Roles Of Putrescine Biosynthetic AtADC Gene In Arabidopsis Root Development

Plant hormones are produced in plant a class of metabolite,which regulates plant growth,development and environment response so on.Polyamines(PAs)as a novel regulator of plant material play important roles in diverse plant growth and development processes,including seed germination,tissue lignifications,organogenesis,flowering,senescence and stress responses.In all these processes,synergistic and antagonistic interactions among various plant hormones have been shown while the molecular mechanisms underlying polyamine action have just begun.The studies using exogenous application of PAs and transcriptome analysis have revealed complex and differential relationships among the three main PAs-putrescine,spermidine and spermine-in regard to regulation of plant hormone biosynthesis and signaling.Little is known or understood about the signal transduction pathways regulating a myriad of PA effects.The model plant Arabidopsis thaliana,with simplified root system,plays an irreplaceable role in plant developmental research.Arginine decarboxylase,the synthesis of putrescine key rate-limiting enzyme,plays a key role in the polyamine synthesis pathway.We focused on the molecular mechanism of the function of Arginine decarboxylase in root system morphogenesis to provide a reference about the interaction between polyamines and other hormones.Function of AtADC,which is associated with root morphogenesis,was elucidated by molecular genetic,cytological,physiological strategies.The main results are as follows:1.D-Arg,arginine decarboxylase inhibitor,inhibits the primary root growth of Col-0 with dose effect.The lower concentrations putrescine promotes the primary root growth of Col-0,while the higher putrescine concentration inhibits it.When treated with D-Arg,there is a decrease in meristematic zone in both length and cell number,but the meristematic cells size remain the same.2.By marking the cell cycle with the reporter CYCB1;1::GUS,D-Arg treatment inhibits cell division in meristem zone while putrescine promotes it.Semi-quantitative RT-PCR analysis showed the expression of AtCDKA;1,AtCDKB2;1,AtCYCB1;1 and AtCYCB2;1 were inhibited by D-Arg.It indicates that D-Arg affects the cell cycle G1/S,G2/M conversion through regulations of certain gene expression,leading to the effects on meristematic zone cells division.3.By treating auxin content with the reporter DR5::GUS,D-Arg and putrescine treatment affect auxin accumulation in the root tip.D-Arg affects auxin accumulation in the root tip by regulating the polar auxin transport through research on auxin-related reporter lines and real-time quantitative PCR analysis.4.By querying the database of TAIR and promoter fusion GUS analysis,we found that AtADC2 mainly expressed in mature and elongation region of the leaf tip,especially the latter.In addition,AtADC::GUS with auxin treatment demonstrates that auxin induces ADC2 expression.5.Under normal growth conditions,no significant difference was observed in plant development between single mutant and Col-0 plants,while double mutant is lethal.With low D-Arg concentration treatment,adc2-3 primary root growth was inhibited significantly while there was no difference in adcl-2 compared with Col-0.It suggests that AtADC2,rather than AtADC1,is a key gene for production of putrescine in Arabidopsis.6.The expression of CYCB1;1::GUS/GFP in mutant significantly decreased compared with that in the wild type,indicating that ADC deletion affects the population of division cells in the root meristem.7.The expression of DR5::GUS/GFP in mutant significantly decreased compared with that in the wild type,indicating that ADC deletion affects auxin distribution.Analysis of PIN gene family changes showed that the decrease of PIN7 gene expression contributes,at least in part,to the altered auxin distribution in adc2-3.8.Under normal growth conditions,no significant difference was observed in overexpression AtADC1 transgenic plant development compared with Col-0 plants,while overexpression AtADC2 transgenic plants show dwarfism,late-flowering,more branches,partial stamen developmental disorder and the longer primary root.We found that the number of meristematic zone cells in overexpression AtADC2 transgenic families increase through DIC observation on root.Real-time quantitative PCR analysis shows that cell division-related genes,auxin early response genes and auxin polar transport genes were up-regulated while auxin synthesis-related genes were down-regulated in overexpression AtADC2 transgenic plants.In summary,cell cycle process is inhibited in exogenous application of putrescine synthesis inhibitor treatment or AtADC function deletion mutant.In the meanwhile,the expression of AtADC affects directly or indirectly cell cycle and auxin related-genes to regulate cell divison in meristem zone.In addition,auxin also induces AtADC expression to influence polyamine synthetic metabolism.