Effects Of Overexpressing Phospholipase C Gene On Resistance To Abiotic Stress Of Transgenic Maize Plants

Early studies have indicated phospholipid signaling plays a key role in plant growth, development and in responses to environmental stress.Phosphoinositide-specific phospholipase C(PI-PLC)is a critical enzyme in the phospholipid signaling pathway. Signals outside the cell can be perceived at the cell membrane by receptors linked to a variety of signaling pathways,especially phospholipid signaling pathway.In this pathway,signals activate PI-PLC,which catalyzes the hydrolysis of phosphatidylinositol (4,5)bisphosphate[PtdIns(4,5)P₂]to form diacylglycerol(DAG)and Ins(1,4,5)P₃ (IP₃).In plants,DAG is rapidly phosphorylated by the diacylglycerol kinase(DGK)to phosphatidic acid(PA).IP₃ and PA,both as second messenger,play important roles on various stress signaling in plants.Compared with animal systems,the physiology roles of PI-PLC in plants are not well clear now.The purpose of the present study is to explore the role of a maize PI-PLC(ZmPLC1)gene on abiotic stress responses using the transgenic maize plants constitutively expressing sense or antisense ZmPLC1 gene.Abiotic stresses such as drought,salt and low temperature are main factors limiting the yield and quality of crops.So it is important step to breed new crop varieties that are more tolerant to these abiotic stresses so that new land can be brought under cultivation to meet the progressively demand of the society on farm produce.Maize(Zea mays L.)is an important crop all over the world.It is a profound work to enhance the resistance of maize to abiotic stress by transgenic approach.In this paper,transgenic maize plants expressing ZmPLC1 transgene in sense or antisense orientation were generated from the transformed embryogenic(Typeâ…¡)calli by Agrobacterium tumefaciens-mediated. Molecular analyses revealed that the sense or antisense ZmPLC1 transgene has been integrated into maize genome,and inherited into T1 to T2 progenies as a Mendelian pattern.High levels expression of the transgene was confirmed by real-time RT-PCR and PI-PLC activity assay.Moveover,the resistance of transgenic plants to various abiotic stresses was also studied.The main results presented in this dissertation are as follows: 1.Agrobacterium tumefaciens-mediated transformation of elite maize inbred lines and molecular analyses of transgenic plantsIn this study,to generate transgenic maize plants expressing the ZmPLC1 gene in sense or antisense orientation,embryogenic(Typeâ…¡)calli derived from the immature embryos of maize was used as donor cells for Agrobacteriurn tumefaciens-mediated transformation.A.tumefaciens strain LBA4404 containing the binary vector pCUA-ZmPLC1-als(The T-DNA region of the vector containing sense or antisense ZmPLC1 gene and mutational chlorsulfuron-resistance acetolactate synthase gene als.) was used in all experiments.Transformants were selected on the selective medium containing chlorsulfuron at 0.5-1.0 mg/L,and transgenic plantlets were regenerated from those resistant calli.T0(plant regenerated from transgenic Typeâ…¡callus),T1 and T2 plants were artificially selfed to obtain the next generation.Molecular analyses such as polymerase chain reaction(PCR),Southern blot and real-time RT-PCR were performed. The results showed that the sense or antisense ZmPLC1 gene had been integrated into maize genome and inherited to the following generations,and expressed normally.In most of the transformants,a single copy insertion of the sense or antisense ZmPLC1 occurred.Homogenous transgenic lines carrying the transgene were identified in T2 generations.The real-time RT-PCR was performed on single-copy insertion events using total RNA from transgenic lines,and the RNA from WT as negative control to monitor the relative expression levels of the transgene.The results indicated that the expression levels of the transgene were much higher than that of endogenous ZmPLC1 gene.For the PI-PLC assay,four homogenous transgenic lines,including two ZmPLC1 sense transgenic lines(line 1,line 2)and two antisense lines(line 5,line 6)were selected,WT served as control.The activity of ZmPLC1 was determined by scintillation counting of water-soluble radioactivity after incubation of micellar[³H]PI 4,5-P₂ with highly purified plasma membranes separated