| Tree peony is an important economical crop, with high medicinal, ornamental and edible value. Seed sowing is important propagation way in tree peony, which is always used in large-scale production of oil and medicinal tree peony. It will cost about six months under field conditions from sowing to germination when the seeds were sown in proper time, while it needs 1- 2 years when the seeds are not sown in proper time(or cold storage seeds), which seriously affects the breeding efficiency and restricts the industrial development. The seeds of Paeonia ostii ‘Fengdan’, which are widely sown in seedling production, were used as materials in this work. The sowing seeds of Paeonia ostii ‘Fengdan’ were grew under 25 °C and 4 °C conditions, respectively. The mechanism of seed hypocotyl germination was explored by physiological substance measurement, starch grains observation, dynamic proteome and gene expression analysis. The main results were as follows:1. The seed hypocotyl of Peaonia ostti ‘Fengdan’ could germinate under 25 °C treatment, while they delayed germination under 4 °C treatment. Under 25 °C treatment, the moisture content, longitudinal diameter and fresh weight of seeds increased, while the starch and soluble sugar content decreased, and starch grains fast degradated; soluble protein content showed a down to up pattern, while ABA and GA3 content change showed a decreased trend and an up-to-down trend respectively. Under 4 °C treatment, the moisture content, longitudinal diameter and fresh weight of seeds did not change significantly any more after seed swelling, while the starch content remained stable and starch grains degradated much slower compared with that under 25 °C treatment; the trends of soluble sugar, soluble protein and GA content under 4 °C treatment were all similar to that under 25 °C treatment, and the GA3 content of 17 d after sowing(DAS) seeds under 4 °C treatment was significantly lower than that under 25 °C treatment(the testa rupture stage) and the ABA content of seeds under 4 °C treatment increased rapidly after a short time decrease, and it was significantly higher than that under 25 °C treatment. It was deduced that the degradation of starch grains could provide energy for seed germination; the high level of ABA content(under 4 °C treatment) and GA3 content(in the testa rupture stage under 25 °C treatment) delayed and promoted seed hypocotyl germination respectively.2. A total of 100 protein spots were recongised from 2-DE gels and identified by mass spectrometry in Paeonia ostii ‘Fengdan’ seed during 8 seed growth stages under 25 °C and 4 °C conditions, with 35 differential expression proteins, 39 specific proteins under 25 °C treatment and 27 specific proteins under 4 °C treatment. The proteins were identified by MALDI-TOF/TOF MS. A total of 97 protein spots were identified, which involved in 9 main categories, such as carbohydrate metabolism, proteometabolism and amino acid metabolism, lipid metabolism, stress-related proteins and etc. Further analysis showed that a lot of proteins appeared at the testa rupture stage under 25 °C treatment, and most of the proteins involved in the carbohydrate metabolism. A large number of stress-related proteins appeared under 4 °C treatment. A GA synthesis associated protein and an ABA-induced protein kinase were found under 25 °C and 4 °C treatments respectively. It was deduced that seed germination might be regulated by ABA and GA. In addition, the glucose and ribose alcohol dehydrogenase content and glycolytic enzymes content showed a down-up-down and an up-to-down trend respectively. It was suggested that there was a basic pattern of oxygen consumption in seed germination and the seed germination was regulated by the oxidative pathways.3. Four carbohydrate metabolism and two hormone related genes expression were studied. The expression of enolase gene and GAPC both showed an up-to-down pattern under 25 °C and 4 °C treatments, and it is significantly higher at the early stages of seed growth under 25 °C treatment than that under 4 °C treatment. It was deduced that two high expressed enzymes could promote glycolysis and support high energy for seed hypocotyls germination. The expression of malic enzyme gene and endochitinase gene continuously decreased under 25 °C treatment, while malic enzyme gene showed a down-to-up trend under 4 °C treatment with a higher level at the late stages of seed growth. So it was surmised that 4 °C low-temperature delayed the oxidation pathway and inhibited seed germination. The expression of auxin related gene PCNT115 showed a down-to-up trend under 25 °C and 4 °C treatments; the expression of FPPS showed a low level at early stages of seed growth and then dramatically increased in 17 DAS under 25 °C treatment(significantly higher than that under 4 °C treatment), and it showed an up to down trend under 4 °C treatment. It was suggested that FPPS could positively regulated GA and promoted germination. Combined with the expression under the protein level, the expression trend of GAPC was similar to that under protein level, while the expression trends of PCNT115 and endochitinase gene were slightly different from that on protein level.4. The expression of four ABA and GA signal transduction key genes were tested. The results showed that the expression pattern of Pob ZIP was similar to ABA content change, and the expression level of Pob ZIP under 25 °C treatment was significantly lower than that under 4 °C treatment. The expression of Po GID1 a and Po GAI both showed an up-to-down trend, which was similar to the trend of GA3 content. The expression of Po GID1 a at early and middle stages of seed growth under 25 °C treatment was significantly higher than that under 4 °C treatment and the expression of Po GAI under 25 °C treatment(21 DAS expected) was significantly lower than that under 4 °C treatment. The expression of Po MYB4 showed an increasing trend under 25 °C treatment, while it increased slightly at the early stages of seed growth and then rapidly decreased under 4 °C treatment. On the whole, the expression level of Po MYB4 under 25 °C treatment was significantly higher than that under 4 °C treatment, at 7- 100 DAS. This study suggested that ABA inhibited seed germination by promoting the expression of Pob ZIP; GA promoted seed germination by regulating signal transduction genes(Po GID1 a, Po GAI and Po GAMYB4). |
