In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco ( Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)-mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast-targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non-transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.
Six peroxidase (POD) cDNAs were isolated from suspension cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and their expression was investigated with a view to understanding the physiological functions of each POD in relation to environmental stress. The gene products encoded by these cDNAs could be divided into two groups, anionic PODs (SWPA4, SWPA5, SWPA6) and basic PODs (SWPB1, SWPB2, SWPB3), on the basis of the predicted pI values of the mature proteins. RT-PCR analysis revealed that the six POD genes showed diverse expression patterns in various tissues of intact plants, a various stages of growth in suspension cultures, and in leaf tissues exposed to different stresses. The six genes from which they were derived are predominantly expressed in cultured cells of sweetpotato. Thus, transcripts of swpa4 were not detected in any tissues of the intact plant. The genes swpa6 and swpb1 were highly expressed in root tissues, whereas swpa6 and swpb3 were highly expressed in stem tissues. During suspension culture, the expression patterns of the six genes differed from each other. The level of swpa4, swpa5, swpb2 and swpb3 transcripts progressively increased during culture, whereas swpa6 and swpb1 showed high expression levels regardless of the age of the culture. In leaf tissues the six POD genes responded differently to various abiotic stresses. In particular, swpa4 was highly induced by several abiotic stresses, including exposure to hydrogen peroxide (440 mM) or NaCl (100 mM), and wounding of leaf tissues, suggesting that this POD gene is inducible by many stresses. Based on the different expression patterns of these POD genes, we propose that each POD may have different enzymatic properties and physiological functions during cell growth and development.
We investigated the expression patterns of diverse genes at various time points after gamma irradiation of young tobacco plants. The first group of genes showed stimulation of transcript levels upon gamma irradiation, although their induction patterns varied. This group included glutathione-S-transferase, peroxidase, superoxide dismutase, and catalase. A second group, with post-irradiation reduction of transcripts, included genes encoding cytosolic ascorbate peroxidase, stromal ascorbate peroxidase, and a TMK1 receptor-like kinase. The third group of genes either showed no change in transcript levels or exhibited irregular patterns. These included genes encoding PRla (pathogenesisrelated protein), tobacco Ca++-dependent protein kinase, the ~-subunit of translational initiation factor 2B, and CHRK1, a chitinase-related receptor-like kinase. Thus, various genes displayed differential patterns of gene expression in response to gamma irradiation in tobacco plants, thereby suggesting a complex signaling mechanism is involved in the irradiation-induced defense by plants. In addition, many stress-responsive genes exhibited gene expression pattems upon gamma irradiation that differed from those resulting from other biotic and abiotic stresses. With the knowledge of distinctive expression patterns of diverse genes, irradiation-indicating marker plants could be developed by engineering and monitoring multiple radiation-responsive genes.
A cDNA, mSOD1, encoding cytosolic copper/ zinc superoxide dismutase (CuZnSOD) was cloned and characterized from cell cultures of cassava (Manihot esculenta Crantz) which produce a high yield of SOD. mSOD1 encodes a 152-amino acid polypeptide with a pI value of 5.84. Southern analysis using an mSOD1-speci®c probe indicated that a single copy of the mSOD1 gene is present in the cassava genome. The mSOD1 gene is highly expressed in cultured cells, as well as in intact stems and tuberous roots. It is expressed at a low level in leaves and petioles. Transcripts of mSOD1 were not detected in nontuberous roots. Transcriptional level of mSOD1 reaches a high level at stationary phase, and then sharply decreases during further culture. In excised cassava leaves, the mSOD1 gene responded to various stresses in dierent ways. The stresses tested included changes in temperature and exposure to stress-inducing chemicals. Levels of mSOD1 transcript increased dramatically a few hours after heat stress at 37°C and showed a synergistic eect with wounding stress. Levels decreased in response to chilling stress at 4°C and showed an antagonistic eect with wounding stress. The gene was induced by abscisic acid, ethephon, NaCl, sucrose, and methyl viologen. These results indicate that the mSOD1 gene is involved in the response to oxidative stress induced by environmental change.
Cotyledonary expiants of ginseng zygotic embryos were cocultured with Agrobacterium tumefadens strain LBA4404 harboring the binary vector pBI121 for 48 h and transferred onto MS medium supplemented with 1 mgl(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mgl(-1) kinetin, and 100 mgl(-1) kanamycin. After 8 weeks of culture, kanamycin-resistant calli formed on the cut surfaces of cotyledonary expiants and subsequently they gave rise to numerous somatic embryos. Eight weeks after transfer onto medium containing 1 mgl(-1) each of 6-benzyladenine (BA) and gibberellic acid, most of them developed into plantlets. Southern analysis confirmed that the β-glucuronidase (GUS) gene was incorporated into the genomic DNA of regenerants. Protoplasts were enzymatically isolated from transformed somatic embryo segments and cultured in liquid medium containing 60 gl(-1) myo-inositol, 1 mgl(-1) 2,4-D, 0.5 mgl(-1) BA, and 0.5 mgl(-1) kinetin. Plants were regenerated from protoplasts via somatic embryogenesis. The polymerase chain reaction method revealed that 92% of the regenerants retained the GUS gene. When treated with X-glucuronide, 78% of the regenerants showed a GUS-positive response. The overall results indicate that the transgene is stably transmitted during somatic ontogeny and stably expressed in most the regenerants, whereas it may be deleted or impaired in some portion of them.
A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the beta-glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the -1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the -1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.
Two cDNAs for anionic peroxidase (PODs), swpa2 and swpa3, were isolated from suspension cultures of sweet potato (Ipomoea batatas), and their expression was investigated with a view to understanding the physiological function of PODs in relation to environmental stresses. Swpa2 (whose putative mature protein product would have a pI value of 4.1) and swpa3 (4.3) encode polypeptides of 358 and 349 amino acids, respectively. The genes from which they were derived are predominantly expressed in cultured cells of sweet potato; transcripts of swpa2 were not detected in any tissues of the intact plant, and transcripts of swpa3 were detected at a low level only in the stem tissue. During cell culture, the expression patterns of the two genes differed; the level of swpa2 RNA progressively increased during cell growth, whereas that of swpa3 reached a maximum at the stationary phase and decreased on further culture. The two genes responded differently to stresses such as wounding or chilling of leaves. Swpa2 was strongly induced 48 h after wounding, but swpa3 was not affected by this treatment. The two genes were also highly expressed upon chilling (4 degrees C), but expression was reduced by prior acclimation at 15 degrees C. In addition, both genes were strongly induced immediately after treatment with ozone, and expression had decreased to the basal level 12 h after treatment. The response of these two genes to stresses such as aging, wounding, and chilling are different from those of the POD genes (swpa1 encoding an anionic product and swpn1 a neutral peroxidase) that we described previously. The responses of the two genes were also different from each other. These results suggest that the two new POD genes are involved in overcoming oxidative environmental stress, and each POD gene may be regulated by cell growth and environmental stress in different ways.
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