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Keywords:

  • rice;
  • VIRESCENT 2;
  • plastid and mitochondrial guanylate kinase;
  • chloroplast development;
  • cytosolic guanylate kinase;
  • Arabidopsis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Guanine nucleotides are the major energy sources for many chemical reactions, and are also the building blocks for DNA, RNA and some signaling molecules (e.g. cGMP). It has been assumed that the biosynthetic processes for guanine nucleotides in higher plants are generally similar to those of animals and micro-organisms (Stasolla et al., 2003; Zrenner et al., 2006). GMP is synthesized through two pathways: a de novo pathway that uses amino acids and other small molecules, and a salvage pathway that uses pre-formed guanine and guanosine. GMP can subsequently be phosphorylated to GDP and GTP. However, the regulatory network for these pathways in higher plants is much more complex than that in animals. This increased complexity arises from the additional presence of plastids in plant cells that can differentiate into various types of organelle, such as chloroplasts, amyloplasts and chromoplasts (Stasolla et al., 2003; Zrenner et al., 2006).

Guanylate kinase (GK, ATP:GMP phosphotransferase; EC 2.7.4.8) is an essential enzyme for the biosynthesis of guanine nucleotides by catalysis of the reaction (d)GMP + ATP [RIGHTWARDS ARROW] (d)GDP + ADP, where (d)GMP indicates GMP or dGMP. GK also plays an important role in the recycling of the second messenger cGMP (cGMP [RIGHTWARDS ARROW] GMP [RIGHTWARDS ARROW] GDP [RIGHTWARDS ARROW]GTP [RIGHTWARDS ARROW] cGMP), and is thought to regulate the supply of guanine nucleotides to signal transduction pathway components (Gaidarov et al., 1993). In addition to these physiological roles, the biomedical role of human GK in the activation of pro-drugs used for the treatment of cancers and viral infections has attracted significant attention (Brady et al., 1996; Stolworthy et al., 2003). Thus, GK is the subject of considerable interest. As a result, a number of studies have been performed on the biochemistry, genetics and regulation of GK in animals and micro-organisms (Beck et al., 2003; Brady et al., 1996; Gentry et al., 1993; Konrad, 1992; Ray et al., 2005; Stolworthy et al., 2003). Similarly, several studies have attempted to identify the GK gene/protein of higher plants (Kumar, 2000; Kumar et al., 2000; Stasolla et al., 2003). However, despite the obvious importance of GK, there has been a dearth of studies on the physiological roles of GK in plants.

The rice mutant virescent 2 (v2) has a mutation of a single nuclear gene. The mutation is temperature-sensitive, and mutant plants develop chlorotic leaves at the restrictive temperature (20°C), but develop nearly normal green leaves at the permissive temperature (30°C) (Iba et al., 1991). A temperature-shift analysis of v2 mutant plants revealed that they have a temperature-sensitive period at the P4 stage of leaf development (Iba et al., 1991). This is the leaf developmental stage immediately after formation of the basic leaf structure, but just before the onset of leaf elongation (Nemoto and Yamazaki, 1993). Moreover, our recent work has shown that the v2 mutation affects activation of the chloroplast translation machinery and/or expression of the plastid-to-nucleus signaling pathway during this leaf developmental stage (Sugimoto et al., 2004).

In this study, we show that rice plants have two types of enzymatically active GK isozyme, which play distinct roles in different subcellular compartments. Our first step was to identify the V2 gene using positional cloning. Next, through studies of subcellular localization and in vitro enzymatic activity, and a yeast complementation test, we show that the V2 gene encodes a novel type of GK (designated pt/mtGK) that is targeted to plastids and mitochondria. Rice plants have a second type of GK (designated cGK) localized in the cytoplasm. In various species, cGK functions in guanine nucleotide biosynthesis in fundamental cellular processes, such as nucleic acid metabolism and signal transduction. Our investigation has identified a new factor, pt/mtGK, that is a prerequisite for the early stage of chloroplast differentiation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The V2 gene functions at an early stage of chloroplast and leaf development

The V2 locus was previously mapped as a classical phenotypic marker on the short arm of chromosome 3 (Yoshimura et al., 1997). Fine mapping pinpointed the V2 locus to a 20 kb genomic region (Figure 1b), and identified one base substitution in a putative gene. A BLAST search (Altschul et al., 1990) revealed that this putative gene had high similarity to a guanylate kinase (GK) gene (Figure 2). To determine whether this GK homolog gene represents the V2 gene, we constructed transgenic v2 mutants carrying the candidate cDNA under the control of the CaMV 35S promoter (pPro35S:V2). Six independent transgenic plants were generated. Four plants grown at the restrictive temperature (20°C) developed normal green leaves with mature chloroplasts (Figure 1a), suggesting that the candidate GK gene is indeed the V2 gene. Sequence analyses revealed that the V2 gene consists of four exons and encodes a 285 amino acid protein. In the v2 mutant allele, the valine at position 162 is replaced by an isoleucine (Figures 1c and 2).

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Figure 1.  Positional cloning of the V2 gene. (a) Comparison of the phenotypes of the wild-type (left), v2 mutant, transgenic v2 mutant containing the empty vector, and transgenic v2 mutant over-expressing wild-type V2 protein (right) grown at the restrictive temperature (20°C). (b) High-resolution genetic and physical maps of the V2 locus. The upper part shows the genetic linkage map representing the relative position of V2 to the RFLP markers on chromosome 3. The central parts show high-resolution genetic linkage maps of the V2 region developed by the analysis of 3286 F2 chromosomes and the establishment of YAC (Y), BAC (OSJNBa or OSJNBb) and PAC (P) clone contigs spanning the V2 region. The bottom part shows a fine-scale genetic and physical map of the 20 kb V2 region. The numbers under the linkage map indicate the numbers of recombinants in the marker intervals. Circles indicate CAPS markers generated from the RFLP markers and the YAC/BAC end sequences. Rectangles indicate YAC/BAC end markers. a and b indicate the CAPS markers. (c) Structure of the V2 gene. Boxes and thin lines represent exons and introns, respectively. The V2 open reading frame is depicted as gray boxes. The position of the amino acid substitution in the v2 mutant is indicated. The complete nucleotide sequence of the V2 cDNA has been deposited in DDBJ/EMBL/Genbank, accession number AB267728. (d) Diagram of a rice seedling with a fully emerged third leaf. L1, L2 and L3 indicate the 1st, 2nd and 3rd leaves, respectively. The 3rd leaf was removed first and divided into two parts, the basal and upper halves of the blade. Next, the root, albumin, coleoptile, 1st leaf and 2nd leaf were removed. The 3rd leaf sheath was divided into two parts, shoot base (an 8 mm piece from the bottom of the 3rd leaf sheath) and L4 (the 4th leaf above the shoot base). The plant parts used for sample preparation are indicated on the right. R, root; SB, shoot base; L4, 4th leaf; L3 L, basal half of the 3rd leaf; L3U, upper half of the 3rd leaf. (e) RT-PCR analysis of V2 gene expression. Total RNA was extracted from seedlings of the wild-type and v2 mutant grown at the restrictive temperature (20°C) and amplified with gene-specific primers as described in Experimental procedures. The total RNA samples used as template for RT-PCR were run on a gel and stained with ethidium bromide (EtBr) to allow comparison of the sample concentrations (see bottom panel). (f) Immunoblot analysis of the V2 protein. Total soluble protein was extracted from wild-type and v2 mutant seedlings grown at the restrictive temperature (20°C). Equal amounts of total soluble protein (40 μg per lane) were loaded onto the gel. The V2 protein was detected using an anti-V2 protein antibody. Band ‘a’, at 27.5 kDa, corresponds to the V2 protein. (g) V2 protein expression in the transgene-complemented v2 mutant. Total soluble protein was extracted from the roots (R) and upper half of the 3rd leaves (L3U) of the wild-type (lane 1) and the transgene-complemented v2 mutants (lane 2) grown at the restrictive temperature (20°C). Equal amounts of total soluble protein (40 μg per lane) were loaded onto the gel. V2 protein was detected using an anti-V2 protein antibody. Band ‘a’, at 27.5 kDa, corresponds to the V2 protein.

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Figure 2.  Multiple sequence alignments of the V2 protein with the most closely related GKs. Amino acid sequence alignment was performed using CLUSTAL X (Thompson et al., 1997). The alignment includes Arabidopsis V2 ortholog AtGK3 (accession number AF378877), OsGK1 of Oryza sativa (accession number AB267729), the Arabidopsis GKs AtGK1 (accession number AF204675) and AtGK2 (accession number AF204676), ScGUK1 of Saccharomyces cerevisiae (accession number L04683) and HsGK of Homo sapiens (accession number U66895). Residues in black boxes are identical in at least three of the seven proteins, and those in shaded boxes share similarity with conserved residues. Numbers on the left indicate residue numbers in the predicted polypeptides. The position of the amino acid substitution in the v2 mutant is indicated. We propose to rename the V2 protein OsGKpm to conform with standard nomenclature.

