Characteristics of ethylene biosynthesis-inducing xylanase movement in tobacco leaves.

Department of Horticulture, University of Maryland, Beltsville, Maryland 20705.
Plant physiology (Impact Factor: 6.56). 01/1993; 100(4):2059-65. DOI: 10.1104/pp.100.4.2059
Source: PubMed

ABSTRACT (125)I-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active (125)I-labeled EIX was obtained using chloramine-T as the oxidizing agent. Labeled EIX was detected in the far most edges of the leaf 5 min after it was applied to the petiole of a detached leaf. EIX was distributed uniformly throughout the leaf, including the mesophyll area within 5 to 15 min, after which there was only little change in the distribution of radioactivity in the leaf. (125)I-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincubation of labeled EIX with anti-EIX antibodies, indicating that the intact peptide moves in the leaf. Injection of anti-EIX antibodies into the intercellular spaces of the leaf mesophyll prevented induction of necrosis by EIX, suggesting the mesophyll as the site of EIX action. EIX was translocated both to upper and lower parts of the plant when applied to a whole plant through the petiole of a cut leaf. Radioactivity was found in all leaves and in the stem, although some leaves accumulated much more EIX than others; EIX was not found in the roots. There was no difference between the accumulation pattern of EIX in fresh and ethylene-treated leaves or between sensitive (Xanthi) and insensitive (Hicks) tobacco cultivars. These data support the hypothesis that intact EIX protein is translocated to the leaf mesophyll, where it directly elicits plant defense responses.

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    ABSTRACT: In cultured XD6S tobacco cells, xylanase from Trichoderma viride (TvX) induced the expression of a luciferase reporter gene that was under the control of a GCC box, which is an 11 bp sequence (TAAGAGCCGCC) that is found in the 5'-upstream region of pathogen-responsive defence genes that include genes for class I basic chitinase. TvX-induced biosynthesis of ethylene was not required for the TvX-activated transcription. The TvX-induced, GCC box-mediated transcription of the reporter gene was completely blocked not only by staurosporine, an inhibitor of serine/threonine protein kinases, at 1 microM, but also by calyculin A, an inhibitor of protein phosphatases 1 and 2A, at 0.2 microM. It appeared also that protein synthesis de novo was required for the GCC box-mediated transcription of the reporter gene. Accumulation of mRNAs for various ERFs (ethylene-responsive transcription factors), which have been shown to bind specifically to the GCC box, was also induced by TvX prior to increases in the level of mRNA for a class I basic chitinase. In particular, the level of mRNA for EFR2 reached a maximum from 3 to 6 h, whereas levels of mRNAs for ERF3 and ERF4 were highest 0.5 h after the start of treatment of TvX and decreased thereafter. Moreover, induction of accumulation of the mRNA for ERF2 was inhibited by staurosporine and calyculin A. These results suggest that ERF2 might play a major role in TvX-induced, GCC box-mediated transcription of genes and that both protein kinase(s) and protein phosphatase(s) might be involved, as positive regulators, in the signal transduction pathway that leads to expression of ERF2 and subsequent GCC box-mediated transcription of genes.
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    ABSTRACT: Fusariumoxysporum produces a 24-kDa protein, Nep1, which induces necrosis and ethylene production in leaves of many dicot plant species. Detached Nicotiana tabacum L. cv. Xanthi leaves respond with concentration-dependent necrosis after infiltration with Nep1 or when Nep1 is taken up by the vascular tissue. This response follows the induction of ethylene biosynthesis and accumulation of ACC synthase and ACC oxidase transcripts. Pretreating the leaves with 100 μl/l ethylene prior to elicitation enhanced Nep1-induced ethylene production. Nep1 (208 nM) causes extensive necrosis of mature tobacco leaf tissue when applied to Xanthi tobacco as a foliar spray (129 ml/m2). Tobacco cell cultures respond to Nep1 by alkalization of the culture media, the accumulation of potassium in the media, oxygen uptake, induction of active oxygen species, and eventual cell death. The response of cultured tobacco cells to Nep1 is time- and concentration-dependent. Cell death was the same at 300 min for 5 ng/ml and higher concentrations, while 0.5 ng/ml had no effect on cell death. In the case of O2 uptake, cells responded to 0.5 ng/ml within minutes of treatment, but at a rate lower than 5 ng/ml. The lower concentration of Nep1 did not induce an increase in pH, K+ efflux, or increasing H2O2 accumulation in the culture media.
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