G. H. M. Sagor

Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana

The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress.

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C18 reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 degrees C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.

The polyamine spermine protects Arabidopsis from heat stress-induced damage by increasing expression of heat shock-related genes

It is known that the polyamine (PA) biosynthetic pathway is modulated at the transcriptional level during abiotic stresses. Here we studied the expression of PA biosynthetic pathway genes upon exposure to heat shock (HS) in Arabidopsis and showed that the spermine (Spm) synthase gene (SPMS) and S-adenosylmethionine decarboxylase 2 gene are induced at the earliest stage, followed by the induction of the arginine decarboxylase 2 gene. Correspondingly, Spm content increased linearly upon HS, and putrescine (Put) and spermidine (Spd) content also increased but not thermospermine (T-Spm) content. Exogenously applied Spm had a potential to protect Arabidopsis plants from HS-induced damage. Such protection was also observed to the same extent with T-Spm and by Spd to a lesser extent but not by Put. Then we tested whether altered endogenous Spm content affects sensitivity to HS using both transgenic plants overexpressing SPMS and a Spm deficient (spms) mutant plant. The result revealed that the higher the Spm content the higher the thermotolerance. Even in the spms plant, representative genes encoding heat shock proteins (HSPs) and heat shock transcription factors were upregulated upon HS, while the expression of such genes was increased in a positively correlated manner with Spm content. Furthermore four kinds of HSPs (HSP101, HSP90, HSP70 and HSP17.6) were detected proportionally with the levels of their respective transcripts upon HS. We propose that Spm increases the HS response at transcriptional and translational levels and protects host plants from HS-induced damage.

Exogenous thermospermine has an activity to induce a subset of the defense genes and restrict cucumber mosaic virus multiplication in Arabidopsis thaliana.

We previously proposed the defensive role of a signal pathway triggered by the polyamine spermine (Spm) in the reaction against avirulent viral pathogens in Nicotiana tabacum and Arabidopsis thaliana. In this study, we showed that thermospermine (T-Spm), an isomer of Spm, is also active in inducing the expression of the genes involved in the Spm-signal pathway at a similar dose as Spm. Furthermore, we found that T-Spm enhances the expression of a subset of pathogenesis-related genes whose expression is induced during cucumber mosaic virus (CMV)-triggered hypersensitive response. In consistent with the above observation, we further showed that exogenous T-Spm can repress CMV multiplication with same efficiency as Spm.Key messagePolyamine thermospermine, an isomer of spermine, is able to induce a subset of hypersensitive response-related defense genes and can suppress cucumber mosaic virus multiplication in Arabidopsis thaliana.

Polyamines in Plant Stress Response

Polyamines (PAs) are ubiquitous biogenic amines that have been implicated in diverse cellular functions in all organisms. Recent work has demonstrated that mainly the three prominent PAs in plants, putrescine, spermidine, and spermine, have a role as part of the complex signaling networks activated in response to abiotic stresses and pathogen attack. In a network comprising the PAs itself, their catabolic products such as H2O2 produced by action of diamine oxidases (DAO) and polyamine oxidases (PAO), and other compounds such as γ-aminobutyric acid (GABA), abscisic acid (ABA), and nitric oxide (NO), are part of interlaced cellular pathways. Current knowledge about PAs in plant stress responses is summarized in this chapter.

Reducing Cytoplasmic Polyamine Oxidase Activity in Arabidopsis Increases Salt and Drought Tolerance by Reducing Reactive Oxygen Species Production and Increasing Defense Gene Expression

The link between polyamine oxidases (PAOs), which function in polyamine catabolism, and stress responses remains elusive. Here, we address this issue using Arabidopsis pao mutants in which the expression of the five PAO genes is knocked-out or knocked-down. As the five single pao mutants and wild type (WT) showed similar response to salt stress, we tried to generate the mutants that have either the cytoplasmic PAO pathway (pao1 pao5) or the peroxisomal PAO pathway (pao2 pao3 pao4) silenced. However, the latter triple mutant was not obtained. Thus, in this study, we used two double mutants, pao1 pao5 and pao2 pao4. Of interest, pao1 pao5 mutant was NaCl- and drought-tolerant, whereas pao2 pao4 showed similar sensitivity to those stresses as WT. To reveal the underlying mechanism of salt tolerance, further analyses were performed. Na uptake of the mutant (pao1 pao5) decreased to 75% of WT. PAO activity of the mutant was reduced to 62% of WT. The content of reactive oxygen species (ROS) such as hydrogen peroxide, a reaction product of PAO action, and superoxide anion in the mutant became 81 and 72% of the levels in WT upon salt treatment. The mutant contained 2.8-fold higher thermospermine compared to WT. Moreover, the mutant induced the genes of salt overly sensitive-, abscisic acid (ABA)-dependent- and ABA-independent- pathways more strongly than WT upon salt treatment. The results suggest that the Arabidopsis plant silencing cytoplasmic PAOs shows salinity tolerance by reducing ROS production and strongly inducing subsets of stress-responsive genes under stress conditions.

