RESUMO
Using primers designed on the basis of sequence homologies in the copper-binding domains for a number of plant and fungal tyrosinases, two tyrosinase encoding cDNAs were cloned from an Agaricus bisporus U1 cDNA-library. The sequences AbPPO1 and AbPPO2 were, respectively, 1.9 and 1.8 kb in size and encoded proteins of approximately 64 kDa. The cDNAs represent different loci. Both AbPPO1 and AbPPO2 occur as single copies on the genomes of the U1 parental strains H39 and H97. The genomic size of AbPPO1 and AbPPO2 is minimally 2.3 and 2.2 kb, respectively. Alignment and phylogenetic analysis of 35 tyrosinase and polyphenol oxidase sequences of animal, plant, fungal, and bacterial origin indicated conserved copper-binding domains, and stronger conservation within genera than between them. The translation products of AbPPO1 and AbPPO2 possess putative N-glycosylation and phosphorylation sites and are recognised by antibodies directed against a 43-kDa tyrosinase. The observations are consistent with previously proposed maturation and activation models for plant and fungal tyrosinases.
Assuntos
Agaricus/enzimologia , Agaricus/genética , Clonagem Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Sequência de Aminoácidos , Cromossomos Fúngicos/genética , DNA Complementar , Genes Fúngicos , Glicosilação , Dados de Sequência Molecular , Fosforilação , Filogenia , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
We investigated changes in gene expression in Iris hollandica flowers by microarray technology. Flag tepals were sampled daily, from three days prior to flower opening to the onset of visible senescence symptoms. Gene expression profiles were compared with biochemical data including lipid and protein degradation and DNA coiling, and with morphological data. Plasmodesmata of mesophyll cells closed about two days before flower opening, while in the epidermis they closed concomitant with opening. Similarly, the onset of visible senescence in the epidermis cells occurred about two days later than in the mesophyll. About 1400 PCR-amplified clones, derived from a subtractive cDNA library enriched for tepal-specific genes, were spotted and about 240 clones, including 200 that were expressed most differentially, were sequenced. The expression patterns showed three main clusters. One exhibited high expression during tepal growth (cluster A). These genes were putatively associated with pigmentation, cell wall synthesis and metabolism of lipids and proteins. The second cluster (B) was highly expressed during flower opening. The third cluster (C) related to the final stages of senescence, with genes putatively involved in signal transduction, and the remobilization of phospholipids, proteins, and cell wall compounds. Throughout the sampling period, numerous plant defence genes were highly expressed. We identified an ion channel protein putatively involved in senescence, and some putative regulators of transcription and translation, including a MADS-domain factor.
Assuntos
Flores/genética , Perfilação da Expressão Gênica , Magnoliopsida/genética , Northern Blotting , Análise por Conglomerados , DNA Complementar/química , DNA Complementar/genética , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Magnoliopsida/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase 1. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-I gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.
Assuntos
Apoptose/genética , Proteínas de Caenorhabditis elegans , Regulação da Expressão Gênica de Plantas , Proteínas Repressoras/genética , Solanum lycopersicum/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Sequência de Bases , Camptotecina/metabolismo , Camptotecina/farmacologia , Células Cultivadas , Clonagem Molecular , DNA de Plantas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lantânio/farmacologia , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants.
