Plant Physiol. (1999) 119: 989-1000

Antisense Expression of the CK2 -Subunit Gene in Arabidopsis. Effects on Light-Regulated Gene Expression and Plant Growth¹

Yew Lee, Alan M. Lloyd, and Stanley J. Roux^*

Department of Botany and Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78713

	ABSTRACT

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The protein kinase CK2 (formerly casein kinase II) is thought to be involved in light-regulated gene expression in plants because of its ability to phosphorylate transcription factors that bind to the promoter regions of light-regulated genes in vitro. To address this possibility in vivo and to learn more about the potential physiological roles of CK2 in plants, we transformed Arabidopsis with an antisense construct of the CK2 -subunit gene and investigated both morphological and molecular phenotypes. Antisense transformants had a smaller adult leaf size and showed increased expression of chs in darkness and of cab and rbcS after red-light treatment. The latter molecular phenotype implied that CK2 might serve as one of several negative and quantitative effectors in light-regulated gene expression. The possible mechanism of CK2 action and its involvement in the phytochrome signal transduction pathway are discussed.

	INTRODUCTION

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One of the most important mechanisms in signal transduction is protein phosphorylation. This reaction is mediated by several groups of protein kinases in the cell and controls cellular processes that determine the state and rate of cell growth, metabolism, and differentiation (Krebs and Beavo, 1979; Trewavas and Gilroy, 1991; Hunter, 1995; Stone and Walker, 1995). Light signals that induce changes in growth and development also stimulate phosphorylation in plant cells (Datta et al., 1985; Otto and Schäfer, 1988; Reymond et al., 1992; Biermann et al., 1994; Fallon and Trewavas, 1994; Tong et al., 1996).

CK2 (formerly casein kinase II) is one of the major multifunctional protein kinases in cells. It recognizes Ser/Thr residues situated in an acidic environment in the substrate (X-S/T-X-X-E/D) (Pinna, 1990; Litchfield and Lüscher, 1993; Allende and Allende, 1995). This enzyme occurs ubiquitously and is essential for survival (Padmanabha et al., 1990). It is involved in the control of DNA replication and transcription, RNA processing and translation, cell metabolism, and motility of cells (Litchfield and Lüscher, 1993). The activity of CK2 is not directly affected by any known secondary messenger, but it can be stimulated or inhibited by extracellular signals (Tuazon and Traugh, 1991). To understand the regulation and functional significance of CK2, it is necessary to identify downstream and/or upstream components of the signaling pathways involving this kinase.

CK2 is composed of two -subunits ( and ) and two autophosphorylated -subunits (Tuazon and Traugh, 1991). Their primary and quaternary structures are highly conserved and are readily evident in the structure of CK2 genes from a variety of organisms (Wirkner et al., 1992). All CK2s have several biochemical characteristics in common: a high sensitivity to polyanions such as heparin, the ability to use GTP as well as ATP as phosphoryl donors, a preference for acidic substrates such as phosvitin and casein, and being stimulated by polyamines (for review, see Pinna, 1990).

Several laboratories are trying to identify the signal transduction pathways that are initiated by light. One of the key issues in this field is to find the physiological significance of phosphorylation/dephosphorylation events in the light-triggered signal transduction pathway. In this context, CK2s are of particular interest, not only because of increasing recognition of the major role of protein phosphorylation in regulating plant metabolism but also because these kinases have been specifically implicated in the phosphorylation of trans-acting factors that regulate gene expression (Roux, 1994).

Phosphorylation of the transcription factors AT-1 and ATBP-1 by CK2 was thought to inhibit their binding to AT-rich regions because this binding was decreased in nuclear extracts treated with Mg²⁺ and ATP or GTP (Datta and Cashmore, 1989; Tjaden and Coruzzi, 1994; for review, see Terzaghi and Cashmore, 1995). In another study, dephosphorylation reduced G-box binding by GBFs, and this binding could be restored by phosphorylation by CK2 (Klimczak et al., 1992). This result was confirmed by in vitro studies using recombinant GBF1 and CK2 reconstituted from recombinant plant catalytic subunits and regulatory subunits (Klimczak et al., 1995) and by inhibitor studies using protein kinases and phosphatase inhibitors that affected the activity of cytosolic GBFs in parsley (Harter et al., 1994). However, there is no in vivo evidence, to our knowledge, that the phosphorylation of transcription factors affects the expression of light-regulated genes.

