Physiological And Quantitative Proteomics Analysis Of Brassica Napus Seed Germination In Response To NaCl

Seeds are the basis of plant regeneration, and seed germination is one of the most important phases of plant life cycle. Seed germination is very sensitive to salt stress. Salinity is one of the most significant abiotic stresses and it limits the productivity and geographical distribution of plants. Salt stress affects seed germination firstly, by impeding water uptake. Secondly, once salts have entered the seed, ion excess can lead to cellular injury, further reducing seed germination. At the cellular level, uptake of ions may disturb ion homeostasis and elevate the production of reactive oxygen species (ROS). ROS can induce oxidative stress which could damage the structure of biomacromolecules with potentially deleterious consequences on seed metabolism.Brasscia napus seed is a major soure of vegetable oil. In the present study, we use B. napus seeds as materials to investigate the molecular mechanisms of seed germination response to salinity tolerance. The germination rate and shoot and root length of seeds under NaCl (0,50.100and200mM) and mannitol (0,100.200and400mM) were investigated. The water content, membrane permselectivity, osmolystes, ROS, antioxidant enzymes and antioxidants were investigated in mature and germinatd seeds under NaCl (0,100and200mM) and mannitol (0,200and400mM). We also compare the contents of Na+, K+and Ca2+of mature seeds and germinated seeds under NaCl (0,100and200mM). Meanwhile,54differential expression proteins in mature and germinated seeds,46differential expression proteins in salt-stressed germinated seeds were identified by high throughput proteomics technology.Physiological results indicated that the contents of proline and betaine were increased in B. napus germinated seeds without treatment, but the content of soluble sugar was decreased. In the presence of100mM NaCl, the contents of soluble sugar, proline and betaine were increased. But in germinated seeds treated by200mM NaCl the contents of proline and betaine were increased. Under200mM NaCl. Na+content was increased dramaticalv. but that of K+and Ca2+were decreased. In the germinated seeds that without NaCl stress, malonaldehyde (MDA) content was increased. But under NaCl stress, both the MDA content and relative electrolyte leakge were increased in germinated seeds. ROS content was increased in germinated seeds in the presence of NaCl or not. The activitiies of enzymes that involved in superoxide dismutase (SOD) pathway, catalase (CAT) pathway, ascorbate-glutathione (AsA-GSH) cycle, glutathione peroxidase (GPX) pathway, and peroxidase (POD) pathway were increased in germinated seeds in the presence of100mM NaCl or not, and the substrates of AsA-GSH cycle were also increased. Under200mM NaCl, activities of most of the antioxidant enzyme showed no obverious changes or showed lower activities, compared with that in germinated seeds without NaCl treatment. Under the same condition, the acitivities of most antioxidant enzymes was not higher than that of the control, and the antioxidant enzymes activities under400mM mannitol were higher than that under200mM NaCl.Meanwhile, the protein profiles of mature seeds and germinated seeds under0,100and200mM NaCl were obtained by using techniques of proteomics. On the2-DE gels of mature seeds and germinated seeds,69reproducibly matched spots showed differential expression in abundance. After submitted to MALDI-TOF-TOF MS for identification,59spots were identified. Among these protein spots,54protein spots were identified as unique proteins in each spot,5spots were identified containing as more than one protein in each spot. On the2-DE gels of germinated seeds response to different NaCl concentrations,53reproducibly matched spots showed differential expression in abundance. After submitted to MALDI-TOF-TOF MS for identification,50spots were identified. Among these protein spots,46protein spots were identified as unique proteins in each spot,4spots were identified containing as more than one protein in each spot. During seed germination, the abundance of aldose reductase (AR) was increased, but that of late-embryogenesis abundant (LEA) proteins was decreased. Also. LEA abundance was decreased under100mM NaCl. But LEA abundance showed no obverious changes under200mM NaCl. compared with the control. The expression abundance of SOD, peroxiredoxin (Prx), and dehydroascorbate reductase (DHAR) was increased during seed germination. Also, the abundance of SOD and glyoxalase I (GLO) was increased under200mM NaCl. During seed germination, the abundance of RNA processing and protein fate related proteins was increased. But under200mM NaCl. the abundance of heat shock protein (HSP)70, unnamed protein containing ACD domain, and20S proteasome beta subunit was decreased. However, the abundance of heat shock cognate (HSC)70.1protein, HSP101, mitochondrial processing peptidase (MPP). HSC70-2, HSP60. and HSP90was increased. In addition, the abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) that involved in glycolysis, and malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and aconitate hydratase that involved in TCA cycle was also increased during seed germination. Under salt stress, the expression abundance of GAPDH and aconitate hydratase was increased too. During seed germination, methionine synthase (MES), S-adenosylmethionine synthetase (SAMS) and adenosylhomocysteinase (AdoHcyase) was up-regulated, but under salt stress the expression abundance of these proteins showed various changes.By integrating the analysis of physiological and proteomic results, we can conclude that (1) Under salt stress, B.napus germinated seeds maintain osmotic homeostasis by synthesis of soluble sugar, proline and betaine. Under200mM NaCl, ionic homeostasis in B.napus germinated seeds was broken and ionic toxicity was enhanced. Under100mM NaCl, the degradation of LEA proteins was not influenced, which was good for seed germination. But under200mM NaCl, the degradation of LEA proteins may be inhibited.(2) ROS scavenging in B.napus germinated seeds in the presence of100mM NaCl or not was through SOD pathway, CAT pathway, AsA-GSH cycle, GPX pathway and POD pathway. The up-regulation of SOD, Prx and DHAR suggests that these proteins may play important roles in ROS scavenging. Under200mM NaCl, ROS scavenging in germinated seeds was mainly through GST and POD pathways. And the up-regulation of GLO and SOD may helpful in scavenging hazardous substance.(3) During B.napus seed germination, RNA processing and protein fate were active. And under salt stress conditions, cytosolic protein synthesis metabolism and that in mitochondrion in B.napus germinated seeds were both enhanced to cope with salt stress.(4) During B.napus seed germination, soluble sugar was quickly mobilized, and provid energy for seed germination through glycolysis and TCA cycle. And the up-regulation of GAPDH and aconitate hydratase under salt stress may provid energy for germinated seeds to cope with salt stress.(5) During B.napus seed germination, methionine cycle was enhanced, but under salt stress conditions methionine cycle was disturbed seriously, affecting the resistance of seeds. These results may provide new clues for further research on salt-tolerant mechanisms in seed germination.