Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds.
During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Jaeju
J
Yu
Adolfo
A
Saiardi
John S
JS
Greenwood
J Derek
JD
Bewley
D001483:Base Sequence; D015139:Blotting, Southern; D002367:Ricinus communis / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003198:Computer Simulation; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010833:Phytic Acid / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012639:Seeds / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
D012333:RNA, Messenger; D010833:Phytic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C502806:inositol 1,3,4,5,6-pentakisphosphate 2-kinase