The polyamine oxidase from lycophyte Selaginella lepidophylla (SelPAO5), unlike that of angiosperms, back-converts thermospermine to norspermidine

In the phylogeny of plant polyamine oxidases (PAOs), clade III members from angiosperms, such asArabidopsis thaliana PAO5 andOryza sativa PAO1, prefer spermine and thermospermine as substrates and back‐convert both of these substrates to spermidine in vitro. A clade III representative of lycophytes, SelPAO5 fromSelaginella lepidophylla, also prefers spermine and thermospermine but instead back‐converts these substrates to spermidine and norspermidine, respectively. This finding indicates that the clade III PAOs of lycophytes and angiosperms oxidize thermospermine at different carbon positions. We discuss the physiological significance of this difference.

Longer uncommon polyamines have a stronger defense gene-induction activity and a higher suppressing activity of Cucumber mosaic virus multiplication compared to that of spermine in Arabidopsis thaliana

Key messageOur work suggests that long chain polyamines and their derivatives are potential chemicals to control viral pathogens for crop production.AbstractPreviously we showed that two tetraamines, spermine (Spm) and thermospermine (T-Spm), induce the expression of a subset of defense-related genes and repress proliferation of Cucumber mosaic virus (CMV) in Arabidopsis. Here we tested whether the longer uncommon polyamines (LUPAs) such as caldopentamine, caldohexamine, homocaldopentamine and homocaldohexamine have such the activity. LUPAs had higher gene induction activity than Spm and T-Spm. Interestingly the genes induced by LUPAs could be classified into two groups: the one group was most responsive to caldohexamine while the other one was most responsive to homocaldopentamine. In both the cases, the inducing activity was dose-dependent. LUPAs caused local cell death and repressed CMV multiplication more efficiently as compared to Spm. LUPAs inhibited the viral multiplication of not only avirulent CMV but also of virulent CMV in a dose-dependent manner. Furthermore, LUPAs can activate the systemic acquired resistance against CMV more efficiently as compared to Spm. When Arabidopsis leaves were incubated with LUPAs, the putative polyamine oxidase (PAO)-mediated catabolites were detected even though the conversion rate was very low. In addition, we found that LUPAs induced the expression of three NADPH oxidase genes (rbohC, rbohE and rbohH) among ten isoforms. Taken together, we propose that LUPAs activate two alternative reactive oxygen species evoked pathways, a PAO-mediated one and an NADPH-oxidase-mediated one, which lead to induce defense-related genes and restrict CMV multiplication.

Spermine signaling in defense reaction against avirulent viral pathogen in Arabidopsis thaliana.

We have proposed that the polyamine spermine functions as a signaling molecule to evoke defense reactions/cell death in N-gene bearing Nicotiana tabacum infected with tobacco mosaic virus (TMV). In this work we examined whether this spermine-based signal transduction pathway is present in other plants such as Arabidopsis thaliana. For this purpose, we used the experimental system of cucumber mosaic virus-yellow strain (CMV-Y) and A. thaliana ecotype C24 carrying the RCY-1 resistance gene, in which a hypersensitive response is triggered. Almost all spermine-responsive genes in Arabidopsis were similarly regulated as in the TMV-Nicotiana tabacum system. In addition to the formerly identified spermine-signal component genes such as alternative oxidase- and mitogen-activated protein kinase 3 (WIPK orthologue)-genes, AtbZIP60, encoding an endoplasmic reticulum (ER)-localized transcription factor, was identified as a spermine-responsive gene, indicating that the ER may be involved in a spermine-signaling pathway. Treatment with polyamine oxidase inhibitors prior to CMV inoculation suppressed the expression of the genes mentioned above and compromised the host defense against CMV. Furthermore, exogenously added spermine suppressed the multiplication of CMV in Arabidopsis as did spermidine but less efficiently, while putrescine did not show such effects. Collectively, we conclude that the spermine-signaling pathway plays an important defensive role in A. thaliana as well as in N. tabacum when those plants are attacked by incompatible viral pathogens.

Arabidopsis mutant plants with diverse defects in polyamine metabolism show unequal sensitivity to exogenous cadaverine probably based on their spermine content

Arabidopsis plants do not synthesize the polyamine cadaverine, a five carbon-chain diamine and structural analog of putrescine. Mutants defective in polyamine metabolic genes were exposed to exogenous cadaverine. Spermine-deficient spms mutant grew well while a T-DNA insertion mutant (pao4-1) of polyamine oxidase (PAO) 4 was severely inhibited in root growth compared to wild type (WT) or other pao loss-of-function mutants. To understand the molecular basis of this phenomenon, polyamine contents of WT, spms and pao4-1 plants treated with cadaverine were analyzed. Putrescine contents increased in all the three plants, and spermidine contents decreased in WT and pao4-1 but not in spms. Spermine contents increased in WT and pao4-1. As there were good correlations between putrescine (or spermine) contents and the degree of root growth inhibition, effects of exogenously added putrescine and spermine were examined. Spermine mimicked the original phenomenon, whereas high levels of putrescine evenly inhibited root growth, suggesting that cadaverine-induced spermine accumulation may explain the phenomenon. We also tested growth response of cadaverine-treated WT and pao4-1 plants to NaCl and found that spermine-accumulated pao4-1 plant was not NaCl tolerant. Based on the results, the effect of cadaverine on Arabidopsis growth and the role of PAO during NaCl stress are discussed.