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 message

Our work suggests that long chain polyamines and their derivatives are potential chemicals to control viral pathogens for crop production.

Abstract

Previously 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.     

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S-adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock-out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.     

Identification of Tobacco HIN1 and Two Closely Related Genes as Spermine-Responsive Genes and their Differential Expression During the Tobacco Mosaic Virus-Induced Hypersensitive Response and During Leaf- and Flower-Senescence

Previously we showed that the polyamine spermine (Spm) specifically leads to mitochondrial dysfunction in tobacco that is followed by the activation of salicylic acid-induced protein kinase and wound-induced protein kinase. To identify the possible downstream components of the Spm signalling pathway, we isolated Spm-responsive genes by a differential hybridization approach. This showed that the harpin-induced 1 (HIN1) gene is responsive to Spm. Genomic Southern analysis showed that HIN1 constitutes a multi-gene family and this led to the isolation of two novel HIN1 -like tobacco cDNAs that we designated as HIN9 and HIN18. Both genes are also responsive to Spm, albeit HIN18 is induced weakly compared to HIN1 and HIN9. As HIN1 is up-regulated both during the hypersensitive response (HR) generated by an incompatible plant-pathogen interaction and during senescence, we compared the expression of the three HIN1 family genes in these situations. All three were responsive to HR due to Tobacco mosaic virus infection, although HIN18 was less efficiently induced, and HIN1 and HIN18 were both strongly up-regulated during leaf- and flower-senescence. This suggests that the signalling pathways in the HR and senescence overlap somehow but are distinct. That HIN1 and its closely related genes are Spm-responsive genes also supports the idea that Spm plays a role as a signal transmitter in the HR process.     

Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus interactions

Plant viruses elicit the expression of common sets of genes in susceptible hosts. Studies in Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) indicate that at least one-third of the genes induced in common by viruses have been previously associated with plant defense and stress responses. The genetic and molecular requirements for the induction of these stress and defense-related genes during compatible host-virus interactions were investigated with a panel of Arabidopsis mutant and transgenic plants defective in one or more defense signaling pathways. pad4, eds5, NahG, npr1, jar1, ein2, sid2, eds1, and wild-type Columbia-0 and Wassilewskija-2 plants were infected with two different viruses, cucumber mosaic virus and oilseed rape mosaic virus. Gene expression was assayed by a high-throughput fiber-optic bead array consisting of 388 genes and by RNA gel blots. These analyses demonstrated that, in compatible host-virus interactions, the expression of the majority of defense-related genes is induced by a salicylic acid-dependent, NPR1-independent signaling pathway with a few notable exceptions that did require NPR1. Interestingly, none of the mutant or transgenic plants showed enhanced susceptibility to either cucumber mosaic virus or oilseed rape mosaic virus based on both symptoms and virus accumulation. This observation is in contrast to the enhanced disease susceptibility phenotypes that these mutations or transgenes confer to some bacterial and fungal pathogens. These experimental results suggest that expression of many defense-related genes in compatible host plants might share components of signaling pathways involved in incompatible host-pathogen interactions, but their increased expression has no negative effect on viral infection.     

A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco

A cellular signal transduction pathway induced by the polyamine, spermine (Spm), and transmitted by mitochondrial dysfunction is proposed in tobacco. In this investigation, we further resolve the pathway by identifying a subset of hypersensitive response (HR) marker genes as downstream components. In a previous report, we identified harpin-induced 1 (HIN1) and two closely related genes as responsive to Spm. Other HR marker genes, HSR203J, HMGR, HSR201, and HSR515, are also Spm-responsive. Induction of these HR marker genes, including HIN1, by Spm was suppressed by pre-treatment with antioxidants, calcium channel blockers, inhibitor of mitochondrial permeability transition pore openings, and blockers of amine oxidase/polyamine oxidase. Such quenching is also observed for Spm-induced activation of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK), and upregulation of the WIPK gene, suggesting that all these components are part of the same signaling pathway. Furthermore, gain-of-function and loss-of-function studies on MAPK cascade members reveal that the expression of Spm-induced HR marker genes varies with respect to involvement of SIPK/WIPK activation.     

