Fossdal et al 2001 PMB Peroxidase (original) (raw)
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Plant Molecular Biology, 2001
Peroxidases are associated with the active defence reactions in higher plants in response to foreign organisms. They are involved in the oxidation of phenolic compounds in cell walls, polymerization of lignin and suberin, and in several other oxidation processes but the exact function of individual peroxidases is not known. We have isolated a cDNA encoding the putative defence-related and basic plant peroxidase SPI2 (spruce pathogen-induced 2), with an estimated molecular mass of 34 kDa, from roots of Norway spruce (Picea abies) seedlings. This is the first description of the isolation of a complete cDNA encoding a putative peroxidase from a gymnosperm. The transcript was present in the roots of healthy seedlings, and during infection with the pathogen Pythium dimorphum there was a rapid initial increase followed by a dramatic reduction of the transcript. The 34 kDa mature SPI2 protein was detected in both the developing root and shoot of healthy seedlings and increased amounts of SPI2 and increased accumulation of highly basic peroxidase isoforms was observed in roots after infection. In addition, two SPI2-related proteins with apparent molecular masses of 38 and 39 kDa, were also detected. Both these proteins accumulated in roots only after infection, and the 39 kDa protein was in addition detected in shoots of root-infected seedlings. Thus, both SPI2 and the SPI2-related proteins accumulate as a local response, in roots, and as a systemic response to infection the 39 kDa protein accumulates in the shoot.
Plant Cell Reports, 2001
Peroxidases constitute a large family of proteins found in all higher plants. Owing to the complexity of the peroxidase isoenzyme family it has been difficult to assess the precise function of individual peroxidase enzymes. In this work we have studied the effects of an endogenous peroxidase-like gene from Norway spruce [Picea abies (L.) Karst], spi 2, on the development and growth of Norway spruce somatic embryo plants. Embryogenic cells of Norway spruce transformed with spi 2 under control of the maize ubi-1 promoter showed up to 40 times higher total peroxidase activity than the control cells; regenerated plants overexpressing spi 2 showed an increased total peroxidase activity. Based on these results and the overall sequence similarity with cationic peroxidases we conclude that spi 2 encodes a peroxidase. Overexpression of spi 2 resulted in increased sensitivity to stress, leading to a reduction in epicotyl formation and in height growth compared with control plants. The plants overexpressing spi 2 also showed a deeper phloroglucinol staining but similar levels of Klason lignin.
Molecular cloning of a peroxidase gene from poplar and its expression in response to stress
Tree Physiology, 2006
To elucidate the precise functions of peroxidase in poplar (Populus alba × P. tremula var. glandulosa), we cloned a peroxidase gene (PoPOD1) from poplar suspension culture cells and examined its expression pattern in response to various stresses. PoPOD1 showed the highest homology with a bacterial-induced peroxidase gene from cotton (Gossypium hirsutum L.). The PoPOD1 gene encodes a putative 316 amino acid protein with an N-terminal signal peptide of 23 residues. The DNA blot analysis indicated that PoPOD1 is a single copy gene in the poplar genome. The RNA blot analyses indicated that PoPOD1 shows cell-culture-specific expression. Expression of PoPOD1 is down-regulated by various treatments including treatment with some metals, NaCl, methyl viologen and polyethylene glycol, and by the plant growth regulators, jasmonic acid (JA) and gibberellic acid (GA 3). The gene is significantly up-regulated by the bacterial-elicitor laminarin and by wounding. Thus, PoPOD1 gene expression is sensitively and specifically regulated at the transcription level. Because both JA and GA 3 appear to be involved in the regulation of PoPOD1 expression in poplar cells, we postulate that the peroxidase encoded by PoPOD1 plays a pivotal role in defense against pathogen invasion, possibly through the formation of a cell wall barrier over the wound.
Physiologia Plantarum, 2004
Seedlings of Norway spruce were exposed to fungal infection and drought in order to investigate differences in their stress responses on the enzymatic level. Six-week-old seedlings were infected with the root rot fungus Rhizoctonia, or subjected to drought, respectively. Changes at the enzymatic level were more rapid and significantly higher in infected plants in comparison with drought-stressed spruce plants. Rhizoctonia infection resulted in early local and systemic increase in peroxidase and chitinase activity. The most prominent isoforms responding were highly basic peroxidases and chitinases (pI 9-9.5) and several acidic chitinases (pI3-4). An increased intensity of similar peroxidase isoforms was found in drought-affected plants. Two peroxidase isoforms (with pI < 9) accumulated exclusively in response to drought. These results suggest that at an early stage of infection and drought stress, the two stresses can be distinguished by the temporal appearance and isoform profile of peroxidases and chitinases. Changes in enzyme activity appeared before changes in physiological parameters, thus these isoform profiles could be used as early markers of stress conditions in spruce. Periodic acid Schiff's; Spi2, Spruce pathogen induced number 2.
Physiological and Molecular Plant Pathology, 1992
A cDNA library of RNA from barley leaves inoculated with Ervsiphe graminis was screened using labelled cDNA enriched for specific sequences by subtractive hybridization against RNA from non-inoculated leaves. This resulted in isolation of several clones representing pathogen induced genes. By cross-hybridization and sequence analysis, one of the cDNAs (pBT6-3) was found to be a partial clone representing a putative peroxidase, for which a full-length eDNA clone (pBH6-301) was subsequently isolated. The predicted amino acid sequence revealed a 21 amino acid signal peptide and a 294 amino acid mature protein I3I kDa1 and shows 56 t 0 amino acid identity to a basic peroxidase from turnip, 89 % to a putative peroxidase from wheat, but only 38 % to the amino acid sequence derived from the eDNA clone 1pcD1311) of a second putative barley peroxidase expressed in leaves. Northern blot analysis showed that the pBT6-3 (pBH6-301) transcript is elevated as early as 4 h after inoculation with E. graminis f. sp. harder and that two maxima in transcript levels appear, which can be correlated with penetration attempts by the fungus. The amount of the pcD 131 1 transcript was also found to increase in inoculated leaves but at a later time point .
Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens
Plant Physiology, 1999
A cDNA clone encoding the Prx7 peroxidase from barley (Hordeum vulgare L.) predicted a 341-amino acid protein with a molecular weight of 36,515. N-and C-terminal putative signal peptides were present, suggesting a vacuolar location of the peroxidase. Immunoblotting and reverse-transcriptase polymerase chain reaction showed that the Prx7 protein and mRNA accumulated abundantly in barley coleoptiles and in leaf epidermis inoculated with powdery mildew fungus (Blumeria graminis). Two isoperoxidases with isoelectric points of 9.3 and 7.3 (P9.3 and P7.3, respectively) were purified to homogeneity from barley coleoptiles. P9.3 and P7.3 had Reinheitszahl values of 3.31 and 2.85 and specific activities (with 2,2-azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid], pH 5.5, as the substrate) of 11 and 79 units/mg, respectively. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry peptide analysis identified the P9.3 peroxidase activity as due to Prx7. Tissue and subcellular accumulation of Prx7 was studied using activity-stained isoelectric focusing gels and immunoblotting. The peroxidase activity due to Prx7 accumulated in barley leaves 24 h after inoculation with powdery mildew spores or by wounding of epidermal cells. Prx7 accumulated predominantly in the epidermis, apparently in the vacuole, and appeared to be the only pathogen-induced vacuolar peroxidase expressed in barley tissues. The data presented here suggest that Prx7 is responsible for the biosynthesis of antifungal compounds known as hordatines, which accumulate abundantly in barley coleoptiles.
Plant Physiology, 1988
A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidasebinding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls. Plant peroxidases (EC 1.1 1.1.7) are of importance in a variety of cellular functions such as lignin biosynthesis, NADH-dependent hydrogen peroxide formation, suberization, metabolism of aromatic compounds, and ethylene production (1 1). Peroxidases are found in both the cytoplasm and the cell wall of plant cells, and the cell wall isozymes differ from the cytoplasmic ones (9). Most of the literature on wall peroxidases concerns factors that induce changes in their activity, as measured by colorimetric assays, or studies of their localization. For example, several authors have tested the possibility that peroxidase changes are involved in the control of cell elongation and have found that there is an inverse relationship between peroxidase activity levels and growth (2). The localization studies utilize a variety of different techniques, including cytochemical staining (13), radioactive labeling (31), and immunofluorescence assays with polyclonal antibodies (8). A number of wall peroxidase isoenzymes have been identified and partially purified from different plant species and tissues. Despite these studies, very little is known about the protein chemistry of wall peroxidases. As van Huystee reviewed (28), the most appealing approach for studying the various functions of plant peroxidases in detail is to employ immunological assays. Efforts to quantitate, purify, and biochemically characterize wall peroxidases would be greatly aided by the availability of monoclonal antibodies against them. In this paper, we describe the production of monoclonal antibodies against cell wall antigens 'This work was supported in part by a grant, NSG 7480, from the
Physiologia Plantarum, 1994
1993. Purification and characterization of an anionic peroxidase from sycamore maple (Acer pseudoplatanus ) cell suspension cultures. --Physiol. Plant. 00:000-000 A 42 kDa anionic peroxidase (EC 1.11.1.7) having a pI of 3.6 was purified from suspension cultures of dark-grown sycamore maple (Acer pseudoplatanus L.) cells by a combination of lectin-affinity, anion-exchange and gel permeation chromatography. The enzyme had an amino acid composition similar to that found for other anionic plant peroxidases, but the protein was blocked to amino-terminal protein sequencing. Commercially available antibodies against horseradish peroxidase were shown to cross-react with the sycamore maple enzyme on immunoblots. The purified peroxidase displayed differences in its affinity for each of the three monolignols, and these differences were compared to those found for a commercial preparation of horseradish peroxidase, as well as a laccase (p-diphenol:O 2 oxidoreductase; EC 1.10.3.1) purified from sycamore maple cell suspension cultures. These results are discussed with respect to the role played by peroxidases in lignin deposition and host-pathogen response.
Plant peroxidases : biochemistry and physiology
1996
Resistant (Reba B50) and susceptible (Acala 44) cotton plants were investigated for intratissular growth of bacterial populations and peroxidase (POx) activity, after infection of cotyledons with races 18 or 20 from #Xanthomonas (#Axonopodis$) campestris$ pv. #malvacearum$. Considerable multiplication of the bacterial population was noticed in the compatible interaction (Acala 44 / Xcm race 18) ; it was much lower during the incompatible interaction when race 18 was infiltrated into cotyledons of Reba B50. An intermediate level of bacterial growth was obtained when Reba B50 was infiltrated with race known to overcome resistance of this line. High increase in POx activity occurred into the infected cotyledons during incompatible interaction, while the increase was much lower when the interactions were compatible. On leaves, a similar and significant difference in enzyme activity was also observed indicating that the "peroxidase response" was systemically induced in entire r...