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

GmPep914, an Eight-Amino Acid Peptide Isolated from Soybean Leaves, Activates Defense-Related Genes1[W][OA]

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

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 designated NtC7 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 NtC7 normally germinated and grew in the presence of 500 mm mannitol, 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

Role of a Nonselective de Novo DNA Methyltransferase in Maternal Inheritance of Chloroplast Genes in the Green Alga, Chlamydomonas reinhardtii

In the green alga, Chlamydomonas, chloroplast DNA is maternally transmitted to the offspring. We previously hypothesized that the underlying molecular mechanism involves specific methylation of maternal gamete DNA before mating, protecting against degradation. To obtain direct evidence for this, we focused on a DNA methyltransferase, DMT1, which was previously shown to be localized in chloroplasts. The full-length DMT1 protein with a molecular mass of 150 kD was expressed in insect cells, and its catalytic activity was determined. In vitro assays using synthetic DNA indicated methylation of all cytosine residues, with no clear selectivity in terms of the neighboring nucleotides. Subsequently, transgenic paternal cells constitutively expressing DMT1 were constructed and direct methylation mapping assays of their DNA showed a clear nonselective methylation of chloroplast DNA. When transgenic paternal cells were crossed with wild-type maternal cells, the frequency of biparental and paternal offspring of chloroplasts increased up to 23% while between wild-type strains it was ∼3%. The results indicate that DMT1 is a novel type of DNA methyltransferase with a nonselective cytosine methylation activity, and that chloroplast DNA methylation by DMT1 is one of factors influencing maternal inheritance of chloroplast genes

PEPR2 Is a Second Receptor for the Pep1 and Pep2 Peptides and Contributes to Defense Responses in Arabidopsis[W]

This work identifies Arabidopsis PEPR2 as a second receptor for the defense-related Pep peptides. PEPR2 expression patterns and its binding properties to Pep peptides were compared with those of PEPR1, and it is shown that both PEPR1 and PEPR2 are required to activate defense responses after Pep treatment

Isolation and Characterization of Hydroxyproline-Rich Glycopeptide Signals in Black Nightshade Leaves1[OA]

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants

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