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To examine stage-specific expression of the V2 gene during chloroplast and leaf development, we carried out RT-PCR analysis of various tissues of wild-type and v2 mutant seedlings that had been grown at the restrictive temperature until they had a fully emerged 3rd leaf. In rice, successive leaves emerge from the sheath in an ordered manner with respect to relative timing, and leaf cells divide, elongate and enlarge in a longitudinal manner (Nemoto and Yamazaki, 1993). In general, when a rice leaf begins to emerge from the sheath, the shoot contains four immature leaves. Therefore, when the 3rd leaf has fully emerged, the shoot also contains the 4th to the 7th immature leaves. Thus the leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the SB and L4 samples contain proplastids and early developing chloroplasts (Figure 1d) (Kusumi et al., 1997; Sugimoto et al., 2004). RT-PCR analysis revealed that, in the wild-type seedlings, V2 transcripts are much more abundant at an early stage of leaf development than at the later stages (Figure 1e). In contrast, V2 transcripts were more abundant at later stages of leaf development in the v2 mutant.

We next examined the levels of V2 protein during leaf development by immunoblot analysis. Three candidate V2 proteins of approximately 26.0, 27.0 and 27.5 kDa (bands a–c, Figure 1f) were detected using an anti-V2 protein antibody. In transgene-complemented v2 mutant seedlings, only the 27.5 kDa protein showed an increased level of expression, suggesting that this protein is the V2 protein (Figure 1g). The amounts of V2 protein in wild-type and v2 mutant seedlings paralleled those of V2 transcripts during chloroplast and leaf development (Figure 1e,f). At the permissive temperature, the expression profiles of V2 protein in the v2 mutant were similar to those of the wild-type, although the expression levels of V2 protein in the aerial parts of the v2 mutant were higher (data not shown). Taken together, these results suggest that the V2 gene functions at an early stage of chloroplast and leaf development. This conclusion is consistent with a previous temperature-shift experiment in which the V2 gene product was shown to be necessary for chloroplast differentiation during a strictly limited period of early leaf development (Iba et al., 1991).

V2 protein is dual-targeted to chloroplasts and mitochondria

Comparison of the primary structures of GKs of various species showed that the V2 protein carries an N-terminal extension of the GK domain (Figure 2). To determine the subcellular localization of the V2 protein, we generated transgenic Arabidopsis plants expressing GFP fused to the C-terminus of the full-length V2 protein. The translational fusion protein (V2–GFP) was able to rescue the rice v2 mutant phenotype (data not shown), indicating that the fusion protein was functional. A confocal microscopy analysis of V2–GFP expression showed that GFP fluorescence was mostly detected in multiple small intracellular compartments in root cells of transgenic Arabidopsis plants (Figure 3a). This fluorescence pattern of V2–GFP overlapped that of mitochondria stained by MitoTracker Red (Figure 3a), indicating that V2–GFP is targeted to mitochondria. In protoplasts isolated from mature leaves of this transgenic plant, we detected strong GFP fluorescence in mitochondria. Additionally, we identified weak GFP fluorescence in structures much larger than mitochondria. These structures showed GFP fluorescence that matches chlorophyll autofluorescence (Figure 3b). A similar fluorescence pattern has been described for other dual-targeted proteins (Duchêne et al., 2005; Taira et al., 2004). Furthermore, we carried out an immunoblot analysis of V2–GFP on chloroplasts and mitochondria isolated from the same transgenic Arabidopsis plants expressing V2–GFP. As expected, a V2–GFP band with the measured molecular mass of 58 kDa was detected using an anti-V2 protein antibody in both chloroplasts and mitochondria (Figure 3c). This band was also recognized using an anti-GFP antibody (data not shown).

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Figure 3.  Dual targeting of the V2 protein. (a, b) Root hair cells (a) and protoplasts isolated from mature leaves (b) of the transgenic Arabidopsis plant carrying a construct expressing the V2–GFP fusion protein (pPZP2Ha3(−)V2–GFP). Mitochondria were specifically stained with MitoTracker Red. Chloroplasts were visualized by chlorophyll autofluorescence. Scale bars = 10 μm. (c) Immunoblot analysis of V2–GFP fusion protein isolated from chloroplasts and mitochondria of mature leaves of transgenic Arabidopsis plants expressing V2–GFP. Equal amounts of total chloroplast (Cp) or mitochondria (Mt) extracts isolated from two independent transgenic Arabidopsis plants expressing V2–GFP (line 110, lanes 1 and 4, and line 132, lanes 2 and 5), and a transgenic Arabidopsis plant carrying a construct expressing non-targeted GFP (pPZP2H-lacGFP) (lanes 3 and 6) (1 μg per lane) were loaded onto the gel. The V2–GFP fusion protein was detected using an anti-V2 protein antibody. Purity of the chloroplasts and mitochondria was tested using antibodies raised against organelle-specific proteins: chlorophyll a/b binding protein of light-harvesting complex II (LHCPII) of chloroplasts, and prohibitin 5 (Phb5) of mitochondria. (d) Immunoblot analysis of the V2 protein with isolated rice chloroplasts and mitochondria. Equal amounts of total chloroplast (Cp) or mitochondria (Mt) extracts isolated from wild-type rice plants (1 μg per lane) were loaded onto the gel. The V2 protein was detected using an anti-V2 protein antibody.

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To verify the dual targeting of the V2 proteins, we performed an immunoblot analysis of chloroplasts and mitochondria isolated from wild-type rice plants (Figure 3d). A V2 protein band with an apparent molecular mass of 27.5 kDa was detected in both chloroplasts and mitochondria, indicating that the V2 protein was localized in both organelles. Thus, subcellular localization of V2 protein in rice plants was the same as that of V2–GFP in the transgenic Arabidopsis plants described above, confirming that dual targeting is indeed a general property of the V2 protein.

The V2 gene encodes plastid and mitochondrial guanylate kinase (pt/mtGK)

To confirm that the V2 protein has a GK activity, we assessed the in vitro enzymatic activities of the V2 protein using various (d)NMP substrates (Table 1). The N-terminally truncated wild-type V2 protein (residues 92-285) with a His-tag (t-V2) had the highest substrate specificity for GMP and dGMP. The specific activities for the substrates GMP and dGMP were 26- and 8-fold lower, respectively, compared to those of purified yeast GK (GUK1) fused to His-tag, which was used as a positive control. In contrast, the N-terminally truncated mutant recombinant protein (t-v2), in which the Val162 residue is replaced by Ile, showed a 20- and 28-fold reduction in specific activity for the substrates GMP and dGMP, respectively, compared with the t-V2 protein (Table 1). This indicates the importance of the Val162 residue for enzymatic activity. These results suggest that the V2 gene encodes an enzymatically active GK, and that the v2 mutation has a drastically decreased GK activity.

Table 1.   Guanylate kinase activity of N-terminally truncated wild-type (t-V2) and mutant (t-v2) proteins
ProteinSubstrateSpecific activity (U mg−1)
  1. Yeast GK (GUK1) activity is shown for comparison. One unit of activity is defined as the consumption of 1 μmol of ATP in 1 min. Mean values (± SE) from three independent experiments are shown. ND, not detected.

t-V2GMP8.61 ± 0.39
dGMP5.07 ± 0.35
AMPND
dAMPND
CMPND
dCMPND
dTMPND
UMPND
dUMPND
t-v2GMP0.42 ± 0.04
dGMP0.18 ± 0.01
GUK1GMP226.25 ± 12.14
dGMP44.05 ± 0.43

The specific activity of bacterially produced and purified recombinant V2 protein was low, and it was possible that this was the result of contamination by trace amounts of highly active Escherichia coli GK (350 U mg−1) (Gentry et al., 1993). To exclude this possibility, we investigated the in vivo GK activity of the V2 protein using a complementation test with a haploid yeast strain lacking (GUK1). Loss of GUK1 function is lethal in yeast because it leads to GDP starvation (Konrad, 1992). Therefore, a guk1 null strain allows tests for survival in the presence of either wild-type or mutant V2 protein, respectively. We generated a yeast guk1 null strain, and refer to this haploid strain as guk1::HIS3/GUK1. As expected from previous studies (Konrad, 1992), the guk1::HIS3/GUK1 cells transformed with empty vector failed to grow on galactose medium containing 5-fluoro-orotic acid (5-FOA) (construct 1, Figure 4), whereas cell viability was restored in the guk1::HIS3/GUK1 cells transformed with the GUK1 expression vector (construct 2). Expression of the N-terminally truncated wild-type V2 protein rescued the lethality of the guk1::HIS3/GUK1 cells (construct 3). In contrast, expression of the N-terminally truncated mutant V2 protein resulted in decreased cell viability compared with expression of the N-terminally truncated wild-type V2 protein, although expression of this protein could rescue the lethal phenotype of this strain (construct 4). These data are consistent with those obtained from the in vitro GK activity assay described above. Here, we propose that the V2 gene should be renamed as Oryza sativa plastid and mitochondrial guanylate kinase (OsGKpm) to conform with nomenclature conventions. However, for the remainder of this report, we propose to retain the original designation (V2) to conform with that used in previous studies on this gene/protein.

