograms of samples



Fig. 7Effects of osmotic stress with PEG on the secondary metabolites in the S. baicalensis seedlings

Discussion

Seed germination is affected by soil water content. The results showed that the activation treatment of S. baicalensis seeds with PEG6000 (15% and 20%) could greatly improve seed vigor and make the seed rapidly and tidily, germinate and the seedlings were strong. The PEG6000 (15% and 20%) plays an important role in increasing seed germination percentage and shortening the germination time of S. baicalensis.

Water status is one of the most important physiological parameters of plants. Osmotic regulation was an important mode in which plant could reduce osmotic potential and resist adversity stress under water stress．It enhanced cell liquid concentration and reduced osmotic potential mainly through initiatively accumulating various organic and inorganic matters in plant, thereby reducing water potential, so as to continue adsorbing water from outside and maintaining physiological courses of turgor pressure. S. baicalensis reduced water potential and enhanced water keeping capability under the osmotic stress with PEG through enhancing the contents of osmotic adjustment solute such as praline, soluble sugar and soluble protein. In this study, the soluble protein content in S. baicalensis increased along with the increment of stress intensity. It was believed that S. baicalensis induced new protein synthesis in condition of adversity stress. This kind of protein participated in the cell osmotic regulation and promoted the increase of activity of protective enzymes and osmotic adjustment solute syntheses, and finally made total soluble protein content increase.

Plant secondary metabolism is a consequence of the interaction between plants and environments (biotic and abiotic) in the long process of plant evolution. An important step in flavonoid accumulation is the phenylpropanoid pathway. The first step towards the phenylpropanoid biosynthetic pathway is catalyzed by PAL, which converts Lphenylalanine to transcinnamic acid. The second step is catalyzed by C4H, which converts Lphenylalanine transcinnamic acid to 4coumarate. Through the osmotic stress treatment experiment, we learned that the PAL and C4H significantly decreased after osmotic stress which is in accordance with the secondary metabolites. There is a positive correlation between the activity of flavonoids and PAL and C4H.

In this article, the research method of water physiology was applied to S. baicalensis to investigate its responses to water stress. The theoretic basis could be provided on the standard production of the Radix Scutellariae through research on the physiological and biochemical characteristics of plants at different water stress.

References

［1］ BOHNERT HJ, JENSEN RG. Strategies for engineering water stress tolerance in plants［J］. Trends in Biotechnology, 1996, 14: 89-97.

［2］ SHINOZAKI K, YAMAGUCHISHINOZAKI K. Gene expression and signal transduction in waterstress response［J］. Plant Physiology, 1997, 115: 327-334.

［3］ ZHANG GJ. Identificology of Chinese Materia Medica［M］. science press, 2002, 167-170.

［4］ WANG M, SUN ZX. Research situation of Scutellaria Baicalensis Georgi［J］. Huaihai medicine, 2007, 25: 484-485.

［5］ SUN XY. A Survey of Studies on Scutellaria Baicalensis Georgi［J］. Information on Traditional Chinese Medicine, 1993, 5: 32-34.

［6］ HUANG YH, CHEN XS. Research progress of water stress in fruit trees［J］. Journal of Shandong Agricultural University, 2005, 36: 481-484.

［7］ SHIMONO A, WASHITANI I. Seedling emergence patterns and dormancy/germination physiology of Primula modesta in a subalpine region［J］. Ecol Res, 2004, 19:541–551.

［8］ ZHAO SJ, LIU HS, DONG XC. Phytophysiologica experiment guidance［M］. Beijing: China Agricultural Science and Technology Press, 1998.

［9］ JIN LP. Effect of drought stress in different ecological conditions of the Prunus mongolica maxim almond leaves in PAL and C4H activity［J］. Acta Agriculture Borealisinica, 2009, 24(5):118-122.