Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases

Abstract

Phytate (inositol hexakisphosphate, IP6) is a regulator of intracellular signaling, a highly abundant animal antinutrient, and a phosphate store in plant seeds. Here, we report a requirement for inositol polyphosphate kinases, AtIPK1 and AtIPK2beta, for the later steps of phytate synthesis in Arabidopsis thaliana. Coincident disruption of these kinases nearly ablates seed phytate without accumulation of phytate precursors, increases seed-free phosphate by 10-fold, and has normal seed yield. Additionally, we find a requirement for inositol tetrakisphosphate (IP4)/inositol pentakisphosphate (IP5) 2-kinase activity in phosphate sensing and root hair elongation. Our results define a commercially viable strategy for the genetic engineering of phytate-free grain and provide insights into the role of inositol polyphosphate kinases in phosphate signaling biology.

Fig. 1.

Biosynthetic pathways of phytate in plants. (A) Three distinct pathways have been proposed for the synthesis of IP₆. Pathways I and II originate from the hydrolysis of PI(4,5)P₂ by phospholipase C and are therefore lipid-dependent. Pathway III is a lipid-independent pathway that involves the sequential phosphorylation of I(3)P or inositol (21, 38). The majority of the participating kinases in this pathway are unknown at the molecular level. Gray arrows designate AtIpk2 activities; black arrows designate AtIpk1 activities; striped arrows designate I(1,3,4)P₃ 5/6-kinase/I(3,4,5,6)P₄ 1-kinase activities; the thin dashed arrow designates the I(1,4,5)P₃ 3-kinase that has not been identified in plants but exists in other higher eukaryotes; the thin solid arrow represents IP 5-phosphatase; IP₆ kinase is indicated for which no gene has been formally identified but which are likely to exist given their conservation; the horizontal strip indicates myo-inositol kinase; and white arrows indicate unknown or tentatively assigned kinases.

Fig. 2.

IP metabolism of IPK mutants. (A) Nonradioactive mass IP analysis of mature dessicated seed extracts. Seed extracts were separated by an IonPac AS7 anion-exchange column, detected by metal-dye chelation, and measured at 550 nm. Data points are plotted as negative absorbance values. (B) Wild-type and IPK mutant seeds were germinated in liquid MS salts with 400 μCi/ml [³H]-myo-inositol for 6 days, and IPs were harvested from seedlings and analyzed by Partisphere strong anion exchange HPLC. Representative chromatograms for each genotype are shown and indicated at the right. The chemical identity of IP₃ a is currently unknown. Eighty percent of IP₄ a is I(3,4,5,6)P₄, and 20% is I(1,3,4,6)P₄ and/or I(1,4,5,6)P₄. IP₅ a is I(1,3,4,5,6)P₅. IP₄b is I(1,3,4,6)P₄ and/or I(1,4,5,6)P₄. IP₅b is I(1,2,3,4,6)P₅. IP₅c is either I(1,2,4,5,6)P₅ or its enantiomer I(2,3,4,5,6)P₅. Sometimes slight alterations in IP migration were observed and are due to different strong anion exchange columns. In all cases, the IPs are compared with known standards.

Fig. 3.

Phenotypic analysis of IPK mutants. Seeds were germinated on a mixture of sand and vermiculite through subirrigation until the cotyledons had emerged. Plants were then top-watered with either 0.1 × or 0.5 × Hoagland's solution daily and were grown at 20°C under a 14-hr light (150 μmol/m²·s^-1) and 10-hr dark cycle in the Duke University Phytotron. Top view of 5-week-old plants watered with 0.1 × Hoagland's solution (A) or 0.5 × Hoagland's solution (B). Genotypes are indicated. (C) Complementation of IP synthesis and growth phenotype in atipk1-1 by constitutive expression of AtIPK1 through a 35S promoter. (Left) HPLC chromatograms of IP profiles from [³H]-inositol-labeled 5-day-old seedlings. (Right) Leaves from 5-week-old plants grown in half-strength Hoagland's solution as described. Upper and leaf row are wild-type plants transformed with the empty expression vector. Middle and leaf row are atipk1-1 plants transformed with the empty expression vector. Bottom and leaf row are atipk1-1 plants transformed with the expression vector harboring the AtIPK1 gene.

Fig. 4.

Aberrant phosphate sensing and root hair growth in atipk1-1 mutants. (A) Leaves from 5-week-old wild-type and atipk1-1 plants grown as described above in half-strength Hoagland's solution with variable phosphate concentrations as indicated. (B) Inorganic phosphate concentrations in the leaves from plants grown at various phosphate concentrations. (C) Representative images of main root and root hairs from wild-type (Left) and atipk1-1 (Right) plants grown vertically on MS-agar plates containing 10 μM phosphate. (Bar, 0.2 mm.) (D) Root hair lengths of the indicated genotypes grown vertically on MS-agar plates containing various phosphate concentrations as indicated. n = 12 for each genotype.