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Figure 4.  Complementation of the yeast guk1 mutation by the V2 gene or OsGK1. To generate a guk1 null allele, the yeast haploid YPH499 strain was transformed with a URA3 plasmid encoding the GUK1 gene (pRS316GUK1), and the genomic GUK1 locus was then replaced with the HIS3 gene. This haploid strain is referred to as guk1::HIS3/GUK1. 5-FOA is a pyrimidine analog that reacts with orotidine-5′-phosphate decarboxylase (the URA3 gene product) to produce a toxic product, 5-fluorouracil, and provides a strong selection pressure in favor of cells that have lost the URA3 plasmid. Therefore, haploid guk1::HIS3/GUK1 cells fail to grow on medium containing 5-FOA. Next, the guk1::HIS3/GUK1 strain was transformed with constructs expressing GUK1 (construct 2: YEpGAL112GUK1; positive control), the N-terminally truncated wild-type V2 protein (construct 3: YEpGAL112V2-N), the N-terminally truncated mutant V2 protein (construct 4: YEpGAL112v2-N), the full-length OsGK1 (construct 5: YEpGAL112OsGK1) or the N-/C-terminally truncated OsGK1 (construct 6: YEpGAL112OsGK1-N/C) under the control of the galactose-inducible GAL10 promoter. As a negative control, the guk1::HIS3/GUK1 strain was also transformed with empty vector (construct 1: YEpGAL112). Serial dilutions of exponentially growing cell cultures were spotted on an SG plate lacking histidine, uracil and tryptophan (SG-HUW, left) or a 5-FOA-containing SG plate lacking tryptophan (5-FOA (SG-W), right) and the growth on these plates was observed after 4 days at 30°C.

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The v2 mutation does not decrease the copy number of organellar DNA

In humans, it has been reported that deficiency of essential enzymes in the mitochondrial salvage pathway causes a quantitative defect of mitochondrial DNA (mtDNA) (Elpeleg et al., 2002). As the V2 protein has pt/mtGK functions, it is possible that the v2 mutation causes organellar DNA depletion in mutant cells. To investigate this possibility, we carried out Southern analysis of organellar DNA levels.

Chloroplast DNA (cpDNA) was identified using two probes, trnK-rps16 and rpl20-5-rps12 (Figure 5a). mtDNA was identified using two probes, rps13-rps4 and rpl2 (intron) (Figure 5b). In order to quantify organellar DNA levels, we compared the relative intensities of two bands, one representing organellar DNA and the other representing nuclear DNA (25S rRNA gene). cpDNA levels increased approximately 4.5-fold during wild-type and v2 mutant leaf development, and no significant differences in mtDNA levels were detected between wild-type and v2 mutant seedlings at an early stage of leaf development. However, at a later stage, mtDNA levels increased approximately threefold in the v2 mutant, whereas mtDNA levels did not change significantly during wild-type leaf development. These results indicate that the V2 protein is not primarily involved in the synthesis and maintenance of the organellar DNA during leaf development.

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Figure 5.  Southern blot analysis of organellar genomes relative to the nuclear-encoded 25S rRNA gene. (a) Southern blots of chloroplast DNA (cpDNA). (b) Southern blots of mitochondrial DNA (mtDNA). The seedlings were grown at the restrictive temperature (20°C) and had fully emerged 3rd leaves. Total DNA (2 μg) was obtained from all sampling locations on wild-type and v2 seedlings, digested with EcoRI, electrophoresed, blotted, and hybridized with the probes indicated on the left. Sample designations are as described in Figure 1(d). Top, representative blots. Bottom, the intensity of each band quantified using Scion Image. The ratio of organellar DNA to nuclear DNA was calculated for each sample, and normalized against the value for the SB sample from wild-type seedlings. Values are means and SE from three independent experiments.

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Rice has another enzymatically active GK, OsGK1, localized in the cytoplasm

It is reasonable to predict that other GK-related enzymes must function in organellar DNA synthesis and maintenance, as organellar DNA synthesis in rice leaf cells is not dependent on the V2 protein. A BLAST search of rice databases (available at http://signal.salk.edu/cgi-bin/RiceGE) identified another GK gene, designated Oryza sativa guanylate kinase 1 (OsGK1), on chromosome 12 (Figure 6a). The deduced amino acid sequence of OsGK1 exhibited significant sequence similarities to two Arabidopsis guanylate kinase (AtGK) isozymes, AtGK1 and AtGK2 (Figure 2). AtGK1 and AtGK2 were formerly reported as AGK1 and AGK2, respectively (Kumar et al., 2000). Like AtGK1 and AtGK2, OsGK1 has N- and C-terminal extensions surrounding the central GK domain (Figure 2). AtGK1 has been reported to have an active GK domain (Kumar et al., 2000). As expected, both full-length and N-/C-terminally truncated OsGK1 rescued the lethality of the guk1::HIS3/GUK1 cells (constructs 5 and 6, Figure 4), suggesting that OsGK1 has GK activity.

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Figure 6.  Functional analysis of OsGK1. (a) Structure of OsGK1. Boxes and thin lines represent exons and introns, respectively. The OsGK1 open reading frame is depicted by the gray boxes. (b) Subcellular localization of OsGK1. The atgk2 mutant plant was transformed with a construct expressing GFP–OsGK1 fusion protein under the control of the CaMV 35S promoter (pPZP2H-lacGFP-OsGK1). This translational fusion protein complements the atgk2 mutant phenotype. A wild-type Arabidopsis plant (Col-0) was transformed with a construct expressing non-targeted GFP (pPZP2H-lacGFP) and used as a control. GFP fluorescence was visualized in root cells of 5-day-old seedlings expressing GFP–OsGK1 or non-targeted GFP. Note that GFP fluorescence in each of these transgenic seedlings was distributed throughout the cytoplasm. Scale bars = 50 μm. (c) RT-PCR analysis of OsGK1 expression. Total RNA was extracted from wild-type and v2 mutant seedlings grown at the restrictive temperature (20°C) and amplified with gene-specific primers as described in Experimental procedures. The total RNA samples used as template for RT-PCR were run out on a gel and stained with EtBr to allow comparison of the sample concentrations (see bottom panel). Sample designations are as described in Figure 1(d). (d) Phenotype of transgenic rice seedlings with RNAi knockdown of OsGK1. Each line carrying a single construct (pOsGK1RNAi1, pOsGK1RNAi2, pOsGK1RNAi3 or the empty vector) was grown at 20°C (the restrictive temperature for the v2 mutant). Three representative and independent OsGK1 RNAi seedlings for each construct were photographed. Scale bars = 30 mm. (e) RT-PCR analysis of OsGK1 expression in OsGK1 RNAi line seedlings. Total RNA was extracted from the 3rd leaf of OsGK1 RNAi line seedlings grown at 20°C (the restrictive temperature for the v2 mutant) and amplified with gene-specific primers as described in Experimental procedures. Ubiquitin (Ubq) was used as a control. The total RNA samples used as template for RT-PCR were run out on a gel and stained with EtBr to allow comparison of the sample concentrations (see bottom panel).

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No significant sequence conservation, predictable motifs or domains for specific subcellular targeting were detectable within the N- and C-terminal extensions of both AtGK1 and AtGK2 (Kumar et al., 2000). We used the TargetP program (Emanuelsson et al., 2000) to confirm that OsGK1 likewise has no features specific to a transit peptide or an organellar targeting signal (data not shown). To determine the subcellular localization of OsGK1, we generated atgk2 seedlings expressing GFP fused to the N-terminus of full-length OsGK1. Plants homozygous for the atgk2 mutation showed growth retardation (described below in more detail). The translational fusion protein (GFP–OsGK1) was able to rescue this mutant phenotype (data not shown), indicating that GFP–OsGK1 was functional. A confocal microscopy analysis showed that the GFP fluorescence of GFP–OsGK1 was predominantly detected in the cytoplasm, with only a low level in the nucleus (Figure 6b). This localization pattern was the same as that of non-targeted GFP (Figure 6b), indicating that OsGK1 is a cytosolic GK (cGK).