Other evidence for phosphorylation of a transcription factor by CK2 can be found in maize seed development (Ciceri et al., 1997). A basic Leu-zipper transcriptional activator, O2 (Opaque 2), regulates the expression of a storage protein zein that is the major product during endosperm development. This transcription factor can be phosphorylated by CK2, and its dephosphorylated and hypophosphorylated forms are able to bind to the zein promoter. The activity of the hyperphosphorylated form of O2 is increased during the night and decreased during the day, indicating that light may regulate the phosphorylating activity of CK2.

This report investigates the regulation and function of CK2 in plants, with a focus on further clarifying its role in gene regulation by light. As a step toward accomplishing this goal, we transformed Arabidopsis with an antisense construct of one of the CK2 -subunit genes, ATHCK2A1. This allowed physiological and phenotypic comparisons between the transformants and wild-type plants and provided further evidence for the involvement of CK2 in the regulation of light-modulated genes.

	MATERIALS AND METHODS

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Arabidopsis Strain and Growth Conditions

Construction of Recombinant ATHCK2A1 Antisense Binary Vector

View larger version (22K): [in this window] [in a new window]   Figure 1. Structure of the ATHCK2A antisense chimeric gene used to transform Arabidopsis. The PCR product amplified from the coding region of the ATHCK2A cDNA was inserted into pCRII (lower diagram). The 5 primer had an XbaI site for further cloning. The recombinant pCRII vector was cut with XbaI and XhoI, and the insert was ligated into a similarly cut binary vector, pKYLX71 (upper diagram). This process gave the antisense orientation of ATHCK2A under the control of the cauliflower mosaic virus (CaMV) 35S promoter. MCS, Multiple cloning sites; nos, nopaline synthase gene; KmR, kanamycin-resistance gene; TcR, tetracyclin-resistance gene; RK2 ori, bacterial replication origin; TR and TL, right and left border of T-DNA, respectively.

Transformation of Arabidopsis

Isolation of Genomic DNA and Southern-Blot Hybridization

Isolation of RNA and Northern-Blot Hybridization

RNA (20 µg) was denatured in 50% formamide, 5.9% formaldehyde, and Mops buffer (20 mM Mops, pH 7.0, 10 mM sodium acetate, and 3 mM EDTA) at 65°C for 10 min and fractionated on a 1.2% agarose gel containing 2.2 M formaldehyde in Mops buffer. To ensure that equal amounts of RNA were loaded in each well, ethidium bromide was included in the RNA-loading buffer (50% glycerol, 1 mM EDTA, pH 8.0, 0.25% bromphenol blue, and 0.25% xylene cyanol FF) or hybridized with radiolabeled rDNA probes after transfer. After checking for equal loading, the RNA was transferred to a Zeta-Probe membrane (Bio-Rad) in 10× SSC (1.5 M NaCl and 0.15 M sodium citrate). The bound RNA was cross-linked using a Stratalinker (Stratagene).

The RNA-bound membrane was hybridized with the ³²P-labeled coding region of each cDNA. Labeling was performed by a random-primed method using a DNA-labeling kit (DECAprime, Ambion, Austin, TX) and [-³²P]dATP.

After a 2-h prehybridization (0.5 M Na₂HPO₄, pH 7.2, and 7% SDS), hybridization with labeled probe was carried out for 18 h at 60°C in a buffer identical to the prehybridization solution. After hybridization, washes were in 25 mM Na₂HPO₄, pH 7.2, containing 5% SDS at 60°C for 1 h, and then in the same phosphate buffer including 1% SDS at 60°C for 1 h. The washed membrane was exposed to radiographic film at 70°C with intensifying screens. The developed film was scanned into NIH Image software (National Institutes of Health, Bethesda, MD), and the density of the bands was measured.

