Expression profile of seven polyamine oxidase genes in rice (Oryza sativa) in response to abiotic stresses, phytohormones and polyamines.

Polyamine levels are controlled by biosynthesis, intra- and inter-cellular flux by the respective transporters, and catabolism. The catabolism is catalyzed by two groups of enzymes. One is copper-containing amine oxidases and the other is polyamine oxidases (PAOs). In Oryza sativa, seven PAO genes exist and they are termed as OsPAO1 to OsPAO7. However, their physiological function has not been elucidated yet. Here, we examined the expressional changes of seven OsPAO genes upon abiotic and oxidative stress, phytohormone, and exogenous polyamines application. The transcript of extracellular polyamine oxidase OsPAO2 and OsPAO6 are strongly induced upon wounding, drought, salinity, oxidative stress (H2O2), and exogenous application of jasmonic acid, spermidine, spermine, thermospermine and negatively regulated upon indole acetic acid, isopentenyl adenine (iPT), gibberellic acid (GA), abscisic acid; OsPAO7 is to iPT, GA and all polyamines; OsPAO4 and OsPAO5 are mildly responsive to heat, cold, oxidative stress. These results suggest that polyamine oxidase encoding extracellular enzyme may play a pivotal role during exogenous stimulus to protect the plant cell. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01006-1.

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.

Scots pine aminopropyltransferases shed new light on evolution of the polyamine biosynthesis pathway in seed plants.

Background and Aims: Polyamines are small metabolites present in all living cells and play fundamental roles in numerous physiological events in plants. The aminopropyltransferases (APTs), spermidine synthase (SPDS), spermine synthase (SPMS) and thermospermine synthase (ACL5), are essential enzymes in the polyamine biosynthesis pathway. In angiosperms, SPMS has evolved from SPDS via gene duplication, whereas in gymnosperms APTs are mostly unexplored and no SPMS gene has been reported. The present study aimed to investigate the functional properties of the SPDS and ACL5 proteins of Scots pine (Pinus sylvestris L.) in order to elucidate the role and evolution of APTs in higher plants. Methods: Germinating Scots pine seeds and seedlings were analysed for polyamines by high-performance liquid chromatography (HPLC) and the expression of PsSPDS and PsACL5 genes by in situ hybridization. Recombinant proteins of PsSPDS and PsACL5 were produced and investigated for functional properties. Also gene structures, promoter regions and phylogenetic relationships of PsSPDS and PsACL5 genes were analysed. Key Results: Scots pine tissues were found to contain spermidine, spermine and thermospermine. PsSPDS enzyme catalysed synthesis of both spermidine and spermine. PsACL5 was found to produce thermospermine, and PsACL5 gene expression was localized in the developing procambium in embryos and tracheary elements in seedlings. Conclusions: Contrary to previous views, our results demonstrate that SPMS activity is not a novel feature developed solely in the angiosperm lineage of seed plants but also exists as a secondary property in the Scots pine SPDS enzyme. The discovery of bifunctional SPDS from an evolutionarily old conifer reveals the missing link in the evolution of the polyamine biosynthesis pathway. The finding emphasizes the importance of pre-existing secondary functions in the evolution of new enzyme activities via gene duplication. Our results also associate PsACL5 with the development of vascular structures in Scots pine.

Outer Membrane Proteins Derived from Non-cyanobacterial Lineage Cover the Peptidoglycan of Cyanophora paradoxa Cyanelles and Serve as a Cyanelle Diffusion Channel.