RT-PCR analysis also revealed that OsGK1 transcripts were ubiquitously expressed in all tissues of wild-type seedlings, with higher levels of expression in roots and young leaves (Figure 6c). In the v2 mutant seedlings, the expression profile of OsGK1 transcripts was similar to that in wild-type seedlings, although the levels of OsGK1 transcripts in v2 mutant seedlings were significantly higher during later leaf development compared to wild-type.

Rice plants with reduced expression of OsGK1 are not phenotypically different to normal plants

To gain insight into the physiological roles of OsGK1, we used the RNAi technique to knockdown the expression of endogenous OsGK1. The efficacy and specificity of the RNAi knockdown was evaluated using three constructs: pOsGK1RNAi1, corresponding to the N-terminal extension of OsGK1; pOsGK1RNAi2, corresponding to the GK domain of OsGK1; and pOsGK1RNAi3, corresponding to the C-terminal extension of OsGK1. We generated 16 independent transgenic plants. RT-PCR analysis revealed that none of the OsGK1 RNAi line seedlings generated completely suppressed the expression of OsGK1. However, a weak knockdown effect on OsGK1 mRNA expression was observed (Figure 6e). In addition, there was little variation in the degree of knockdown between the constructs (Figure 6e). At 20°C, the restrictive temperature for the v2 mutant, the OsGK1 RNAi line seedlings showed the same growth rate, with normal green leaves, as control line seedlings transformed with the empty vector (Figure 6d). It has been proposed that complete loss of function of a critical enzyme involved in nucleotide biosynthesis is lethal if the enzyme is encoded by a single-copy gene (Schröder et al., 2005). Analyses of the fully sequenced rice genome (International Rice Genome Sequencing Project, 2005) and of the rice full-length cDNA database (Rice Full-Length cDNA Consortium, 2003) suggested that OsGK1 is encoded by a single-copy gene. Thus it might be possible that complete loss of OsGK1 leads to the death of rice plants. Our results suggest that OsGK1 might play an important role in the de novo and salvage pathways of guanine nucleotide biosynthesis in whole rice cells.

Arabidopsis has two types of enzymatically active GK

To answer the question of whether other plant species possess these two types of active GK, we performed a search of the fully sequenced Arabidopsis genome to identify potential homologs and/or orthologs. We identified three GKs in the Arabidopsis genome: AtGK1 (At2 g41880), AtGK2 (At3 g57550) and a pt/mtGK-like gene designated AtGK3 (At3 g06200), which is highly homologous to the rice V2 gene (Figure 2).

We confirmed that the rice v2 phenotype was complemented by over-expression of AtGK3 cDNA, suggesting that AtGK3 is a functional ortholog of the V2 gene (data not shown). Next, to investigate the physiological roles of AtGK3 in Arabidopsis, we generated two AtGK3 RNAi lines: one line was generated using a construct, pAtGK3RNAi1, corresponding to the GK domain of AtGK3; the second was generated using a construct, pAtGK3RNAi2, corresponding to the organelle-targeting signal sequence of AtGK3. RT-PCR analysis revealed that expression of AtGK3 was strongly suppressed in both AtGK3 RNAi line seedlings, although the pAtGK3RNAi1 construct caused a more severe suppression than did pAtGK3RNAi2 (Figure 7j). The AtGK3 RNAi line seedlings generally exhibited a moderately dwarfed phenotype and developed albino, or more rarely pale-green or variegated (green/white) cotyledons, and pale-green or variegated (pale-green/white) true leaves (Figure 7b,c,f–i, and data not shown). A few seedlings carrying the pAtGK3RNAi1 construct showed an extreme dwarfed phenotype and developed albino cotyledons and true leaves (Figure 7e). Seedlings transformed with pAtGK3RNAi1 had a more severe phenotype than those transformed with pAtGK3RNAi2. Thus, it seems that the severity of phenotypic effect was correlated with the degree of knockdown of AtGK3 by the RNAi constructs. These results suggest that AtGK3 expression is a prerequisite for normal chloroplast development in Arabidopsis.

image

Figure 7.  Molecular analysis of AtGKs. (a–c) Representative 7-day-old Arabidopsis transgenic line seedlings carrying a single construct: the empty vector (a), pAtGK3RNAi1 (b) or pAtGK3RNAi2 (c). Scale bars = 1 mm. (d–i) Representative 3- and 4-week-old Arabidopsis transgenic line seedlings carrying a single construct: the empty vector (d), pAtGK3RNAi1 (e–g) or pAtGK3RNAi2 (h,i). On the basis of phenotypic differences, AtGK3 RNAi line seedlings carrying an RNAi construct, pAtGK3RNAi1, were divided into three groups (AtGK3 RNAi1-a, -b, -c). Similarly, AtGK3 RNAi line seedlings carrying an RNAi construct, pAtGK3RNAi2, were divided into two groups (AtGK3 RNAi2-a, -b). Line AtGK3 RNAi1-a seedlings (e) had a severe dwarf phenotype with albino true leaves. Line AtGK3 RNAi1-b seedlings (f) had a dwarf phenotype with pale-green or variegated (pale-green/white) true leaves. AtGK3 RNAi1-c and RNAi2-a line seedlings (g, h) had pale-green true leaves, while line AtGK3RNAi2-b seedlings (i) had slightly pale-green true leaves. Scale bars = 10 mm (d, f–i) and 1 mm (e). (j) RT-PCR analysis of AtGK expression in AtGK3 RNAi line seedlings. Total RNA was extracted from 3-week-old AtGK3 RNAi line seedlings and amplified with gene-specific primers as described in Experimental procedures. The phenotypic differences between AtGK3 RNAi seedlings as described above are indicated by a, b and c, respectively. EF1α was used as a control. The total RNA samples used as template for RT-PCR were run out on a gel and stained with EtBr to allow comparison of the sample concentrations (see bottom panel). (k) The structures of AtGK1 and AtGK2. Boxes and thin lines represent exons and introns, respectively. The AtGK1 and AtGK2 open reading frames are depicted as gray boxes. The position of the T-DNA insertion in each AtGK is shown. The T-DNA is not drawn to scale. (l) RT-PCR analysis of AtGK expression in atgk1 and atgk2 mutant seedlings. Total RNA was extracted from 3-week-old seedlings of Col-0, atgk1 and atgk2 mutants and amplified with gene-specific primers as described in Experimental procedures. EF1α was used as a control. The total RNA samples used as template for RT-PCR were run out on a gel and stained with EtBr to allow comparison of the sample concentrations (see bottom panel). (m–p) Three-week-old seedlings of Col-0 (m), atgk1 (n), atgk2 (o) and atgk1/AtGK1 atgk2/atgk2 (p). Scale bars = 10 mm. (q, r) Siliques of the wild-type (q) and atgk1/atgk1 atgk2/AtGK2 (r) seedlings. In atgk1/atgk1 atgk2/AtGK2 siliques, aborted ovule-like structures (arrows) are visible, whereas wild-type siliques show full seed set. Scale bars = 1 mm.

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AtGK1 and AtGK2 share 81% identity at the amino acid level (Kumar et al., 2000). The structural and enzymatic similarities between OsGK1 and AtGK1 and AtGK2 described above (Figures 2 and 4) led us to investigate whether these proteins are functionally related. Loss of AtGK2 function results in growth retardation (described below in more detail). This mutant phenotype was complemented by over-expression of the OsGK1 cDNA (data not shown), suggesting that rice OsGK1 is a functional ortholog of AtGK1 and AtGK2.

To investigate the functions of AtGK1 and AtGK2, we obtained T-DNA insertional lines from the SALK Institute and the SAIL collection (SALK_017051 for AtGK1 and SAIL_847_E10 for AtGK2) (Alonso et al., 2003; Sessions et al., 2002). The SALK_017051 line, designated atgk1, has a T-DNA inserted at exon 4 and a 17 bp deletion (Figure 7k). The SAIL_847_E10 line, designated atgk2, has a T-DNA inserted at the boundary of intron 3 and exon 4 and a deletion of 12 bp, possibly compromising correct mRNA splicing (Figure 7k). Segregation analyses showed that both T-DNA insertions behave as single Mendelian loci, suggesting that each mutant has a single T-DNA insertion (data not shown). RT-PCR analysis showed that the atgk1 mutant completely lacked AtGK1 transcripts, indicating that atgk1 is a transcriptional knockout (Figure 7l). Similarly, no AtGK2 transcripts were detected in the atgk2 mutant by RT-PCR using primers spanning the T-DNA insertion (Figure 7l), although reduced levels of AtGK2 transcripts were detected when using primers that amplify the 3′ sequences downstream of the insertion (data not shown). As the T-DNA insertion at the AtGK2 locus eliminated the acceptor splicing site of intron 3, this insertion probably causes a severe reduction in the function of AtGK2. This suggests that the T-DNA insertion in the AtGK2 locus resulted in a strong and presumably null mutation.

