Regulatory Function of Polyamine Oxidase-Generated Hydrogen Peroxide in Ethylene-Induced Stomatal Closure in Arabidopsis thaliana

Hydrogen peroxide （H2O2） is an important signaling molecule in ethylene-induced stomatal closure in Arabidopsis thaliana. Early studies on the sources of H2O2 mainly focused on NADPH oxidases and cell-wall peroxidases. Here, we report the involvement of polyamine oxidases （PAOs） in ethylene-induced H2O2 production in guard cells. In Arabidopsis epidermal peels, application of PAO inhibitors caused the failure of ethylene to induce H2O2 production and stomatal closure. Results of quantitative RT-PCR analysis and pharmacological experiments showed that AtPAO2 and AtPAO4 transcripts and activities of PAOs were both induced by ethylene. In transgenic Arabidopsis plants over-expressing AtPAO2 and AtPAO4, stomatal movement was more sensitive to ethylene treatment and H2O2 production was also significantly induced. The increased H2O2 production in the transgenic lines compared to the wild-type plants suggests that AtPAO2 and AtPAO4 probably are involved in ethylene-induced H2O2 production. Several factors which induce stomatal closure such as dehydration and high salinity all enhanced the expression of AtPAO2 and AtPAO4 to different degrees. Moreover, GFP- AtPAOs fusion protein localized in the nucleus, cytoplasm, and cell wall of the guard cells. Therefore, our results strongly indicated that PAO is a source of H2O2 generation in Arabidopsis guard cells and plays crucial roles in stomatal movement.

Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield

Under conditions of labor or resource scarcity,direct seeding,rather than transplantation,is the preferred mode of rice(Oryza sativa)cultivation.This approach requires varieties that exhibit uniform seedling emergence.Mesocotyl elongation(ME),the main driver of rapid emergence of rice seedlings from soil,is enhanced by darkness and inhibited by light.Plant polyamine oxidases(PAOs)oxidize polyamines(PAs)and release H2O2,Here,we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness.To determine its role in ME,we created OsPAO5 mutants using CRISPR/Cas9.Compared with the wild type,pao5 mutants had longer mesocotyls,released less H2O2,and synthesized more ethylene.The mutant seedlings emerged at a higher and more uniform rate,indicating their potential for use in direct seeding.Nucleotide polymorphism analysis revealed that an SNP(PAO5-578G/A)located 578 bp upstream of the OsPAO5 start codon alters its expression,and was selected during rice mesocotyl domestication.The PAO5-578G genotype conferring a long mesocotyl mainly exists in wild rice,most Aus accessions,and some Geng(Japonica)accessions.Intriguingly,knocking out OsPAO5 can remarkably increase the grain weight,grain number,and yield potential.In summary,we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential,which can be conveniently and widely used to breed varieties of direct-seeding rice.

Polyamine oxidase 3 is involved in salt tolerance at the germination stage in rice

Soil salinity inhibits seed germination and reduces seedling survival rate,resulting in significant yield reductions in crops.Here,we report the identification of a polyamine oxidase,OsPAO3,conferring salt tolerance at the germination stage in rice(Oryza sativa L.),through map-based cloning approach.OsPAO3 is up-regulated under salt stress at the germination stage and highly expressed in various organs.Overexpression of OsPAO3 increases activity of polyamine oxidases,enhancing the polyamine content in seed coleoptiles.Increased polyamine may lead to the enhance of the activity of ROS-scavenging enzymes to eliminate over-accumulated H;O;and to reduce Na;content in seed coleoptiles to maintain ion homeostasis and weaken Na;damage.These changes resulted in stronger salt tolerance at the germination stage in rice.Our findings not only provide a unique gene for breeding new salt-tolerant rice cultivars but also help to elucidate the mechanism of salt tolerance in rice.

