Phosphorylation of wheat chloroplast-targeting COR/LEA proteins via 50-kDa protein kinase

 

Ryoko Ohno, Shigeo Takumi and Chiharu Nakamura

Laboratory of Plant Genetics, Department of Biological and Environmental Science, Faculty of Agriculture, and Graduate School of Science and Technology, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan

Corresponding author: Chiharu Nakamura

Laboratory of Plant Genetics, Faculty of Agriculture, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan

E-mail: nakamura@kobe-u.ac.jp


Abstract

Cold acclimation, an adaptive process for developing low temperature/freezing tolerance, is an important agronomic trait that is regulated by the concerted expression of a series of Cor (cold responsive)/Lea (late embryogenesis abundant) genes.  To study biochemical characteristics of paralogous wheat COR/LEA proteins, WCOR14 and WCOR15, their mode of phosphorylation was examined by in vitro and in-gel assays.  The assays showed that these COR/LEA proteins were phosphorylated by 50-kDa wheat protein kinase(s), the process of which likely contributes to their chloroplast targeting.

 

Cold acclimation is an adaptive process for developing low temperature (LT)/freezing tolerance in over-wintering plants.  The process involves a number of biochemical and physiological changes (Levitt 1980; Guy et al. 1985), which are regulated by LT through changes in gene expression.  Among a number of LT-responsive genes, the Cor (cold-responsive)/Lea (late-embryogenesis-abundant) family is the most well characterized gene family that comprises major LT-signaling components and can contribute to the significant development of freezing tolerance (Thomashow 1999).  Two wheat Cor/Lea genes, Wcor14 and Wcor15, encode chloroplast-targeting proteins and are associated with cold acclimation (Tsvetanov et al. 2001; Takumi et al. 2003; Shimamura et al. 2005).  Wcor15 shows a high level of homology with Wcs19, whose transcript level is positively correlated with the relative reduction state of photosystem II in wheat (Gray et al. 1997).  WCOR14, an ortholog of barley BCOR14b (Crosatti et al. 1999), contains the same signal peptide for chloroplast targeting at the N-terminal as WCOR15. 

NDong et al. (2002) reported that the highly homologous signal peptides of WCOR14 and WCS19 contained one putative 14-3-3 protein recognition motif.  This amino acid motif was conserved in WCOR15 (Fig. 1).  In this motif, an S-residue was predicted as a phosphorylation site by the NetPhos version 2.0 software (http://genome.cbs.dtu.dk/services/NetPhos).  Besides this, there were four and two other putative phosphorylation sites in WCOR14 and WCOR15, respectively (Fig. 1). 

To study whether WCOR14 and WCOR15 are the phosphorylation targets, in vitro phosphorylation assay was performed.  A winter wheat cultivar ‘Mironovskaya 808’ (abbreviated as M808) was grown in a controlled-climate cabinet at 25 ˚C with a 16 h photoperiod at a light intensity of 110-120 µm photons m-2 s-1 provided by cool white fluorescence lamps (the standard condition).  Conditions for cold acclimation were according to Ohno et al. (2001).  Total proteins (10 µg) from cold-acclimated and non-acclimated M808 seedlings were mixed with 10 µg of the recombinant WCOR14 and WCOR15 proteins that were synthesized in E. coli and purified by affinity chromatography according to Kobayashi et al. (2004).  The phosphorylation reaction was performed for 2 h at 25 ˚C in solution containing 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 2 mM MnCl2 and 10 µCi [γ-32P] ATP.  An aliquot (10 µL) of each reaction was analyzed by SDS-PAGE and visualized by autoradiography.  This in vitro phosphorylation assay clearly showed that both WCOR proteins were phosphorylated (Fig. 2A).  The level of phosphorylation was much higher in WCOR14 than in WCOR15, which seemed to reflect the number of putative phosphorylation sites in these WCOR proteins.  A weak signal was observed at the position of ca. 50-kDa, suggesting that this 50-kDa protein(s) is the putative kinase with multifunctional and autophosphorylation activity.

To confirm that the 50-kDa protein(s) represents the wheat kinase(s) associated with the phosphorylation of WCOR14 and WCOR15, in-gel assay was conducted.  Total proteins (20 µg) from cold-acclimated and non-acclimated M808 seedlings were fractionated on a SDS-PAGE gel containing 0.1 mg mL-1 of WCOR14 or WCOR15 proteins.  Renaturable in-gel protein kinase activity (Usami et al. 1995) was assayed by incubating the gel for 1 h in buffer containing 40 mM Tris-HCl (pH 8.0), 50 mM NaCl, 20 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 2mM DTT, 50 mM ATP, and 10 µCi [γ-32P] ATP.  Phosphorylated proteins were detected by autoradiography.  The assay showed that the two WCOR proteins in fact were phosphorylated by kinase with 50-kDa in size.  LT treatment however did not affect the level of phosphorylation of these COR proteins (Fig. 2B), which agreed with the result of in vitro assay (Fig. 2A).  Under the LT condition, this kinase was active irrespective of the light conditions, which seemed to be in contrast with the LT-inducible and light-stimulated expression of these COR proteins (Shimamura et al. 2005).  Under the non-acclimated condition, however, the light illumination enhanced the level of phosphoryaltion of both WCOR proteins.  The regulation of this kinase activity by LT and light has to be further clarified. 

Generally, the 14-3-3 recognition motifs are phosphorylated and continuously interact with the 14-3-3 proteins (May and Soll 2000).  The binding of the 14-3-3 proteins to the signal peptides is necessary for the chloroplast precursor proteins to be efficiently transported into chloroplasts (May and Soll 2000).  Since both of the WCOR14 and WCOR15 proteins contain a putative 14-3-3 recognition motif in the chloroplast-targeting signal and WCOR15 at least is targeted into chloroplasts in transgenic tobacco plants (Takumi et al. 2003), these phosphorylated proteins might interact with the 14-3-3 proteins to be efficiently transported into chloroplasts of both monocotyledonous and dicotyledonous plants.

 

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