Identification of the conserved serine/threonine residues important for gibberellin-sensitivity of Arabidopsis RGL2 protein.
The DELLA proteins GAI, RGA, RGL1 and RGL2 in Arabidopsis are plant growth repressors, repressing diverse developmental processes. Studies have shown that gibberellin (GA) attenuates the repressive function of DELLA proteins by triggering their degradation via the proteasome pathway. However, it is not known if GA-induced protein degradation is the only pathway for regulating the bioactivity of DELLA proteins. We show here that tobacco BY2 cells represent a suitable system for studying GA signaling. RGL2 exists in a phosphorylated form in BY2 cells. RGL2 undergoes GA-induced degradation, and this process is blocked by proteasome inhibitors and serine/threonine phosphatase inhibitors; however, serine/threonine kinase inhibitors had no detectable effect, suggesting that dephosphorylation of serine/threonine is probably a prerequisite for degradation of RGL2 via the proteasome pathway. Site-directed substitution of all 17 conserved serine and threonine residues showed that six mutants (RGL2(S441D, RGL2(S542D), RGL2(T271E), RGL2(T319E), RGL2(T411E) and RGL2(T535E)) mimicking the status of constitutive phosphorylation are resistant to GA-induced degradation. This suggests that these sites are potential phosphorylation sites. A functional assay based on the expression of GA 20-oxidase revealed that RGL2(T271E) is probably a null mutant, RGL2(S441D), RGL2(S542D), RGL2(T319E) and RGL2(T411E) only retained about 4-17% of the activity of the wild type RGL2, whereas RGL2(T535E) retained about 66% of the activity of the wild type RGL2. However, expression of GA 20-oxidase in BY2 cells expressing these mutant proteins is still responsive to GA, suggesting that the stabilization of RGL2 protein is not the only pathway for regulating its bioactivity.
Hussain
Alamgir
A
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.
Cao
Dongni
D
Cheng
Hui
H
Wen
Zilong
Z
Peng
Jinrong
J
D017360:Arabidopsis / Q000187:drug effects* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017124:Conserved Sequence; D015536:Down-Regulation; D018506:Gene Expression Regulation, Plant; D005875:Gibberellins / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D030821:Plants, Genetically Modified; D061988:Proteasome Inhibitors; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D005875:Gibberellins; D061988:Proteasome Inhibitors; C450823:RGL2 protein, Arabidopsis; D014157:Transcription Factors; D013912:Threonine; D012694:Serine; C007842:gibberellic acid; D006899:Mixed Function Oxygenases; C089210:gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing); D010749:Phosphoprotein Phosphatases