A new P450 involved in the furanocoumarin pathway underlies a recent case of convergent evolution.

Furanocoumarins are phytoalexins often cited as an example to illustrate the arms race between plants and herbivorous insects. They are distributed in a limited number of phylogenetically distant plant lineages, but synthesized through a similar pathway, which raised the question of a unique or multiple emergence in higher plants. The furanocoumarin pathway was investigated in the fig tree (Ficus carica, Moraceae). Transcriptomic and metabolomic approaches led to the identification of CYP76F112, a cytochrome P450 catalyzing an original reaction. CYP76F112 emergence was inquired using phylogenetics combined with in silico modeling and site-directed mutagenesis. CYP76F112 was found to convert demethylsuberosin into marmesin with a very high affinity. This atypical cyclization reaction represents a key step within the polyphenol biosynthesis pathway. CYP76F112 evolutionary patterns suggests that the marmesin synthase activity appeared recently in the Moraceae family, through a lineage-specific expansion and diversification. The characterization of CYP76F112 as the first known marmesin synthase opens new prospects for the use of the furanocoumarin pathway. It also supports the multiple acquisition of furanocoumarin in angiosperms by convergent evolution, and opens new perspectives regarding the ability of cytochromes P450 to evolve new functions related to plant adaptation to their environment.

Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei × N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37 °C, but after shifting to 26 °C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Radish sprouts as an efficient and rapidly available host for an agroinfiltration-based transient gene expression system.

Agroinfiltration, the infiltration of plants with Agrobacterium harboring a plasmid that contains a specific gene, is used to transiently express a gene in a heterologous organism. Using the "Tsukuba system", greater amounts of target protein accumulate compared with usual expression plasmids. Reported host plants, including Nicotiana benthamiana, a common plant for agroinfiltration, need several weeks after sowing to grow enough for infection. To shorten the culture period and, thereby, improve target protein production, we tested sprouts as host plants. Sprouts were grown in the dark to encourage elongation so that vacuum infiltration becomes easier, and this was followed by a few days of exposure to illumination before infection with pBYR2HS-EGFP, the EGFP expression plasmid of the Tsukuba system. Among six tested species of Fabaceae and Brassicaceae, radish showed the highest transient expression. Among six tested radish cultivars, Kaiware, Hakata, and Banryoku provided the best results. Culturing for 5 day, including 1 day of imbibition and 1 to 2 day of exposure to illumination resulted in EGFP fluorescence in 80% of the cotyledon area. Thus, a remarkable amount of EGFP was obtained only 8 day after seed imbibition. The EGFP amount in Kaiware cotyledons was comparable with Rubisco at ∼0.7 mg/g fresh weight. Kaiware sold in supermarkets could also be used, but resulted in lower expression levels.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

A simple and highly sensitive method of measuring heme oxygenase activity.

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in microorganisms, insects, plants, and mammals. Previously used assays of HO activity were complicated and had low sensitivity. We found that the use of an eel bilirubin-bound fluorescent protein, UnaG, can achieve a highly sensitive and simple assay of HO activity. Using several enzyme sources including human culture cells, homogenates of plant tissues, and recombinant yeast HO, data were successfully obtained. The present method can facilitate the examination of HO in various organisms.

Continuous de novo biosynthesis of haem and its rapid turnover to bilirubin are necessary for cytoprotection against cell damage.

It is well known that haem serves as the prosthetic group of various haemoproteins that function in oxygen transport, respiratory chain, and drug metabolism. However, much less is known about the functions of the catabolites of haem in mammalian cells. Haem is enzymatically degraded to iron, carbon monoxide (CO), and biliverdin, which is then converted to bilirubin. Owing to difficulties in measuring bilirubin, however, the generation and transport of this end product remain unclear despite its clinical importance. Here, we used UnaG, the recently identified bilirubin-binding fluorescent protein, to analyse bilirubin production in a variety of human cell lines. We detected a significant amount of bilirubin with many non-blood cell types, which was sensitive to inhibitors of haem metabolism. These results suggest that there is a basal level of haem synthesis and its conversion into bilirubin. Remarkably, substantial changes were observed in the bilirubin generation when cells were exposed to stress insults. Since the stress-induced cell damage was exacerbated by the pharmacological blockade of haem metabolism but was ameliorated by the addition of biliverdin and bilirubin, it is likely that the de novo synthesis of haem and subsequent conversion to bilirubin play indispensable cytoprotective roles against cell damage.

