Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants)

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

Regulation of expression of a wound-inducible tomato inhibitor I gene in transgenic nightshade plants

A wound-inducible proteinase Inhibitor I gene from tomato containing 725 bp of the 5 region and 2.5 kbp of the 3 region was stably incorporated into the genome of black nightshade plants (Solanum nigrum) using an Agrobacterium Ti plasmid-derived vector. Transgenic nightshade plants were selected that expressed the tomato Inhibitor I protein in leaf tissue. The leaves of the plants contained constitutive levels of the inhibitor protein of up to 60 g/g tissue. These levels increased by a factor of about two in response to severe wounding. Only leaves and petioles exhibited the presence of the inhibitor, indicating that the gene exhibited the same tissue specificity of expression found in situ in wounded tomato leaves. Inhibitor I was extracted from leaves of wounded transformed nightshade plants and was partially purified by affinity chromatography on a chymotrypsin-Sepharose column. The affinity-purified protein was identical to the native tomato Inhibitor I in its immunological reactivity and in its inhibitory activity against chymotrypsin. The protein exhibited the same M _r of 8 kDa as the native tomato Inhibitor I and its N-terminal amino acid sequence was identical to that of the native tomato inhibitor I, indicating that the protein was properly processed in nightshade plants. These expriments are the first report of the expression of a member of the wound-inducible tomato Inhibitor I gene family in transgenic plants. The results demonstrate that the gene contains elements that can be regulated in a wound-inducible, tissuespecific manner in nightshade plants.     

The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S ₂ -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S ₂ RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S ₂ -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S ₂ polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S ₂ -RNase protein in pollen.     

Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity

GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of l-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than those of WT, while the contents of H₂O₂ and O₂ ^−· were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net photosynthetic rate (P _n) and the maximal photochemical efficiency of PSII (F _v/F _m) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants.     

Responses of Ribulose-1,5-Bisphosphate Carboxylase,Protein Content,and Stomatal Conductance to Water Deficit in Maize,Tomato, and Bean

We compared responses of maize, tomato, and bean plants to water stress. Maize reached a severe water deficit (leaf water potential –1.90 MPa) in a longer period of time as compared with tomato and bean plants. Maize stomatal conductance (g _s) decreased at mild water deficit. g _s of tomato and bean decreased gradually and did not reach values as low as in maize. The protein content was maintained in maize and decreased at low water potential (_w); in tomato it fluctuated and also decreased at low _w; in bean it gradually decreased. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity remained high at mild and moderate stress in maize and tomato plants; in bean it remained high only at mild stress.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> Alleviates Salinity by Boosting Redox Poise and Antioxidative Potential of Tomato

More than 20% of irrigated land has been influenced by salt stress, decreasing crop production. In this research, we investigated the effect of different levels of salinity (0, 50, 100 and 150 mM NaCl) and the efficiency of Piriformospora indica on growth, biochemical traits, antioxidative defense system in tomato (Solanum lycopersicum L.). NaCl stress reduced chlorophyll content, height and biomass of plants. Higher level of salinity (150 mM) declined the plant height by 22.65%, total dry weight by 56.44% and total chlorophyll by 44.34%, however, P. indica inoculation raised plant height by 43.47%, dry weight by 69.23% and total chlorophyll content by 48.09%. Salinity stress increased H₂O₂, malondialdehyde (MDA), superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) level in leaves and roots tomato seedlings. However, P. indica inoculation reduced H₂O₂, MDA and superoxide anion and enhanced DPPH compared to non-inoculated plants at all NaCl levels. The total phenol and flavonoids increased with NaCl treatment. On the other hand, the total phenolic and flavonoid increased more in P. indica inoculated plants compared to non-inoculated ones. Moreover, inoculation of P. indica implicated noteworthy improvement of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) activity in tomato upon salinity. Notably, colonization with P. indica significantly improved the content of reduced ascorbic acid (AsA), glutathione (GSH) and redox ratio in the tomato plants under salinity resulting in reduced redox state. Our findings confirmed that salinity had negative effect on tomato seedling; however, P. indica inoculation increased tolerance to salinity by improving the content of phenolic compounds, non-enzymatic antioxidants, and increasing the activity of antioxidant enzymes.     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

Structure of the dehydrin tas 14 gene of tomato and its developmental and environmental regulation in transgenic tobacco

