Molecular Mechanism Underlying The Regulation Of Lateral Root Formation By Multi-walled Carbon Nanotubes And Magnesium Hydride

Multi-walled carbon nanotubes（MWCNTs）have unique physical and chemical properties,and certain biological functions,including promoting the formation of plant roots.In recent years,the biological effects of hydrogen gas（H₂）have attracted attention.It has been found that H₂ participates in the processes of plant resistance,growth and development.Magnesium hydride（Mg H₂）is a kind of hydrogen storage material.The latest research has found that it can delay the senescence of cut flowers and promote the preservation of cut flowers.However,there are few reports on its effects on plant growth and development.In order to investigate the botanic effects of MWCNTs and Mg H₂,this study used alfalfa and Arabidopsis as research materials to explore the underlying molecular mechanisms of MWCNTs and Mg H₂ in inducing the formation of lateral root through the methods of pharmacology,anatomy,and genetics.The main results are as follows:1.MWCNTs induced the formation of alfalfa lateral root in a concentration-dependent manner,and 50 mg/L MWCNTs had the best effect.Using spectrophotometer and laser scanning confocal microscope（LSCM）analysis,it is found that MWCNTs can rapidly induce the production of hydrogen sulfide（H₂S）in alfalfa root.After 12 h treatment,the H₂S content reached the peak,earlier than the occurrence of lateral roots.These results indicate that H₂S may be as the downstream signaling molecule of the above-mentioned MWCNTs-induced effect.It is known that H₂S is an important signal molecule for inducing root morphogenesis.In order to further investigate whether it is involved in the process of MWCNTs inducing lateral root formation of alfalfa.The experiment further used H₂S scavenger hypotaurine（HT）and synthetic inhibitor _DL-propargylglycine（PAG）alone or in combination,and exogenous sodium hydrosulfide（Na HS,a H₂S donor）was used as a positive control.The results showed that the effect of MWCNTs and Na HS on lateral root formation of alfalfa could be inhibited by HT and PAG,accompanied by the decrease of endogenous H₂S.Further anatomical microscopic analysis showed that HT and PAG could also reverse the formation of lateral root primordial induced by MWCNTs and Na HS.The q PCR analysis found that both Na HS and MWCNTs can increase the expression of cell cycle genes（Ms CDKB1;1 and Ms CDKB2;1）in alfalfa roots related to lateral root formation.The combination of HT or PAG can reverse the promotion effect.To further explore the molecular mechanism,the model plant Arabidopsis was used for genetic verification.The results showed that compared with wild-type（WT）,the endogenous H₂S content and lateral root formation of the Arabidopsis cysteine desulfhydrase（DES）deficient mutant Atdes1 were inhibited,it could be reversed by external Na HS,but adding MWCNTs has no such effect.Further evidence showed that,compared with WT,the expression of mutant At CDKB1;1 and At CDKB2;1 were down-regulated,which could also be reversed by exogenous Na HS,while the addition of MWCNTs had no reversal effect.The above results indicate that H₂S is the downstream signal of MWCNTs induced lateral root formation.2.Pharmacological experiments showed that the appropriate concentration of Mg H₂（0.01 g/L）could induce the development of lateral root of alfalfa.Then,q PCR and ELISA kit were used to detected,it was found that exogenous addition of Mg H₂ could up-regulate SNAT gene expression and promote MT accumulation.SNAT expression reached a peak after 9 h,and MT content reached a peak at 12 h.Therefore,it can be preliminarily judged that MT may be the downstream signal molecule of Mg H₂ induced lateral root formation.It has been known that Melatonin（MT）can promote the formation of lateral root,and it has been found that it can be used as a downstream signal molecule to participate in the process of H₂ to improve the salt tolerance of Arabidopsis.However,it is not clear whether MT is involved in the development of lateral roots induced by Mg H₂ and its possible mechanism.Therefore,the MT synthesis inhibitor p-chlorophenylalanine（p-CPA）was further treated alone or in combination,and exogenous MT was used as positive control.It was found that p-CPA could reverse the formation of lateral root induced by Mg H₂ and MT,accompanied by the decrease of endogenous MT.Anatomical experiments found that Mg H₂ and MT can promote the formation of alfalfa lateral root primordial,and the addition of p-CPA also reversed the promotion effect,which is consistent with the phenotypic results.The result of molecular biology experiments showed that Mg H₂ up-regulated the expression of cell cycle genes related to the formation of lateral root,while p-CPA inhibited its regulatory effect.To further verify the results,Arabidopsis serotonin N-acetyltransferase（SNAT）deficient mutant Atsnat was used in our study.Compared with wild type,endogenous MT content,lateral root formation and cell cycle gene expression（At CDKB1;1 and At CDKB2;1）in Atsnat were inhibited.Exogenous Mg H₂ could not reverse the inhibition effect,which was consistent with the results in alfalfa.It is suggested that MT is involved in lateral root formation induced by Mg H₂.In summary,MWCNTs and Mg H₂ can induce the production of H₂S and MT to promote lateral root formation,and the regulation of cell cycle genes expression may be an indispensable key strategy in these processes.The above results provide a preliminary theoretical basis for the biological applications of MWCNTs and Mg H₂.