Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase‐like proteins

Abstract

In Lycopersicon esculentum, two cDNAs encoding glutathione peroxidase (GPX)-like proteins were isolated, sequenced and characterized as putative phospholipid hydroperoxide :glutathione peroxidases. After mechanical stimulation (rubbing) of the plant, each GPX tomato gene was found to behave differently in terms of inducibility and tissue distribution. The accumulation of GPX transcripts suggests that reactive oxygen species are involved in the plant response to a mechanical stress.Keywords : Lycopersicon esculentum; free-radical scavenger; gene expression ; glutathione peroxidase ; mechanical stimulation.The consumption of oxygen in normal respiratory processes ing are expressed in tomato. Two GPX-like cDNAs were cloned and the tissue distribution and accumulation of the correspondand the production of oxygen during photosynthesis results in the generation of toxic reactive oxygen species (ROS) [1,2]. ing mRNAs were investigated. Biotic and abiotic stresses can give rise to further increases in the ROS level [3,4] ; this phenomenon is described as oxidative burst. The primary constituents of protective mechanisms MATERIALS AND METHODS against ROS include enzymes, such as superoxide dismutase, Plant material, culture conditions and stimulation. Culcatalase and peroxidase, and free-radical scavengers, such as ca-ture conditions. Tomato (Lycopersicon esculentulum Mill. cv. rotenoid, tocopherol, ascorbate and glutathione [5]. VFN8) plants were raised from seeds in moist vermiculite in a Glutathione peroxidases (GPX) belong to a family of iso-controlled environment room: 16 h daylight at 60 µmol · m Ϫ2 · zymes that catalyses the reduction of H 2 O 2 , organic hydroper-s Ϫ1 , photosynthetically active radiation provided by 40-W white oxides and lipid hydroperoxides using GSH as a reducing agent daylight tubes (Mazda LDL, TF 40), 23Ϯ1°C (day) and and thus help to protect the cells against oxidative damage [6].19Ϯ1°C (night), 60Ϯ 10% relative humidity. At the cotyledon In animals, where these enzymes have been studied extensively, stage, plants were transferred to a mineral solution [13]. both selenium-dependent and selenium-independent GPX have Mechanical stimulation. Mechanical stimulus was applied to been described [7,8]. Much less is known about these enzymes 3-week-old plants, with six developed internodes. The young in plants. Recent reports have shown increases of the steady-growing internode [14] was held between the thumb and forefinstate level of mRNA or protein corresponding to GPX, and ger and rubbed back and forth. stress conditions, such as pathogenic attack, salt treatment and RNA and DNA isolation, northern blotting and Southern osmotic stress [9Ϫ11], seem to be involved. blot hybridizations. Total RNA was extracted from different The present paper reports that rubbing of tomato plants initi-organs (roots, internodes, mature and young leaves) of 3-weekates an oxidative response. Previous data in Bryonia dioica have old tomato plants by the borate method [15]. Extractions were suggested that the mechanica...

Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways

“…Gene expression analyses of several GPXs have been conducted, and the results showed that GPX mRNA steadystate levels increase under different environmental stresses, such as pathogen infections (Criqui et al, 1992), high salt and metal concentrations (Sugimoto and Sakamoto, 1997), mechanical stimulation (Depe Á ge et al, 1998), and aluminum toxicity (Rodriguez Milla et al, 2002). In addition, the activity of a GPX was found to increase under drought stress in the moss Tortula ruralis (Dhindsa, 1991), and salt treatment induced the accumulation of a citrus PHGPX protein (Avsian-Kretchmer et al, 1999).…”

Multigene families encode the major enzymes of antioxidant metabolism in Eucalyptus grandis L

“…A cytosolic PhGPx encoding cDNA from tomato (Depège et al, 1998) and chloroplastic PhGPx encoding cDNAs from pea and Arabidopsis (Mullineaux et al, 1998) were previously reported. Furthermore, citrus PhGPx has been purified and the enzyme activity has been determined (Beeor-Tzahar et al, 1995).…”

The importance ofArabidopsisglutathione peroxidase 8 for protectingArabidopsisplant andE. colicells against oxidative stress

“…[11][12][13] Members in the family could be divided into two groups; the first group (GPX1 to GPX4) has selenium-dependent glutathione peroxidase activity and contains the amino acid selenocysteine (SeC) in their primary sequence, whereas the other group (GPX5 to GPX8) is without selenoenzymes activity because their primary sequence contains cysteine as active site instead of SeC. 13,14 In plants, GPX genes are responsive to abiotic stress, 15,17 hormone treatments, 16 pathogens, 18 wounding, 19 and immune response. 20 Plants GPXs catalyze the reduction of H2O2 as well as different kinds of lipid peroxides by using GSH or thioredoxin as an electron donor.…”