The atgk1 mutant did not exhibit a whole-plant phenotype different to that of the wild-type (Figure 7n). In contrast, the atgk2 mutant showed growth retardation, narrow and dark-green leaves with elongated petioles, root growth inhibition and reduced fertility (Figure 7o). These observations suggest that significant functional redundancy exists between the two AtGK isozymes, and that AtGK2 is the predominant isoform. Furthermore, atgk1/AtGK1 atgk2/atgk2 plants showed a more severe phenotype than the atgk2 mutant, suggesting a gene-dosage effect on AtGK1 activity (Figure 7p). No atgk1 atgk2 double mutant was obtained in the F1 populations of self-fertilized atgk1/atgk1 atgk2/AtGK2 plants (Table 2). Visual analysis of the siliques of self-fertilized atgk1/atgk1 atgk2/AtGK2 plants showed that they contained aborted ovule-like structures (arrows on Figure 7r). Similarly, the siliques of self-fertilized atgk2 plants included aborted ovule-like structures and abnormal seeds (data not shown). However, seeds produced from atgk2/AtGK2 plants were indistinguishable from wild-type seeds in size, shape and germination rates, and segregated in a 3:1 ratio (wild-type:mutant) (data not shown), suggesting that a single atgk2 mutation did not affect gametogenesis or embryogenesis. Taken together, our results indicate that the defects in both AtGK1 and AtGK2 functions cause lethality. Notably, segregation analysis on the F1 populations of self-fertilized atgk1/atgk1 atgk2/AtGK2 plants showed that the numbers of atgk1/atgk1 atgk2/AtGK2 plants were lower than expected for a 2:1 (atgk1/atgk1 atgk2/AtGK2:atgk1/atgk1 AtGK2/AtGK2) segregation ratio (Table 2). In addition, the F1 populations obtained from the crosses atgk1/atgk1 atgk2/AtGK2 (♀) × atgk1/atgk1 AtGK2/AtGK2 (♂) and atgk1/atgk1 AtGK2/AtGK2 (♀) × atgk1/atgk1 atgk2/AtGK2 (♂) exhibited a marked deviation from the expected 1:1 ratio (Table 2), suggesting that the disruption of both AtGKs might cause defects in male and female gametogenesis. These results suggest that AtGK1 and AtGK2 are required for normal development of the gametophyte and embryo, and that OsGK1-type GK might actively participate in guanine nucleotide biosynthesis and metabolism, in a similar fashion to cGKs found in animals and micro-organisms.

Table 2.   Genotypes of progeny in various atgk1 atgk2 crosses
F1 progenyGenotypes of adult plants (observed/expected)Totalχ2
AtGK2/AtGK2atgk2/AtGK2atgk2/atgk2
  1. Only the genotype at the AtGK2 locus is shown, as all plants are also atgk1/atgk1. As atgk1 atgk2 double mutants are lethal, the χ2 values for atgk1/atgk1 atgk2/AtGK2:atgk1/atgk1 AtGK2/AtGK2 segregations of 2:1 (a) and 1:1 (b) were calculated using only two genotypic classes (one degree of freedom). The 95% confidence limit for rejecting the expected 2:1 or 1:1 segregation is ≥3.84 and the 99% limit is ≥6.64.

Self-fertilized atgk2/AtGK2144/5056/100 0/50 200134.55a
atgk2/AtGK2 (♀) × AtGK2/AtGK2 (♂)109/58 7/58 −/−11689.69b
AtGK2/AtGK2 (♀) × atgk2/AtGK2 (♂)136/7820/78−/−156 89.28b

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this study, we provide evidence of the physiological significance of a plastid and mitochondrial guanylate kinase (pt/mtGK). In rice and Arabidopsis cells, there are two types of GK, pt/mtGK, which is targeted to both chloroplasts and mitochondria, and cytosolic guanylate kinase (cGK). Disruption of the function of rice or Arabidopsis pt/mtGK (V2 and AtGK3, respectively) results in severe defects in chloroplast differentiation. In contrast, knockout of the function of Arabidopsis cGKs (AtGK1 and AtGK2) causes lethality. Although rice cGK (OsGK1) RNAi seedlings were viable and developed normal green leaves, it is possible that this was due to incomplete suppression of OsGK1 expression. The phenotypic differences resulting from mutations of pt/mtGKs compared to cGKs are caused by differences in the physiological and developmental functions of these GKs in plant cells.

The GK family can be divided into two distinct groups: the low-molecular-weight forms of GK and the high-molecular-weight and membrane-associated GK homologs (MAGUKs). The low-molecular-weight forms are catalytically active and primarily involved in guanine nucleotide metabolism and cell signal transduction pathways. MAGUKs share a common modular structure that consists of either one or three PDZ domains, a single Src homology 3 (SH3) domain, and a single region of similarity to yeast GK (GUK1), known as the GK domain, and are implicated in the assembly of synapses and tight junctions (Funke et al., 2005). However, unlike the low-molecular-weight forms of GK, the GK domain in MAGUKs is catalytically inactive, although nucleotide-binding sites are conserved in some MAGUKs (Kuhlendahl et al., 1998; Olsen and Bredt, 2003).

Based on amino acid sequence comparison of GKs from different species and the criterion of catalytic activity of the GK domain, rice V2 protein and OsGK1 are classified within the group of low-molecular-weight forms of GK (Table 1 and Figures 2 and 4). Interestingly, unlike GKs from other species, OsGK1 carries N- and C-terminal extensions surrounding the central GK domain, suggesting functional conservation but structural divergence through evolution. In a range of species, the low-molecular-weight forms of GK appear to be localized to the cytoplasm. However, V2 protein is localized in both chloroplasts and mitochondria, although OsGK1 is present in the cytoplasm (Figures 3 and 6b). Thus, rice has two types of enzymatically active GK, pt/mtGK encoded by the V2 gene and cGK encoded by OsGK1. Arabidopsis also has two types of enzymatically active GK, the V2-type GK, AtGK3, and the OsGK1-type GKs, AtGK1 and AtGK2 (Figure 7). These results suggest that, in higher plants, two structurally different types of enzymatically active GK might be conserved and play distinct roles in different subcellular compartments of the plant cell.

OsGK1 transcripts were present in all tissues and showed particularly high expression in roots and young leaves, suggesting a ubiquitous function of OsGK1 (Figure 6c). Expression of OsGK1 was not completely suppressed in any of the OsGK1 RNAi line seedlings examined in this study (Figure 6d,e). It has been suggested that the complete disruption of an enzyme essential for nucleotide metabolism could lead to death of the plant if the enzyme is encoded by a single-copy gene (Schröder et al., 2005). This suggestion is supported by the results of phenotypic analyses of several mutants (Xu et al., 2005; Zhang et al., 2002) and sense (co-suppression) and antisense lines (Moffatt et al., 2002; Schröder et al., 2005) for the critical enzymes involved in nucleotide metabolism. Thus, our observation of incomplete inhibition of OsGK1 expression might be due to the fact that rice plants have only a single copy of OsGK1. If OsGK1 were not an essential gene, we would expect to obtain OsGK1 RNAi line seedlings with complete or severe loss of OsGK1 expression. However, in our RNAi experiment using three constructs corresponding to the different regions of OsGK1, OsGK1 RNAi line seedlings with a relatively small reduction in OsGK1 expression were generated (Figure 6e). From the outcome of this experiment, we conclude that OsGK1 is an essential gene that is crucial for guanine nucleotide biosynthesis and metabolism. This interpretation gains some support from our genetic analyses of AtGK1 and AtGK2. Segregation analyses of F1 populations from various atgk1 atgk2 crosses suggested that atgk1 atgk2 double mutant plants were lethal and that both AtGKs were required for gametogenesis and embryogenesis (Table 2 and Figure 7q,r). This observation is consistent with an Affymetrix gene expression profiling study (Schmid et al., 2005), which showed that AtGK2 is more abundantly and widely expressed than AtGK1, and that both AtGKs are highly expressed in mature pollen. Overall, therefore, we conclude that the OsGK1-type GK functions primarily as a central enzyme for guanine nucleotide metabolism in plant cells, just as cGK does in animals and micro-organisms.

In contrast, pt/mtGK appears to play a determinant role at an early stage of chloroplast differentiation and shows no evidence of being functionally redundant. This conclusion is supported by the following observations. First, at the restrictive temperature, the v2 mutant seedlings developed chlorotic, chloroplast-deficient leaves (Figure 1a). Second, in wild-type seedlings, V2 transcripts and proteins accumulated dramatically at an early stage of leaf development, indicating that V2 proteins have specific roles during early chloroplast differentiation (Figure 1e,f). Third, in humans, mutations in several enzymes involved in mtDNA synthesis and maintenance result in mtDNA depletion (Elpeleg et al., 2002). However, we found that the v2 mutation did not decrease the copy number of either cpDNA or mtDNA (Figure 5). It is interesting that the mtDNA copy number in the v2 mutant as well as in the barley albostrians albino mutant, which is phenotypically similar to v2 mutant, is increased approximately threefold at a later stage of leaf development (Hedtke et al., 1999). These observations indicate that pt/mtGK performs additional plant-specific functions.

