Os03g0320900

• The rice gene Os03g0320900 was reported as v2 in 2007.

Annotated Information

Function

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

• Rice has another enzymatically active GK, OsGK1, localized in the cytoplasm, although V2 protein is localized in both chloroplasts and mitochondria. Thus, rice has two types of enzymatically active GK, pt/mtGK encoded by the V2 gene and cGK encoded by OsGK1.

• pt/mtGK appears to play a determinant role at an early stage of chloroplast differentiation and shows no evidence of being functionally redundant.

• The 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.

• The v2 mutation did not decrease the copy number of either cpDNA or mtDNA.

Mutation

• To examine stage-specific expression of the V2 gene during chloroplast and leaf development, the researchers 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. 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 1). In contrast, V2 transcripts were more abundant at later stages of leaf development in the v2 mutant.

Figure 1. 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℃) 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). .

• The researchers next examined the levels of V2 protein during leaf development by immunoblot analysis. The amounts of V2 protein in wild-type and v2 mutant seedlings paralleled those of V2 transcripts during chloroplast and leaf development (Figure 1,2). 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.

Figure 2. Immunoblot analysis of the V2 protein. Total soluble protein was extracted from wild-type and v2 mutant seedlings grown at the restrictive temperature (20℃). Equal amounts of total soluble protein (40 lg 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. .

• 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.

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). .

Expression Pattern

• In the wild-type seedlings, V2 transcripts are much more abundant at an early stage of leaf development than at the later stages.

Subcellular localization

• 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 3). 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 4a). This fluorescence pattern of V2–GFP overlapped that of mitochondria stained by MitoTracker Red (Figure 4a), indicating that V2–GFP is targeted to mitochondria. In protoplasts isolated from mature leaves of this transgenic plant, the researchers detected strong GFP fluorescence in mitochondria. Additionally, the researchers identified weak GFP fluorescence in structures much larger than mitochondria. These structures showed GFP fluorescence that matches chlorophyll autofluorescence (Figure 4b). Furthermore, the researchers 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 4c). This band was also recognized using an anti-GFP antibody.

Figure 3. Multiple sequence alignments of the V2 protein with the most closely related GKs. .

Figure 4. Dual targeting of the V2 protein. .

Labs working on this gene

• Department of Biological Sciences, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan,
• Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan,
• National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan, and
• Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan