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Reversible heat-induced inactivation of chimeric beta-glucuronidase in transgenic plants

Author:
Almoguera, C., Rojas, A., Jordano, J.
Source:
Plant physiology 2002 v.129 no.1 pp. 333-341
ISSN:
0032-0889
Subject:
Helianthus annuus, Nicotiana tabacum, tobacco, transgenic plants, seedlings, seeds, plant proteins, heat shock proteins, recombinant proteins, gene expression, messenger RNA, biosynthesis, heat stress, beta-glucuronidase, genetic markers, protein products, enzyme activity, temperature, plant extracts, amino acid sequences, nucleotide sequences, chemical constituents of plants, transcription (genetics), stress response
Abstract:
We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to beta-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5'-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions.
Agid:
670885