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Title: Peroxidase-induced wilting in transgenic tobacco plants

Abstract

Peroxidases are a family of isoenzymes found in all higher plants. However, little is known concerning their role in growth, development or response to stress. Plant peroxidases are heme-containing monomeric glycoproteins that utilize either H{sub 2}O{sub 2} or O{sub 2} to oxidize a wide variety of molecules. To obtain more information on possible in planta functions of peroxidases, the authors have used a cDNA clone for the primary isoenzyme form of peroxidase to synthesize high levels of this enzyme in transgenic plants. They were able to obtain Nicotiana tabacum and N. sylvestris transformed plants with peroxidase activity that is 10-fold higher than in wild-type plants by introducing a chimeric gene composed of the cauliflower mosaic virus 35S promoter and the tobacco anionic peroxidase cDNA. The elevated peroxidase activity was a result of increased levels of two anionic peroxidases in N. tabacum, which apparently differ in post-translational modification. Transformed plants of both species have the unique phenotype of chronic severe wilting through loss of turgor in leaves, which was initiated a the time of flowering. The peroxidase-induced wilting was shown not to be an effect of diminished water uptake through the roots, decreased conductance of water through the xylem, or increasedmore » water loss through the leaf surface of stomata. Possible explanations for the loss of turgor, and the significance of these types of experiments in studying isoenzyme families, are discussed.« less

Authors:
;  [1];  [2]
  1. (Ohio State Univ., Columbus (United States))
  2. (Univ. of Guelph, Ontario (Canada))
Publication Date:
OSTI Identifier:
7303010
DOE Contract Number:
FG02-89ER14004
Resource Type:
Journal Article
Resource Relation:
Journal Name: Plant Cell; (United States); Journal Volume: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; NICOTIANA; BIOLOGICAL STRESS; PEROXIDASES; BIOLOGICAL FUNCTIONS; DNA-CLONING; ENZYME ACTIVITY; PHENOTYPE; POST-TRANSLATION MODIFICATION; CLONING; DNA HYBRIDIZATION; ENZYMES; HYBRIDIZATION; MAGNOLIOPHYTA; MAGNOLIOPSIDA; ORGANIC COMPOUNDS; OXIDOREDUCTASES; PLANTS; PROTEINS; 550200* - Biochemistry

Citation Formats

Lagrimini, L.M., Bradford, S., and Rothstein, S. Peroxidase-induced wilting in transgenic tobacco plants. United States: N. p., 1990. Web. doi:10.2307/3869046.
Lagrimini, L.M., Bradford, S., & Rothstein, S. Peroxidase-induced wilting in transgenic tobacco plants. United States. doi:10.2307/3869046.
Lagrimini, L.M., Bradford, S., and Rothstein, S. 1990. "Peroxidase-induced wilting in transgenic tobacco plants". United States. doi:10.2307/3869046.
@article{osti_7303010,
title = {Peroxidase-induced wilting in transgenic tobacco plants},
author = {Lagrimini, L.M. and Bradford, S. and Rothstein, S.},
abstractNote = {Peroxidases are a family of isoenzymes found in all higher plants. However, little is known concerning their role in growth, development or response to stress. Plant peroxidases are heme-containing monomeric glycoproteins that utilize either H{sub 2}O{sub 2} or O{sub 2} to oxidize a wide variety of molecules. To obtain more information on possible in planta functions of peroxidases, the authors have used a cDNA clone for the primary isoenzyme form of peroxidase to synthesize high levels of this enzyme in transgenic plants. They were able to obtain Nicotiana tabacum and N. sylvestris transformed plants with peroxidase activity that is 10-fold higher than in wild-type plants by introducing a chimeric gene composed of the cauliflower mosaic virus 35S promoter and the tobacco anionic peroxidase cDNA. The elevated peroxidase activity was a result of increased levels of two anionic peroxidases in N. tabacum, which apparently differ in post-translational modification. Transformed plants of both species have the unique phenotype of chronic severe wilting through loss of turgor in leaves, which was initiated a the time of flowering. The peroxidase-induced wilting was shown not to be an effect of diminished water uptake through the roots, decreased conductance of water through the xylem, or increased water loss through the leaf surface of stomata. Possible explanations for the loss of turgor, and the significance of these types of experiments in studying isoenzyme families, are discussed.},
doi = {10.2307/3869046},
journal = {Plant Cell; (United States)},
number = ,
volume = 2,
place = {United States},
year = 1990,
month = 1
}
  • Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionicmore » peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H{sub 2}O{sub 2} via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.« less
  • Various targeting motifs have been identified for plant proteins delivered to the vacuole. For barley (Hordeum vulgare) lectin, a typical Gramineae lectin and defense-related protein, the vacuolar information is contained in a carboxyl-terminal propeptide. In contrast, the vacuolar targeting information of sporamin, a storage protein from the tuberous roots of the sweet potato (Ipomoea batatas), is encoded in an amino-terminal propeptide. Both proteins were expressed simultaneously in transgenic tobacco plants to enable analysis of their posttranslational processing and subcellular localization by pulse-chase labeling and electron-microscopic immunocytochemical methods. The pulse-chase experiments demonstrated that processing and delivery to the vacuole are notmore » impaired by the simultaneous expression of barley lectin and sporamin. Both proteins were targeted quantitatively to the vacuole, indication that the carboxyl-terminal and amino-terminal propeptided are equally recognized by the vacuolar protein-sorting machinery. Double-labeling experiments showed that barley lectin and sporamin accumulate in the same vacuole of transgenic tobacco (Nicotiana tabacum) leaf and root cells. 35 refs., 5 figs., 3 tabs.« less
  • A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and tobacco cells on a disarmed Ti plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a nuclear trait, and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100{mu}M CdCl{sub 2}, whereas, control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicatemore » that agriculturally important plants such a B. napus can be genetically engineered for heavy metals tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.« less
  • Biochemical characteristics of Acidothermus cellulolyticus endoglucanase (E1) and its physiological effects in transgenic tobacco (Nicotiana tabacum) has been studied previously. In an attempt to obtain a high level of production of intact E1 in transgenic plants, the E1 gene was expressed under the control of strong Mac promoter (a hybrid promoter of manopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region) or tomato Rubisco small subunit (RbcS-3C) promoter with different 5’ untranslated leader (UTL) sequence and targeted to different subcellular comartmentations with various transit peptides. The expression of E1 protein in transgenic tobacco plants was determined via E1more » activity, protein immunobloting, and RNA gel-blotting analyses. Effects of different transit peptides on E1 protein production and its stability were examined in transgenic tobacco plants carrying one of six transgene expression vectors with the same (Mac) promoter and transcription terminator (Tmas). Transgenic tobacco plants with apoplast transit peptide (Mm-apo) had the highest average E1 activity and protein accumulation , while E1 protein was more stable in transgenic plants with no transit peptide (Mm) than others. The E1 expression under tomato RbcS-3C promoter was higher than that under Mac promoter based on the average E1 activity, E1 protein accumulation, and RNA gel-blotting. The E1 expression was increased more than two fold when the 5’-UTL of alfalfa mosaic virus RNA4 gene replaced the UTL of RbcS-3C promoter, while the UTL of alfalfa mosaic virus RNA4 gene was less effective than the UTL of Mac promoter. The optimal combination of promoter, 5’-UTL, and subcellular compartmentation (transit peptide) for E1 protein production in transgenic tobacco plants are discussed.« less
  • Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and diseasemore » resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.« less