The Role Of G?, NO And CGMP In The Occurrence Of Adventitious Roots Of Arabidopsis Thaliana Cuttings And The Relationship Between ROS And Systemic ROS

The cutting is a classic system of adventitious roots,and its cutting process can cause damage to the cutting site and the interruption of the association with the whole plant.A large number of studies have shown that plant hormone auxin plays a key role in the formation of adventitious roots.And cutting treatment will produce a large number of injury signals,such as ethylene,salicylic acid,reactive oxygen species(ROS)and so on.Research by our laboratory have shown that ROS as a system of injury signal quickly generate at the incision and upward conduction to the stem tip regulating the transport of auxin to control the rooting process.How does ROS work,and is there any other signal molecule involved?We intend to study this.In this paper,the effect of the heterotrimeric G protein ? subunit(Ga),NO,cyclic guanacetic acid(cGMP)in the adventitious root of Arabidopsis thaliana and the relationship between the injury signal ROS were studied by means of pharmacological treatment,laser confocal microscopy and gene expression analysis,and using wild type Col-0 and related mutant cuttings of Arabidopsis thaliana as experimental materials.The results are as follows:1.G? activator CTX promoted the adventitious root production of wild-type Col-0 cuttings,whereas Ga-repressor PTX significantly inhibited rooting.G? overexpression mutant wGa had a significant increase in adventitious root and G?-deficient mutant gpal-3 and gpal-4 were reduced in rooting,suggesting that G? may positively control the adventitious root of Arabidopsis thaliana.2.NO scavenger c-PTIO,NR inhibitor Na2WO4 inhibited the rooting of wild-type cuttings.NO donor SNP promoted rooting and the rooting of NR mutant Nia2-1,Nia1-2/Nia2-5 after being cutted reduced,but rooting change of Nia1-2 was not obvious.The results of endogenous NO showed that NO significantly increased at 20 min after Col-0 striation and gradually transported along the upper part of the embryo axis.The NO production of Nia2-1,Nia1-2/Nia2-5 were significantly inhibited,indicating that NO originated from the Nia2 gene as a system signal participate in the cutting root.3.Guanylate cyclase inhibitor LY83583 significantly inhibited Col-0 rhizome rooting and cGMP analogues 8-Br-cGMP promoted rooting,cGMP expression level of?-FlincG transgenic Arabidopsis thaliana cutting began to significantly increase after the cut 3h and transported along the embryo axial cuttings to the upper,indicating that cGMP is also involved in the occurrence of Arabidopsis thaliana adventitious roots.4.H2O2 could promote the formation of Mutant gpal-4 in adventitious root formation and wGa of endogenous H2O2 production,while ROS accumulation inhibitor DPI,CAT had the opposite effect.Endogenous ROS levels of Mutant gpal-4 intercostal carnosine were significantly reduced and wGa mutants were significantly increased.At the same time,CTX could induce the increase of ROS level and adventitious root number in Co1-0 and mutant AtrbohF cuttings,but the effect of AtrbohD and AtrbohD/F was not obvious.Besides,DPI and CAT inhibited the effect of CTX on rooting.Compared with the control,the expression of RBOHD gene was significantly up-regulated in wGa cuttings,and the expression of gpal-4 was down-regulated.The above results indicated that Ga regulated the adventitious root formation by regulating the signaling signal ROS of RBOHD gene.5.H2O2 could promote the formation of Col-0 and Nia1-2 mutants in somatic shoots but could not induce Nia2-1 and Nia1-2/Nia2-5 mutants in vivo.At the same time,DPI and CAT could inhibit NR-related mutations Body cuttings take root.However,SNP reversed the reduction of ROS levels resulting to rooting inhibition.H2O2 could induce the production and conduction of endogenous NO in the hypocotyls,DPI and CAT inhibited the level of endogenous NO,but the NO-related reagents had no significant effect on the production and conduction of ROS.In addition,NO production of AtrbohD and AtrbohD/F was inhibited,but the levels of ROS were not inhibited in Nia2-1 and Nia1-2/Nia2-5 cuttings.These results indicate that NO is involved in the regulation of Arabidopsis thaliana rooting as a downstream signal of the system injury signal ROS.6.8-Br-cGMP could significantly reversed the rooting inhibition caused by ROS reduction,but LY83583,8-Br-cGMP did not affect the outbreak and conduction of ROS in Col-0.Exogenous H2O2 could increase the level of cGMP in the hypocotyls of?