Analysis On The Allelopathic Effects And Differential Proteome Of The Different Chinese Fir Clones Under Salicylic Acid Stress

Plant allelopathy is refered to any beneficial or harmful effect by one plant releasing specific secondary matters on other plants neighborhood. Autotoxicity, as one special manifestation of allelopathy, widely exists in many species in nature. By secreting, dripping and decomposing ways, plant organs such as leaves and roots release autotoxins to the environment and result in stress when t he autotoxins accumulate to a certain degree in the soil, which reduce the ability of metabolic regulation and inhibit seed germination and seedling growth of the same plants. Such phenomena are caused by autotoxicity.Autotoxicity caused by Chinese fir monoculture has been confirmed by the domestic scholars. The accumulation of autoxins can result in land degradation and productivity decline in Chinese fir plantations. Salicylic acid is one of the Chinese fir phenolic acids autotoxins. In this study by adding the exogenous phenolic acid into MS culture medium, the stress experiment have been carried out to research the different allelopathic effects of salicylic acid stress on some physiological processes such as osmotic factors, anti oxidase system, nucleic acids and proteins and endogenous hormones of the two kinds of Chinese fir clones (including tolerant type and sensitive type) leaves. Further more the differential proteome of two kinds of Chinese fir clones leaves was analyzed under salicylic acid stre ss.The main results were as follows:1．Allelopathic effects of salicylic acid stress on the osmotic factors in different Chinese fir clones leavesUnder low concentration stress, the degree of membrane lipid peroxidation appeared to be inhibited at the prophase, and the stimulated effect strengthened with the time prolonging and the concentration increasing. The stimulated effect of No.01 shows weaker than No.02, the MDA accumulation speed is also slower relatively.Plasma-membrane permeability appeared to be promoted at the prophase except N o.01 appeared to be inhibited under 40mg/L concentration. With the time prolonging, the stimulated effect on two clones decreased. But at the later period the stimulated effect enhanced largely and No.02 always appeared higher promoted effect than No.01 at all concentration levels. The stress destroyed cell membrane structure of No.02 seriously.Compared with the stimulatory effect on the soluble sugar content of No.02, that of No.01 came earlier and effect index also is bigger, but its inhibited effect is smaller at high concentration. The total free amino acids content of Chinese fir leaves manifested the stimulated effect throughout the stress period. On the whole the stimulated effect of No.01 is bigger than No.02．At 40mg/L, 80mg/L,120mg/L concentration, the stimulated effect indexes of No.01 are respectively 4,8, 3 times greater than No.02 at later period. The salicylic acid stress exert insensitive impact on the accumulation of praline.2．Allelopathic effects of salicylic acid stress on the antioxidant enzyme activities in different Chinese fir clones leavesUnder all concentration stress level the stimulatory effects on SOD activities occurred at the initial period and the intermediate stage, but transformed into inhibitory effect at the later period. The allelopathic effects on SOD activities of two clones showed differences from the intermediate stage mainly. The stimulatory effect on SOD activities of No.02 falls largely until the allelopathic effect on it vanished at high concentration, while No.01 still maintains the stimulated effect; at the later period, the effect on No.01 is lower than No.02 when appearing the inhibited effect.Salicylic acid stress performed stimulatory impact on POD activities of two clones leaves throughout the whole stress period. At the 10th day and 20th day, the stimulatory effect on the POD activity of No.Ol obviously bigger than No.02, but the stimulatory effect on No.02 is stronger at the 30th. Above results indicated that POD of No.01 clones leaves participated in el iminating H₂O₂ earlier.At the initial period, salicylic acid exerted an overall stimulatory impact on the CAT activities of two clones leaves and the stress led to higher stimulation for No.01 than No.02．At the later period salicylic acid still maintained to stimulate the CAT activity of No.01, but also at the higher the concentration, the smaller the stimulatory effect; while the stress has the inhibitory effect on No.02, and the higher the concentration, the bigger the inhabited effectSalicylic acid stress performed stimulatory impact on AsA-POD activities of two clones leaves throughout the whole stress period. At the initial period, No.01 more rapidly started the AsA-POD function to eliminate H₂O₂ and appeared higher stimulated effect than No.02 at all concentration level. Although at the intermediate stage the stimulated effect of No.02 is stronger than No.01, but the difference was not distinctive. At the later period under stress, the stimulated effect of No.01 raised again, but No.02 dropped largely.To the PPO activity, salicylic acid stress performed stimulatory effect on No.01 but the effect on No.02 displayed the characteristics of inhabitation-stimulation-inhabitation with the prolonging time. So the result indicated that No.01 started the PPO to anti-oxidation, positively participated in the resistance to poor surroundings but the PPO of No.02 participated in oxide compound eliminates is later than No.01, and is temporary.3．Allelopathic effects of salicylic acid stress on the nucleic acid and protein contents in different Chinese fir clones leavesIn most situations, for No.01, salicylic acid stress inhabited its DNA content synthesis and promoted that its DNA content synthesis but the effect characteristics on No.02 was opposite to No.01．It is thus clear that the salicylic acid stress performed different mechanism to nucleic acids of different clones. The allelopathic effect on nucleic acids mainly manifested at the RNA synthesis and decomposition mechanism of No.01, but t at the DNA synthesis and decomposition mechanism of No.02．