Yube Yamaguchi

Periodic DNA Methylation in Maize Nucleosomes and Demethylation by Environmental Stress

When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990)Mol. Gen. Genet. 220, 441–447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.

Induction of Hypersensitive Cell Death by Hydrogen Peroxide Produced through Polyamine Degradation in Tobacco Plants

Screening immediate-early responding genes during the hypersensitive response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana tabacum) plants, we identified a gene encoding ornithine decarboxylase. Subsequent analyses showed that other genes involved in polyamine biosynthesis were also up-regulated, resulting in the accumulation of polyamines in apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine biosynthesis, α-difluoromethyl-ornithine, however, suppressed accumulation of polyamines, and the rate of HR was reduced. In contrast, polyamine infiltration into a healthy leaf induced the generation of hydrogen peroxide and simultaneously caused HR-like cell death. Polyamine oxidase activity in the apoplast increased up to 3-fold that of the basal level during the HR, and its suppression with a specific inhibitor, guazatine, resulted in reduced HR. Because it is established that hydrogen peroxide is one of the degradation products of polyamines, these results indicate that one of the biochemical events in the HR is production of polyamines, whose degradation induces hydrogen peroxide, eventually resulting in hypersensitive cell death.

Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca2+ permeable channels in mesophyll cells, resulting in cytosolic Ca2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca2+ conductance and resulting cytosolic Ca2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca2+-dependent manner.

Osmotic Stress Tolerance of Transgenic Tobacco Expressing a Gene Encoding a Membrane-Located Receptor-Like Protein from Tobacco Plants

Tobacco (Nicotiana tabacum) genes regulated during the early stage of responses to wounding were screened by a modified fluorescence differential display method. Among 28 genes initially identified, a particular clone designatedNtC7 was subjected to further analysis. Its transcripts were found to accumulate rapidly and transiently within 1 h upon treatments with not only wounding but also salt and osmotic stresses. However, jasmonic and abscisic acids and ethylene did not effectively induce NtC7 transcripts. Amino acid sequence analysis suggested NtC7 to be a new type of transmembrane protein that belongs to the receptor-like protein family, and a membrane location was confirmed in onion (Allium cepa) epidermis cells transiently expressing an NtC7-green fluorescent protein fusion protein. Seeds of transgenic tobacco overexpressing NtC7normally germinated and grew in the presence of 500 mmmannitol, but not in the presence of 220 mm sodium chloride or 60 mm lithium chloride. Cuttings of mature transgenic leaf exhibited a marked tolerance upon treatment with 500 mm mannitol for 12 h, at which concentration wild-type counterparts were seriously damaged. These results suggested that NtC7 predominantly functions in maintenance of osmotic adjustment independently of ion homeostasis.

Direct interaction between the tobacco mosaic virus helicase domain and the ATP-bound resistance protein, N factor during the hypersensitive response in tobacco plants.

Plants cope with pathogens with distinct mechanisms. One example is a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was pre-requisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50. Moreover, we found that intra- and/or inter-molecular interactions take place in the N factor molecule. This interaction required ATP, and was disrupted by the p50. Based on these results, we propose a following model for the TMV recognition mechanism in tobacco plants. The N factor forms a complex with ATP, to which the helicase domain interacts, and enhances ATP hydrolysis. The resulting ADP/N factor complex then changes its conformation, thereby facilitating further interaction with the down-stream signaling factor(s). This model is consistent with the idea of ‘protein machine’.

Molecular Cloning and Functional Characterization of Three Distinct N -Methyltransferases Involved in the Caffeine Biosynthetic Pathway in Coffee Plants

Caffeine is synthesized from xanthosine throughN-methylation and ribose removal steps. In the present study, three types of cDNAs encodingN-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated asCaXMT1, CaMXMT2, andCaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with aK m value of 78 μm, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K m of 251 μm, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with aK m of 1,222 μm. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified threeN-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.

Cadmium stress induces production of thiol compounds and transcripts for enzymes involved in sulfur assimilation pathways in Arabidopsis

Summary One of the defense systems against toxic heavy metals in plants is to employ cysteine-rich chelating compounds, phytochelatins, which are polymeric forms of glutathione. In the present study cadmium stressed Arabidopsis was employed in order to investigate the response of genes involved in glutathione biosynthesis via cysteine and production of thiol compounds. The results showed a 13-fold increase in transcripts for ATP sulfurylase, 6 to 10-fold for APS reductase and 2-fold for sulfite reductase, all of which are involved in cysteine synthesis. In addition, a 3-fold increase in total thiols was noted, mainly of glutathione, phytochelatins and cysteine. It is suggested that, to cope with toxic heavy metals, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of glutathione for phytochelatin biosynthesis.