Mapping the virus and host genes involved in the resistance response in cucumber mosaic virus-Infected Arabidopsis thaliana

A yellow strain of cucumber mosaic virus (CMV) [CMV(Y)] induces a resistance response characterized by inhibition of virus systemic movement with development of necrotic local lesions in the virus-inoculated leaves of Arabidopsis thaliana ecotype C24. In this report, the avirulence determinant in the virus genome was defined and the resistance gene (RCY1) of C24 was genetically mapped. The response of C24 to CMV containing the chimeric RNA3 between CMV(Y) and a virulent strain of CMV indicated that the coat protein gene of CMV(Y) determined the localization of the virus in the inoculated leaves of C24. The RCY1 locus was mapped between two CAPS markers, DFR and T43968, which were located in the region containing genetically defined disease resistance genes and their homologues. These results indicate that the resistance response to CMV(Y) in C24 is determined by the combination of the coat protein gene and RCY1 on chromosome 5.     

Functional diversity inside the Arabidopsis polyamine oxidase gene family

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.     

Oryza sativa polyamine oxidase 1 back-converts tetraamines, spermine and thermospermine, to spermidine

Key message

Oryza sativa polyamine oxidase 1 back-converts spermine (or thermospermine) to spermidine. Considering the previous work, major path of polyamine catabolism in rice plant is suggestive to be back-conversion but not terminal catabolism.

Abstract

Rice (Oryza sativa) contains seven genes encoding polyamine oxidases (PAOs), termed OsPAO1 to OsPAO7, based on their chromosomal number and gene ID number. We previously showed that three of these members, OsPAO3, OsPAO4 and OsPAO5, are abundantly expressed, that their products localize to peroxisomes and that they catalyze the polyamine back-conversion reaction. Here, we have focused on OsPAO1. The OsPAO1 gene product shares a high level of identity with those of Arabidopsis PAO5 and Brassica juncea PAO. Expression of OsPAO1 appears to be quite low under physiological conditions, but is markedly induced in rice roots by spermine (Spm) or T-Spm treatment. Consistent with the above finding, the recombinant OsPAO1 prefers T-Spm as a substrate at pH 6.0 and Spm at pH 8.5 and, in both cases, back-converts these tetraamines to spermidine, but not to putrescine. OsPAO1 localizes to the cytoplasm of onion epidermal cells. Differing in subcellular localization, four out of seven rice PAOs, OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze back-conversion reactions of PAs. Based on the results, we discuss the catabolic path(s) of PAs in rice plant.     

Antagonistic interactions between the SA and JA signaling pathways in Arabidopsis modulate expression of defense genes and gene-for-gene resistance to cucumber mosaic virus

Gene-for-gene resistance to a yellow strain of cucumber mosaic virus [CMV(Y)] is conferred by the dominant RESISTANCE to CMV(Y) (RCY1) allele in the Arabidopsis thaliana ecotype C24. RCY1-conferred resistance to CMV(Y) and expression of the Pathogenesis-related 1 (PR-1) and PR-5 genes are partially compromised by the eds5 mutation and the nahG transgene that block accumulation of salicylic acid (SA). In contrast, the RCY1-conferred resistance to CMV(Y) is not affected by the jasmonic acid (JA)-insensitive coi1 and jar1 mutations. Interestingly, we report here that in contrast to the eds5 RCY1 plant, the eds5 coi1 RCY1 double-mutant plant exhibited a higher level of resistance to CMV(Y). Presence of the coi1 mutant allele also restored the CMV(Y)-activated expression of the PR-1 and PR-5 gene in the eds5 coi1 RCY1 plant. In contrast to the PR-1 and PR-5 genes, expression of the JA-dependent PLANT DEFENSIN 1.2 (PDF1.2) and HEVEIN-LIKE PROTEIN (HEL) genes was elevated in the CMV(Y)-inoculated leaves of the eds5 RCY1 plant, but not in the virus-inoculated leaves of the wild-type RCY1 and coi1 RCY1 plants. We propose that antagonistic interactions between the SA and JA signaling mechanisms modulate defense gene expression and the activation of RCY1-conferred gene-for-gene resistance to CMV(Y).     

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.     

