Reversible heat-induced inactivation of chimeric beta-glucuronidase in transgenic plants.
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.
Almoguera
Concepción
C
Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones Científicas, Apartado 1052, 41080 Sevilla, Spain.
Rojas
Anabel
A
Jordano
Juan
J
D000222:Adaptation, Physiological / Q000235:genetics; D001483:Base Sequence; D004789:Enzyme Activation; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006368:Helianthus / Q000235:genetics*; D006358:Hot Temperature; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*
D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase