Os03g52760

The rice gene Os03g52760 was reported as OsMIK in 2008.

Annotated Information

Function

• MIK encodes a myo-inositol kinase. MIK phosphorylates myo-inositol to form myo-inositol monophosphate and is believed to contribute to the lipid-independent biosynthesis of phytic acid in plants.

• MIK activity has been reported to be active in the aleurone layers of rice seed, which is the predominant site of phytic acid biosynthesis and accumulation. In general, myo-inositol serves as a precursor of several metabolic pathways including cell wall biogenesis through UDP-glucuronate by myo-inositol oxidation, auxin storage and transport though auxin-inositol conjugation, phosphatidylinositol signaling pathway through inositol phospholipd and synthesis of phytic acid through sequential phosphorylation. MIK could determine the fate of myo-inositol especially in seed.

• OsMIK is necessary for wild type levels of seed phytic acid, but not essential for viability.

Mutation

• In order to conWrm that these two mutants are lpa mutants, the seed total P, phytic acid-P (PA-P) and inorganic P (Pi) contents were examined (Table 1).

Table 1 Comparison of seed myo-inositol, phytic acid and inorganic P contents of lpa mutants and wild-types. .

• Seed extracts of lpa mutants were subjected to HPLC analysis to determine if the other inositol phosphates were aVected by the mutation. Comparison to wild-type Nipponbare and inositol phosphate standards revealed that N15- 186 seeds are signiWcantly reduced in PA and inositol monophosphate while exhibiting an increase in Pi (Fig. 1). No signiWcant accumulation of other inositol phosphate intermediates (InsP2 to InsP5) was detected under the conditions used. The HPLC elution proWle of N15-375 showed an increase in Pi and a decrease in PA, but no other signiWcant changes were observed (Fig. 1).

Fig. 1 HPLC chromatograms of inositol phosphates. a Inositol phosphate standards, retention times for the various inositol phosphates were: Ins(2)P1, 9.5 min; Pi, 15.5 min, Ins(1,4)P2, 21 min; Ins(1,3,4)P3, 32 min; Ins(13,4,5)P4, 33 min; Ins(1,3,4,5,6)P5, 35 min; and Ins(1,2,3,4,5,6)P6, 29 min. b Nipponbare wild-type, c N15-186 lpa mutant, d N15-375 lpa mutant .

• The GC–MS analysis revealed that the myo-inositol content, as well as those of other sugars (glucose, fructose and galactose), was increased in the N15-186 mutant compared to the wild-type (Fig. 2). The myo-inositol content of N15-186 seed was ninefold greater than wild-type, while the other sugars examined were 2–3 fold higher. In contrast, the myo-inositol content of N15-375 mutant seeds did not diVer from wild-type.

Fig. 2 GC–MS analysis of myoinositol and other carbohydrates as alditol hexaacetate derivatives from seed extracts of wild-type, N-186 and N-375 mutants. .

Expression

• RT-PCR analysis of total RNA from various tissues including shoot, root, and panicle indicated that OsMIK is expressed in all tissues (Fig. 6). Higher expression was detected in the vegetative shoot tissue compared to the reproductive tissue with relatively lower expression in the roots. A search of publicly available rice EST data indicated that OsMIK is expressed in callus, leaf, root, shoot and panicle tissues (http://www.tigr.org/).

Fig. 6 Expression analysis of OsMIK in diVerent tissues of wild-type plant. RT-PCR analysis was performed using total RNA from various tissues [shoot (B) = leaf blade, shoot (s) = leaf sheath/stem, root and panicle] of wild-type Nipponbare and primers for OsMIK and actin (control) .

Evolution

• A BLASTP search revealed that OsMIK is a highly conserved protein among higher plants (Fig. 5). Maize LPA3 (AAX14809) is 80% identical to OsMIK at the amino acid level. Sorghum has a theoretical protein (Sbi_0.14483), which exhibits 88% identity to OsMIK. Populus has two OsMIK orthologues showing 53% identity (gw1.IX.3754.1) and 51% identity (gw1.I.898.1), respectively. The Vitis genome has a protein (CAO21458) showing 50% amino acid identity and the Arabidopsis protein (NP_125260) encoded by the At5g5873 has 46% identity.

Fig. 5 Alignment of the deduced amino acid sequences from rice OsMIK (AAP03418), Sorghum bicolor (Sbi_0.14483) maize Lpa3 (AAX14809), Vitis (CAO21458), Arabidopsis thaliana (NP_125260) and Populus (gw1.I.898.1). .

Labs working on this gene

• USDA-ARS Crops Pathology and Genetics Research Unit, Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
• Department of Plant Sciences, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
• USDA-ARS Western Human Nutrition Research Center, Department of Nutrition, University of California at Davis, 430 West Health Sciences Drive, Davis, CA 95616, USA