Involvement of Polyamine Oxidase in Abscisic Acidinduced Cytosolic Antioxidant Defense in Leaves of Maize

Using pharmacological and biochemical approaches, the role of maize polyamine oxidase （MPAO） in abscisic acid （ABA）- induced antioxidant defense in leaves of maize （Zea mays L.） plants was investigated. Exogenous ABA treatment enhanced the expression of the MPAO gene and the activities of apoplastic MPAO. Pretreatment with two different inhibitors for apoplastic MPAO partly reduced hydrogen peroxide （H202） accumulation induced by ABA and blocked the ABA-induced expression of the antioxidant genes superoxide dismutase 4 and cytosolic ascorbate peroxidase and the activities of the cytosolic antioxidant enzymes. Treatment with spermidine, the optimum substrate of MPAO, also induced the expression and the activities of the antioxidant enzymes, and the upregulation of the antioxidant enzymes was prevented by two inhibitors of MPAO and two scavengers of H202. These results suggest that MPAO contributes to ABA-induced cytosolic antioxidant defense through H202, a Spd catabolic product.

Thymol ameliorates ammonium toxicity via repressing polyamine oxidase-derived hydrogen peroxide and modulating ammonium transporters in rice root

Background:Ammonium is an indispensable nutrient for crop growth,but anoxic conditions or inappropriate fertilizer usage result in the increase in ammonium content in soil.Excessive ammonium causes phytotoxicity.Thymol is a kind of natural phenolic compound with anti-microbial properties.However,little is known about the role of thymol in modulating plant physiology.Here we find the novel role of thymol in protecting rice from ammonium toxicity.Results:Thymol remarkably rescued rice seedlings growth from ammonium stress,which may resulted from the attenuation of reactive oxygen species(ROS)accumulation,oxidative injury,and cell death in both shoots and roots.Polyamine oxidase(PAO)metabolizes polyamines to produce ROS in plants in response to stress conditions.Thymol blocked ammonium-induced upregulation of a set of rice PAOs,which contributed to the decrease in ROS content.In rice seedlings upon ammonium stress,thymol downregulate the expression of ammonium transporters(AMT1;1 and AMT1;2);thymol upregulated the expression of calcineurin B-like interacting protein kinase 23(CIPK23)and calcineurin B-like binding protein 1(CBL1),two negative regulators of AMTs.This may help rice avoid ammonium overload in excessive ammonium environment.Correlation analysis indicated that PAOs,AMTs,and CBL1 were the targets of thymol in the detoxification of excessive ammonium.Conclusion:Thymol facilitates rice tolerance against ammonium toxicity by decreasing PAO-derived ROS and modulating ammonium transporters.Such findings may be applicable in the modulation of nutrient acquisition during crop production.

The Role of Polyamine Oxidative Degradation in the Process of Lettuce Seed Germination

[Objective]The aim of this study was to understand the role of polyamine oxidative degradation in the process of lettuce seed germination. [Method]After lettuce seed soaking treatments with aminoguanidine （AG,a specific inhibitor of polyamine oxidases）,seed germination rate,activities of polyamine oxidase （PAO） and diamine oxidase （DAO）,change of endogenous polyamine and H2O2 content were determined. [Result]Compared with the control,AG treatment strongly inhibited the seed germination,which also had an extremely significant difference in seed germination rate after incubation for 12 h. During the seed germination,activities of PAO and DAO significantly changed,while their activities firstly increased and then decreased,then DAO and PAO reached peaks at 24 h and 48 h respectively. AG treatment was strongly inhibitory for activities of DAO and PAO,whose activities even disappeared after incubation with AG for 24 h and 36 h. During the seed germination,endogenous Put reduced sharply in the first 24 h,then reduced slowly in 24-60 h,while Spd decreased slowly in the early stage of germination,and then sharply declined after 48 h. However,Spm content was low but slightly increased,and the total polyamine gradually decreased. AG treatment could significantly increase endogenous polyamines,especially Put and Spd contents. During the seed germination,H2O2 content gradually increased,and had a peak from 36 to 48 h,then kept a high level at last. AG treatment could significantly reduce H2O2 content. [Conclusion]During the seed germination,the changes of endogenous polyamine and H2O2 content correspond with the changes of PAO and DAO activities,which indicate that there is an active polyamine metabolism of oxidative degradation during the lettuce seed germination.