For northern-blot analyses using riboprobes, the probes were synthesized by PCR. For the CK2 -subunit, a 928-bp riboprobe was synthesized using a sense primer (5-TAATACGACTCACTATAGGGATGTCGAAAGCTCGTGTTTA-3; the T7 promoter site is underlined) and an antisense primer (5-ATTGAATTTAGGTGACACTATAGGCAGTTAATCTGTCTTG-3; the SP6 promoter site is underlined), the region of which is the same as that of the antisense construct. T7 DNA polymerase was used for making antisense-strand-specific CK2 riboprobe, and SP6 DNA polymerase was used for synthesizing sense-strand-specific CK2 riboprobe. For cdc2, a 511-bp riboprobe (from Glu-41 to Gln-213) was made using a sense primer (5-AAGGTGTTCCTAGCACAGCAA-3, EGVPSTA) and an antisense primer (5-GAATCCATTGAATTTAGGTGACACTATAGGATCAATCTCGGAGTCTCC-3, GDSEIDQ; the SP6 promoter region is underlined). Riboprobes were synthesized using a DNA probe synthesis and removal kit (Strip-EZ, Ambion) using T7 or SP6 polymerase and [-³²P]UTP (800 mCi/mL).

Northern-blot transfer was performed as described above, and the hybridization was done following the Zeta-Probe membrane manufacturer's manual. In this experiment, 50% formamide was included in the prehybridization solution, and prehybridization was performed at 60°C for 18 h. Washing was performed using 2× SSC, 0.5× SSC including 0.1% SDS at room temperature and 0.1× SSC including 0.1% SDS at room temperature for CK2 and at 60°C for cdc2. After it was washed, the blot was exposed to a phosphor imager (Molecular Dynamics, Sunnyvale, CA) screen for 1 week.

Dot-Blot Analysis

Probes Used for Northern- and Dot-Blot Analyses

Measurement of Leaf Area and Length of Hypocotyls and Roots

View larger version (26K): [in this window] [in a new window]   Figure 5. Leaf size in Arabidopsis ATHCK2A1 antisense transformants. A, Measurement of juvenile leaves of the wild-type (WT) and antisense transformants (D, F, G, H, and M). B, Measurement of adult leaves of the wild type and antisense transformants taken from 21-d-old plants grown under continuous white light. Data are means of more than 15 plants of each line. Error bars show +SD of measurements. C, Schematic representation of the lower part of an Arabidopsis plant. A cauline leaf (cross-hatched) between the floral stem (FS) and the secondary inflorescence stem (SIS) was taken as an adult leaf. Juvenile leaves (striped) were taken from the rosette (R). PIS, Primary inflorescence stem. The leaves from plants transformed with the plasmid vector only were indistinguishable from wild-type leaves with regard to size.

To measure the length of the hypocotyl and root, 5-d-old etiolated seedlings were used. These seedlings were also treated with red light to test for phytochrome effects. For the hypocotyl-elongation experiment, Suc was deleted from the germination medium to maximize the effect of red light. Hypocotyl length was measured with a metric ruler. At least 40 seedlings were measured for each data point.

Light Treatment

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CK2 Activity Assay

The CK2 activity assay was carried out according to the method of Litchfield et al. (1990). Crude extract (10 µL) was incubated at room temperature or 37°C for 10 min with 0.73 mM peptide substrate RRREEETEEE (Promega) in 20 µL of kinase buffer (100 mM Tris-HCl, pH 7.6, 20 mM MgCl₂, 150 mM NaCl, 5 mM NaF, and 0.1 mM [-³²P]GTP; 550 cpm/pmol). After the reaction, the reaction mixture was spotted onto a P81 membrane (Whatman), washed with 75 mM phosphoric acid five times, and dried in the air. The dried membranes were put into scintillation fluid, and their bound radioactivity was measured with a liquid-scintillation counter.

