metallothionein and ethylene

Surface signaling plays a major role in fungal infection. Topographical features of the plant surface and chemicals on the surface can trigger germination of fungal spores and differentiation of the germ tubes into appressoria. Ethylene, the fruit-ripening hormone, triggers germination of conidia, branching of hyphae, and multiple appressoria formation in Colletotrichum, thus allowing fungi to time their infection to coincide with ripening of the host. Genes uniquely expressed during appressoria formation induced by topography and surface chemicals have been isolated. Disruption of some of them has been shown to decrease virulence on the hosts. Penetration of the cuticle by the fungus is assisted by fungal cutinase secreted at the penetration structure of the fungus. Disruption of cutinase gene in Fusarium solani pisi drastically decreased its virulence. Small amounts of cutinase carried by spores of virulent pathogens, upon contact with plant surface, release small amounts of cutin monomers that trigger cutinase gene expression. The promoter elements involved in this process in F. solani pisi were identified, and transcription factors that bind these elements were cloned. One of them, cutinase transcription factor 1, expressed in Escherichia coli, is phosphorylated. Several protein kinases from F. solani pisi were cloned. The kinase involved in phosphorylation of specific transcription factors and the precise role of phosphorylation in regulating cutinase gene transcription remain to be elucidated.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carboxylic Ester Hydrolases; Ethylenes; Fusarium; Genes, Plant; Metallothionein; Mice; Mitosporic Fungi; Molecular Sequence Data; Neurospora crassa; Plant Physiological Phenomena; Plants; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Signal Transduction; Virulence; Waxes; Xenopus

Mango (Mangifera indica L. cv. Alphonso) development and ripening are the programmed processes; conventional indices and volatile markers help to determine agronomically important stages of fruit life (fruit-setting, harvesting maturity and ripening climacteric). However, more and precise markers are required to understand this programming; apparently, fruit's transcriptome can be a good source of such markers. Therefore, we isolated 18 genes related to the physiology and biochemistry of the fruit and profiled their expression in developing and ripening fruits, flowers and leaves of mango using relative quantitation PCR. In most of the tissues, genes related to primary metabolism, abiotic stress, ethylene response and protein turnover showed high expression as compared to that of the genes related to flavor production. Metallothionin and/or ethylene-response transcription factor showed highest level of transcript abundance in all the tissues. Expressions of mono- and sesquiterpene synthases and 14-3-3 lowered during ripening; whereas, that of lipoxygenase, ethylene-response factor and ubiquitin-protein ligase increased during ripening. Based on these expression profiles, flower showed better positive correlation with developing and ripening fruits than leaf. Most of the genes showed their least expression on the second day of harvest, suggesting that harvesting signals significantly affect the fruit metabolism. Important stages in the fruit life were clearly indicated by the significant changes in the expression levels of various genes. These indications complemented those from the previous analyses of fruit development, ripening and volatile emission, revealing the harmony between physiological, biochemical and molecular activities of the fruit.

Topics: 14-3-3 Proteins; Enzymes; Ethylenes; Flowers; Fruit; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Mangifera; Metallothionein; Plant Leaves; Plant Proteins; Polymerase Chain Reaction; Signal Transduction; Transcription Factors

The 1,053-bp promoter of the oil palm metallothionein gene (so-called MSP1) and its 5' deletions were fused to the GUS reporter gene, and analysed in transiently transformed oil palm tissues. The full length promoter showed sevenfold higher activity in the mesocarp than in leaves and 1.5-fold more activity than the CaMV35S promoter in the mesocarp. The 1,053-bp region containing the 5' untranslated region (UTR) gave the highest activity in the mesocarp, while the 148-bp region was required for minimal promoter activity. Two positive regulatory regions were identified at nucleotides (nt) -953 to -619 and -420 to -256 regions. Fine-tune deletion of the -619 to -420 nt region led to the identification of a 21-bp negative regulatory sequence in the -598 to -577 nt region, which is involved in mesocarp-specific expression. Gel mobility shift assay revealed a strong interaction of the leaf nuclear extract with the 21-bp region. An AGTTAGG core-sequence within this region was identified as a novel negative regulatory element controlling fruit-specificity of the MSP1 promoter. Abscisic acid (ABA) and copper (Cu(2+)) induced the activity of the promoter and its 5' deletions more effectively than methyl jasmonate (MeJa) and ethylene. In the mesocarp, the full length promoter showed stronger inducibility in response to ABA and Cu(2+) than its 5' deletions, while in leaves, the -420 nt fragment was the most inducible by ABA and Cu(2+). These results suggest that the MSP1 promoter and its regulatory regions are potentially useful for engineering fruit-specific and inducible gene expression in oil palm.

Topics: 5' Untranslated Regions; Abscisic Acid; Arecaceae; Electrophoretic Mobility Shift Assay; Ethylenes; Fruit; Gene Expression Regulation, Plant; Metallothionein; Metals, Heavy; Plant Leaves; Promoter Regions, Genetic

Physcomitrella patens is a model plant for studying gene function using a knockout strategy. To establish a proteome database for P. patens, we resolved over 1,500 soluble proteins from gametophore and protonema tissues by two-dimensional electrophoresis (2-DE) and obtained peptide mass fingerprints (PMFs) by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Using expressed sequence tags (ESTs), we were able to predict the identities of 90 protein spots. Most of these were related to energy or primary metabolism. Comparative proteome analysis was used to identify proteins specific for each of the tissue types. One of these was a metallothionein type-2 (PpMT2) protein that was highly upregulated in gametophore tissue. PpMT2 was induced in both the gametophore and protonema following culture on solid media and in response to various abiotic stresses such as copper, cadmium, cold, indole-3-acetic acid, and ethylene. We suggest that PpMT2 is not only involved in metal binding and detoxification, but also in many biological aspects as a metal messenger or a protein with additional functions.