A previous phenotypic analysis showed that activation of the plastid translation machinery during early chloroplast differentiation was severely suppressed in v2 mutants grown at a restrictive temperature (Sugimoto et al., 2004). This deficiency in the plastid translation machinery results from a defect at the level of initiation of plastid translation or at an early elongation step. Plastid rRNA levels were found to be depleted throughout leaf development in the v2 mutant, except in the L3L sample, which has some green tissues in the basal parts of the fully expanded third leaf (Figure 1e). This indicates that the v2 mutant lacks functional ribosomes in its plastids. Therefore, our findings raise the possibility that pt/mtGK in plastids might function in the synthesis or assembly of one or more component of the plastid translation machinery, such as plastid ribosomes or the translation initiation complex. Several studies provide indirect support for this interpretation. For example, similarly to the expression of the V2 gene, the induction of expression of nuclear transcripts encoding the plastid translation machinery precedes that of plastid photosynthetic genes during early chloroplast and leaf development (Harrak et al., 1995). Moreover, the temperature-dependent leaf chlorophyll content observed in the v2 mutant is consistent with that of plants with mutations that directly or indirectly affect plastid ribosome integrity and translational activity (Barkan, 1993; Schultes et al., 2000; Tokuhisa et al., 1998). Some TRAFAC GTPases of the P-loop GTPase super-class are involved in the biogenesis of ribosomes and the function of the translation apparatus (Leipe et al., 2002). Although we have no direct evidence, it is possible that the GDP produced by pt/mtGK is phosphorylated to GTP. In turn, hydrolysis of this GTP by TRAFAC GTPases might be required for activation of the plastid translation capacity. As the v2 mutation drastically decreases the GK activity of pt/mtGK (Table 1 and Figure 4), it is possible that the plastids of the v2 mutant have a shortage of the GTP necessary for the assembly and function of the plastid translation machinery. This would result in a deficiency of plastid ribosomes or the translation initiation complex in mutant plastids. This speculation is supported by the fact that the phenotype of the Arabidopsis mutant deficient in chloroplast elongation factor G, which is a TRAFAC GTPase, resembles that of Arabidopsis AtGK3 RNAi lines (Figure 7) (Albrecht et al., 2006). An alternative explanation is that pt/mtGK might catalyze the phosphorylation of unidentified molecules or proteins that are required for construction of the plastid translation system.

One unanswered question relates to the physiological role of pt/mtGK in plant mitochondria. It has been reported that simultaneous defects in plastid and mitochondria translation results in strong suppression of expression of nuclear photosynthetic genes; this defect is not seen if the defect is limited to one or other organelle (Pesaresi et al., 2006). In microarray experiments, we previously observed that the v2 mutation inhibits expression of many nuclear photosynthetic genes (Sugimoto et al., 2004). As the v2 mutation disrupts plastid translation capacity, it is reasonable to propose that pt/mtGK in mitochondria might be involved in activation of the mitochondrial translation system.

Dual targeting of the V2 protein provides direct evidence for the importance of coordination of the biogenesis of plastids and mitochondria during early leaf development. It is interesting that many nuclear proteins targeted to chloroplasts and mitochondria are involved in these organellar genetic systems (Duchêne et al., 2005; Silva-Filho, 2003). Further analyses are necessary to elucidate the physiological relationship between plastids and mitochondria during the development of photosynthetic competence, and the function of pt/mtGK in this process.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant materials and growth conditions

The virescent 2 (v2) mutant line of Oryza sativa, KL515, was obtained from the Institute of Genetic Resources, Kyushu University, Japan. KL515 is a spontaneous mutant of the Japanese paddy rice variety ‘Yaeho’. As a japonica rice variety ‘Taichung 65’ was used as the wild-type reference strain, KL515 was first back-crossed five times to the ‘Taichung 65’ line to remove the ‘Yaeho’ background before use in this study. Rice seedlings were grown on either plant nutrient medium (Yoshida et al., 1976) solidified with 0.6% w/v agar or on soil until full expansion of the 3rd leaf. The seedlings were maintained under contrast cool-white fluorescent light (1500 lux) at 65% humidity and at 20°C.

The Arabidopsis thaliana Columbia (Col-0) ecotype was used as the wild-type. The atgk1 T-DNA insertional line was obtained from the SALK collection of T-DNA insertions in Arabidopsis (SALK_017051) (Alonso et al., 2003). The atgk2 T-DNA insertional line was obtained from the Syngenta Arabidopsis Insertion Library (SAIL) (SAIL_847_E10) (Sessions et al., 2002). A. thaliana seedlings were grown on half-strength Murashige and Skoog (MS) medium containing 1% w/v sucrose under continuous light (230 μmol m−2 sec−1) at 22°C.

High-resolution mapping of the V2 locus

For genetic linkage mapping of the V2 locus, all RFLP markers, BAC and YAC end-fragment sequences were converted into CAPS markers as described previously (Michaels and Amasino, 1998). To identify genomic contigs containing V2 locus, we screened BAC and PAC libraries provided by the Clemson University Genomic Institute and the Rice Genome Research Program of the National Institute of Agrobiological Sciences (Tsukuba, Japan).

Cloning of the full-length V2 cDNA

RNA extraction was carried out as described previously (Sugimoto et al., 2004). To isolate a full-length V2 cDNA, first-strand cDNA synthesis and RACE PCR were performed using the BD SMART™ RACE cDNA amplification kit (Clontech, http://www.clontech.com/).

Semi-quantitative RT-PCR analysis

cDNAs corresponding to 63 ng of total RNA were used in each RT-PCR amplificaton. The following primers were used for RT-PCR: V2, 5′-GAGGAGTTCCTCACGATGAT-3′ and 5′-CAGCATCAATGATAGACTCC-3′; OsGK1, 5′-GCCCTGATCACGGCGCATTTTATGGG-3′ and 5′-TGGAGCTCTTGTGGTGTGGCTAACAG-3′. PCR conditions were as follows: 19 cycles for V2 and 17 cycles for OsGK1; annealing temperature of 50°C for V2 and 60°C for OsGK1. Southern hybridization was performed using 32P-labelled V2 and OsGK1 cDNA fragments.

Immunoblot analysis

Frozen tissue in liquid nitrogen was ground with a pestle to a fine powder, thawed in extraction buffer (50 mm Tris/HCl, pH 8.0, 7 mm EDTA, pH 8.0, 0.1% Triton X-100, 3 μg ml−1 leupeptin, 3 μg ml−1 antipain, 1 mm PMSF, 5 μg ml−1 aprotinin and 1 mm DTT), filtered through two layers of Miracloth (Calbiochem; http://www.emdbiosciences.com), and centrifuged at 8000 g for 10 min. The supernatant was used as the total soluble protein.

Chloroplasts and mitochondria were isolated from rice and Arabidopsis plants as described previously (Kusumi et al., 2004).

Immunoblot analysis was carried out essentially as described previously (Kusumi et al., 2004). A rabbit antiserum was raised against the N-terminally truncated V2 protein (92-285 residues). Immunodetection was performed with the ECL plus Western blotting detection system (Amersham Biosciences, http://www5.amershambiosciences.com/). The antiserum to Phb5 (Kato et al., 2007) was kindly provided by Wataru Sakamoto (Okayama University).

Complementation test

For pPro35S:V2, a V2 cDNA fragment with a BglII site at the 5′ end and a SalI site at the 3′ end was inserted into the BamHI/SalI sites of pPZP2Ha3(+) (Fuse et al., 2001). This plasmid was introduced into Agrobacterium tumefaciens (strain EHA101). Rice transformation was performed as described previously (Yara et al., 2001).