Western-Blot Analysis

	RESULTS

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Antisense Transformation of the CK2 -Subunit Gene in Arabidopsis

View larger version (85K): [in this window] [in a new window]   Figure 2. Southern-blot hybridization of genomic DNA from antisense ATHCK2A1 transformants. Genomic DNA was digested with HindIII, separated by electrophoresis, transferred to a nylon membrane, and hybridized with a 32P-labeled cauliflower mosaic virus 35S promoter region cut from pBI121 (Clontech). D, F, G, H, and M, Five different antisense transformants.

View larger version (29K): [in this window] [in a new window]   Figure 3. Results of CK2 activity test. The phosphotransferase activity of the wild-type (WT) and antisense transformant lines (D, F, G, H, and M) was measured using a synthetic peptide RRREEETEEE (0.73 mM) and radiolabeled GTP as substrates. Two independent assays were performed for each line and each kinase assay was carried out with triplicate samples. The data shown are calculated average values ± SD.

Transformants Express Wild-Type Levels of cdc2 mRNA and Protein

View larger version (60K): [in this window] [in a new window]   Figure 4. Dot-blot analysis of ATHCKA1 binding to cDNA inserts of other protein kinases (A) and northern-blot analyses of cdc2 in wild type (WT) and the transformants (D, F, G, H, and M) (B). A, Either 100 ng (1×) or 200 ng (2×) of the 1.2-kb ATHCK2A1 cDNA insert (column 3, second lane) was loaded, and the amount of other DNAs (the number of molecules) was adjusted according to their size. As a negative control, the cDNA insert of CK2B1 and the plasmid 100 ng of pUC18 was loaded. Lane 1, CK2B1 insert (1.1 kb, SalI/XbaI cut); lane 2, pUC18; lane 3, CK2A1 insert (1.2 kb, SalI/XbaI cut); lane 4, cdc2a (1.4 kb, EcoRI cut); lane 5, clone 2F1T7P Arabidopsis protein kinase homolog (0.7 kb, SalI/XbaI cut); and lane 6, plant-specific protein-kinase clone CD-18 (0.63 kb, EcoRI cut). B, Northern-blot analyses of the expression of cdc2 kinase in the wild type and the transformants. Poly(A+) RNA was isolated from the total RNA extracted from 21-d-old light-grown plants and 0.5 µg was loaded in each lane. Riboprobes were synthesized as described in ``Materials and Methods''. The probed blot was stripped off using a kit and reprobed.

Antisense Effect Seen in the Adult Leaves of Transformants

When the wild type and the transformants were grown under continuous white light for 21 d, flowers showed no significant differences between the transgenic lines and the wild type, although the bolting time was slightly slower in the antisense transformants than in the wild type (data not shown). When we investigated the root growth of 5-d-old etiolated seedlings, we saw no significant difference in root length between the wild type and the antisense transformants.

There was a significant difference in the size of adult leaves (P < 0.05), but there was no difference in the size of juvenile leaves in the rosette (Fig. 5, A and B). We found no difference in the leaf shapes.

Effect of Red Light on Hypocotyl Elongation of Etiolated Seedlings

Differential Effect of Antisense Transformation on the Expression of Light-Regulated Genes

