Functional Study Of Arabidopsis FAE1 And EXP2 In Seed Germination

Seed dormancy and germination are adaptive features of plants to adapt to environmental changes, seed germination is a very complex physiological process, regulated by both internal factors and external environmental factors. The main endogenous hormones involved in the regulation of seed germination are gibberellins and abscisic acid. Light and temperature are the two most critical environmental factors in controlling seed germination. Light and cold stratification could promote seed germination. Recently a large number of long-term in-depth studies have been made to investigate the molecular mechanism underline seed germination in response to light and temperature, identified a number of key genes involved in the light signaling pathways and temperature signaling pathways. In this study, according to the previous work and the information in the database, we identified several genes may be involved in regulating seed germination in response to light and temperature, we used genetics and molecular biology methods to analyse the expression pattern of these genes, its interaction with GA and ABA, and the role in the regulation of seed germination, meanwhile, we used molecular cloning and transgenic methods to verify the gene function. The main results are as follows:1. We used qRT-PCR and GUS staining methods to analyse the expression pattern of FAE1, found that FAE1 mainly expressed in siliques and germinating seeds. Germination assay revealed that the germination rate of fael is significantly higher than the wild-type, suggesting that FAE1 may act as a negative regulatory factor in seed germination. Hormone induction assay showed that FAE1 expression was repressed by GA. Freshly matured fael seeds were significantly less dormant than the wild-type, and less sensitive to cold stratification than wild-type. qRT-PCR analysis had shown that the expression level of FAE1 was down-regulated by cold stratification, suggested that cold stratification promoted seed germination by repressing FAE1. In germinating seeds, the expression of FAE1 was down-regulated by SPT. qRT-PCR analysis showed that FAE1 repressed the expression of GA3ox1, GA3ox2, GA20ox1, GA20ox2, GA20ox3 and CYP707A2, suggested that FAE1 could maintain seed dormancy by repressing GA biosynthesis and ABA catabolism. Germination test under salt stress and osmotic stress showed that fael was less sensitive to stress treatment, while FAEl overexpression line was more sensitive to stress than WT, suggested that FAE1 may reduce seed tolerance to salt stress and osmotic stress.2. qRT-PCR and GUS staining analysis showed that exp2 specifically expressed in germinating seed and radical, and after 24h imbibition, the expression of EXP2 reached its peak. GA and ABA induction assays showed that the expression of EXP2 was up-regulated by GA, but not influenced by ABA. We then analysed the expression of EXP2 in DELLA mutants, and found that almost all the DELLAs have a role in the inhibition of the EXP2 expression, including RGA, GA1, RGL1 and RGL2, and the RGL1 played the most dominant role. Germination assay revealed that exp2 germinated slower than WT, PAC treatment suggested that factors other than GA involved in the regulation of EXP2. In the stress treatment, exp2 showed higher sensitivity than WT, while the overexpression line was less sensitive compared to WT, suggested that EXP2 could improve seed tolerance to salt stress and osmotic stress.3. By searching the database we found that PIL5 have two splicing variants, one splicing variant lack the APB motif, we named it PIL5a, another variant contain the full length CDS, named PIL5β, qRT-PCR analysis showed that PIL5β expression level was higher than PIL5a. We then constructed the PIL5a overexpression line and PIL5β overexpression line respectively. Germination assay showed that PIL5a participated in the inhibition of seed germination. The full length splicing variant P1L5β overexpression line showed lower germination rate than WT under light treatment, suggested that the binding capacity of Phytochrome to PIL5 is limited. After both cold stratification and light treatment, all the overexpression lines showed almost 100% germination rate, suggested that other regulators would involved in the light signal pathway besides PIL5, and we presumed that the post-transcriptional level of PIL5 would be negatively regulated and leaded to an interrupt in the accumulation of PIL5 protein.4. We used spt-1, spt-2, spt-3,phyB-1 and their double mutants spt-1phyB-1, spt-2phyB-1 and spt-3phyB-1 to assess the relationship between SPT and PHYB in the regulation of seed germination in response to light and cold stratification, the results showed that the high dormancy of spt mutants were rescued to the wild-type level in the phyB-1 null mutant background, suggested that the control of seed germination by SPT is PHYB dependent. qRT-PCR analysis found that the GASox1 and GA3ox2 expression level were higher in double mutants than spt single mutants, suggested that the regulation of seed germination by SPT and PHYB converged to the regulation of GA metabolism.Through this research, our new results provide new evidences for us to have a better understanding of the molecular mechanism underline seed germination in response to light and temperature. The accumulated experimental evidence for the study of the physiological and biochemical characteristics of seed germination from this study will be important for us to understand the gene regulatory networks of seed germination, which will have important theoretical significance and important practical application value in the future.