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.

Keywords: Triticum aestivum; gene silencing; inositol pentakisphosphate kinase; phytic acid; wheat transformation.

FIGURE 1

Differential expression analysis of three homoeologs of TaIPK1 at two seed developmental stages (14 and 21 DAA). Transcript specific primers were designed for 2AL, 2BL, 2DL of TaIPK1 homoeologs based on genomic information available at IWGSC. gDNA free cDNA was prepared using 2 μg of RNA. qRT-PCR assays were performed using SYBR green and C_t values were normalized against wheat ADP-ribosylation factor 1 (ARF1) as an internal control. The indicated error bars represents the standard deviation from three independent replicates.

FIGURE 2

Schematic representation and confirmation of the RNAi construct in wheat for targeted gene silencing of TaIPK1. (A) Vector backbone of pMCG161 was utilized to clone fragments of TaIPK1 gene in the sense and antisense orientations. bar gene was used as a plant selection marker (B) Representative picture for the screening of the putative transgenics to confirm the genomic integration of the RNAi constructs in wheat. PCR amplification of bar gene from the genomic DNA of 11 independent integration events (T₀ stage) recovered after hardening procedure (S1–S6, S8–S11, S16 are putative transgenic plants, C306, control plant, +ve, plasmid pMCG161, NTC, no template control).

FIGURE 3

Confirmation of silencing in TaIPK1: RNAi lines in T₄ seeds. (A) Free Pi content of control C306 and TaIPK1:RNAi lines were estimated using colorimetric based assays. (B) Relative fold change of TaIPK1 expression in wheat transgenic lines. RNAi lines from three independent events were subjected to expression analysis at 14 DAA stage. The cDNA templates were prepared from 2 μg of DNase free RNA. qRT-PCR assays were performed using SYBR green and C_t values were normalized against wheat ADP-ribosylation factor 1 (ARF1) as an internal control. (C) Total phytic acid in mature wheat grains of transgenic lines (T₄). PA was measured in the mature seeds collected from the primary tiller of each line. ^#Indicates significant differences at p < 0.05.

FIGURE 4

Phenotypic characteristics of TaIPK1:RNAi lines. (A) Representative pictures for grain filling at the spike head on wheat RNAi lines and control (C306) and (B) Representative pictures of seeds collected from wheat RNAi lines and control (C306) at T₂ and T₃ stages. Eight seeds were selected randomly from C306 and TaIPK1:RNAi lines and images were captured using a light microscope (Leica Microscope). (C) Representative pictures of wheat caryopsis on the onset of flowering of control C306 and TaIPK1:RNAi lines at T₄. (D) Spike length of the primary tiller of transgenic and control plants representing T₄ stage. Each bar indicates the mean of eight biological replicates.

FIGURE 5

Spike characteristics of wheat transgenic plants. (A) Awn length from primary tiller of control C306 and TaIPK1:RNAi lines. (B) Spikelet count from the primary tiller of TaIPK1:RNAi lines and control C306 and at T₄ stage. (C) Seed weight of control C306 and TaIPK1:RNAi lines. Average seed weight was measured by weighing 50 random seeds from each line. The data shown here were collected from T₃ progenies. Each bar indicates the mean of three biological replicates (three technical replicates). (D) Seed count from the primary tiller of control C306 and TaIPK1:RNAi lines for T₃ progenies. Each bar indicates the mean of three biological replicates. The data in (A,B) indicate the means of eight biological replicates. ^#Indicates significant differences at p < 0.05.

FIGURE 6

Molar ratio of Fe:PA and Zn:PA in different transgenic lines of wheat. (A,B) Molar ratios of Fe:PA and Zn:PA was calculated of four different transgenic wheat lines and were compared to the control (C306) seeds. The standard bar indicates average of four technical plants for each of the respective lines. Mean values showed a significant difference at p < 0.05 (#) with respect to their control.