RNAi-Mediated Downregulation of Inositol Pentakisphosphate Kinase (IPK1) in Wheat Grains Decreases Phytic Acid Levels and Increases Fe and Zn Accumulation

Abstract

Enhancement of micronutrient bioavailability is crucial to address the malnutrition in the developing countries. Various approaches employed to address the micronutrient bioavailability are showing promising signs, especially in cereal crops. Phytic acid (PA) is considered as a major antinutrient due to its ability to chelate important micronutrients and thereby restricting their bioavailability. Therefore, manipulating PA biosynthesis pathway has largely been explored to overcome the pleiotropic effect in different crop species. Recently, we reported that functional wheat inositol pentakisphosphate kinase (TaIPK1) is involved in PA biosynthesis, however, the functional roles of the IPK1 gene in wheat remains elusive. In this study, RNAi-mediated gene silencing was performed for IPK1 transcripts in hexaploid wheat. Four non-segregating RNAi lines of wheat were selected for detailed study (S3-D-6-1; S6-K-3-3; S6-K-6-10 and S16-D-9-5). Homozygous transgenic RNAi lines at T4 seeds with a decreased transcript of TaIPK1 showed 28–56% reduction of the PA. Silencing of IPK1 also resulted in increased free phosphate in mature grains. Although, no phenotypic changes in the spike was observed but, lowering of grain PA resulted in the reduced number of seeds per spikelet. The lowering of grain PA was also accompanied by a significant increase in iron (Fe) and zinc (Zn) content, thereby enhancing their molar ratios (Zn:PA and Fe:PA). Overall, this work suggests that IPK1 is a promising candidate for employing genome editing tools to address the mineral accumulation in wheat grains.

Gene Expression Pattern of Vacuolar-Iron Transporter-Like (VTL) Genes in Hexaploid Wheat during Metal Stress

“…Moreover, the Fe is mobilized through a multistep process that overcomes transport bottlenecks and eventually is loaded in the developing grains [10][11][12][13]. Researchers worldwide are utilizing multiple approaches to either enrich Fe in grains or their storage with enhanced bioavailability [14][15][16][17]. To improve Fe content in cereal grains, multiple transporters and chelators have been targeted through multiple molecular approaches [14,15,18].…”

“…Researchers worldwide are utilizing multiple approaches to either enrich Fe in grains or their storage with enhanced bioavailability [14][15][16][17]. To improve Fe content in cereal grains, multiple transporters and chelators have been targeted through multiple molecular approaches [14,15,18]. A number of additional micronutrient transporters have been identified, those are good candidates for micronutrient biofortification, including transporters belonging to the major facilitator superfamily (MFS) gene family [19,20].…”

“…Previously, vacuolar iron transporters (VIT) were shown to be play an important role in maintaining Fe in the optimal physiological range and prevent cellular toxicity [14]. VIT genes from multiple plant species have been characterized and assessed for their ability to enhance Fe content in cereal crops [15]. These VIT genes show high homology with a small family of nodulin like protein containing a CCC-1 (Ca 2+ -Sensitive Cross Complementer) like domain with yeast CCC1p1 [29].…”

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean

“…Research in the past has evidenced the potential of RNAi technique to generate Ipa rice 2,18 , wheat 31 and soybean 22 by effectively down regulating PA pathway genes. Down regulation of MIPS gene expression under CaMV35S promoter using self - complementary hairpin RNA was also carried out in soybean 17 resulting in a drastic reduction (~94.5%) of phytate content in the developed transgenic lines.…”

Gene expression pattern of vacuolar-iron transporter-like (VTL) genes in hexaploid wheat during metal stress

“…Researchers worldwide is generating information for the means to enrich Fe rich grains and their storage with enhanced bioavailability. To improve Fe content in cereal grains, multiple transporters and chelators have been targeted including molecular approaches [14–16]. In addition to these, a number of genes are still unaddressed that are potential candidates for micronutrient biofortification, including transporters belong to the inventory of the Major facilitator superfamily (MFS) gene family [17].…”