A regulatory cascade involving class II ETHYLENE RESPONSE FACTOR transcriptional repressors operates in the progression of leaf senescence.

Leaf senescence is the final process of leaf development that involves the mobilization of nutrients from old leaves to newly growing tissues. Despite the identification of several transcription factors involved in the regulation of this process, the mechanisms underlying the progression of leaf senescence are largely unknown. Herein, we describe the proteasome-mediated regulation of class II ETHYLENE RESPONSE FACTOR (ERF) transcriptional repressors and involvement of these factors in the progression of leaf senescence in Arabidopsis (Arabidopsis thaliana). Based on previous results showing that the tobacco (Nicotiana tabacum) ERF3 (NtERF3) specifically interacts with a ubiquitin-conjugating enzyme, we examined the stability of NtERF3 in vitro and confirmed its rapid degradation by plant protein extracts. Furthermore, NtERF3 accumulated in plants treated with a proteasome inhibitor. The Arabidopsis class II ERFs AtERF4 and AtERF8 were also regulated by the proteasome and increased with plant aging. Transgenic Arabidopsis plants with enhanced expression of NtERF3, AtERF4, or AtERF8 showed precocious leaf senescence. Our gene expression and chromatin immunoprecipitation analyses suggest that AtERF4 and AtERF8 targeted the EPITHIOSPECIFIER PROTEIN/EPITHIOSPECIFYING SENESCENCE REGULATOR gene and regulated the expression of many genes involved in the progression of leaf senescence. By contrast, an aterf4 aterf8 double mutant exhibited delayed leaf senescence. Our results provide insight into the important role of class II ERFs in the progression of leaf senescence.

Quinolone compounds enhance delta-aminolevulinic acid-induced accumulation of protoporphyrin IX and photosensitivity of tumour cells.

Exogenous δ-aminolevulinic acid (ALA)-induced photodynamic therapy (PDT) has been used in the treatment of cancer. To obtain a high efficacy of ALA-PDT, we have screened various chemicals affecting ALA-induced accumulation of protoporphyrin in cancerous cells. When HeLa cells were treated with quinolone chemicals including enoxacin, ciprofloxacin or norfloxacin, the ALA-induced photodamage accompanied by the accumulation of protoporphyrin was stronger than that with ALA alone. Thus, quinolone compounds such as enoxacin, ciprofloxacin and norfloxacin enhanced ALA-induced photodamage. The increased ALA-induced photodamage in enoxacin-treated HeLa cells was decreased by haemin or ferric-nitrilotriacetate (Fe-NTA), suggesting that an increase in iron supply cancels the accumulation of protoporphyrin. On the other hand, the treatment of the cells with ALA plus an inhibitor of haem oxygenase, Sn-protoporphyrin, led to an increase in the photodamage and the accumulation of protoporphyrin compared with those upon treatment with ALA alone, indicating that the cessation of recycling of iron from haem augments the accumulation. The use of quinolones plus Sn-protoporphyrin strongly enhances ALA-induced photodamage. To examine the mechanisms involved in the increased accumulation of protoporphyrin, we incubated ferric chloride with an equivalent amount of quinolones. Iron-quinolone complexes with visible colours with a maximum at 450 nm were formed. The levels of iron-metabolizing proteins in enoxacin- or ciprofloxacin-treated cells changed, indicating that quinolones decrease iron utilization for haem biosynthesis. Hence, we now propose that the use of quinolones in combination with ALA may be an extremely effective approach for the treatment modalities for PDT of various tumour tissues in clinical practice.

Dual mitochondrial localization and different roles of the reversible reaction of mammalian ferrochelatase.

Ferrochelatase catalyzes the insertion of ferrous ions into protoporphyrin IX to produce heme. Previously, it was found that this enzyme also participates in the reverse reaction of iron removal from heme. To clarify the role of the reverse reaction of ferrochelatase in cells, mouse liver mitochondria were fractionated to examine the localization of ferrochelatase, and it was found that the enzyme localizes not only to the inner membrane, but also to the outer membrane. Observations by immunoelectron microscopy confirmed the dual localization of ferrochelatase in ferrochelatase-expressing human embryonic kidney cells and mouse liver mitochondria. The conventional (zinc-insertion) activities of the enzyme in the inner and outer membranes were similar, whereas the iron-removal activity was high in the outer membrane. 2D gel analysis revealed that two types of the enzyme with different isoelectric points were present in mitochondria, and the acidic form, which was enriched in the outer membrane, was found to be phosphorylated. Mutation of human ferrochelatase showed that serine residues at positions 130 and 303 were phosphorylated, and serine at position 130 may be involved in the balance of the reversible catalytic reaction. When mouse erythroleukemia cells were treated with 12-O-tetradecanoyl-phorbol 13-acetate, an activator of protein kinase C, or hemin, phospho-ferrochelatase levels increased, with a concomitant decrease in zinc-insertion activity and a slight increase in iron-removal activity. These results suggest that ferrochelatase localizes to both the mitochondrial outer and inner membranes and that the change in the equilibrium position of the forward and reverse activities may be regulated by the phosphorylation of ferrochelatase.