-FlincG transgenic Arabidopsis thaliana in vivo and ROS accumulation inhibitor inhibited the level of endogenous cGMP in the embryos of the cuttings,indicating that cGMP is involved in regulating the rooting of the cuttings as a downstream signal of ROS.7.CTX induced rooting and increased endogenous NO in hypocotyl of Co1-0 and Nia1-2,but did not promote the adventitious root formation and endogenous NO of NR mutant Nia2-1,Nia1-2/Nia2-5 and reverse the inhibition of c-PTIO and Na2WO4 on rooting.SNP could reverse the gpal-4 adventitious root inhibition effect.The level of endogenous NO in wGa was higher than that in Co1-0,but the level of gpa1-4 was lower than that of Col-0.It was also found that CTX increased the level of cGMP in the hypocotyls of ?-FlincG transgenic Arabidopsis thaliana,whereas PTX had the opposite effect.8-Br-cGMP could reverse the adventitious root inhibitory effect of gpa1-4,and LY83583 also inhibited CTX on Col-0 cutting adventitious root.The promotion effect SNP on the expression of cGMP in hypocotyls of ?-FlincG transgenic Arabidopsis thaliana was promoted and the effect of c-PTIO and Na2WO4 on the upper part of the embryo axis was significantly inhibited.However,8-Br-cGMP and LY83583 could not affect the NO level.The results showed that 8-Br-cGMP could reverse the accumulation of adventitious roots caused by NO accumulation,but SNP could not release the inhibitory effect of LY83583 on adventitious roots.These results indicated that Ga is located upstream of the system signal NO and cGMP in Arabidopsis thaliana,whereas cGMP work downstream of NO.8.CTX could significantly increase IAA level at incision and the up of DR5rev::GFP after the cuttings 5 h,PTX had inhibitory effect.The IAA synthesis and transport-related genes in gpa1-4 was significantly lower than that in Co1-0 and wGa,and only the number of PIN3 gene was decreased increase.The expression level of the related gene of wGa was higher than that of Col-0 and gpa1-4,especially the expression of PIN3 gene in 2 h was significantly highest.These results suggest that Ga may be involved in the early regulation of IAA synthesis and transpor in the adventitious roots of Arabidopsis thaliana.9.Na2WO4 and c-PTIO could significantly inhibit IAA level in the incision and the upper,while SNP was opposite.RT-PCR results showed that the expression level of IAA-related genes in Nia2-1 mutant was slightly weaker than that of the control group.SNP treatment significantly up-regulated the expression of YUCCA2,PIN3 and ABCB19 and slightly up-regulated TAA1,TAR2,PIN1,PIN7,AUX1 gene expression.While c-PTIO,Na2WO4 treatment could down-regulated IAA-related gene levels.These results suggest that NO can participate in the regulation of early IAA synthesis and transportation in the adventitious root of Arabidopsis thaliana.10.LY83583 significantly inhibited the level of cuttings IAA,while 8-Br-cGMP had a role in promoting.The level of cGMP in the upper part of the transgenic Arabidopsis thaliana inserted by ?-FlincG increased significantly after 4 h and increased with time.The level of cGMP at the incision significantly increased at about 3 h,and the horizontal effect was similar to that of IAA except earlier than IAA changes.Compared with the control,8-Br-cGMP significantly up-regulated the expression of YUCCA2,PIN3,AUX1 and ABCB19 genes,while LY83583 significantly down-regulated the expression of these genes.These results suggest that cGMP can be involved in the regulation of IAA leveling in adventitious roots of Arabidopsis thaliana.The above results indicate that Ga up-regulates the system signaling signal ROS controlled by RBOHD gene,and the signal signal ROS induced by Nia2 is induced by injury signal ROS.NO regulates cGMP and finally regulates the synthesis and transportation of IAA in the upper part of the cuttings to control adventitious root formation.