Novel allelic variant of Lpa1 gene associated with a significant reduction in seed phytic acid content in rice (Oryza sativa L.)

Abstract

21In plants, 2,3,4,5, ), also known as phytic acid 22 (PA), is a major component of organic phosphorus (P), and accounts for up to 85% of the total 2 23 P in seeds. In rice (Oryza sativa L.), PA mainly accumulates in rice bran, and chelates mineral 24 cations, resulting in mineral deficiencies among brown rice consumers. Therefore, 25 considerable efforts have been focused on the development of low PA (LPA) rice cultivars. In 26 this study, we performed genetic and molecular analyses of OsLpa1, a major PA biosynthesis 27 gene, in Sanggol, a low PA mutant variety developed via chemical mutagenesis of Ilpum rice 28 cultivar. Genetic segregation and sequencing analyses revealed that a recessive allele, lpa1-3, 29 at the OsLpa1 locus (Os02g0819400) was responsible for a significant reduction in seed PA 30 content in Sanggol. The lpa1-3 gene harboured a point mutation (C623T) in the fourth exon of 31 the predicted coding region, resulting in threonine (Thr) to isoleucine (Ile) amino acid 32 substitution at position 208 (Thr208Ile). Three-dimensional analysis of Lpa1 protein structure 33 indicated that myo-inositol 3-monophosphate [Ins(3)P 1 ] kinase binds to the active site of Lpa1, 34 with ATP as a cofactor for catalysis. Furthermore, the presence of Thr208 in the loop adjacent 35 to the entry site of the binding pocket suggests that Thr208Ile substitution is involved in 36 regulating enzyme activity via phosphorylation. Therefore, we propose that Thr208Ile 37 substitution in lpa1-3 reduces Lpa1 enzyme activity in Sanggol, resulting in reduced PA 38 biosynthesis. 39 40 42 phytic acid (PA), is considered a major source of phosphorus (P) available in the form of 43 phytate, and accounts for 65-85% of the total P in seeds [1]. Monogastric animals poorly digest 44 PA, as they lack the phytase enzyme, which is responsible for the release of phosphate residues 45 [2]. PA is an efficient chelator of mineral cations, such as zinc (Zn 2+ ), iron (Fe 2+ ), magnesium 46 (Mg 2+ ), potassium (K 2+ ), and calcium (Ca 2+ ), in the nutritional tract. Because of these attributes, 3 47 PA is considered as an antinutrient [3, 4]. Hence, there is a need to develop low PA (LPA) crop 48 cultivars to maximize the nutritional benefits of grains. 49 Mutants associated with the LPA phenotype have been identified in several crop plants 50 including maize (Zea mays) [5, 6], barley (Hordeum vulgare) [7], soyabean (Glycine max) [8], 51 rice (Oryza sativa) [9], and wheat (Triticum aestivum) [10]. Although, LPA mutants are 52identified primarily on the basis of percentage reduction of PA and high inorganic P (P i ) content 53 in seeds [5, 11], some mutants show a significant accumulation of myo-inositol and inositol 54 phosphate [Ins(1,3,4)P 3 5-/6] intermediates in seeds [12, 13]. 55Previously, the LPA phenotype of seeds has been associated with reduced agronomic 56 performance of mutant crop plants in the field [5, 14]. It is important to understand the genetic 57 and molecular bases of reduced agronomic performance of LPA mutants for ef...

Identification of phytic acid mutants in oilseed rape (Brassica napus) by large‐scale screening of mutant populations through amplicon sequencing

“…It was hypothesized that this enzyme, in addition to producing 2,3‐bisphosphoglycerate, uses myo ‐inositol monophosphate as a substrate, resulting in the synthesis of inositol bisphosphate (Raboy, ; Lu et al , ). The enzyme was initially identified in rice, and knockout mutants are available from rice and Arabidopsis, which showed a phytic acid reduction of 45% and 57%, respectively (Kim et al , ; Kim & Tai, ; Kishor et al , ). Paralogues of Bn2‐PGK2 showed a conserved P‐loop kinase domain with two conserved motifs (Walker A and Walker B; Fig.…”

“…Since the biochemical function of the 2‐PGK2 protein is largely unknown in plant species (Lu et al , ; Kishor et al , ), the novel bn2‐pgk2 mutants can be used to investigate its biochemical properties in the phytic acid pathway in B. napus . Since we have not seen pleiotropic effects on plant growth and thousand kernel weights between the mutants and the controls there are no adverse effects of the induced mutations (Table S4).…”

Mutational Analysis of OsPLDα1 Reveals Its Involvement in Phytic Acid Biosynthesis in Rice Grains

“…RNA Extraction and qRT-PCR Assay. In rice, phytic acid accumulation starts after flowering and continues until 25 d after flowering (DAF), 27 so we extracted total RNA from seeds at 20 DAF by using the OMEGA plant RNA kit (Catalog No. R6827-27) following the manufacturer's protocol.…”

“…Cloning and characterization of numerous genes involved in the synthesis of phytic acid through lipidindependent pathway has been well-documented in cereal plants. 25,26 However, until now the lipid-dependent pathway has not been well-studied, 27 due to which its specific biosynthetic steps and genes involved in the network remains ambiguous and needs to be explored.…”