Fluorescence microscopy

For pPZP2Ha3(−)V2-GFP, a modified gfp ORF fragment with a glycine linker obtained by PCR using primers 5′-GGTCGCGATGGAGGTGGAGGTGCTATGGTGAGCAAGGGC-3′ and 5′-CGGGCGGCCGCTTTACTTGTA-3′ (the glycine linker site is underlined) was inserted into pGEM-T Easy vector (Promega, http://www.promega.com/) to produce pG-GFP1. A DNA fragment containing the V2 ORF, obtained by PCR using primers 5′-AGTCGACCAAACAAATCACTCCAATCCC-3′ and 5′-GAGCATTGAGCTCATATTAACCGTA-3′, was inserted into the pGEM-T Easy vector to produce pG-V2-C. The AatII–Ecl136II fragment of pG-V2-C was inserted into the AatII/NruI sites of pG-GFP1 to produce pG-V2-GFP. pG-V2-GFP was digested with NotI, filled in using the Klenow fragment, and digested with SalI. This fragment containing the V2–GFP translation fusion was inserted into the Ecl136II/XhoI sites of pPZP2Ha3(−) (Fuse et al., 2001). For pPZP2H-lacGFP-OsGK1, a modified gfp ORF fragment with a glycine linker obtained by PCR using primers 5′- ACCATGGTGAGCAAGGGCGA-3′ and 5′- ACATATGAGCACCTCCACCTCCCTTATACAGCTCGTC-3′ (the glycine linker site is underlined) was inserted into the pGEM-T Easy vector to produce pG-GFP2. A DNA fragment containing the OsGK1 ORF, obtained by PCR using primers 5′-TCATATGGGTGAAGAGGCTCCGGA-3′ and 5′-ACCAACGCGTTGGTGTACAAGGACTACATGAATTCTTTT-3′, was inserted into the pGEM-T Easy vector to produce pG-OsGK1-C. The NdeI–BstXI fragment of pG-OsGK1-C was inserted into the NdeI/BstXI sites of pG-GFP2 to produce pG-GFP-OsGK1. A HindIII–EcoRI fragment of pGFP(S65T) (Chiu et al., 1996) containing the CaMV 35S promoter and gfp cDNA, was inserted into the HindIII/EcoRI sites of pBluescript II KS+ (Stratagene, http://www.stratagene.com/) to produce pKS(+)GFP. The NcoI–BsrGI fragment of pG-GFP-OsGK1 was inserted into the NcoI/BsrGI sites of pKS(+)GFP to produce pKS(+)GFP-OsGK1. The ApaI–SmaI fragment of pKS(+)GFP-OsGK1 was inserted into the ApaI/SmaI sites of pPZP2H-lac (Fuse et al., 2001). For pPZP2H-lacGFP, pGFP(S65T) was digested with EcoRI, filled in using the Klenow fragment, and digested with HindIII. This fragment, containing the gfp ORF, was inserted into the HindIII/Ecl136II sites of pPZP2H-lac.

The plasmids were introduced into A. tumefaciens (strain C58). Arabidopsis transformation was performed by the floral dip method (Clough and Bent, 1998).

Root tissues and leaf protoplasts of the transgenic Arabidopsis plants were observed using a confocal laser scanning microscope (LSM510, Carl Zeiss, http://www.zeiss.com/). Preparation of protoplasts was performed as described previously (Abel and Theologis, 1994). Mitochondrial staining was performed using MitoTracker Red (Molecular Probes; http://www.probes.invitrogen.com). GFP fluorescence was stimulated using an excitation wavelength of 488 nm and detected between 500 and 530 nm using an emission band pass filter. Chlorophyll autofluorescence and MitoTracker Red fluorescence were detected using an excitation wavelength of 543 nm and an emission long-pass filter at 560 nm.

Purification of V2 protein and guanylate kinase activity assay

To construct plasmids for recombinant N-terminally truncated wild-type and mutant V2 proteins with a six-histidine tag at the N-terminus (t-V2 and t-v2, respectively), DNA fragments containing the N-terminally truncated wild-type and mutant V2 ORF, obtained by PCR using primers 5′-CAGCATATGATCCTCGTCATC-3′ and 5′-AGAGCTCATGGAAAAGGAAATATCCGTAGC-3′, were digested with the enzymes NdeI and SacI, and inserted into the NdeI/SacI sites of the pET28-a vector (Novagen; http://www.merckbiosciences.com). To construct a plasmid for recombinant GUK1 with a six-histidine tag at the N-terminus, a DNA fragment containing the GUK1 ORF, obtained by PCR using primers 5′-AGCTAGCATGTCCCGTCCTATCGTAAT-3′ and 5′-TCTCGAGAGAGAAAGGAGAGCCTCGC-3′, was digested with the enzymes NheI and XhoI, and inserted into the NheI/XhoI sites of the pET28-a. These constructs were introduced into E. coli BL21(DE3) (Novagen). Recombinant fusion proteins were purified using a HisTrap HP kit (Amersham Biosciences). Protein concentration was determined using the BCA™ protein assay kit (Pierce; http://www.piercenet.com).

Guanylate kinase activities were measured at 30°C using spectrophotometric assays in 1 ml of reaction medium containing 100 mm Tris/HCl, pH 7.5, 100 mm KCl, 10 mm MgCl2, 2 mm ATP, 2 mm nucleoside monophosphate, 0.25 mm NADH, 0.5 mm phosphoenolpyruvate, 5 units of lactate dehydrogenase and 4 units of pyruvate kinase (Agarwal et al., 1978; Kumar et al., 2000).

Yeast strain, media and genetic methods

The haploid Saccharomyces cerevisiae strain YPH499 (MATa, ura3-52, lys2-801anber, ade2-101ochre, trp1-Δ63, his3-Δ200, leu2-Δ1) was used in this study. For pRS316GUK1, a 2.58 kb S. cerevisiae genomic fragment containing the GUK1 ORF, obtained by PCR using primers 5′-ACTCGAGGAATATAATGCCCGGGGGAC-3′ and 5′-TGGATCCTCCCTTCGGGTCGATTATGA-3′ was digested with BamHI and XhoI, and inserted into the BamHI/XhoI sites of pRS316. For pSKGUK1HIS3, the BamHI-digested HIS3 gene was inserted into the BamHI site of pBluescript II SK+ (Stratagene) to produce pSKHIS3. The 3′-end fragment of GUK1, obtained by PCR using primers 5′-AGGATCCACAAGGAATTGAAGGATTTTAT-3′ and 5′-TCTCGAGAGAGAAAGGAGAGCCTCGC-3′, was inserted into the pGEM-T Easy vector to produce pG-GUK1C. The 5′-end fragment of GUK1, obtained by PCR using primers 5′-AGAGCTCTGATTGTCAAAACTGCGACG-3′ and 5′-TACTAGTGGTTCTAGTAGTGGATGAAAC-3′, was inserted into the pGEM-T Easy vector to produce pG-GUK1 N. The EcoRI–XhoI fragment of pG-GUK1C was inserted into the EcoRI/SalI sites of pSKHIS3 to produce pSKHIS3GUK1C. The NotI–SacI fragment of pG-GUK1 N was inserted into the NotI/SacI sites of pSKHIS3GUK1C. To generate a guk1 null allele, the ApaI–SacI fragment of pSKGUK1HIS3 containing the disrupted GUK1 gene was introduced into YPH499 possessing pRS316GUK1, resulting in the haploid guk1::HIS3/GUK1 strain. For YEpGAL112GUK1, a DNA fragment containing the GUK1 ORF, obtained by PCR using primers 5′-ACCATGGCCCGTCCTATCGTAAT-3′ and 5′- AGAGCTCAGATCTTGAGGATGACCAACC-3′, was digested with NcoI and SacI, and inserted into the NcoI/SacI sites of YEpGAL112 (Saitoh et al., 2005). For YEpGAL112 V2-N and YEpGAL112v2-N, DNA fragments containing the N-terminally truncated wild-type and mutant V2 cDNAs, obtained by PCR using primers 5′-CAGCATATGATCCTCGTCATC-3′ and 5′-AGAGCTCATGGAAAAGGAAATATCCGTAGC-3′, were digested with NdeI and SacI, and inserted into the NdeI/SacI sites of YEpGAL112. For YEpGAL112OsGK1, a DNA fragment containing the OsGK1 ORF, obtained by PCR using primers 5′-TCATATGGGTGAAGAGGCTCCGGA-3′ and 5′-TGCGGCCGCAGGACTACATGAATTCTTTTAC-3′, was digested with NdeI and NotI, and inserted into the NdeI/NotI sites of YEpGAL112. For YEpGAL112OsGK1-N/C, an N- and C-terminally truncated fragment of the OsGK1 ORF, obtained by PCR using primers 5′-ACCATGGAAAAGCCAATTGTGATTAGCG-3′ and 5′-ATCAGCAACAATGTTTTGAGTTATCTAAGAATCATTTGAATCTTCG-3′, was inserted into the pGEM-T Easy vector to produce pG-OsGK1-N/C. The EcoRI–NcoI fragment of pG-OsGK1-N/C was inserted into the EcoRI/NcoI sites of YEpGAL112.

Yeast cells were grown and maintained in minimal synthetic medium containing either glucose (SD) or galactose (SG), or in complete medium containing either glucose or galactose. Strains carrying the URA3 plasmid were selected by culturing on solid synthetic media containing 1 mg ml−1 5-FOA.

