Seeds were obtained from Funiu Mountain of Henan province, China, on the hillside with the altitude of 700 meters. After being collected from adult trees in September 2014, the seeds were air-dried at room temperature and stored containers at 2˚C - 5˚C.

The experiments were conducted in test plots of Shandong Agricultural University. To determine the response of seeds to cold stratification, seeds were soaked for 24 h with water, 200, 400, 600 mg/L of GA₃, respectively, and mixed with moistened sand in a ratio of 1:3. After that, the mixture of seeds and sand was buried in leeward side for the depth of 50 cm for 5 months. The moisture of seeds and sand were checked to avoid oxygen deprivation and water shortage of sand so that the seeds would not get mouldy during the entire period of stratification. Experiments were conducted every 15 days.

Soluble sugar was extracted from 1 g FM of seed in 25 ml of boiling water. After heating the seeds for 10 minutes at 100˚C, the extract was filtered through filter paper, diluted with water to 50 ml and mixed. Then 0.5 ml of extract, 1.5 ml of distilled water, 0.5 ml of 2% anthrone solution, and 5 ml of H₂SO₄ were mixed for 10 minutes. The soluble sugar was measured by a spectrophotometer at a wavelength of 620 nm, following the methods for extraction and preparation of test solutions and standards of Li [19] . There were three replications of each sample.

After heating the remaining seeds with 20 ml water for 15 minutes at 100˚C, the extract was mixed with 2 ml of 9.2 mol/L per chloric acid. Then the remaining steps are the same as soluble sugar.

Extraction procedures of Li [19] were adopted for the determination of amylase activity. For the preparation of enzyme extract, 1 g seeds were homogenized with 8 ml of water. After 20 minutes^, standing, the extract was centrifuged at 10,000 r/min for 20 min, filtered through two layers of filter paper to remove impurities. All the preparations were carried out at 4˚C. The supernatant obtained was used as crude enzyme extract for α-amylase and α + β-amylase assay. Then the detailed steps are shown in Table 1.

After heating the mixture for 5 min at 100˚C, the enzyme activity was measured by a spectrophotometer at a wavelength of 540 nm.

The soluble protein content was measured by using Coomassie brilliant blue staining referring to extraction procedures of Li [19] . 1 g seeds were ground with 8 ml of water and then the extract was centrifuged at 10,000 r/min for 20 min. 1ml of protein stock solution was mixed with 5 ml of Coomassie brilliant blue, measured by a spectrophotometer at a wavelength of 595 nm.

1 g seeds were ground with 5 ml of 10% acetic acid, diluted with water to 100 ml, and the extract was filtered through a layer of filter paper to remove impurities. Then 1 ml of filtrate, 1 ml of ammonia-free distilled water, 3 ml of ninhydrin, 0.1 ml of 0.1% ascorbic acid were mixed and heated for 15 min at 100˚C. After diluting with 60% ethanol to 20 ml, the mixture was measured by a spectrophotometer at a wavelength of 570 nm.

Crude fat content was analyzed by the Soxhlet extraction method with absolute ether as solvent following the methods of Li [19] . The method was modified by our practice experience. The detailed steps are described as follows:

1) The filter paper was folded to make a bag and the bag was air-dried for 2 h in an oven at 105˚C. After drying, the filter paper was cooled to room temperature in desiccators. About 30 min later, the filter paper was weighed in an analytical balance, and the weight was noted as W₀.

2) 1 g seeds were ground and loaded into the filter paper bag. Then the bag was weighed in an analytical balance, and the weight was noted as W₁.

3) The bags were put into the Soxhlet extractor with absolute ether for 8h. After that, the paper bag was air-dried for 2 h in an oven at 105˚C and was weighed in an analytical balance, and the weight was noted as W₂.

4) Crude fat content = (W₁ − W₂)/(W₁ − W₀) × 100%.

Lipase activity was analyzed by basic titration. 3 g seeds were homogenized with 5 ml 0.1 mol∙L⁻¹ of Phosphate-citrate buffer, the homogenate was used for lipase assay. The detailed steps were described as follows Table 2.

Then use 0.1 mol∙L⁻¹ of NaOH to titrate the solution to reddish color.

As shown in Figure 1. Seed germination was affected by cold stratification. Seeds started to germinate when they were stratified for 90days. And the finial germination of seeds with no GA₃ treatment was 12.7%. In contrast, GA₃ significantly increased seed

germination depending on concentration. At the concentration of 200, 400, 600 mg/L, seed had reached a germination to 29.2%, 21.7%, and 18.4%, increasing by 16.5%, 9%, and 5.7% compared to those without GA₃ treatment, respectively. Obviously, the germination of seeds treated with 200 mg/L GA₃ was higher than others, which explaining that cold stratification with 200 mg /L GA₃ treatment was an effective method to break seed dormancy.