Effect of thermospermine on expression profiling of different gene using massive analysis of cDNA ends (MACE) and vascular maintenance in Arabidopsis

Arabidopsis thaliana polyamine oxidase 5 gene (AtPAO5) functions as a thermospermine (T-Spm) oxidase. Aerial growth of its knock-out mutant (Atpao5-2) was significantly repressed by low dose(s) of T-Spm but not by other polyamines. To figure out the underlying mechanism, massive analysis of 3′-cDNA ends was performed. Low dose of T-Spm treatment modulates more than two fold expression 1,398 genes in WT compared to 3186 genes in Atpao5-2. Cell wall, lipid and secondary metabolisms were dramatically affected in low dose T-Spm-treated Atpao5-2, in comparison to other pathways such as TCA cycle-, amino acid- metabolisms and photosynthesis. The cell wall pectin metabolism, cell wall proteins and degradation process were highly modulated. Intriguingly Fe-deficiency responsive genes and drought stress-induced genes were also up-regulated, suggesting the importance of thermospermi′ne flux on regulation of gene network. Histological observation showed that the vascular system of the joint part between stem and leaves was structurally dissociated, indicating its involvement in vascular maintenance. Endogenous increase in T-Spm and reduction in H₂O₂ contents were found in mutant grown in T-Spm containing media. The results indicate that T-Spm homeostasis by a fine tuned balance of its synthesis and catabolism is important for maintaining gene regulation network and the vascular system in plants.     

Isolation of an Arabidopsis thaliana Mutant in Which the Multiplication of both Cucumber Mosaic Virus and Turnip Crinkle Virus Is Affected

During the systemic infection of plants by viruses, host factors play an important role in supporting virus multiplication. To identify and characterize the host factors involved in this process, we isolated an Arabidopsis thaliana mutant named RB663, in which accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in RB663 plants was controlled by a monogenic, recessive mutation designated cum2-1, which is located on chromosome III and is distinct from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants, whereas the multiplication in RB663 protoplasts was similar to that in wild-type protoplasts. This suggests that the cum2-1 mutation affects the cell-to-cell movement of CMV rather than CMV replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of RB663 plants compared to that in wild-type plants. Furthermore, the phenotype of delayed TCV multiplication cosegregated with the cum2-1 mutation as far as we examined. Therefore, the cum2-1 mutation is likely to affect the cell-to-cell movement of both CMV and TCV, implying a common aspect to the mechanisms of cell-to-cell movement in these two distinct viruses.     

Suppression of antiviral silencing by cucumber mosaic virus 2b protein in Arabidopsis is associated with drastically reduced accumulation of three classes of viral small interfering RNAs

We investigated the genetic pathway in Arabidopsis thaliana targeted during infection by cucumber mosaic virus (CMV) 2b protein, known to suppress non-cell-autonomous transgene silencing and salicylic acid (SA)-mediated virus resistance. We show that 2b expressed from the CMV genome drastically reduced the accumulation of 21-, 22-, and 24-nucleotide classes of viral small interfering RNAs (siRNAs) produced by Dicer-like4 (DCL4), DCL2, and DCL3, respectively. The defect of a CMV 2b-deletion mutant (CMV-Delta2b) in plant infection was efficiently rescued in Arabidopsis mutants producing neither 21- nor 22-nucleotide viral siRNAs. Since genetic analysis further identifies a unique antiviral role for DCL3 upstream of DCL4, our data indicate that inhibition of the accumulation of distinct viral siRNAs plays a key role in 2b suppression of antiviral silencing. Strikingly, disease symptoms caused by CMV-Delta2b in Arabidopsis mutants defective in antiviral silencing were as severe as those caused by CMV, demonstrating an indirect role for the silencing suppressor activity in virus virulence. We found that production of CMV siRNAs without 2b interference depended largely on RNA-dependent RNA polymerase 1 (RDR1) inducible by SA. Given the known role of RDR6-dependent transgene siRNAs in non-cell-autonomous silencing, our results suggest a model in which 2b inhibits the production of RDR1-dependent viral siRNAs that confer SA-dependent virus resistance by directing non-cell-autonomous antiviral silencing.