Studies On Molecular Ecological Mechanisms Of Rice Allelotathy In The Suppression Of Weed Mediated By Nitrogen Nutrient

As one of the most important chemoecological phenomenon,allelopathy refers to the favorable or deleterious effects of one plant on its neighboring plants(including microbes)through the release of secondary metabolism chemical compounds into the environment via volatilization,leaching,excretion,and decomposition.Using rice allelopathy to control weeds in agroecological system has been one of the hottest researching fields,which will be a key technology to control weed safely and friendly in 21^stcentury. The previous study mainly focused on the biological characteristics of alleopathy such as the properties of genetics,physiology and isolation and identification of allelochemicals.Research has indicated that allelopathy is a quantitative trait susceptible to environmental regulation.However,the change of allelopathy potential in variable environment is always a controversial issue.Nutrient stress is a distinct problem in productive practice,therefore,the paper used the universally acknowledged allelopathic PI312777 and non-allelopathic rice Lemont as materials and maked some studies on the molecular mechanism of allelopathy potential change in allelopathic rice accession under low nitrogen supply,which will be the base of manipulating allelopathy potential by molecular technology and screening allelochemicals.At first,in order to estimate allelopathy potential resonably,and seperate allelopathy potential and resource competition includedin the biointerference previously,a new bioassay methodology, allelopathy-competition separation based(ACS)was designed,and ACS was introduced to estimate the allelopathy potential under nitrogen stress,rooting ability and microscopy of bamyardgrass root tip were executed to explore the influence of the used hydroponic solution from allelopathic and non-allelopathic rice accessions on barnygrass(Echinochloa crusgalli L.).The result showed that the resource competition of PI312777 caused 26.34%-29.14%reduction in IR and its allelopathic effect on the target weed was 38.5%-51.4%in terms of inhibition rate(IR)under different nitrogen treatments.The difference is significant between allelopathic effect and resource competition,the effect of allelopathy is greater than that of resouce competition.However,the small deviation of the two curves in Lemont was found under normal or rich nitrogen conditions(10-20mg/L)in hydroponic culture,showing the lower values of resource competition and allelopathy as well as insignificantly different between them.Reverse was true in lower N supply,performing the stronger resource competition in Lemont.These findings suggested that rice aeeession PI312777 had higher ability in the interference with the target weed,barnyardgrass,performing stable and higher ability in resource competition as well as the increasing tendency in allelopathic potential in the suppression of the companied weed,barnyardgrass as N supplies decreased in the cocultured system. The reverse was true in the ease of Lemont,flee accession,exhibiting its lower allelopathic potential in all nitrogen treatments,and higher resource competition ability as N supplies decreased and resulted in reduced growth of barnyardgrass(47.99%),which indicated that resource competition ability was contributed to the barnyardgrass inhibition ability.The stronger resource competition ability may be induced by the limited resource in riee/barnyardgrass co-culture system under N deficiency.Allelopathy originated the synergistic effect of several allelochemicals,many enzymes and its interaction were involved.Previous studies mainly analysised the action of individual enzyme from the view of physiology.The differential proteomics can reveal all of the enzymes and proteins related with the allelopathy,and display the higher systemic.So,differential proteomics was used to analysis the differential expression of proteins in allelopathic and non-allelopathic dee accessions under low N supply.Twenty-one different proteins were detected because of the nitrogen effect by differential proteornics,of which,twelve proteins were from allelopathic rice,and three proteins were decreased functioning in signal transduction, ATP synthesis,nucleic acids synthesis,and nine proteins were upregulated including allelochemical synthesis related protein,signal transduction,phytoalexin,phenolic synthesis,gene expression and regulation.Nine genes are from non-allelopathic rice,of which,the expression of five proteins was decreased as N supply decreased including allelochemieal synthesis related,cell division related,growth regulation related,fatty acid synthesis related,light defence related,polyamine synthesis related.And the expression of four proteins was increase including ATP synthesis,phenolic acids synthesis,terpene degradation,which indicated the proteins of nutrient metabolism related were decreased and proteins of defence were increased including phenolic acid synthesis in allelopathic dee under low N supply,the reverse was true in non-allelopathic rice.Phenolic acids can act as the function of stress defense in previous studies.In the experiment,phenolic acids were increased in rice accessions under low N condition.However, there was different in quantity,two proteins were detected in non-allelopathic rice,and four in allelopathic rice,which indicated the phenolic acids were the stress responsive element in plant,and in another hand, phenolic acids were related with the allelopathy characteristic.Researches have suggested the allelopathy ability of phenolic acids is primarily evidenced.The paper revealed the expression of phenolic acids was enhanced under low N supply by differential proteomics. However,the probability of the false positive may occur in any