Molecular Cloning and Tissue-Specific Expression of an Anionic Peroxidase in Zucchini1

Abstract

A calcium-pectate-binding anionic isoperoxidase (APRX) from zucchini (Cucurbita pepo) was purified and subjected to N-terminal amino acid microsequencing. The cDNA encoding this enzyme was obtained by reverse transcriptase polymerase chain reaction from a cDNA library. It encoded a mature protein of 309 amino acids exhibiting all of the sequence characteristics of a plant peroxidase. Despite the presence of a C-terminal propeptide, APRX was found in the apoplast. APRX protein and mRNA were found in the root, hypocotyls, and cotyledons. In situ hybridization showed that the APRX-encoding gene was expressed in many different tissues. The strongest expression was observed in root epidermis and in some cells of the stele, in differentiating tracheary elements of hypocotyl, in the lower and upper epidermis, in the palisade parenchyma of cotyledons, and in lateral and adventitious root primordia. In the hypocotyl hook there was an asymmetric expression, with the inner part containing more transcripts than the outer part. Treatment with 2,3,5-triiodobenzoic acid reduced the expression of the APRXencoding gene in the lower part of the hypocotyl. Our observations suggest that APRX could be involved in lignin formation and that the transcription of its gene was related to auxin level.Plant peroxidases (EC 1.11.1.7) exist in numerous molecular forms. For example, more than 50 different sequences encoding peroxidase have been identified in Arabidopsis. These enzymes are mainly located in cell walls and vacuoles (Mäder, 1992) and catalyze the reduction of hydrogen peroxide into water using electrons from various donor molecules. This redox activity allows them to oxidize many substances, such as polyphenols, flavonoids (Gaspar et al., 1982), and alkaloids (Blom et al., 1991), or to promote the oxidative cross-linking of cell wall polymers (Fry, 1986). Their substrate specificity is generally considered to be low, except toward some electron donors such as scopoletin (Reigh et al., 1973) and extensin (Brownleader et al., 1995), which are oxidized by specific isoforms. In some cases, they can also oxidize molecules such as IAA (Gazaryan et al., 1996) through catalytic mechanisms that differ from the classical peroxidative cycle. They have also been shown to produce hydrogen peroxide in the presence of reducers such as NADH (Elstner and Heupel, 1976) and Cys (Pichorner et al., 1992).The wide spectrum of biochemical reactions that peroxidases are able to catalyze and the great number of molecular isoforms explain the difficulty encountered in the study of plant peroxidases. Studies combining the techniques of molecular biology and biochemistry are necessary to get an insight into their precise function in plants. It is also essential to identify the mechanisms that determine their microlocalization in cells, particularly within the extracellular matrix network. Most of the known peroxidase mRNAs encode a signal peptide that targets the neosynthesized protein to the secretory pathway. Some sequences also encode a C-terminal ...

An anionic class III peroxidase from zucchini may regulate hypocotyl elongation through its auxin oxidase activity

“…DiVerent GFP constructs were used in order to asses the APRX cellular localization. APRX was detected in the cell wall as expected based both on the fact that APRX was Wrst isolated from the apoplast (Carpin et al 1999) and its capacity to bind the calcium-pectate complex (Carpin et al 2001). Nevertheless, mutation in the pectate binding site did not modiWed the eVect of APRX ectopic expression in the Arabidopsis transgenic lines studied here, suggesting that its binding ability is not essential to APRX function.…”

“…To assess the role of APRX in the regulation of auxin levels, APRX was puriWed from zucchini seedling based on its pectin binding ability (Carpin et al 1999). The puriWed protein was assayed for its auxin oxidase activity.…”

“…APRX mRNA accumulated strongly in hypocotyls (Carpin et al 1999) particularly in the elongation area (Dunand et al 2002). APRX was shown to speciWcally bind a complex of calcium-pectate through a motif of four clustered arginines (Carpin et al 2001) The localization of APRX related to its binding ability could for example indicate the involvement of APRX in the control of the cell elongation in the hypocotyl.…”

Growth regulators and guaiacol peroxidase activity during the induction phase of somatic embryogenesis in Picea species

“…Peroxidases (EC 1.11.1.7) belong to the group of oxidoreductases, catalyzing hydrogen peroxide oxidation of various substrates. In plant cells, peroxidase isoenzymes are located in the cell wall and vacuole (Carpin et al 1999). They participate in the antioxidant system, protecting cells from excessive accumulation of hydrogen peroxide and pathogen attack (Sutherland 1991) as well as in plant cell development (Brownleader et al 2000;Cordewener et al 1991), e.g.…”

Alterações ligno-anatômicas em Solanum gilo Raddi por aplicação de cálcio e boro como estratégia de defesa

“…Na realidade, o íon Ca ++ forma ligações cruzadas entre pectatos através de grupos carboxílicos de ácidos urônicos, portanto elevadas doses de Ca ++ podem inferir baixa afinidade às substâncias pécticas, aumentando e diminuindo as interações entre pectinas/ peroxidase/Ca ++ . Isto sugere que a rigidez da parede celular provavelmente aumentou em decorrência do aumento do teor de lignina sob este tratamento (CARPIN et al, 1999).…”