Topics: Amino Acid Sequence; Bryopsida; Cadmium; Cold Temperature; Copper; Electrophoresis, Gel, Two-Dimensional; Ethylenes; Expressed Sequence Tags; Gene Expression Regulation, Plant; Indoleacetic Acids; Metallothionein; Molecular Sequence Data; Plant Proteins; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The metallothionein gene, LSC54, shows increased expression during leaf senescence in Brassica napus and Arabidopsis thaliana. A number of abiotic and biotic stresses have been shown to induce senescence-like symptoms in plants and, to investigate this further, the promoter of the LSC54 gene was cloned and fused to the GUS gene and transformed into Arabidopsis. The promoter was highly induced during leaf senescence and also in response to wounding; histochemical analysis indicated that this induction was localised to a few cells close to the wound site. The transgenic Arabidopsis tissue was infected with compatible and incompatible isolates of both the fungal biotroph, Peronospora parasitica and the bacterial necrotroph, Pseudomonas syringae. Incompatible isolates induced rapid cell death (the hypersensitive response) at the site of infection and, with both pathogens, early, localised expression of the GUS gene was observed. In contrast, relatively slow induction of the GUS gene was seen in the compatible interaction and this was correlated with the appearance of senescence-like symptoms in the biotrophic interaction and cell death by necrosis that occurred in response to the necrotrophic pathogen. These results suggest that there are common steps in the signalling pathways that lead to cell death in the hypersensitive response, pathogen induced necrosis and senescence.

Topics: Arabidopsis; Base Sequence; Cloning, Molecular; Enzyme Induction; Ethylenes; Fungi; Gene Expression; Genes, Reporter; Glucuronidase; Metallothionein; Metals; Molecular Sequence Data; Plant Diseases; Plants, Genetically Modified; Promoter Regions, Genetic; Pseudomonas; Time Factors

Two cDNAs encoding putative metallothionein (MT)-like peptides have been isolated from tomato (L. esculentum L.). The predicted protein products of these two cDNAs (LeMT(A) and LeMT(B)) are 72 and 83 amino acids respectively and both encode peptides with arrangements of cysteine residues characteristic of type II plant MTs. In other plants which possess more than one gene expressing MT proteins of the same type, the products are closely related or identical, but LeMT(A) and LeMT(B) constitute two different classes of message, and encode two different protein products. Northern blot analysis of LeMT(A) and LeMT(B) showed that transcripts of both MT-like genes were more abundant in leaves than roots in tomato plants grown without addition of extra metal ions, a characteristic of type II MTs. A genomic clone corresponding to LeMT(B) (LeMT(B)) was isolated and sequenced. The 5'-flanking region of LeMT(B) was shown to contain a putative metal regulatory element (MRE) which suggests the possibility of metal-regulated transcription. In addition, the upstream region also contains a G-box like motif (CACGTG) and an 8 bp sequence (AATTCAAA) found within the promoters of genes shown to be ethylene-responsive.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Ethylenes; Genes, Plant; Metallothionein; Metals; Molecular Sequence Data; Plant Proteins; Regulatory Sequences, Nucleic Acid; Sequence Analysis, DNA; Solanum lycopersicum

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Complementary; Ethylenes; Genes, Plant; Immunity, Innate; Metallothionein; Molecular Sequence Data; Nicotiana; Open Reading Frames; Plant Diseases; Plant Proteins; Plants, Toxic; Sequence Homology, Amino Acid; Tobacco Mosaic Virus; Transcription, Genetic; Wounds and Injuries

A cDNA encoding a metallothionein-like protein has been isolated from a cDNA library from the abscission zones of ethylene-treated Sambucus nigra leaflets. The precise function of this group of proteins in plants has yet to be confirmed but in animals there is convincing evidence that they bind heavy metals. Several of these proteins have recently been characterised from plants and it has been demonstrated that heavy metals have no stimulatory effect on their expression. In this paper we describe the isolation and characterisation of a metallothionein-like mRNA identified as a consequence of differentially screening a cDNA library for messages up-regulated during abscission. The accumulation of the mRNA occurred in the abscission zone tissue within 18 h of exposure to ethylene while, in contrast, no expression was detectable in adjacent non-abscission-zone tissue. The transcript size of the message was approximately 0.6 kb. Northern analysis revealed that the cDNA insert (JET12) did not hybridise to mRNA from either green or senescing leaflets but a signal was detectable with mRNA extracted from senescent tissue. The size of this hybridising transcript was approximately 0.5 kb. The predicted metallothionein-like protein encoded by JET12 was cysteine-rich (18.4%) and had a molecular weight of approximately 7.5 kDa. Southern analysis of S. nigra genomic DNA showed that the mRNA was encoded by a small gene family. The protein exhibited greatest homology to other metallothioneins belonging to the Type 2 family including those from Mimulus (62%) and Arabidopsis (57%). This homology was greatest around the cysteine-rich amino and carboxy termini.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Base Sequence; Blotting, Southern; Cloning, Molecular; DNA, Plant; Ethylenes; Metallothionein; Molecular Sequence Data; Plant Leaves; Plant Proteins; RNA, Messenger; RNA, Plant