The cyanelle is a primitive chloroplast that contains a peptidoglycan layer between its inner and outer membranes. Despite the fact that the envelope structure of the cyanelle is reminiscent of Gram-negative bacteria, the Cyanophora paradoxa genome appears to lack genes encoding homologs of putative peptidoglycan-associated outer membrane proteins and outer membrane channels. These are key components of Gram-negative bacterial membranes, maintaining structural stability and regulating permeability of outer membrane, respectively. Here, we discovered and characterized two dominant peptidoglycan-associated outer membrane proteins of the cyanelle (∼2 × 10(6) molecules per cyanelle). We named these proteins CppF and CppS (cyanelle peptidoglycan-associated proteins). They are homologous to each other and function as a diffusion channel that allows the permeation of compounds with Mr <1,000 as revealed by permeability measurements using proteoliposomes reconstituted with purified CppS and CppF. Unexpectedly, amino acid sequence analysis revealed no evolutionary linkage to cyanobacteria, showing only a moderate similarity to cell surface proteins of bacteria belonging to Planctomycetes phylum. Our findings suggest that the C. paradoxa cyanelle adopted non-cyanobacterial lineage proteins as its main outer membrane components, providing a physical link with the underlying peptidoglycan layer and functioning as a diffusion route for various small substances across the outer membrane.

A novel strategy to produce sweeter tomato fruits with high sugar contents by fruit-specific expression of a single bZIP transcription factor gene.

Enhancement of sugar content and sweetness is desirable in some vegetables and in almost all fruits; however, biotechnological methods to increase sugar content are limited. Here, a completely novel methodological approach is presented that produces sweeter tomato fruits but does not have any negative effects on plant growth. Sucrose-induced repression of translation (SIRT), which is mediated by upstream open reading frames (uORFs), was initially reported in Arabidopsis AtbZIP11, a class S basic region leucine zipper (bZIP) transcription factor gene. Here, two AtbZIP11 orthologous genes, SlbZIP1 and SlbZIP2, were identified in tomato (Solanum lycopersicum). SlbZIP1 and SlbZIP2 contained four and three uORFs, respectively, in the cDNA 5'-leader regions. The second uORFs from the 5' cDNA end were conserved and involved in SIRT. Tomato plants were transformed with binary vectors in which only the main open reading frames (ORFs) of SlbZIP1 and SlbZIP2, without the SIRT-responsive uORFs, were placed under the control of the fruit-specific E8 promoter. Growth and morphology of the resulting transgenic tomato plants were comparable to those of wild-type plants. Transgenic fruits were approximately 1.5-fold higher in sugar content (sucrose/glucose/fructose) than nontransgenic tomato fruits. In addition, the levels of several amino acids, such as asparagine and glutamine, were higher in transgenic fruits than in wild-type fruits. This was expected because SlbZIP transactivates the asparagine synthase and proline dehydrogenase genes. This 'sweetening' technology is broadly applicable to other plants that utilize sucrose as a major translocation sugar.

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

KEY MESSAGE: Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity.

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.

Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifically expressed in anthers.

Polyamine oxidase (PAO), which requires FAD as a cofactor, functions in polyamine catabolism. Plant PAOs are classified into two groups based on their reaction modes. The terminal catabolism (TC) reaction always produces 1,3-diaminopropane (DAP), H2O2, and the respective aldehydes, while the back-conversion (BC) reaction produces spermidine (Spd) from tetraamines, spermine (Spm) and thermospermine (T-Spm) and/or putrescine from Spd, along with 3-aminopropanal and H2O2. The Oryza sativa genome contains seven PAO-encoded genes termed OsPAO1-OsPAO7. To date, we have characterized four OsPAO genes. The products of these genes, i.e. OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze BC-type reactions. Whereas OsPAO1 remains in the cytoplasm, the other three PAOs localize to peroxisomes. Here, we examined OsPAO7 and its gene product. OsPAO7 shows high identity to maize ZmPAO1, the best characterized plant PAO having TC-type activity. OsPAO7 seems to remain in a peripheral layer of the plant cell with the aid of its predicted signal peptide and transmembrane domain. Recombinant OsPAO7 prefers Spm and Spd as substrates, and it produces DAP from both substrates in a time-dependent manner, indicating that OsPAO7 is the first TC-type enzyme identified in O. sativa. The results clearly show that two types of PAOs co-exist in O. sativa. Furthermore, OsPAO7 is specifically expressed in anthers, with an expressional peak at the bicellular pollen stage. The physiological function of OsPAO7 in anthers is discussed.

