The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene

Abstract

Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P(3) 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P(3), Ins(3,5,6)P(3), Ins(3,4,5,6)P(4), and Ins(1,2,5,6)P(4). The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F(2) family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.

Figure 1

Amino acid sequence alignment of the maize ZmIpk, Arabidopsis, and human Ins(1,3,4)P₃ 5/6-kinase. Identical amino acids are shaded in black. AtItpk, Arabidopsis Ins(1,3,4)P₃ 5/6-kinase; HsItpk, human Ins(1,3,4)P₃ 5/6-kinase; AtItpk-1, GenBank accession number JC5401; AtItpk-2, T10544; AtItpk-3, NP_195103; ZmIpk, AY172635; and HsItpk, U51336.

Figure 2

Inositol phosphate kinase activity assay with [γ-³²P]ATP. A, Radioautograph of inositol phosphate kinase assay. The reaction mixtures contained 5 μL of the recombinant ZmIpk protein, 40 μm inositol phosphate as indicated in each lane, 40 μm ATP, and 0.5 μL of [γ-³²P]ATP (3,000 Ci mmol⁻¹) in a total volume of 20 μL. The reaction product was separated on a PEI-cellulose-coated TLC plate. Lane 1, Ins(1,3,4)P₃, the enzyme was inactivated by boiling before added to the reaction mixture; lane 2, Ins(1,3,4)P₃; lane 3, Ins(1,3,4,5)P₄; lane 4, Ins(1,3,4,6)P₄; lane 5, Ins(1,3,5,6)P₄; lane 6, Ins(3,4,5,6)P₄; and lane 7, Ins(3,5,6)P₃. B, Authentic inositol phosphates purchased from Sigma-Aldrich (St. Louis) were loaded on the TLC plate and developed with 0.5 n HCl as in A. The plate then was sprayed with ammonium molybdate/HClO₄/HCl solution to show the migration of the inositol phosphates. Lane 1, Ins(1,3,4,5)P₄; lane 2, Ins(1,3,4,6)P₄; lane 3, Ins(1,3,5,6)P₄; lane 4, Ins(3,4,5,6)P₄; and lane 5, Ins(1,3,4,5,6)P₅.

Figure 3

Mu transposon insertion sites in ZmIpk-mum mutants and nucleotide mutation in lpa2-2 allele. The Mu-ZmIpk junction region in ZmIpk-mum alleles and ZmIpk gene in lpa2-2 allele were PCR-amplified and sequenced. The rectangular box represents the open reading frame of the ZmIpk gene. The triangle marks the Mu insertion site. *, The site of mutated nucleotide in lpa2-2 allele. The underlined sequences represent the characteristic duplication associated with Mu insertion.

Figure 4

Inositol phosphate accumulation in ZmIpk Mu insertion mutants. HPLC analysis of inositol phosphates in mature seeds of ZmIpk Mu insertion mutants (A) and nonmutant control (B).

Figure 5

Southern-blot analysis of the ZmIpk gene in the lpa2-1 mutant. A, Diagram of the ZmIpk gene restriction sites. The rectangular box represents the open reading frame of the ZmIpk gene. B, Southern-blot analysis of the lpa2-1 mutant line (lanes 1, 3, 5, 7, and 9) and near-isogenic nonmutant line (lanes 2, 4, 6, 8, and 10).

Figure 6

Northern-blot analysis of the ZmIpk gene. Total RNA from different tissues of B73 was separated on 1% (w/v) gel. After transfer, the blot was probed with the maize ZmIpk. Ethidium bromide-stained gel was shown at bottom as a control for loading. Each lane contained 10 μg of total RNA.