Triple mutation of Cys26, Trp35, and Cys126 in stromal ascorbate peroxidase confers H2O2 tolerance comparable to that of the cytosolic isoform.

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid of the chloroplast play a principle role in detoxifying hydrogen peroxide (H(2)O(2)) generated in photosystem I; however, once the ascorbate is depleted, the enzyme is attacked by H(2)O(2) and rapidly loses its activity. Here, we report that radical transfer across the porphyrin moiety and amino acid residues in the reaction intermediate and H(2)O(2)-mediated enzyme inactivation involve cooperative interactions of the Cys26, Trp35, and Cys126 residues of stromal APX. The wild-type enzyme had a half-time of inactivation of <10s, while the triple mutant of the three residues retained 50% of the initial activity after H(2)O(2) treatment for 3 min. The H(2)O(2) tolerance of this mutant was comparable to that of the H(2)O(2)-tolerant APX isoform localized in the cytosol.

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase.

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.

Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins.

The red pigments in meat products, including cooked cured ham, arise from the reaction of myoglobin with nitric oxide generated from exogenous nitrite. Since carcinogenic nitrosoamines may be generated by the treatment of meats with nitrite, the production of nitrite-free meat products is an attractive alternative. Raw dry-cured (Parma) hams are produced by the treatment of meats with salts other than nitrite. Analysis of pigments in raw dry-cured hams reveals that the main pigment is zinc protoporphyrin, suggesting that the conversion of heme to zinc protoporphyrin occurs via an iron-removal reaction from myoglobin heme during the processing of raw hams. Purification of the iron-removal enzyme showed that it was identical to ferrochelatase. Recombinant ferrochelatase in combination with NADH-cytochrome b5 reductase catalyzed NADH-dependent iron-removal reaction from hemin and hemoproteins. Metal ions such as zinc and cobalt were also removed from the corresponding metalloporphyrins. The addition of zinc ions led to the formation of zinc protoporphyrin. In cultured cells, the conversion of zinc mesoporphyrin to mesoheme was observed to be dependent on ferrochelatase and could be markedly induced during erythroid differentiation. This is the first demonstration of a new enzyme reaction, the reverse reaction of ferrochelatase, which may contribute to a new route of the recycling of protoporphyrin and heme in cells.

Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2.

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from <10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.

The role of nitric oxide in delta-aminolevulinic acid (ALA)-induced photosensitivity of cancerous cells.

Application of delta-aminolevulinic acid (ALA) results in the endogenous accumulation of protoporphyrin IX and is a useful approach in the photodynamic therapy (PDT) of cancers. To investigate the role of nitric oxide (NO) in the specific accumulation of protoporphyrin and ALA-induced PDT of cancerous cells, we transfected inducible-nitric oxide synthase (NOS2) cDNA into human embryonic kidney (HEK) 293T cells and examined the ALA-induced photo-damage as well as the accumulation of porphyrin in the cells. When the NOS2-expressing HEK293T cells were treated with ALA and then exposed to visible light, they became more sensitive to the light with accumulating porphyrins, as compared with the ALA-treated control cells. An increase in the generation of NO in transfected cells led to the accumulation of protoporphyrin with a concomitant decrease of ferrochelatase, the final step enzyme of heme biosynthesis. When mouse macrophage-like RAW264.7 cells were cultured with lipopolysaccharide and interferon-gamma, the expression of NOS2 was induced. The addition of ALA to these cells led to the accumulation of protoporphyrin and cell death upon exposure to light. The treatment of cells with an NOS inhibitor, NG-monomethyl-L-arginine acetate, resulted in the inhibition of protoporphyrin accumulation and cell death. The levels of mitochondrial ferrochelatase and rotenone-sensitive NADH dehydrogenase in the NOS2-induced cells decreased. These results indicated that the generation of NO augments the ALA-induced accumulation of protoporphyrin IX and subsequent photo-damage in cancerous cells by decreasing the levels of mitochondrial iron-containing enzymes. Based on the fact that the production of NO in cancerous cells is elevated, NO in the cells is responsible for susceptibility with ALA-induced PDT.

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation.

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.

Mechanisms involved in delta-aminolevulinic acid (ALA)-induced photosensitivity of tumor cells: relation of ferrochelatase and uptake of ALA to the accumulation of protoporphyrin.

Photodynamic therapy (PDT) using delta-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX is a useful approach to the early detection and treatment of cancers. To investigate the role of ferrochelatase in the accumulation of protoporphyrin, we first made mouse fibroblast Balb/3T3 cells highly expressing ferrochelatase and examined the ALA-induced photo-damage as well as the accumulation of porphyrin in the cells. When the ferrochelatase-transfected cells were treated with ALA and then exposed to visible light, they became resistant to the light without accumulating porphyrins, with a concomitant increase in the formation of heme. The accumulation of protoporphyrin was also abolished in human erythroleukemia K562 cells stably expressing mouse ferrochelatase. When mouse fibrosarcoma MethA cells, mouse fibroblast L929 cells and Balb/3T3 cells were treated with ALA, the greatest accumulation of protoporphyrin and the greatest level of cell death in response to the light were observed in MethA cells. The expression level of ferrochelatase was the lowest in MethA cells, while that of porphobilinogen deaminase was similar among all three cell lines. Moreover, an iron-chelator, desferrioxamine, which sequesters iron preventing the ferrochelatase reaction, enhanced the photo-damage as well as the accumulation of protoporphyrin in ALA-treated L929 cells. Thus, the light-induced cell death was tightly coupled with the accumulation of protoporphyrin caused by a decrease in ferrochelatase. Finally, we examined the uptake of ALA by MethA, L929 and Balb/3T3 cells. The extent of the uptake by MethA and L929 cells was greater, indicating a greater accumulation of protoporphyrin than in the Balb/3T3 cells. Taken together, not only the low level of ferrochelatase but also the augmented uptake of ALA contributes to the ALA-induced accumulation of protoporphyrin IX and subsequent photo-damage in cancer cells.

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori.

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.

Functional analysis of Arabidopsis ethylene-responsive element binding protein conferring resistance to Bax and abiotic stress-induced plant cell death.

Arabidopsis (Arabidopsis thaliana) ethylene-responsive element binding protein (AtEBP) gene was isolated as a suppressor of Bax-induced cell death by functional screening in yeast (Saccharomyces cerevisiae). To further examine the cell death suppressive action of AtEBP in plant cells, we established transgenic tobacco (Nicotiana tabacum) plants overexpressing AtEBP as well as transgenic tobacco plants ectopically expressing mouse Bax protein under a dexamethasone-inducible promoter. We prepared the crosses of the selective lines of each transgenic plant, which were evaluated in terms of cell death suppression activity. Results indicate that AtEBP suppressed Bax-induced cell death in tobacco plants, an action also associated with a lowered level of ion leakage. Furthermore, tobacco Bright Yellow-2 cells overexpressing AtEBP conferred resistance to hydrogen peroxide (H(2)O(2)) and heat treatments. AtEBP protein localized in the nucleus and functioned as an in vivo transcription activator as confirmed in transient assays and experiments using stable transgenic system. Up-regulation of defense genes was observed in transgenic Arabidopsis plants overexpressing AtEBP. Based on the analysis of mRNA accumulation in ethylene-related mutants, the position of AtEBP in signaling pathway is presented.

Isolation of tobacco ubiquitin-conjugating enzyme cDNA in a yeast two-hybrid system with tobacco ERF3 as bait and its characterization of specific interaction.

Tobacco ETHYLENE-RESPONSIVE FACTOR3 (ERF3) is a member of the ERF-domain transcription factors and has a transcriptional repressor activity, whereas other ERF proteins show activation activity. To understand the regulation of ERF3-repressor activity, protein(s) were screened which interact with ERF3 in a yeast two-hybrid system. A partial sequence (B8) of NtUBC2, a tobacco ubiquitin-conjugating enzyme was isolated. This B8 specifically interacted with ERF3 in the yeast two-hybrid system. Further analyses revealed that the region unique to ERF3 interacted with B8. The physiological functions of NtUBC2 and the stability of ERF3 are discussed in relation to the regulation of the repression activity of ERF3.