Southern blot analysis and organellar DNA analysis

Southern blots were performed with the ECL™ Direct Nucleic Acid Labeling System (Amersham Biosciences). cpDNA was detected using two probes corresponding to subcloned sequences between the trnK and rps16 genes (trnKrps16) and between the rpl20 and 5-rps12 genes (rpl205-rps12) of the chloroplast genome. These two probes are respectively localized on 2.38 and 2.91 kb EcoRI fragments that map on the wild-type rice chloroplast genome (Shimada and Sugiura, 1991). mtDNA was detected using two probes corresponding to a subcloned sequence between the rps13 and rps4 genes (rps13rps4) and a cloned fragment of the intron of the rpl2 gene (rpl2 (intron)) of the mitochondrial genome. In the wild-type rice mitochondrial genome, these two probes are respectively localized on 1.81 and 3.05 kb EcoRI fragments that map on the master genome (Notsu et al., 2002). The probes used for Southern blots were obtained by PCR. The following primers were used for PCR: Os25SrRNA, 5′-GGCCGACCCTGATCTTCTGT-3′ and 5′-CACTTGGAGCTCCCGATTCC-3′; trnKrps16, 5′-CGCACACCCCTGTCAAAATC-3′ and 5′-GACTGGATTTATGTCGTGCC-3′; rpl20–5′-rps12, 5′-GGCCCTCTCTATACACCGGA-3′ and 5′-GAAGTATCCATGATCGGTACC-3′; rps13rps4, 5′-TAATAGATCGAGTCAGAATAAATC-3′ and 5′-AGCTTCTGATAACGAGCGGG-3′; rpl2 (intron), 5′-GGCCAAGCATTCTGCCAGAC-3′ and 5′-TCTTATAAGCCCTTGACTATGC-3′.

The intensity of the bands was quantified using Scion Image (Scion Corporation; http://www.scioncorp.com).

Generation and analysis of rice OsGK1 RNAi knockdown lines

Three DNA fragments of OsGK1 cDNA were obtained by PCR using the following primers: OsGK1RNAi1, 5′-CACCATGGGTGAAGAGGCTCCGGA-3′ and 5′-TCCCTTGCTCCATGCAACAA-3′; OsGK1RNAi2, 5′-CACCGGGACATTAATTGCAAAATTAATG-3′ and 5′-TGCTTCAAGAGAAGAAGCCC-3′; OsGK1RNAi3, 5′-CACCTCCAATTCACCAGGTCTTTT-3′ and 5′-GTTAACCGGGGATATCTTGA-3′. The resulting PCR fragments are referred to as OsGK1RNAi1, OsGK1RNAi2 and OsGK1RNAi3, respectively. They were inserted into pANDA according to a published protocol (Miki and Shimamoto, 2004) to produce pOsGK1RNAi1, pOsGK1RNAi2 and pOsGK1RNAi3, respectively. Rice transformation was carried out as described above.

For RT-PCR analysis, total RNA was extracted using Trizol (Invitrogen, http://www.invitrogen.com/). The primers used for PCR amplification of OsGK1 and V2 were the same as described above. The ubiquitin (Ubq) gene was used as an internal control for RT-PCR. The primers used for Ubq were 5′-CCAGGACAAGATGATCTGCC-3′ and 5′-AAGAAGCTGAAGCATCCAGC-3′. PCR conditions were as follows: 29 cycles for OsGK1, 27 cycles for V2, and 25 cycles for Ubq. The annealing temperatures were 60°C for OsGK1, 50°C for V2, and 54°C for Ubq.

Identification and analysis of atgk1 and atgk2 mutants

Mutant lines were genotyped using a PCR-based approach. Wild-type AtGKs were amplified using the following primers: AtGK1, 5′-GGAGAAGCTCCAGCAGTATT-3′ and 5′-CTCGAGGAACCAGATGGAGT-3′; AtGK2, 5′-GTGTCTTCTTGGAGGCTTAG-3′ and 5′-TCCACACCGATGGAACAAAC-3′. T-DNA products were amplified using gene-specific primers in combination with either SALK LBa1 (5′-TGGTTCACGTAGTGGGCCATCG-3′) or SAIL LB3 (5′-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3′).

For RT-PCR analysis, total RNA was extracted using Trizol. EF1α was used as an internal control for RT-PCR. The following primers were used for RT-PCR: AtGK1, 5′-TGACAAAATCAGTAACAACTGGTTT-3′ and 5′-CCCCAAGAGATTCTTGAGCT-3′; AtGK2, 5′-GTGTCTTCTTGGAGGCTTAG-3′ and 5′-TCTCTGTTCCTCGGGCACGA-3′; AtGK3, 5′-GCGGAGAGTGAGCTTGCTAT-3′ and 5′-TTCTCTGTAGTCAGGGGCCA-3′; EF1α, 5′-ATGCCCCAGGACATCGTGATTTCA-3′ and 5′-TTGGCGGCACCCTTAGCTGGATCA-3′. PCR conditions were as follows: 25 cycles for AtGK1, 25 cycles for AtGK2, 25 cycles for AtGK3, and 18 cycles for EF1α. The annealing temperatures were 55°C for AtGK1, 57°C for AtGK2, 56°C for AtGK3, and 58°C for EF1α.

Generation and analysis of Arabidopsis AtGK3 RNAi knockdown lines

For pAtGK3RNAi1, two AtGK3 cDNA fragments obtained by PCR using two sets of primers (5′-GATTCCAAAGAAGCAGATCTAGG-3′ and 5′-TTAACCGCGTCGACAAGCCT-3′; 5′-GATTCCAAAGACTCGAGTTCAGG-3′ and 5′-TTAACCTCTAGATCAAGCCTCC-3′) were inserted into the pGEM-T Easy vector to produce pG-AtGK3 N1 and pG-AtGK3C1, respectively. A GUS cDNA fragment, obtained by PCR using primers 5′-ACTCGAGTCGCGTCGGCATC-3′ and 5′-CACAGATCTTCATGCCAGTCC-3′, was inserted into the pGEM-T Easy vector to produce pG-GUS. The BglII–SalI fragment of pG-AtGK3 N1 was inserted into the BglII/SalI sites of pG-GUS to produce pG-GUSAtGK3 N1. The XhoI–NotI fragment of pG-AtGK3C1 was inserted into the XhoI/NotI sites of pG-GUSAtGK3 N1 to produce pG-AtGK3RNAi1. The XbaI–SalI fragment of pG-AtGK3RNAi1 was inserted into the XbaI/SalI sites of pPZP2Ha3(−). For pAtGK3RNAi2, two AtGK3 cDNA fragments obtained by PCR using two sets of primers (5′-AAGATCTATGATTCGCAAGCTCTGTTC-3′ and 5′-AGTCGACTGACGATCTGATTCGGCGG-3′; 5′-ACTCGAGATGATTCGCAAGCTCTGTTC-3′ and 5′-ATCTAGATGACGATCTGATTCGGCGG-3′) were inserted into the pGEM-T Easy vector to produce pG-AtGK3 N2 and pG-AtGK3C2, respectively. The BglII–SalI fragment of pG-AtGK3 N2 was inserted into the BglII/SalI sites of pG-GUS to produce pG-GUSAtGK3 N2. The XhoI–NotI fragment of pG-AtGK3C2 was inserted into the XhoI/NotI sites of pG-GUSAtGK3 N2 to produce pG-AtGK3RNAi2. The XbaI–SalI fragment of pG-AtGK3RNAi2 was inserted into the XbaI/SalI sites of pPZP2Ha3(+).

RT-PCR analysis was the same as described above. The primers used for AtGK2 were 5′-CAAAGTCACTAACAAGTGGTCC-3′ and 5′-TCTCTGTTCCTCGGGCACGA-3′. Twenty-five cycles of amplification were used for AtGK2. The annealing temperature was 57°C for AtGK2. The PCR conditions used for AtGK1, AtGK3 and EF1α were as described above.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Yoh-hei Saitoh and Takeharu Nishimoto (Kyushu University) for providing vector YEpGAL112 and yeast strain YPH499, Daisuke Miki and Ko Shimamoto (Nara Institute of Science and Technology) for providing vector pANDA, Wataru Sakamoto (Okayama University) for providing the antiserum to Phb5, the Clemson University Genomic Institute for providing filters of BAC library and BAC clones, the Rice Genome Research Program of the National Institute of Agrobiological Sciences for providing restriction fragment length polymorphism probes, BAC and PAC clones, and vectors (pPZP2Ha3(+), pPZP2Ha3(−) and pPZP2H-lac), and for full sequencing of PAC clone P470B02, the Arabidopsis Biological Resource Center for providing the SALK_017051 line, Syngenta Biotechnology for providing the SAIL_847_E10 line, and the AtGenExpress project for availability of the expression atlas of normal Arabidopsis development. This work was supported by CREST, Japan Science and Technology Agency and grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Projects MP-1112 and IP-5005), the Japan Society for the Promotion of Science (13017215, 17370019 and 18657018) and the Ministry of Education, Science and Culture of Japan (17051024).

References

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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