from the roots of maize seedlings.The rate of hydrolysis of[³H]PIP₂ by PI-PLC from sense lines was about 1-2-times higher than that of WT,and the activity of PI-PLC from antisense lines was lower compared with WT. The results showed that the increased PI-PLC activity in sense transgenic lines should result from overexpression of sense ZmPLC1,and the lower PI-PLC activity in antisense transgenic lines derived from the expression of antisense transgene. Based on the data presented above,the four homozygous transgenic lines(two ZmPLC1 sense transgenic lines 1,2 and two ZmPLC1 antisense transgenic lines 5,6) were selected for further study to evaluate the tolerance of transgenic plants to diverse environmental stresses,and WT served as control.2.Drought resistance test of transgenic maizeWhen maize seeds were placed between two layers filter paper soaked by MS salts solution containing 15%or 20%(w/v)PEG-6000 for germination,the germination of the sense ZmPLC1 transgenic seeds was faster and the final germination rate was higher than that of WT;the germination speed of antisense transgenic seeds was comparative with WT while the final germination rate was lower.The differences of final germination rate between the transgenic and WT seeds were more obvious with the stress enhancement.After the maize seedlings grew to the three-leaf stage,the roots of seedlings were placed in MS salt solution containing 12%(w/v)PEG-6000 for 1,3,and 7 days.Under osmotic stress conditions,the sense ZmPLC1 transgenic lines had higher relative water content and chlorophyll content,better osmotic adjustment,lower percentage of ion leakage and less lipid membrane peroxidation than the WT;whereas those expressing the antisense ZmPLC1 transgene exhibited an inferior characters compared with the WT.The osmotic stress had significant effect on the levels of ABA,IAA,ZT and GA₃ in maize leaves.The four endogenous hormones levels were higher in sense transgenic lines than those of WT and antisense transgenic lines.The phenotype of various maize seedlings subjected to osmotic stress suggested that the high levels of IAA,ZT and GA₃ were beneficial on the growth of plants sufferred from osmotic stress.The changes of endogenous hormones contents might be affected by the expression of sense or antisense ZmPLC1 transgene in maize under osmotic stress either directly or indirectly,and the changes were consistent with the alteration of the resistance to osmotic stress.Exogenous application of ZT or GA₃ could partly prevent chlorophyll breakdown,improve the growth of maize seedling subjected to osmotic stress and reduce the differences between the sense or antisense ZmPLC1 transgenic and WT lines,this provided evidence to endogenous hormones could play an important role in the enhancing tolerance of plants to osmotic stress. The maize plants at 10-leaf stage were subjected to drought stress for 3 weeks by withholding irrigation to maintain the relative water content of soil to about 15%,and then watered sufficiently.During the drought stress,the decreases in photosystemâ…¡activity and net photosynthesis rate of sense transgenic lines were less than that of WT, whereas those of antisense transgenic lines were more than that of WT.The plant height, green leaf number and the length of leaf with ear were measured at flowering stage.The data demonstrated that,the sense transgenic plants were higher,they had more green leaves and longer leaf with ear than the WT;whereas those expressing the antisense transgene exhibited an inferior characters compared with the WT.The effect of drought stress on the growth of sense ZmPLC1 transgenic plants was not significant,while those of antisense transgenic and WT plants were inhibited markedly.After the ear maturation, maize plants were harvested and dried for the biomass determination.The yield and the overground total biomass(dry weight)per plant of sense lines were 10.5-17.7%and 11.4-17.8%more than that of WT,while those of antisense lines were 7.5-14.4%and 6.9-12.6%less than that of WT,respectively.Namely,the overexpression of ZmPLC1 gene could enhance the yield and total biomass of maize plants under drought stress, while inhibition expression of ZmPLC1 gene reduced the resistance of plants to drought stress.3.Salt resistance test of transgenic maizeMaize seeds were placed between