The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences

Abstract

The abscisic acid (ABA)-induced protein kinase PKABA1 is present in dormant seeds and is a component of the signal transduction pathway leading to ABA-suppressed gene expression in cereal grains. We have identified a member of the ABA response element-binding factor (ABF) family of basic leucine zipper transcription factors from wheat (Triticum aestivum) that is specifically bound by PKABA1. This protein (TaABF) has highest sequence similarity to the Arabidopsis ABA response protein ABI5. In two-hybrid assays TaABF bound only to PKABA1, but not to a mutant version of PKABA1 lacking the nucleotide binding domain, suggesting that binding of TaABF requires prior binding of ATP as would be expected for binding of a protein substrate by a protein kinase. TaABF mRNA accumulated together with PKABA1 mRNA during wheat grain maturation and dormancy acquisition and TaABF transcripts increased transiently during imbibition of dormant grains. In contrast to PKABA1 mRNA, TaABF mRNA is seed specific and did not accumulate in vegetative tissues in response to stress or ABA application. PKABA1 produced in transformed cell lines was able to phosphorylate synthetic peptides representing three specific regions of TaABF. These data suggest that TaABF may serve as a physiological substrate for PKABA1 in the ABA signal transduction pathway during grain maturation, dormancy expression, and ABA-suppressed gene expression.

Figure 1

Two-hybrid assays showing interaction between PKABA1 and TaABF. A, PJ69-4A yeast strains transformed with plasmids containing the indicated prey and bait constructs were streaked onto medium lacking the indicated nutrients. EV, Use of empty vector encoding the GAL4 BD with no fusion protein. B, Four independent transformants harboring the indicated combinations of prey and bait plasmids were assayed for β-galactosidase activity using a liquid assay with O-nitrophenyl β-d-galactopyranoside as the substrate. The topmost bar indicates the amount of background β-galactosidase activity in untransformed PJ69-4A yeast cells. Error bars, some of which are too small to be seen, indicate se.

Figure 2

Sequence of TaABF. A, Deduced amino acid sequences from TaABF cDNA clones. The TaABFA sequence was obtained by combining the original two-hybrid clone (Br-2H, encoding amino acids 40–336) with an identical overlapping cDNA (CS-46, encoding amino acids 79–391) containing a complete 3′ end. Short conserved sequence blocks present in other ABFs are underlined. The basic domain is indicated by a dashed underline and the Leu zipper region is indicated by a bold wavy underline. Amino acid residues that are identical between TaABFA and TaABFB are indicated by asterisks and similar amino acids are indicated by dots. B, Basic domain of TaABF compared with the basic (DNA-binding) regions of ABI5 (Finkelstein and Lynch, 2000), TRAB1 (Hobo et al., 1999), AtABF2, AtABF1 (Choi et al., 2000), DPBF1 and DPBF2 (Kim et al., 1997), and EmBP1 (Guiltinan et al., 1990). Nucleotide sequences are deposited in GenBank under accession numbers AF519803 (TaABFA) and AF519804 (TaABFB).

Figure 3

Two-hybrid assays of TaABF with PKABA1, nullPKABA1, and TaPK4. A, PJ69-4A yeast strains transformed with plasmids containing the indicated prey and constructs were streaked onto medium lacking the indicated nutrients. The TaABF prey plasmid used was the TaABF(Br-2H) obtained in the two-hybrid screen. EV, Use of empty vector encoding the GAL4 BD with no fusion protein. B, Complete deduced amino acid sequences of TaPK4 and PKABA1. The first 10 amino acids of the PKABA1 clone reconstituted from the genomic clone (see “Materials and Methods”) are italicized. The nucleotide-binding site of PKABA1 that is absent in nullPKABA1 is indicated by a double underline. The C-terminal acidic stretches in TaPK4 and PKABA1 are indicated by a single underline. Amino acid residues that are identical between TaPK4 and PKABA1 are indicated by asterisks. The nucleotide sequence of TaPK4 is deposited in GenBank under accession number AF519805.

Figure 4

Transcript levels of TaABF, PKABA1, and TaPK4 in stressed wheat plants. Seven-day-old wheat seedlings were maintained under control conditions (C) of 22°C and 100% relative humidity or were subjected to a cold treatment of 2°C (2°), application of 250 mm NaCl to the roots (S), application of 25 μm ABA to the roots (A), or removal to a drier chamber maintained at 85% relative humidity (D). After 24 h of the stress treatments, leaves and roots were separately collected for analysis. In a separate experiment, the top 4 cm of leaves was removed from 7-d-old seedlings and placed at 85% relative humidity for the indicated number of hours. RNA was also obtained from whole mature, after-ripened grains. Ten micrograms (20 μg for the TaPK4 blot) of total RNA was electrophoresed, blotted, and hybridized to the cDNA probes indicated on the right side of the figure. The TaABF probe used was the TaABF(Br-2H) partial cDNA, which would be expected to hybridize with both TaABFA and TaABFB transcripts. Total RNA was detected by ethidium bromide fluorescence.

Figure 5

Transcript levels of TaABF and PKABA1 in wheat grains. A, Grains were collected from greenhouse-grown wheat plants at 5 to 45 d after pollination (DAP). Grains collected at 45 DAP were fully mature. B, Grains were stored after harvest for 1 year at room temperature to obtain after-ripened (AR) grains. Grains maintained at −20°C for 1 year after harvest were used for the dormant grains. Grains were placed on moist filter paper and allowed to imbibe for 0 to 48 h. C, After-ripened grains were allowed to imbibe for 3 h before dissection of embryo (emb) and endosperm (end) tissue or collection of whole grains. Ten micrograms of total RNA was electrophoresed, blotted, and hybridized to the cDNA probes indicated on the right side of the figure. The TaABF probe used was the TaABF(Br-2H) partial cDNA. Total RNA was detected by ethidium bromide fluorescence.

Figure 6

In vitro phosphorylation of TaABFA-derived peptides by PKABA1. A, Sequence of synthetic peptides used as substrates in in vitro phosphorylation assays. Residues representing possible phosphorylation sites for PKABA1 (S,T) are in bold. Basic residues added at the end of synthetic peptides for technical reasons, but not present in the TaABFA sequence, are underlined. The MNM peptide represents amino acid residues 60 to 73 of TaABFA as numbered in Figure 2. The other peptides represent residues 102 through 123 (VW), 163 through 178 (GEM), 254 through 274 (SR), 291 through 310 (SCER), and 311 through 326 (BD). B, Phosphorylation of synthetic peptides by control extract and FLAG::PKABA1 extract. A control cell line (C) and a cell line transformed with a gene encoding a FLAG-PKABA1 fusion protein (P) were used for the assays. Crude protein extract was prepared from the cell line and used in in vitro phosphorylation assays in the presence of γ-³²P ATP and 50 μg of the indicated peptide for 15 min. After removal of molecules >10,000 D from the reaction products, phosphorylated peptides were bound to P81 paper and the amount of phosphorylation measured by scintillation counting. Error bars indicate se. D, Phosphorylation of BD peptide by control and PKABA1 extracts. Assays were carried out as in B except that the amount of peptide included was varied.