Figure 6A shows the relative mRNA abundance of all three genes tested in wild-type plants and in antisense transformants. The mean value of the band intensity of transformants was normalized to that of the wild type, vwith the band intensity of the wild type taken as 1 for the dark control and red-light treatment. The expression of all three light-regulated genes was increased by red light, both in the wild type and in the transformant lines (data not shown). However, the transformants showed two gene-expression changes: chs expression was significantly (P < 0.05 by Student's t test) increased in the dark control of the transformants, and after red-light treatment a significantly increased amount of cab and rbcS gene expression was seen in transformants over that of the wild-type control. The wild type and the transformants did not differ in their expression of chs after red-light treatment. The qualitative differences shown as averages in Figure 6A were also seen in each individual transformant line (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 6. Effect of red light on cab, chs, and rbcS gene expression in wild-type and antisense lines. A, Quantitation and normalization of cab, chs, and rbcS gene-expression levels. Density of the bands was measured using NIH Image software and normalized to the intensity of rRNA bands. The expression ratio of each transformant line was calculated with reference to the band intensity of the wild type for each light condition after normalizing for differences in RNA loading, and the mean value of the band intensity of transformants was then calculated. The dotted line shows the value of the wild-type control (1) for dark and red-light treatments. Asterisks indicate that the mean value of gene expression is statistically different (P < 0.05) from that of the wild-type control. B, Red/far-red-light reversibility test of cab expression in the wild type and in the transformants. Five-day-old dark-grown seedlings were frozen at time 0 in the dark (Dk) or were treated with 5 min of red light (R) (1 µmol m2 s1) or 5 min of red light plus 1 min of far-red light (RF) (7 µmol m2 s1) at time 0 and returned to the dark. After 4 h light-treated seedlings were harvested and transferred to liquid N2 in the dark. The two durations of red light given (15 and 5 min) were both above the level needed to fully induce the response (Karlin-Neumann et al., 1988).

Reversibility by Far-Red Light of the Red-Light Effect in the Antisense Transformants

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	DISCUSSION

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Specificity of Antisense RNA of CK2 to CK2 Activity Suppression

Potential Involvement of CK2 in Leaf Growth

Leaf tissue shows a high level of gene expression of the CK2 -subunit gene (Mizoguchi et al., 1993), and here we show a high correlation between reduced kinase activity in the transformants and reduced leaf size. Both findings indicate an involvement of CK2 in leaf physiology. There have been recent reports stating that chloroplast ATP synthase and ribonucleoproteins are phosphorylated by CK2 in spinach chloroplasts in vivo and in vitro (Kanekatsu et al., 1995; Kanekatsu and Hiroshi, 1997), demonstrating the importance of CK2 in chloroplast function.

CK2 May Play a Role in the Repression of Light-Regulated Genes

Gene expression of chs in the CK2 antisense transformants was derepressed in the dark and showed almost no difference after red-light treatment. On the other hand, amplified gene expression of cab and rbcS was seen after red-light treatment (Fig. 6). The fact that an increase in gene expression was seen in both dark and light conditions suggests that CK2 might act as a negative regulator of the expression of genes that are up-regulated by light.

Analysis of the changes in gene expression seen in the nonirradiated and the red-light-treated transformants suggests that CK2 may act differently for regulation of chs than for regulation of cab and rbcS expression. There are several reports that the phytochrome signal transduction pathways that lead to cab gene expression and chs gene expression are different (Neuhaus et al., 1993; Bowler et al., 1994a, 1994b). The pathway that leads to anthocyanin-biosynthesis gene expression is mediated by cGMP, and the other pathway, which affects cab, rbcS gene expression, and chloroplast development, requires calcium and calmodulin for its induction. PSI, Cyt b₆f, and FNR (Fd NADP⁺ reductase) gene expression are induced by both pathways (for review, see Hiratsuka and Chua, 1997).

There are at least two possibilities to explain the changes in light-regulated gene expression in the transformants. One is the regulation of DNA-binding activity of the transcription factors by phosphorylation, and the other is the regulation of their nuclear localization by phosphorylation. The first possibility requires that CK2 primarily localizes in the nucleus and/or can be translocated into the nucleus in response to extracellular signals such as light. In animal systems CK2 is localized primarily in the nucleus (Krek et al., 1992; Gruppuso and Boylan, 1995; Penner et al., 1997), or it can be translocated into the nucleus by mitogenic signaling (Ahmed et al., 1993; Lorenz et al., 1993; Boulikas, 1996). Whether light affects CK2 localization in plants is not known.

