PMC

Determination of Which Amino Acid Species Is Phosphorylated in Response to ABA.
³²P-labeled TRAB1 protein obtained as described for was recovered from the polyacrylamide gel and subjected to phosphoamino acid analysis. The autoradiogram made after two-dimensional thin layer electrophoresis is shown. The spots corresponding to phosphoserine, phosphotyrosine, incompletely digested phosphopeptide, and free phosphate are indicated.

Effects of Mutations at Ser-102 on the Ability of TRAB1 to Mediate ABA-Responsive Transcription.
Cotransfection assays were performed as described for using wild-type (Wt) GBD-TRAB1 or its derivatives that carry mutations at Ser-102. “None” indicates assays using the empty effector plasmid 35S-shΔ-stop. The mutations are illustrated in the sequence at top. The value of induction by ABA is shown with the ABA + bars.
(A) Effect of the S102A mutation.
(B) Effect of the S102D mutation.

ABA-Dependent Phosphorylation of TRAB1-dHA/His Is Abolished by the S102A Mutation.
Protoplast cells transfected with the expression plasmid for wild-type TRAB1-dHA/His (Wt), its S102A mutant derivative (S102A), or the empty vector 35S-shΔ-stop (−) were treated without (−) or with (+) ABA for 1 h. Total cellular extracts were analyzed by immunoblotting with anti-HA antibody. The positions of the S, M, and F bands (see text) are indicated.

Mobility Shift of TRAB1 on SDS-PAGE Caused by ABA-Induced Phosphorylation.
(A) Nuclear extracts of rice suspension-cultured cells (Oc cells) treated with ABA for the indicated times (0 indicates untreated cells) were analyzed by immunoblotting with anti-TRAB1 antibody. The arrow and asterisks indicate TRAB1-specific and nonspecific bands, respectively. Although the expression of the nonspecific bands was affected by ABA, the nature of these polypeptides is unknown.
(B) TRAB1-dHA/His expressed in transgenic cells treated without (−) or with (+) ABA for 30 min was recovered with nickel–nitrilotriacetic acid agarose resin, incubated without (−) or with (+) CIAP, and analyzed by immunoblotting with anti-HA antibody. The signals seen at the top of the +CIAP lanes are parts of bulky CIAP bands that reacted nonspecifically with the anti-HA antibody.

TRAB1 Is Phosphorylated Rapidly in Response to ABA.
Rice suspension-cultured cells (Oc) were labeled with ³²Pi for 1 h. Cells then were treated with 50 μM ABA. Nuclear extracts were prepared from the labeled cells and subjected to immunoprecipitation with anti-TRAB1 antibody followed by SDS-PAGE.
(A) After ABA treatment for 30 min, immunoprecipitation was performed with preimmune serum (P), anti-TRAB1 antibody (AnT), or anti-TRAB1 antibody preincubated with an excess amount of recombinant TRAB1 (AnT+T). The position of the 45-kD TRAB1 band is indicated by the arrow. The smaller minor bands indicated by asterisks are considered to be immunologically related to TRAB1. However, it is not known whether they are partially degraded forms of TRAB1 or the products of related genes.
(B) Immunoprecipitation with anti-TRAB1 antibody using nuclear extracts from the labeled cells treated with ABA for the indicated times.

Effects of Internal Deletions in the N-Terminal Region of TRAB1 on the Ability to Mediate ABA-Responsive Transcription.
Ten micrograms each of the effector plasmid for wild-type (Wt) GBD-TRAB1 or its derivatives with an internal deletion of 20 amino acids (ID1 to ID4) as indicated in the sequence at top and the UAS-TATA-GUS reporter gene containing GAL4 binding sites was cotransfected into protoplasts of rice suspension-cultured cells by electroporation. Each transfection also included 5 μg of a ubiquitin promoter::luciferase (LUC) plasmid as an internal standard. GUS activities were normalized with luciferase activity. The deleted region is indicated by the shaded area in the scheme of each mutant construct. The value of induction by ABA is shown with the ABA + bars. BZ, bZIP.

Conserved Sequence Blocks I to III of the TRAB1 Family Proteins.
The structure of TRAB1 is shown in the scheme at top, where regions I to III and the bZIP are indicated with black and hatched boxes, respectively. Amino acids identical, similar, and dissimilar to those of TRAB1 and gaps are indicated by dashes, uppercase letters, lowercase letters, and underlined letters, respectively. The repeated sequence motifs Thr-hy-ac-ac and QGSLT (see text) are indicated by solid and dotted underlines, respectively. The Ser residue in the protein kinase C phosphorylation signature is indicated by a dot. The arrow points to the Met residue formerly predicted to be the N terminus from the sequence of the TRAB1 cDNA clone. The amino acids of TRAB1 are numbered based on the putative full-length sequence, which includes the N-terminal 56 amino acids predicted from the genome sequence obtained from the Syngenta draft sequence database (see text).

TRAB1 Is Present in the Nucleus Independently of ABA.
(A) Rice suspension-cultured cells (Oc cells) were treated with 50 μM ABA for the indicated times (0 indicates untreated cells). Total cell (T), crude nuclear (N), and postnuclear (PN) fractions were prepared and subjected to immunoblot analysis with anti-TRAB1 antibody. Each lane was loaded with protein corresponding to an approximately equal volume of cells.
(B) Phase contrast and epifluorescence microscopy images deriving from GFP or 4′,6-diamidino-2-phenylindole (DAPI) of a rice protoplast transiently expressing GFP or TRAB1-GFP. Oc cell protoplasts were transformed with a GFP or TRAB1-GFP expression plasmid by electroporation and cultured for 12 h before microscopic observation. Protoplasts were not treated with ABA. DIC, differential interference contrast.