A subtilisin-like protein from soybean contains an embedded, cryptic signal that activates defense-related genes

Among the arsenal of plant-derived compounds activated upon attack by herbivores and pathogens are small peptides that initiate and amplify defense responses. However, only a handful of plant signaling peptides have been reported. Here, we have isolated a 12-aa peptide from soybean (Glycine max) leaves that causes a pH increase of soybean suspension-cultured cell media within 10 min at low nanomolar concentrations, a response that is typical of other endogenous peptide elicitors and pathogen-derived elicitors. The amino acid sequence was determined and was found to be derived from a member of the subtilisin-like protease (subtilase) family. The sequence of the peptide was located within a region of the protein that is unique to subtilases in legume plants and not found within any other plant subtilases thus far identified. We have named this peptide signal Glycine max Subtilase Peptide (GmSubPep). The gene (Glyma18g48580) was expressed in all actively growing tissues of the soybean plant. Although transcription of Glyma18g48580 was not induced by wounding, methyl jasmonate, methyl salicylate, or ethephon, synthetic GmSubPep peptide, when supplied to soybean cultures, induced the expression of known defense-related genes, such as Cyp93A1, Chib-1b, PDR12, and achs. GmSubPep is a unique plant defense peptide signal, cryptically embedded within a plant protein with an independent metabolic role, providing insights into plant defense mechanisms.

Activation of a Novel Transcription Factor through Phosphorylation by WIPK, a Wound-Induced Mitogen-Activated Protein Kinase in Tobacco Plants

Wound-induced protein kinase (WIPK) is a tobacco (Nicotiana tabacum) mitogen-activated protein kinase known to play an essential role in defense against wounding and pathogens, although its downstream targets have yet to be clarified. This study identified a gene encoding a protein of 648 amino acids, which directly interacts with WIPK, designated as N. tabacum WIPK-interacting factor (NtWIF). The N-terminal region with approximately 250 amino acids showed a high similarity to the plant-specific DNA binding domain, B3, but no other similarity with known proteins. The C terminus of approximately 200 amino acids appeared to be essential for the interaction with WIPK, and a Luciferase-reporter gene assay using Bright Yellow 2 cells indicated the full-length protein to possess trans-activation activity, located to the middle region of approximately 200 amino acids. In vitro phosphorylation assays indicated that WIPK efficiently phosphorylates the full-length protein and the N terminus but not the C terminus. When full-length NtWIF was coexpressed with WIPK in Bright Yellow 2 cells, the Luciferase transcriptional activity increased up to 5-fold that of NtWIF alone, whereas no effect was observed with a kinase-deficient WIPK mutant. Transcripts of NtWIF began to simultaneously accumulate with those of WIPK 30 min after wounding and 1 h after the onset of hypersensitive response upon tobacco mosaic virus infection. These results suggest that NtWIF is a transcription factor that is directly phosphorylated by WIPK, thereby being activated for transcription of target gene(s) involved in wound and pathogen responses.

GmPep914, an Eight-Amino Acid Peptide Isolated from Soybean Leaves, Activates Defense-Related Genes

Only a handful of endogenous peptide defense signals have been isolated from plants. Herein, we report a novel peptide from soybean (Glycine max) leaves that is capable of alkalinizing the media of soybean suspension cells, a response that is generally associated with defense peptides. The peptide, DHPRGGNY, was synthesized and found to be active at 0.25 nm and requiring only 5 to 10 min to obtain a maximal pH change. The peptide is located on the carboxy-terminal end of a 52-amino acid precursor protein (Glyma12g00990) deduced from the soybean genome project. A search of the soybean databank revealed a homolog (Glyma09g36370) that contained a similar peptide, DLPRGGNY, which was synthesized and shown to have identical activity. The peptides, designated GmPep914 (DHPRGGNY) and GmPep890 (DLPRGGNY), were capable of inducing the expression of both Glyma12g00990 (GmPROPEP914) and Glyma09g36370 (GmPROPEP890) in cultured soybean suspension cells within 1 h. Both peptides induced the expression of defense genes, including CYP93A1, a cytochrome P450 gene involved in phytoalexin synthesis, chitinaseb1-1, a chitinase involved in pathogen defense, and Glycine max chalcone synthase1 (Gmachs1), chalcone synthase, involved in phytoalexin production. Both GmPROPEP914 and GmPROPEP890 were highly expressed in the roots, relative to the aerial portions of the plant. However, treatment of the aerial portion of soybean plants with hormones involved in elicitation of defense responses revealed a significant increase in expression levels of GmPROPEP914 and GmPROPEP890. A search of gene databases revealed homologous sequences in other members of the Fabales and also in the closely related Cucurbitales but not in any other order of plants.