CK2 is known to phosphorylate GBF1, and this factor is known to bind to the light-responsive elements of phytochrome-regulated genes such as cab and rbcS (Datta and Cashmore, 1989; Klimczak et al., 1992, 1995). The AT-1 box is on the promoter of numerous light-regulated promoters, including pea rbcS-3A and tobacco cab-E genes. The AT-1-box-binding factor is phosphorylated by CK2, and this phosphorylation decreases the AT-1-binding activity (Datta and Cashmore, 1989). However, high-mobility proteins also can bind to the AT-rich regions and can be phosphorylated by CK2 without this affecting their binding affinity to DNA (Klimczak and Cashmore, 1994).

G-box elements are present in the promoters of many genes that are induced by environmental factors such as light, auxin, hypoxia, ethylene, and drought (for review, see Daugherty et al., 1994; Menkens et al., 1995), and the role of these elements in mediating plant responsiveness to light has been reported previously (Block et al., 1990; Donald and Cashmore, 1990). cab, rbcS, and chs, which are up-regulated by phytochrome, have this element (Terzaghi and Cashmore, 1995). The presence of G-box-binding proteins that bind to the G-box in the promoters of chs, rbcS, and cab has been shown by in vitro and in vivo footprinting (Giuliano et al., 1988; Schulze-Lefert et al., 1989; Schindler and Cashmore, 1990; Manzara et al., 1993). One of the G-box-binding proteins that has been functionally identified is O2 in maize (Schmidt et al., 1992; Ueda et al., 1992; Izawa et al., 1993), and this protein is phosphorylated by CK2 in vitro (Ciceri et al., 1997). Phosphorylation of O2 is light dependent and under diurnal control (Ciceri et al., 1997). GBF1 of Arabidopsis can be phosphorylated by a nuclear extract from broccoli that has CK2-like activity, and its phosphorylation increases its G-box-binding activity (Klimczak et al., 1992). This finding has been confirmed in recombinant GBF1 and recombinant CK2 - and -subunits (Klimczak et al., 1995).

Results implicating CK2 in the regulation of transcription factors for light-regulated genes provide a framework for interpreting the transgenic results reported here. Because all three genes tested have a G-box element in their promoters, and because the G-box is commonly found within the 5 upstream region of non-light-regulated genes, it may act as a quantitative element that modulates the expression level of light-responsive genes (Hiratsuka and Chua, 1997). The G-box element itself is not light specific, and an additional element is important for the appropriate response (Menkens et al., 1995).

Interaction between the G-box element and the I-box element in the Arabidopsis rbcS-1A gene is important for light responsiveness (Donald and Cashmore, 1990). The affinity of GBF1 for the G-box element is determined by the flanking sequences (Amstrong et al., 1992; Williams et al., 1992; Foster et al., 1994; Menkens and Cashmore, 1994), and the G-box element does not work solely during inducible gene expression (Daugherty et al., 1994; Terzaghi and Cashmore, 1995). Therefore, there is a high probability that another protein factor that is involved in light-regulated gene expression might be regulated by CK2 phosphorylation and may repress the light-regulated gene expression when it is in a phosphorylated form. This phosphorylated factor should be dephosphorylated by a phosphatase with activity that is activated by red light (or phytochrome). This speculation is consistent with the finding that protein phosphatase activity is required for light-inducible gene expression in maize (Sheen, 1993). To test this idea, it would be necessary to verify whether GBF or AT-1 is hypophosphorylated in the transformants or to find the factor responsible for the repression of chs gene expression in the dark.

Another possibility to be considered is whether the phosphorylation of a transcription factor or a retention factor by CK2 changes the localization of these factors. Gene expression can be regulated by controlling the nucleo-cytoplasmic partitioning of transcription factors (Whiteside and Goodbourn, 1993), and this partitioning can be modulated by the phosphorylation of the amino acids neighboring the nuclear-localization signal (Jans, 1995). For example, the nuclear uptake of the simian virus 40 large T-antigen is enhanced by phosphorylation of a Ser residue by CK2 (Rihs et al., 1991), which increases the binding of this protein to importins (Hübner et al., 1997).