The results of soluble sugar analysis of seeds during cold stratification are presented in Figure 2(a). During the low temperature treatment period from beginning to 45 days, the soluble sugar content in seeds treated with 0, 200, 400, 600 mg/L of GA₃ decreased gently from 2.35, 2.68, 2.60, 2.33 mg・g⁻¹ to 1.38, 1.53, 1.42, and 1.27 mg∙g⁻¹, respectively. After that, soluble sugar content showed a sharp increase until 5 months lamination. At the end of cold stratification, soluble sugar content increased to 3.36, 3.69, 3.38, and 3.68 mg∙g⁻¹. On the contrary, the starch content decreased significantly (Figure 2(b)). During the low temperature treatment period, the starch content in seeds treated with

0, 200, 400, 600 mg/L of GA₃decreased to 2.15, 1.81, 2.61, and 3.18 mg∙g⁻¹, decreased by 65.5%, 72.6%, 59.6%, and 49.4%, compared to those without cold stratification treatment, respectively.

The low temperature treatment showed significant effects on amylase activity. During cold stratification treatment period, both of α-amylase (Figure 2 (c)) and α + β-amy- lase (Figure 2(d)) activity increased dramatically. The α-amylase activity in seeds without GA₃ treatment increased slowly until seeds were laminated for 90days, and then showed a rapid increase at 120, 135 and 150 days, corresponding to 0.391, 0.556, and 0.499 mg∙g⁻¹∙min⁻¹, respectively. The trends of other three treatments were the same as it. Similarly, α + β-amylase activity showed a trend of rising as a whole, despite the fluctuation at the early stage of treatment. After 90 days lamination, α + β-amylase activity in seeds without GA₃ treatment reached to 0.215 mg∙g⁻¹∙min⁻¹, increased by 70.8% compared to the control 0.123 mg∙g⁻¹∙min⁻¹. And the activity at 120, 135 and 150 days increased by 454.5%, 634.1%, and 834.1%, respectively. The maximum activity of α + β-amylase appeared in seeds treated with 200mg/L of GA₃ and it increased to 1.149 mg∙g⁻¹・min⁻¹ at the end of cold stratification treatment. Notably, there was no significant difference between α-amylase and α + β-amylase activity during the treatment period from 0 to 90 days. After 90 days, the activity of α + β-amylase was significantly higher than that of α-amylase during the same period of time.

The results of protein content in seeds during cold stratification are presented in Figure 3(a). There were no obvious changes and differences of protein content in seeds with difference treatments in the early phase of cold stratification. During this time, protein content in seeds treated with 0, 200, 400, and 600 mg/L of GA₃ decreased by 8%, 11%, 4%, and 3%, respectively. As the cold stratification treatment duration increased, the protein content showed a sharp decrease after 75 days, and the most significant decrease in protein content was in 200 mg/L GA₃-treated seeds. In seeds stratified for 90, 120, and 150 days it decreased significantly to 16.4, 15.2, and 7.0 mg∙g⁻¹, with 8%, 11%, and 42% lower than that without GA₃ treatment, respectively. Conversely, free amino acid content showed a trend of rising as a whole in spite of slightly decreased during treatment period from 0 to 30 days (Figure 3(b)). The most significant increase in free amino acid content was in 200 mg/L GA₃-treated seeds.

The results of crude fat content in seeds during cold stratification are presented in Figure 4(a). As a result of low temperature stress, the crude fat content in both treatments increased first then decreased. During the low temperature treatment period (0 - 30 DAT), the crude fat content increased. The content in seeds treated with 200, 400 and

600 mg∙L⁻¹ GA₃ was 55.28%, 61.22% and 57%, higher than that without GA₃ treatment, and the contents all reached the highest point. During the low temperature treatment period (30 - 150 DAT), crude fat content decreased significantly. After 150 days, crude fat contents in different treatments were 32.5%, 24.52%, 32.49% and 29%, respectively. The decrease of crude fat content indicated that crude fat was hydrolyzed to fatty acids and sugars to provide for seed germination.

As the cold treatment duration increased, the lipase activity in both treatments varied (Figure 4(b)). The lipase activity in seeds treated with 400 and 600 mg∙L⁻¹ GA₃ increased steadily. After 150 days, lipase activity was 1.85 and 1.09 u∙(g∙h)⁻¹. However, during the low temperature treatment period (0 - 120 DAT), lipase activity in seeds treated with 0, 200 mg∙L⁻¹ GA₃ increased slowly. Then it increased rapidly. After 150 days, lipase activity was 2.68 and 2.86 u∙(g∙h)⁻¹, higher significantly than other two treatments.