Research On The Production, Conduction And Mechanism Of Injury Signal Reactive Oxygen Species In Arabidopsis Hypocotyl Cuttings

Adventitious root formation is an important theoretical problem in plant developm ental biology,plant rooting of asexual reproduction and industrialization breeding proc-ess is essential,but so far very limited understanding of their regulation.In plant cutting rooting system,adventitious rooting induced condition is to remove the primary root system,which is mainly reflected in the primary root resection root generated-aerial parts material transport disruptions and incision of mechanical injury.Mechanical damage of injuries signals common are Jasmonic acid?Ethylene?Reactive oxygen species(ROS)and so on.In recent years,numerous studies show that ROS as signaling molecules involved in the extensive variety of plant physiological stress response,organ development,stomatal movement and other processes.In this experiment,the model plant Arabidopsis hypocotyl elongation seedlings as experimental material,with pharmacological experiments,confocal laser technology,gas chromatography and gene expression analysis,after cutting cut wound signal generation of reactive oxygen species,conduction and mechanisms have been studied,the main results obtained are as follows:1.Using different kinds of reactive oxygen species-specific probes related combined with confocal laser microscopy,wild-type Col-0 seedlings after root resection of the primary incision and incision above hypocotyl parts of ROS display real-time detection:after the removal of root,hypocotyl cuttings cut at the outbreak can occur rapidly ROS,different kinds of ROS signals between cells can spread rapidly,rapid conduction upwardly through hypocotyl.In addition,ROS signal transduction process in its intensity gradually weakened,cutting the base ROS fluorescence is strongest.This result suggests that mechanical damage can induce rapid cutting incision ROS generation and accumulation,and ROS signal has good conductivity.2.NADPH oxidase inhibitor DPI highly significant inhibition of wild type ROS cuttings upward conduction,but the outbreak of ROS incision less affected,while peroxidase inhibitor SHAM and NaN3 significantly attenuated ROS incision outbreak.Compared to wild type,NADPH oxidase mutant AtrbohD and AtrbohD/F outburst after root resection,hypocotyl cuttings incision can occur ROS broke out,but up significantly inhibited conduction,and AtrbohF cuttings incision of ROS outbreak up and conducting the case of the wild-type little difference.Gene expression analysis results show that the primary root resection,cutting RBOHD gene expression was significantly increased,the increase is most evident when the cut after 0.5h,and gene expression RBOHF rate of increase is small,and the overall amount RBOHD gene expression was significantly higher in RBOHF gene expression.These results indicate that hypocotyl cuttings incision ROS generation may peroxidase pathway followed up ROS signal transduction is through RBOHD gene encoding NADPH oxidase pathway.3.ROS scavengers DMTU,ASA,CAT significantly inhibited Arabidopsis rooting;suitable concentration of exogenous H2O2 in vivo treatment could significantly promote body hypocotyl adventitious root formation.These results suggest the involvement of reactive oxygen species Arabidopsis Cuttings adventitious root formation.DPI,SHAM,NaN3 have the same inhibitory effect Rooting while AtrbohD and AtrbohD/F outburst Rooting number was significantly reduced,suggesting that the inhibition of cuttings incision ROS generation or conduction up inhibited Arabidopsis adventitious root cuttings occurred.Endogenous H202 test results show:exogenous H2O2 treatment can significantly induce the outbreak and seedling hypocotyl up conducting in vivo ROS,DPI suppress this effect,while the SHAM no significant effect.In addition,ROS scavengers DMTU,ASA,CAT treatment can significantly reduce the incision within Arabidopsis hypocotyl source H2O2 levels.These results confirmed the generation of ROS incision upward conduction indeed involved in the regulation of Arabidopsis root cuttings.4.Exogenous IAA treatment can significantly promote the Arabidopsis cuttings generated adventitious roots,polar auxin transport inhibitor TIB A?NPA significantly inhibited rooting cuttings,verify that the endogenous auxin plays a key role in Arabidopsis adventitious roots in cuttings.Use DR5rev::GFP transgenic lines of growth hormone levels after resection of the primary root directly detected,were cut after 5h and 6h detected a significant increase in the auxin reporter DR5 cutting hypocotyl and upper part of the base of expression,and before ROS levels increased significantly within the cuttings.CAT,DMTU,DPI treatment significantly inhibited cutting hypocotyl ghrelin levels and the upper part of the base increases,the impact on the NPA process hypocotyl an upper level of growth hormone is not obvious,but strongly inhibited basal cuttings auxin elevated levels.These results suggest that after removal of the primary root,cut cuttings generated at the synthesis and transport of polar process on to the conduction of ROS may be involved in the regulation of auxin.5.qRT-PCR/RT-PCR results showed that after the removal of root auxin biosynthetic gene TAA1,TAR2,YUCCA2,YUCCA6 from 1h to 6h slight increase in the expression,polar auxin transport-related genes AUX1,PIN1,PIN3,PIN7 expression ABCB19 presented a significant increase,especially PIN3,its expression after cutting cut 2h has nearly doubled;exogenous H2O2 treatment with a similar effect,DMTU?DPI is inhibited these changes.In addition,the polar auxin transport-related gene mutants also showed reduced Rooting Ability.These results confirm that mechanical damage induced by ROS indeed involved in the regulation of expression of synthetic auxin polar transport in particular related genes.6.The wild-type Arabidopsis thaliana(Col-0)and vinyl cutting Mutant(eto3,etrl-3,ein3-1,ein2-5)cut after cutting can normally produce ethylene signal hurt,but cuttings rooting wild-type or less,these results indicate injury may not be apparent signal ethylene in Arabidopsis cuttings adventitious root formation.In summary,the results of this paper show that mechanical damage induced by ROS in Arabidopsis cuttings incision outbreak,followed by NADPH oxidase ROS signaling pathways by hypocotyl conducted to shoot tip,through synthesis of auxin,auxin distribution and pole transportation of final regulatory adventitious root formation.