Os03g0320900

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

Annotated Information

Expression Pattern

• 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. .

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 2). 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, 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 3b). 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 3c). This band was also recognized using an anti-GFP antibody.

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

References

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