Molecular Mechanism Of Multi-walled Carbon Nanotubes And Methane In Regulating Adventitious Rooting

As an important nano-material,multi-walled carbon nanotubes(MWCNTs)have been widely used in various aspects in life because of its unique physical and chemical properties.At the same time,many achievements have been made in the research of MWCNTs.At present,the research on MWCNTs mainly focuses on the toxicity of MWCNTs to animals,plants and microorganisms.Little is known about the mechanism of plant adventitious root formation induced by MWCNTs.Our experimental results show that MWCNTs can induce adventitious root formation in a concentration-dependent manner,which provides an important theoretical basis for studying the role of carbon nanotubes in plant root development.In addition,methane(CH4),which is the main component of natural gas and combustible ice,was found that could also promote the formation of adventitious roots in cucumber,but little is known about its possible molecular mechanism.The following main results have been achieved:MWCNTs can induce the occurrence of cucumber adventitious roots in a concentration dependent manner,and the 5 mg/L MWCNTs has the best induction effect.Using laser confocal and spectrophotometric method,it was found that MWCNTs treatment could change the content of endogenous hydrogen peroxide(H2O2)in cucumber hypocotyl.The analysis of the timing change of endogenous H2O2 showed that there were two different peaks in the timing change,which means the change of endogenous H2O2,and reached the peak after 12 h treatment.In addition,pharmacological experiments showed that the induction effect of MWCNTs and H2O2 could be significantly blocked by H2O2 scavengers dimethylthiourea(DMTU)and ascorbic acid(AsA),suggesting that H2O2 might be involved in the induction of cucumber adventitious roots.Further experiments showed that MWCNTs treatment could up regulate the transcript level of Rboh,up regulate the activity of NADPH oxidase as well as cell-wall POD,suggesting that the main source of endogenous H2O2 were came from NADPH oxidase and cell-wall POD.H2O2 subcellular localization with CeCl3 staining showed that MWCNTs treatment also showed H2O2 accumulation.Using the qRT-PCR method,it showed that MWCNTs and H2O2 also raised the target genes transcription involved in adventitious root formation,after that we have also noticed that MWCNTs could through down-regulation of miR167 and up regulation its target gene ARF8 expression levels which is auxin response depended to further control the regulation of MWCNTs induced adventitious root formation in plants.In order to further verify that whether MWCNTs promoted the adventitious root formation by regulating the change of endogenous H2O2 content or not,we used the H2O2-related mutant AtrbohD and AtrbohF of Arabidopsis to confirm.The results indicate that AtrbohD and Atrboh exposed to MWCNTs were not significantly promote the endogenous production of H2O2 as well as the formation of adventitious roots by using laser scanning confocal microscopy and spectrophotometer.However,when exogenous H2O2 added,it could significantly reverse the status,which reveals that H2O2 were downstream signal molecules in the presence of adventitious root induction exposed to MWCNTs.Therefore,we concluded that the changes of endogenous H2O2 were involved in the development of plant adventitious roots induced by MWCNTs.Using spectrophotometer and LSCM showed that CH4 treatment could promote cucumber hypocotyl endogenous NO production,and promote embryo root primordium changes,then induce the development of adventitious roots at the same time.Above changes were significantly blocked by cPTIO and PTIO.Exogenous addition of CH4 could alleviate the above adventitious root inhibition to a certain extent.Subsequent qRT-PCR experiments showed that CH4 and NO could regulate target genes of adventitious root formation in the process of transcriptional level,including:CsDNAJ-1,CsCDPKl,CsCDPK5,cell cycle gene CsCDC6 and two biotin responsive genes CsAux22D-like and CsAux22B-like,which were relied on the NO signal transduction pathway.The post-translational modification of protein experiment has also showed that CH4 has been involved in the S-nitrosylation.This study showed that NO played a signaling molecule in CH4 induced adventitious roots of cucumbers.Adventitious roots increase the area of nutrient uptake,support and fix plants;on the other hand,they are also important agronomic traits.Our results showed that MWCNTs and CH4 could promote adventitious rooting.It enriches the complex signal transduction networks in which MWCNTs and CH4 are involved,what’s more,it also provides a theoretical basis for us to further explore new plant growth regulators.