The Improvement of Soybean Salt Tolerance by Overexpressed GmPAO1

Polyamines play an important regulatory role during plant growth and development and adversity stress,and polyamine oxidase(PAO)is involved in polyamine catabolism.In this study,an up-regulated polyamine oxidase gene GmPAO1 was obtained by transcriptome sequencing analysis and screening at soybean seedling stages.Also,its expression pattern and function were analyzed.The identification results of transgenic GmPAO1 soybean positive lines showed that the relative expression level of GmPAO1 in the overexpressed lines was increased under salt stress.With increasing stress concentration,the seed germination rate decreased.However,the seed germination rate of the overexpressed lines was significantly higher than that of the control lines,and the phenotypic character of the root systems was also better than that of the control lines.The measurement of superoxide dismutase(SOD)and peroxidase(POD)activities and malondialdehyde and hydrogen peroxide contents revealed that the overexpressed soybean lines significantly increased the SOD and POD activities,significantly reducing the malondialdehyde content.Although the hydrogen peroxide content in the transformed plants gradually increased,the hydrogen peroxide content in the overexpression lines was still lower than that in the gene editing lines.Based on this,it was preliminarily judged that GmPAO1 can improve soybean salt tolerance.

Putrescine, spermidine, and spermine play distinct roles in rice salt tolerance

Polyamines(PAs) play diverse roles in plant growth and development,as well as responses to environmental stimuli.In this study,the effects of PAs on rice salt tolerance were investigated.Salt stress resulted in the alteration of endogenous PAs levels in rice roots and leaves,where spermidine(Spd) and spermine(Spm) contents were increased,and putrescine(Put) content was decreased.RT-qPCR analysis revealed that PAs biosynthesis-related genes ADC1,ODC,and Arginase were significantly downregulated by salt treatment;however,SAMDC transcription was significantly upregulated.Exogenous Spm enhanced rice salt tolerance remarkably;however,exogenous Put and Spd undermined rice salt tolerance.Transgenic rice plants overexpressing SAMDC display a higher ratio of Spm/(Put+Spd) and enhanced salt tolerance.Salt stress also increased polyamine oxidase activities in rice,resulting in elevated reactive oxygen species(ROS) production.Our findings revealed that accumulation of Put and Spd substantially reduced salt tolerance in rice,likely by facilitating ROS production;whereas,conversion of Put and Spd to Spm contributes to rice salt tolerance.

Involvement of Hydrogen Peroxide Generated by Polyamine Oxidative Degradation in the Development of Lateral Roots in Soybean

In order to determine whether hydrogen peroxide （H2O2） generated by polyamlne oxidative degradation Is Involved In the development of lateral roots In soybean, the length and the number of lateral roots, the actlvltlea of polyamlne oxldases and dlamlne oxldases, and the endogenous free polyamlne and H2O2 content were analyzed In soybean （Giycine max （Linn.） Merr.） main roots of 2-d-old seedlings after treatments for 2 d with exogenous β-hydroxyethylhydrazine （an Inhibitor of polyamlne oxldases）, H202, putresclne, cyclohexylamlne （an Inhibitor of spermidine synthase） or N,N＇-dimethylthlourea （a scavenger of hydrogen peroxide）.β-hydroxyethylhydrazlne treatment strongly Inhibited the development of lateral roots In soybean seedlings, reduced the activities of polyamine oxldases and dlamlne oxidases, decreased H2O2 levels, and led to the accumulation of endogenous polyamlnes In the main roots. The inhibitory effect of β-hydroxyethylhydrazlne on root development could be alleviated by exogenously applied 10 μmol/L H2O2 （a major product of polyamlne oxidation）. Treatment with cyclohexylamlne and putresclne promoted root growth slightly, but treatment with cyclohexylamlne plus N,N＇dlmethylthlourea or putresclne plus N,N＇-dlmethylthlourea prevented the development of soybean lateral roots. The effects of these treatments on the development of soybean lateral roots were consistent with the changes In endogenous H2O2 levels. These results suggest that the development of soybean lateral roots Is associated with the oxidative degradation of polyamlnes, and that their products, especially H2O2, are likely to play an Important role In the growth of soybean lateral roots.