molecular technology,differential proteomics is no exception.Some differential proteins may be neglected because of the low contents of proteins or the matching.Hereby,in order to further make clear the change of phenolic acids in allelopathic rice under low N condition,FQ-PCR was applied to compare all of the nine key enzymes involved in the metabolism of phenylalanine that is the main metabolish pathway of phenolic acids.The resulted showed that nine key enzyme genes were differentially expressed when the two rice accessions were exposed to the low nitrogen stress.In roots,all of the nine genes were up-regulated in allelopathic rice accession,and seven genes were down-regulated except PAL and cinnamoyl-CoA with slight increase.The same tendency of the nine genes was found in leaves,which indicated that the increased allelopathic potential induced by low nitrogen supply was related with the phenolic acid synthesis metabolism.The contents of phenolic acids in roots,leaves and exudates by HPLC detection showed that the contents of phenolic acids in roots,leaves and exudates of allelopathic rice accession were higher than that in non-allelopathic rice.And compared with allelopathic rice,no significant change was deceted in the non-allelopathic rice under two nitrogen levels. Regardless in the normal and low nitrogen levels,the higher contents of phenolic acids were detected in the allelopathic rice than that in non-allelopathic rice,the difference in the low nitrogen level was more significant,especially,salicylic acid(SA)exhibited the most increase in all of the detected phenolic acids. All of the results suggested that phenolic acids were one of the main allelochemicals in allelopathic rice accession in the low nitrogen supply.Phenolic acids as main allelochemicals were suspected because of the low concentration caused by the metabolism of soil microbial.However,the terpene can valid in low concentration,so terpenes were thought as main allelochemicals.The paper also examined all of the key enzymes of isoprenoid metabolic pathway related to the terpene metobalism by FQ-PCR to make know if the terpenes play a key role in allelopathic rice under N stress.The result suggested that eleven out of 12 key genes involved in isoprenoid metabolic pathway in rice had the same gene expression patterns under low nitrogen supply in the two rice accessions. The results suggested that the related genes involved in isoprenoid metabolic pathway in the two rice accessions possessed the same or the similar ecological behavior in the responses to the changes in nitrogen supplies in the molecular scale,suggesting that the terpenes was not related closely to the increased allelopathic potential in low nitrogen supply,therefore they could not serve as the main allelochemicals in allelopathic rice PI312777 under low N condition.As is well known,the micro-ecology environment and functional groups of soil microbials will vary according to the quantity and categories of exudates in plants.It is obvious that the increased phenolic acids synthesised and secreted by allelopathic rice in N deficiency can influence the structure of soil microbials because of the chemotaxis of microbial.Thereby,the author studied the structure difference of soil microbial in allelopathic and non-allelopathy rice accessions under normal and low N level,and planed to obtain some knowledge about chemotaxis of soil microbial induced by phenolic acids.The results showed that there were only sixteen differental T-RFs caused by the diffenent of accessions in all of the detected 175 T-RFs,which suggested the main.difference in vertical distribution and horizontal distribution of microbial was induced by N effect.On the whole,the microbial is most rich in low nitrogen supply.And at the same nitrogen supply, the diversity of microbial in rhizosphere of allelopathic rice is greater than that in non-allelopathic rice rhizosphere.The quantity of microbial in the rhizosphere of allelopathic rice is more than that in 0-Scm layer, and the quantity of microbial in non-allelopathic rice and allelopathic rice in normal N supply was on the contrary,that is to say,the quantity of microbial in 0-5cm layer was more than that in the rhizosphere.And the microbial in 0-5cm layer and 5-20cm layer was richer than that in other samples.The different microbial caused by N nutrient was comprised of N cycle related,acids production and eosinophilic microbials related such as propionibacterium and acidithiobacillus,and clostridium related which can produce exotoxin, spirochaeta related which can transduct and defense,and some pathogens such as mycoplasma,phytoplasma and klebsiella,which is correlated to the more C source and corresponding micro-ecology environment provided by phenolic acids in the secretion of allelopathic rice under N deficiency.In summary,the paper introduced a new bioassay,seperated the allelopathy from the resource competetion,and captured the international controversy,and offer the reference of allelopathy evaluation Furthermore,different molecular technologies such as differential proteomics,FQ-PCR,T-RFLP,HPLC were used to explore the molecular mechanism of allelopathy potential change in low N stress.All of the results illuminated the phenolic acids were the key allelochemicals in allelopathic rice PI312777 under low N stress.The combined approach of molecular technologies used for present studies was ont only to effectively control the "false positive" caused by single molecular technology,but also to make our research work systemically in terms of experimental design,which can be considered as the supplement of previous research of physiology.The results supported that allelopathy charateristic can be regulated and will be the base for the isolation and identification of allelochemicals as well as for the specific plant breeding.