two layers filter paper soaked by MS salts solution containing 150 mM or 200 mM NaCl to germinate.The differences in germination rate between transgenic and WT seeds were not obvious under 150 mM NaCl,however,the differences occurred under 200 mM NaCl.The germination rates of sense transgenic lines were higher than that of WT in final,whereas those of antisense lines were slightly lower compared with WT under 200 mM NaCl.The maize plants at four-leaf stage were subjected to salt stress for 6,12,and 18 days by adding 120 mM NaCl in MS salts solution,and then watering with MS salts solution for 6 or 12 days.During salt stress,compared with the WT,the sense ZmPLC1 transgenic lines showed tolerant to salt stress based on the responses to a number of stress-related parameters,including higher relative water content and chlorophyll content, lower cell membrane damage,and more soluble sugars and free amino acids which was in favor of maintaining relatively low osmotic potential;whereas those expressing the antisense transgene exhibited an inferior characters compared with the WT.In recovering from salt stress,the differences of various maize lines were gradually decreased,while the sense ZmPLC1 transgenic lines were still grow better than the WT and antisense transgenic lines.It indicated that the resistance to salt stress was enhanced by overexpression ZmPLC1 gene in maize.4.Cold resistance test of transgenic maizeAt 25℃,the germination rates of maize seeds from different lines were all above 85%,and did not show significant differences.At 15℃,the length of time for germination of transgenic maize seeds was comparative to that of WT,however,the germination rates of the sense transgenic seeds were higher than the WT in final,and those of antisense transgenic seeds were lower than the WT.At 10℃,the length of time for germination and the germination rate in all maize seeds decreased markedly in final at different rates.The germination of sense transgenic seeds was ahead of that of WT and antisense transgenic lines for 3-5 days,and the germination rate in average of sense lines was much higher than the WT and antisense lines in final.In normal conditions(25℃),the differences in the shoot growth rate of geminated seeds were not obvious between transgenic and WT lines.At 15℃,the shoot growth rates were differently decreased in both WT and transgenic lines as compared to 25℃. Compared with that of 25℃,the decrease in shoot growth rate of sense lines was less than that of WT,while those of antisense lines was more than the WT.At 10℃,the shoot growth rates were significantly decreased in all lines.Comparison with that of 25℃,the shoot growth rates at 10℃of sense lines were about 17.4-18.9%,and those of WT and antisense lines were only 11.2%and 5.2-7.5%,respectively.The maize plants at four-leaf stage were subjected to cold stress for 8 days at 10/8℃(day/night),and then recovering for 8 days at 25℃.Under cold stress,the injury of plants from sense transgenic lines was significantly less than those of WT and antisense lines.In addition,the sense transgenic lines showed tolerant to cold stress based on the responses of a number of stress-related parameters,including higher chlorophyll content, lower cell membrane damage and better osmotic adjustment than the WT and antisense plants.When these cold treated seedlings were transferred to 25℃,the recovery of growth occurred in both sense transgenic and WT plants,while the antisense transgenic plants recovered slowly;the differences of different lines were also characterized by the results of related physiological parameters.Based on the data presented above,it indicates that the overexpression of ZmPLC1 gene in maize enhanced the tolerance of plants to abiotic stress.We conclude that an increase or reduction of ZmPLC1 expression impacts the growth of maize plants under abiotic stress,and the ZmPLC1 gene as a stress-inducible gene may play an important role in regulating plant response to abiotic stress.These observations also imply that ZmPLC1 gene is a key factor in signal transduction events under stress conditions. Therefore,it has profound significance to explore the role of ZmPLC1 gene in plant abiotic stress signal transduction and amplification,and could develop a new way to obtain maize varieties with better tolerance to abiotic stress.