Haem Oxygenase/carbon Monoxide-,Hydrogen Gas-,and Multiwall Carbon Nanotube-regulated Lateral Root Formation

Carbon monoxide(CO)is generated from degradation of haem by haem oxygenase(HO,EC 1.14.99.3)in plants.Results obtained from animal research showed that hydrogen gas(H2)alleviated the damage caused by stresses,besides acting as a green fuel.In additional,carbon nanopartical,such as multiwall carbon nanotube(MWCNT),was indicated to promote plant growth.However,they effects on lateral root formation and molecular mechanism are still unclear.We studied the topics and acquired results as follows:1.Firstly,we tested whether haem oxygenase-1(HO-1)is involved in the hydrogen peroxide(H2O2)-induced lateral root formation due to its nature as a signalling system in root organogenesis.The results showed that 0.1 mmol/L H2O2 mimicked the inducible effects of the HO-1 inducer haemin on the up-regulation of tomato HO-1(SIHO1)expression,carbon monoxide(CO)synthesis and thereafter lateral root formation.However,1 mmol/L H2O2 brought about the contrasting responses.The above inducible or inhibitory responses were blocked obviously after the addition of the HO-1 inhibitor zinc proporphyrin IX(ZnPP)or haemin,both of which were reversed by CO or ZnPP.Further results showed that haemin-induced responses were not altered by a H2O2 trap dimethylthiourea(DMTU).When applied alone,DMTU not only decreased H2O2 content,also inhibited SIHO1gene expression and lateral root development,which were recovered by haemin or CO.Molecular evidence indicated that H2O2-modulated expression of target genes responsible for lateral root formation were blocked by ZnPP,but rescued by CO.Further data showed that salinity-induced up-regulation of HO-1 expression and lateral root formation was dependent on the H2O2 generation.In summary,above results demonstrated a novel role of HO-1 in the H2O2-induced tomato lateral root formation.Results would widen the knowledge on the function and molecular of SlHO1 involved in H2O2-modulated tomato lateral root development in details.2.In rapeseed seedlings(Brassica napus),the application of lower doses of NaCl(10 mmol/L)and polyethylene glycol(PEG)(2%)mimicked the inducible effects of naphthylacetic acid and the HO-1 inducer haemin on the up-regulation of BnHO1 and subsequent lateral root formation.Contrasting effects were observed when a higher dose of NaCl or PEG was applied.The above inducible and inhibitory responses were blocked significantly when the HO-1 inhibitor ZnPP or haemin was applied,both of which were reversed by the application of carbon monoxide or ZnPP,respectively.Moreover,the addition of ZnPP at different time points during lateral root formation indicated that BnHO1 might be involved in the early stages of lateral root formation.The auxin response factor transcripts and the auxin content in seedling roots were clearly induced by lower doses of salinity or osmotic stress.However,treatment with the blocker of polar auxin transport N-1-naphthylphthalamic acid prevented the above inducible responses conferred by lower doses of NaCl and PEG,which were further rescued when the treatments were combined with haemin.Taken together,results in this part suggested a novel role of the rapeseed HO-1 gene,as well as its catalysis product CO,in salinity and osmotic stress-induced lateral root formation,with a possible interaction with auxin signalling.Data would make the function and related signal tranducion pathway of BnHO1 clearly in rapeseed lateral root formation modulated by environmental factors.3.Pharmacological text showed exogenous H2-promoted lateral root formation is a universal phenomenon.Further results demonstrated that H2 enhaced H2 production in tomato seedling roots,mimicking the effects of NAA.Interestingly,H2 did not only induce lateral root processing,but also accumulated NO generation in tomato roots,which were sensitive to NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt(cPTIO).Additional data indicated nitrate reductase,rather than NOS-like protein or S-nitrosoglutathione reductase(GSNOR),was the potential enzymatic source of NO biosynthesis responsed to H2.Molecular evidence showed cell cycle regulatory genes responsible for lateral root organogenesis,such as SICDKA1,SlCYCA2;1,and SlCYCD3;1,were H2-modulated target genes.Anatomic and genetic evidences also supported that NR-derived NO mediated H2 signal to regulate lateral root formation.All together,results in this part showed the involvement of H2 in the NAA-induced lateral root formation,which was dependent on NR-triggered NO generation.Results here might indicate the potential function and downstream signal of H2 in plant growth and development.4.Multiwall carbon nanotube(MWCNT)induced lateral root formation in tomato in a dose-dependent manner,in which 5 mg/mL was the optimal concentration.Moreover,MWCNT showed the most obvious effects of lateral root processing comparing with singal-wall carbon nanotube(SWCNT),graphene,and active carbon.Interestingly,further researches showed that NAA and haemin enhanced the induction of MWCNT towards lateral root development,while the blockers of the two compounds N-1-naphthylphthalamic acid(NPA)and ZnPP failed to alter the effects of MWCNT on lateral root formation.By contrast,SNP also strengthened the facilitation effects of MWCNT.However,NO scavenger cPTIO reversed the MWCNT-induced lateral root development and NO accumulation significantly.On the other hand,nitrate reductase(NR)inhibitor tungstate(Tg)mimicked the suppression of cPTIO in comparison with NG-nitro-L-arginine methyl ester hydrochloride(NAME)used as nitric oxide synthase in animals.Furthermore,MWCNT increased NR activity in a time-dependent fusion and reached to a peak after 24-h treatment.Nevertheless,the NR activity peak was restrained by Tg,which was correlated with its inhibitory effects on lateral root formation and NO generation.Thus,results in fourth part indicated that NO generated by NR might be involved in MWCNT-induced lateral root formation in tomato.This study showed the novel capacity of nanoparticals,such as multiwall carbom nanotube,in tomato lateral root processing and possible regulatory mechniam.