Overexpression of rice OsREX1-S, encoding a putative component of the core general transcription and DNA repair factor IIH, renders plant cells tolerant to cadmium- and UV-induced damage by enhancing DNA excision repair.

Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1-S as a transgene. OsREX1-S shows the highest levels of identity to Chlamydomonas reinhardtii REX1-S (referred to as CrREX1-S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1-S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1-S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1-S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1-S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair.

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. 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.

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.

Rice DEP1, encoding a highly cysteine-rich G protein γ subunit, confers cadmium tolerance on yeast cells and plants.

A rice cDNA, OsDEP1, encoding a highly cysteine (Cys)-rich G protein γ subunit, was initially identified as it conferred cadmium (Cd) tolerance on yeast cells. Of the 426 aa constituting OsDEP1, 120 are Cys residues (28.2%), of which 88 are clustered in the C-terminal half region (aa 170-426). To evaluate the independent effects of these two regions, two truncated versions of the OsDEP1-expressing plasmids pOsDEP1(1-169) and pOsDEP1(170-426) were used to examine their effects on yeast Cd tolerance. Although OsDEP1(170-426) conferred a similar level of Cd tolerance as the intact OsDEP1, OsDEP1(1-169) provided no such tolerance, indicating that the tolerance effect is localized to the aa 170-426 C-terminal peptide region. The Cd responses of transgenic Arabidopsis plants constitutively expressing OsDEP1, OsDEP1(1-169) or OsDEP1(170-426), were similar to the observations in yeast cells, with OsDEP1 and OsDEP1(170-426) transgenic plants displaying Cd tolerance but OsDEP1(1-169) plants showing no such tolerance. In addition, a positive correlation between the transcript levels of OsDEP1 or OsDEP1(170-426) in the transgenics and the Cd content of these plants upon Cd application was observed. As several Arabidopsis loss-of-function heterotrimeric G protein ß and γ subunit gene mutants did not show differences in their Cd sensitivity compared with wild-type plants, we propose that the Cys-rich region of OsDEP1 may function directly as a trap for Cd ions.

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.

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. 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.

RCY1-mediated resistance to Cucumber mosaic virus is regulated by LRR domain-mediated interaction with CMV(Y) following degradation of RCY1.

RCY1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (LRR) class R protein, confers the hypersensitive response (HR) to a yellow strain of Cucumber mosaic virus (CMV[Y]) in Arabidopsis thaliana. Nicotiana benthamiana transformed with hemagglutinin (HA) epitope-tagged RCY1 (RCY1-HA) also exhibited a defense response accompanied by HR cell death and induction of defense-related gene expression in response to CMV(Y). Following transient expression of RCY1-HA by agroinfiltration, the defense reaction was induced in N. benthamiana leaves infected with CMV(Y) but not in virulent CMV(B2)-infected N. benthamiana leaves transiently expressing RCY1-HA or CMV(Y)-infected N. benthamiana leaves transiently expressing HA-tagged RPP8 (RPP8-HA), which is allelic to RCY1. This result suggests that Arabidopsis RCY1-conferred resistance to CMV(Y) could be reproduced in N. benthamiana leaves in a gene-for-gene manner. Expression of a series of chimeric constructs between RCY1-HA and RPP8-HA in CMV(Y)-infected N. benthamiana indicated that induction of defense responses to CMV(Y) is regulated by the LRR domain of RCY1. Interestingly, in CMV(Y)-infected N. benthamiana manifesting the defense response, the levels of both RCY1 and chimeric proteins harboring the RCY1 LRR domain were significantly reduced. Taken together, these data indicate that the RCY1-conferred resistance response to CMV(Y) is regulated by an LRR domain-mediated interaction with CMV(Y) and seems to be tightly associated with the degradation of RCY1 in response to CMV(Y).