All four GBFs described in the literature thus far have putative CK2-phosphorylation sites, and a deletion construct of GBF-1 that is missing one of these sites has increased nuclear localization (Terzaghi et al., 1997). In parsley cell cultures, protein kinase inhibitors and phosphatase inhibitors change the activity of a cytosolic GBF whose transport to the nucleus is stimulated by red light (Harter et al., 1994). Therefore, it is reasonable to consider a potential role for CK2 in regulating the nucleo-cytoplasmic partitioning of GBFs and thereby influencing gene expression.

To interpret the gene-expression changes observed in transgenic plants, two things should be pointed out. First, the measured abundance of cab and rbcS must be a cumulative expression of several genes of a family, because we used homologous cDNA probes for this experiment. In chs the major band shown by northern-blot analysis must reflect the expression of a single gene, because it has been found that chs is a single-copy gene in Arabidopsis (Feinbaum and Ausubel, 1988). However, it has been shown that the Arabidopsis rbcS gene family consists of four genes (Krebbers et al., 1988), and five genes have been reported for chlorophyll a/b-binding proteins in Arabidopsis (Leutwiler et al., 1986; Zhang et al., 1991). Genes of both the cab and rbcS families show differential regulation by light (Karlin-Neumann et al., 1988; Dedonder et al., 1993), and our results do not provide information about a specific promoter that is affected by the antisense transformation of the CK2 -subunit. Second, our results give the steady-state level of gene expression; therefore, we cannot exclude the possibility that the changes observed may be attributable to a posttranscriptional control. Gene expression can be affected at a posttranscriptional level by light (Ernst et al., 1988; Jenkins, 1991; Wanner and Gruissem, 1991).

Other light-regulated DNA-binding activities have been found in tobacco and Arabidopsis. 3AF5 and 3AF3 have been found in tobacco, and their binding sites are located at the 5 and 3 ends of box III of the promoter region of the rbcS-3A gene (Sarokin and Chua, 1992). Gel-shift assays showed that DNA-protein complexes moved faster in dark-adapted plants than those from light-grown plants and that the mobility of complexes from light-grown plants was affected by alkaline phosphatase treatment. This finding supports the notion that rbcS gene expression can be regulated by phosphorylation of transcription factors, but there is no evidence regarding which kinase is responsible for this phosphorylation.

In Arabidopsis, CCA-1, which binds to the promoter of the Lhcb*3 gene (formerly cab 140), has been cloned and antisense transformants have been produced (Wang et al., 1997). The antisense transformants have a reduction in red-light-stimulated cab gene expression. CCA-1 is a myb-related protein and has an acidic C-terminal region that has potential CK2-phosphorylation sites. CK2 is an acidophilic kinase (Pinna, 1997) and is known to phosphorylate myb proteins in animals (Lüscher et al., 1990).

HY5, which is a basic Leu zipper DNA-binding protein, has a putative CK2-phosphorylation site (Oyama et al., 1997). The Hy5 mutant has been genetically identified by its insensitivity to the inhibition of hypocotyl elongation by light (Koornneef et al., 1980). It has defects in light-induced chlorophyll accumulation, root development, and the expression of cab expression affecting the circadian control (Ang and Deng, 1994; Anderson et al., 1997; Oyama et al., 1997). It will be useful to determine whether HY5 and CCA-1 are phosphorylated by CK2 and, if so, how this affects the expression of light-regulated genes.

CK2 RNA Suppression Does Not Affect the Reversibility of cab Expression by Far-Red Light

Antisense Transformation Affects Two Red-Light Responses Differently

	FOOTNOTES

   Received July 14, 1998; accepted November 21, 1998.

	ABBREVIATIONS

Abbreviation: GBF, G-box-binding factor.

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