Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds

Abstract

The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

Keywords: Oryza sativa L.; ectopic expression; mineral element; molecular breeding; phosphorus; phytic acid; seed; translocation.

Figure 1

Scheme for biosynthesis of phytic acid (Ins P₆) and inositol in rice.

Figure 2

Gene expression and seed phenotype of RINO1ox and OsIPK1ox transgenic plants. (A) Quantitative RT-PCR analysis of RINO1 (left) and OsIPK1 (right) genes with cDNA templates from leaves of vector control (Vec) and RINO1ox or OsIPK1ox plants 7 d after germination (n = 3); (B) InsP₆ content in mature seeds obtained from non-transformant (NT) and two transgenic plants was determined by ion chromatography (n ≥ 7); (C) Total P content in NT and two transgenic seeds was measured by colorimetric assay (n ≥ 5). Each value (A to C) represents the mean ± SD.

Figure 3

Gene expression and seed phenotype of OsPGK1ox transgenic plants. (A) Semi-quantitative RT-PCR analysis of the expression of OsPGK1. Total RNA was extracted from flowers of non-transformants (NT), azygous plants (Azy), and two independent OsPGK1ox lines (1 and 2) just before flowering. Actin was used as a reference; (B) Seed weights (n = 20); (C) InsP₆ content was determined by ion chromatography (n = 4); (D) Inorganic phosphate content was measured by colorimetric assay using molybdate staining (n = 10); (E) Total P content was measured by ICP-OES analysis (n = 6). Each value (B to E) represents the mean ± SD. * and ** indicate p < 0.05 and p < 0.01, respectively.

Figure 4

Changes in InsP₆ (A) and total P (B) content in immature seeds of OsPGK1ox-1 and NT during seed development from 7 to 25 d after flowering (DAF). InsP₆ and total P contents were determined by ion chromatography and ICP-OES analyses, respectively. Each value represents the mean ± SD of three replicates. * and ** indicate p < 0.05 and p < 0.01, respectively.

Figure 5

Contents of Mg, K, Ca, Fe, and Zn in mature seeds of OsPGK1ox and NT. The mineral contents were measured by ICP-OES analysis. The analysis in Figure 3E and Figure 5 was performed simultaneously using the same seed samples. Each value represents the mean ± SD of six replicates. * indicates p < 0.05.

Figure 6

Semi-quantitative RT-PCR analysis of the expression of 13 phytic acid biosynthesis-related genes in the OsPGK1ox and NT plants. Total RNA was extracted from the roots of non-transformant (NT) and OsPGK1ox-1 3 d after germination. Actin was used as a reference.