An inhibitory effect of the sequence-conserved upstream open-reading frame on the translation of the main open-reading frame of HsfB1 transcripts in Arabidopsis.

Plants have as many as 20 heat shock factors (Hsfs) grouped into three classes, A, B and C, based on sequence similarity and modular structures. Through screening for cell death-inducing factor(s) in Nicotiana benthamiana, we identified Arabidopsis HsfB2b and thus subjected all other members of Arabidopsis Hsf class B (HsfB1, HsfB2a, HsfB2b, HsfB3 and HsfB4) to the same cell death assay. When expressed in N. benthamiana leaves, only HsfB1 and HsfB2b elicited mild cell death. Simultaneously we found that HsfB1 has a post-transcriptional control mechanism, in which a sequence-conserved upstream open-reading frame (sc-uORF) is involved. The known repressor function of the respective HsfBs was confirmed and the difference in cell death-inducing activity of HsfBs was explained by the fact that HsfB1 and HsfB2b are transcriptional repressors but the others are not. Indeed, the cell death symptom by HsfB1 and HsfB2b required not only their repression activity but also their nuclear localization activity. HsfB1 expression was drastically and transiently induced by heat shock (HS) and the intactness of sc-uORF was required for its HS response. Based on the results, the physiological significance of cell death-inducing activity of HsfB1 and HsfB2b and the sc-uORF in the HsfB1 transcript during HS response is discussed.

Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion.

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine (PA) catabolism. Recent studies have revealed that plant PAOs are not only active in the terminal catabolism of PAs as demonstrated for maize apoplastic PAO but also in a polyamine back-conversion pathway as shown for most Arabidopsis PAOs. We have characterized Oryza sativa PAOs at molecular and biochemical levels. The rice genome contains 7 PAO isoforms that are termed OsPAO1 to OsPAO7. Of the seven PAOs, OsPAO3, OsPAO4, and OsPAO5 transcripts were most abundant in 2-week-old seedlings and mature plants, while OsPAO1, OsPAO2, OsPAO6, and OsPAO7 were expressed at very low levels with different tissue specificities. The more abundantly expressed PAOs--OsPAO3, OsPAO4, and OsPAO5--were cloned, and their gene products were produced in Escherichia coli. The enzymatic activities of the purified OsPAO3 to OsPAO5 proteins were examined. OsPAO3 favored spermidine (Spd) as substrate followed by thermospermine (T-Spm) and spermine (Spm) and showed a full PA back-conversion activity. OsPAO4 substrate specificity was similar to that of OsPAO5 preferring Spm and T-Spm but not Spd. Those enzymes also converted Spm and T-Spm to Spd, again indicative of PA back-conversion activities. Lastly, we show that OsPAO3, OsPAO4, and OsPAO5 are localized in peroxisomes. Together, these data revealed that constitutively and highly expressed O. sativa PAOs are localized in peroxisomes and catalyze PA back-conversion processes.

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

UNLABELLED: 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 MESSAGE: Polyamine 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.

Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis.

The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of VDACs in plants is largely unknown. In order to determine the individual function of plant VDACs, molecular and genetic analysis was performed on four VDAC genes, VDAC1-VDAC4, found in Arabidopsis thaliana. VDAC1 and VDAC3 possess the eukaryotic mitochondrial porin signature (MPS) in their C-termini, while VDAC2 and VDAC4 do not. Localization analysis of VDAC-green fluorescent protein (GFP) fusions and their chimeric or mutated derivatives revealed that the MPS sequence is important for mitochondrial localization. Through the functional analysis of vdac knockout mutants due to T-DNA insertion, VDAC2 and VDAC4 which are expressed in the whole plant body are important for various physiological functions such as leaf development, the steady state of the mitochondrial membrane potential, and pollen development. Moreover, it was demonstrated that VDAC1 is not only necessary for normal growth but also important for disease resistance through regulation of hydrogen peroxide generation.