At the initial period all concentration level had higher stimulatory effect on protein content of No.02 than that of No.01．At the intermediate and later periods, the stimulatory effect on No.02 appeared decreasing trend, while the stimulatory effect on No.01 increased continuously with the increasing concentration and its protein synthesis ability enhanced and surpassed No.02 finally. The evidence hinted No.01 aroused more protein to participate in the stress response which enhanced its resistance.4．Allelopathic effects of the endogenous hormones content of different Chinese fir clones leaves under salicylic acid stressOverall salicylic acid stress led to stimulatory effect on the ABA content, higher effect on No.01 than No.02; The contents of IAA, GA₃ and ZR appeared to be inhibited by the stress, inhibitory effect on GA₃, ZR content of No.01 is stronger than No.02; but the difference of the inhibitory effect on IAA content between the two clones is not significant. At the 30th day, the stimulatory effect on ZR content occurred under low and intermediate concentrations but GA₃ content was still inhabited, therefore, we inferred that compared with GA₃, ZR possibly earlier repaired its injured metabolism balance in Chinese fir leaves under salicylic acid stress.5．Optimization of two dimensional gel electrophoresis suitable for proteome of Chinese fir leavesThe results showed that most proteins of Chinese fir leaves were distributed in the pI range from 4 to 7．Lysis buffer with thiourea (2mmol/L) and DTT(60mmol/L) could improved the protein resolution greatly and sample quantities with 1．5mg could generate good indexes of distribution-average, clear and less tail, at the same time the gel could be developed further when iodoacetamide rose to 450mg/15mL in equilibration liquid. When the proteins were visualized with colloidal Coomassie blue which was compatible with Mass Spectrometry, we detected nearly 700 protein spots on the 2-DE gels with pH4-7 gradient IPG strip. Establishing 2-DE of Chinese fir leaves laid a foundation for differential proteomics.6．Map analysis of differential expression proteins in different Chinese fir clone leaves under salicylic acid stress on two-dimensional gel electrophoresisAt the 1st day under salicylic acid stress, 12 differential proteins were examined in No.01, including 6 up-regulated ones, 4 down-regulated ones and 2 added proteins newly under the stress; 2 differential proteins were examined in No.02, including 1 down-regulated protein, and 1 vanished protein under the stress. At the 3rd day under salicylic acid stress, 7 differential proteins were examined in No.01, including 1 up-regulated protein, 5 down-regulated ones and 1 vanished protein under the stress; 2 differential proteins were examined in No.02, including 1 down-regulated protein, 1 added protein newly under the stress. At the 5th day under salicylic acid stress, 12 differential proteins were examined in No.01, including 4 up-regulated ones, 3 down-regulated ones and 1 vanished protein under the stress; only 1 added protein in No.02 under the stress.As mentioned above, the differential expression protein quantities of different clones under salicylic acid stress were not alike at different treated time, when the outside injury produced, the gene regulation system of No.01 Chinese fir clone leaves were active and sensitive to response to the adverse circumstance signal and could comparatively quickly caused many genes to express or to close and led to change protein expression pattern, which benefite d to maintain the normal physiological activity and adapt or resist to outside injury.7．Mass spectrumetry identification and biological functional anlysis of differential expression proteins in different Chinese fir clone leaves under salicylic acid stressMass spectrum MALDI-TOF-TOF/MS and software MASCOT were used to determine and search data and the success ratio was 48%. All 8 proteins successfully identified were belonged to No.01 clones; none differential proteins of No.02 have been successfully identified.+ (1) . 33 kDa subunit of oxygen evolving system of photosystemâ…¡: relating to water decomposition and oxygen evolution of the PSâ…¡system;(2) . Heat Shock Protein: including glucose regulated proteins Grp78 and Hsp cognate70,Hsp90 that belong to the molecular Chaperone. These proteins participate in many processes such as protein folding, renaturation and degeneration of denaturation protein, protein transportation, replication, transcription and signal transduction of genetic material.(3) . Peptidylprolyl cis trans Isomerase: participating in the outset process of unusual protein degeneration and protein folding. This kind of protein is extremely essential for the protein to form spatial structure with the biological function activity.(4) . 26S proteasome : by combining with ubiquitin to degrade wrong folded or damaged proteins, 26S proteasome may prevent the gathering of non-function and potential toxic protein to medicate organism's resistance, which haa special defense function for the cell to resistance adverse circumstance.(5) . Proton shifting membrane ATP enzyme(F1 ATPase): participating in the process that the protons outside membrane enter into cell or the protons in cell are transported reversely out the cell, which is an important adjustment mechanism of the acid tolerant ability in cells.(6) . Glyceraldehyde-3-phosphoric acid Dehydrogenase (GAPDH): playing a vital role in producing the energy as one of the essential core enzymes in plant photosynthesis and breath process, simultaneously, having the functions of transcription promoter,microtubule arrangement and inducing apoptosis and so on.(7) . Microtubule protein: closely relating with morphology, material transportation, signal transduction and cell division and proliferation, being very sensitive to the environmental condition response.(8) . Prolyl tRNA synthetase (ProRS): urging Pro aminoacylation and improving utilization ratio of tRNA to contribute to the protein synthesis. And it may also affect the oxydase transcription and translation and prevent the damage by these enzymes accumulating.