We have isolated a genomic clone encoding tomato TAS14, a dehydrin that accumulates in response to mannitol, NaCl or abscisic acid (ABA) treatment. A fragment of tas14 gene containing the region from –2591 to +162 fused to -glucuronidase gene drives ABA- and osmotic stress-induced GUS expression in transgenic tobacco. Histochemical analysis of salt-, mannitol-and ABA-treated plants showed GUS activity mainly localized to vascular tissues, outer cortex and adventitious root meristems, coinciding with the previously observed distribution of TAS14 protein in salt-stressed tomato plants. In addition, GUS activity was also observed in guard cells, trichomes and leaf axils. Developmentally regulated gus expression was studied in unstressed plants and found to occur not only in embryos, but also in flowers and pollen. Tas14 expression in floral organs was confirmed by northern blots of tomato flowers.     

Iso-superoxide dismutase in <Emphasis Type="Italic">Deinococcus grandis</Emphasis>, a UV resistant bacterium

Deinococcus grandis possesses two types of superoxide dismutase (SOD, E. C. 1.15.1.1.) that show distinct electrophoretic behavior, one that migrates slowly and the other that migrates rapidly (SOD-1 and SOD-2, respectively). In this study, SOD-1 was uniformly and abundantly detected, regardless of growth phase, whereas SOD-2 was not detected during early growth, but was detectable from the exponential growth phase. In addition, a substantial increase in SOD-2 was observed in cells that were treated with potassium superoxide or UV, which suggests that SOD-2 is an inducible protein produced in response to stressful environments. Insensitivity of SOD-1 to both H₂O₂ and cyanide treatment suggests that SOD-1 is MnSOD. However, SOD-2 would be FeSOD, since it lost activity in response to H₂O₂ treatment, but not to cyanide. Localization studies of D. grandis iso-SODs in sucrose-shocked cells suggest that SOD-1 is a membrane-associated enzyme, whereas SOD-2 is a cytosolic enzyme. In conclusion, SOD-1 seems to be an essential constitutive enzyme for viability and SOD-2 appears to be an inducible enzyme that is probably critical for survival upon UV irradiation and oxidative stress.     

Transgene copy number can be positively or negatively associated with transgene expression

Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens. High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and -glucuronidase activity; low-expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression. H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F₁ plants with GUS activity that equalled the parents and individual F₂ plants with 50%, 100%, 150% and 200% of parental values. However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome. Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F₁ and F₂ generations with both types of insert had low GUS activity while F₂ segregants that only had the H-type inserts had high GUS activity again. Suppression of the H-type gene was associated with increased methylation of the insert. Particle acceleration was used to introduce further copies of uidA into tissues of the transformants. Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.     

Sucrose-regulated expression of a chimeric potato tuber gene in leaves of transgenic tobacco plants

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato tubers. Class-I patatin genes encode 98 to 99% of the patatin mRNA in tubers, but are not normally expressed in other tissues. They are not totally tuber-specific; however, since they can be induced to express at high levels in other tissues under conditions of sink limitation or in explants cultured on medium containing elevated levels of sucrose. To examine the evolution of the mechanisms that regulate patatin gene expression, we introduced a chimeric patatin--glucuronidase (GUS) gene containing 2.5 kb of 5 flanking sequence from the Class-I potato patatin gene PS20 into tobacco plants. The construct was not expressed at significant levels in leaves of juvenile plants or plantlets cultured in vitro, but was expressed at high levels in explants cultured on medium containing 0.3 to 0.4 M sucrose. While there were differences in the expression of the chimeric gene between transgenic tobacco and potato plants, the pattern of sucrose induction was very similar. These results suggest that the mechanism that controls patatin gene expression in potato tubers evolved from a widely distributed mechanism in which gene expression is regulated by the level of available photosynthate.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Effects of Foliar and Root Applications of Methanol on the Growth of Arabidopsis, Tobacco, and Tomato Plants

The effects of aqueous methanol solutions applied as a foliar spray or via irrigation were investigated in Arabidopsis, tobacco, and tomato plants. Methanol applied to roots leads to phytotoxic damage in all three species tested. Foliar application causes an increase of fresh and dry weight in Arabidopsis and tobacco plants, but not in tomato plants. The increase in fresh and dry weight of Arabidopsis plants does not correlate with increased levels of soluble sugars, suggesting that increased accumulation of other products is responsible for the differences in the methanol-treated leaves. Foliar application of methanol can induce pectin methylesterase (PME) gene expression in Arabidopsis and tomato plants, activating specific PME genes.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase