Os03g0427300

Glup4 gene encodes small GTPase, Rab5a in rice.

Annotated Information

Function

• From this and other studies, it is apparent that the small GTPase Rab5 plays a role in both storage protein mRNA and protein sorting.

• The results of the microarray and 2D-DIGE analyses of glup4 may be a reflection of Rab5’s role in RNA and protein sorting. Over 50 genes were identified whose transcript or protein expression was affected in the glup4 mutant by at least 2-fold (Table 1–Table 4).

• The identification of a number of genes having roles in carbon metabolism, and in particular starch, as being differentially expressed in glup4 supports the idea that the syntheses of nitrogen and carbon storage reserves in seeds are interrelated processes.

Mutation

• Using RT-PCR in the presence of dUTPOregon Green to label RNAs and rhodamine B hexyl ester to stain prolamine protein bodies, glutelin RNAs are found to be preferentially transported to the cisternal-ER (Cis-ER) in wildtype endosperm (Fig. 1). Prolamine RNAs, on the other hand, are targeted to ER that delimits the spherical prolamine protein bodies (PB-ER). In the glup4 mutant, however, glutelin RNAs are partially misdirected from the Cis-ER to the PB-ER (Fig. 1), as well as to large dilated multivesicular body-like structures located near the plasma membrane (data not shown). The glutelin RNA mistargeting was not only observed in one gene (Gt-2), but also in others (GluA-1, GluA- 3 and GluB-1, data not shown). Prolamine RNA localization to the PB-ER is unaffected (Fig. 1), suggesting that Rab5 is involved in glutelin RNA sorting only.

Fig. 1. Localization of prolamine and glutelin mRNAs in wildtype and glup4 developing rice endosperm. .

• Microarray analysis of transcripts isolated from glup4 and wildtype developing seeds identified 35 transcripts that were upregulated and 10 that were downregulated by at least 2-fold in the glup4 mutant using a stringent P value (<0.001) to avoid false positives (Table 1 and Table 2).

Table 1. Transcripts upregulated in the glup4 mutant compared to wildtype (P < 0.001) .

Table 2. Transcripts downregulated in the glup4 mutant compared to wildtype (P < 0.001) .

• The researchers utilized 2D-DIGE to analyze the protein expression profiles of soluble extracts from 12–14 DAF wildtype and glup4 rice seeds (Fig. 3). Twenty-one protein spots were found to be differentially expressed (P < 0.05) with at least a 2-fold change in expression when analyzed on pH 3–10 non-linear immobilized pH gradient strips. Only two proteins were found to have at least a 2-fold increase in expression in the glup4 mutant (Table 3). One of these proteins, annotated as a glycosyl hydrolase, has similarity to xylanase inhibitor (XI) proteins, which function in cell wall restructuring. Although not the same protein, xylanase inhibitor TAXI-VI mRNA was also found to be upregulated in the mutant (Table 1).

Fig. 3. Two-dimensional difference in gel electrophoresis (2D-DIGE) analysis comparing the soluble protein fraction from glup4 and wildtype developing rice seeds using pH 3-10 nonlinear immobilized pH gradient strips. .

Table 3. Proteins upregulated in the glup4 mutant compared to wildtype (P < 0.05) .

• A number of proteins involved in other aspects of carbon metabolism were found to be downregulated in glup4,

including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate, orthophosphate dikinase (PPDK, Table 4).

Table 4. Proteins downregulated in the glup4 mutant compared to wildtype (P < 0.05) .

Labs working on this gene

• Institute of Biological Chemistry, Washington State University, Pullman WA, 99164-6340 USA
• Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan
• School of Molecular Biosciences, Washington State University, Pullman WA, 99164-7520 USA
• Present Address: International Liberal Arts Program, Akita International University, Akita 010-1292, Japan
• Present Address: Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan