Low Phytic Acid Soybean Research Articles

Aberrant RNA splicing of the phytic acid synthesis gene inositol-1,3,4 trisphosphate 5/6-kinase in a low phytic acid soybean line

ABSTRACT Most phosphorus is stored as phytic acid in plant seed, although it is considered an anti-nutrient compound that reduces the bioavailability of minerals in monogastric animals. Low-phytic acid (LP) crops improve mineral bioavailability in monogastric animals, which can decrease environmental pollution due to phosphorus. Recently, we reported a newly developed LP soybean line by crossing the LP line CX1834 and Japanese normal phytic acid cultivar Natto-kotsubu (Natto), which showed superior traits in seedling emergence, growth, and yield compared with CX1834. The molecular mechanism underlying the LP trait in the LP line remains unclear. The LP line was used for a de novo RNA-Seq analysis, and we illustrated the aberrant RNA splicing of the phytic acid synthase gene inositol-1,3,4 trisphosphate 5/6-kinase 3 (GmITPK3) in the LP line. The expression level of phytic acid biosynthesis-related genes and inositol phosphate isomer concentrations were investigated. A de novo RNA-Seq analysis revealed that the intron retention (IR) transcript of GmITPK3 is highly expressed in the leaves of the LP line. Although expressed in both, the relative abundances of multiple IR transcripts were significantly higher in the LP line than that in the Natto cultivar. The IR transcripts of GmITPK3 encoding truncated proteins were missing an essential domain for their activity, while one possibly contained an abnormal ligand binding site. The relative abundance of IR transcripts was 1.9- to 5.4-times higher in the LP line than that in the Natto cultivar during seed development; accordingly, the production of phytic acid and its precursors in the developing seeds was lower in the LP line than that in the Natto cultivar. No mutation was detected at the exon–intron junction, where a splicing error occurs in GmITPK3 of the LP line, implying that a splicing error is attributed to unknown splicing factor genes involved in the splicing regulation of GmITPK3. Our results provide insights into the possible involvement of splicing errors in GmITPK3 for the trait of low phytic acid production in the LP line.

Network Inference of Transcriptional Regulation in Germinating Low Phytic Acid Soybean Seeds.

The low phytic acid (lpa) trait in soybeans can be conferred by loss-of-function mutations in genes encoding myo-inositol phosphate synthase and two epistatically interacting genes encoding multidrug-resistance protein ATP-binding cassette (ABC) transporters. However, perturbations in phytic acid biosynthesis are associated with poor seed vigor. Since the benefits of the lpa trait, in terms of end-use quality and sustainability, far outweigh the negatives associated with poor seed performance, a fuller understanding of the molecular basis behind the negatives will assist crop breeders and engineers in producing variates with lpa and better germination rate. The gene regulatory network (GRN) for developing low and normal phytic acid soybean seeds was previously constructed, with genes modulating a variety of processes pertinent to phytic acid metabolism and seed viability being identified. In this study, a comparative time series analysis of low and normal phytic acid soybeans was carried out to investigate the transcriptional regulatory elements governing the transitional dynamics from dry seed to germinated seed. GRNs were reverse engineered from time series transcriptomic data of three distinct genotypic subsets composed of lpa soybean lines and their normal phytic acid sibling lines. Using a robust unsupervised network inference scheme, putative regulatory interactions were inferred for each subset of genotypes. These interactions were further validated by published regulatory interactions found in Arabidopsis thaliana and motif sequence analysis. Results indicate that lpa seeds have increased sensitivity to stress, which could be due to changes in phytic acid levels, disrupted inositol phosphate signaling, disrupted phosphate ion (Pi) homeostasis, and altered myo-inositol metabolism. Putative regulatory interactions were identified for the latter two processes. Changes in abscisic acid (ABA) signaling candidate transcription factors (TFs) putatively regulating genes in this process were identified as well. Analysis of the GRNs reveal altered regulation in processes that may be affecting the germination of lpa soybean seeds. Therefore, this work contributes to the ongoing effort to elucidate molecular mechanisms underlying altered seed viability, germination and field emergence of lpa crops, understanding of which is necessary in order to mitigate these problems.

Transcriptome analysis identifies differentially expressed genes in the progenies of a cross between two low phytic acid soybean mutants

Phytic acid (PA) is a major antinutrient that cannot be digested by monogastric animals, but it can decrease the bioavailability of micronutrients (e.g., Zn and Fe). Lowering the PA content of crop seeds will lead to enhanced nutritional traits. Low-PA mutant crop lines carrying more than one mutated gene (lpa) have lower PA contents than mutants with a single lpa mutant gene. However, little is known about the link between PA pathway intermediates and downstream regulatory activities following the mutation of these genes in soybean. Consequently, we performed a comparative transcriptome analysis using an advanced generation recombinant inbred line with low PA levels [2mlpa (mips1/ipk1)] and a sibling line with homozygous non-mutant alleles and normal PA contents [2MWT (MIPS1/IPK1)]. An RNA sequencing analysis of five seed developmental stages revealed 7945 differentially expressed genes (DEGs) between the 2mlpa and 2MWT seeds. Moreover, 3316 DEGs were associated with 128 metabolic and signal transduction pathways and 4980 DEGs were annotated with 345 Gene Ontology terms related to biological processes. Genes associated with PA metabolism, photosynthesis, starch and sucrose metabolism, and defense mechanisms were among the DEGs in 2mlpa. Of these genes, 36 contributed to PA metabolism, including 22 genes possibly mediating the low-PA phenotype of 2mlpa. The expression of most of the genes associated with photosynthesis (81 of 117) was down-regulated in 2mlpa at the late seed developmental stage. In contrast, the expression of three genes involved in sucrose metabolism was up-regulated at the late seed developmental stage, which might explain the high sucrose content of 2mlpa soybeans. Furthermore, 604 genes related to defense mechanisms were differentially expressed between 2mlpa and 2MWT. In this study, we detected a low PA content as well as changes to multiple metabolites in the 2mlpa mutant. These results may help elucidate the regulation of metabolic events in 2mlpa. Many genes involved in PA metabolism may contribute to the substantial decrease in the PA content and the moderate accumulation of InsP3–InsP5 in the 2mlpa mutant. The other regulated genes related to photosynthesis, starch and sucrose metabolism, and defense mechanisms may provide additional insights into the nutritional and agronomic performance of 2mlpa seeds.

Evaluating Genotypes and Seed Treatments to Increase Field Emergence of Low Phytic Acid Soybeans

Low phytic acid (LPA) soybean [Glycine max (L.) Merr] genotypes reduce indigestible PA in soybean seeds in order to improve feeding efficiency of mono- and agastric animals, but often exhibit low field emergence, resulting in reduced yield. In this study, four LPA soybean varieties with two different genetic backgrounds were studied to assess their emergence and yield characters under 12 seed treatment combinations including two broad-spectrum, preplant fungicides (i.e., ApronMaxx (mefenoxam: (R,S)-2-[(2,6-dimethylphenyl)-methoxyacetylamino]-propionic acid methyl ester; fludioxonil: 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile) and Rancona Summit (ipconazole: 2-[(4-chlorophenyl)methyl]-5-(1-methylethyl)-1-(1H-1,2,4-triazol-1-ylmethyl) cyclopentanol; metalaxyl: N-(methooxyacetyl)-N-(2,6-xylyl)-DL-alaninate)), osmotic priming, and MicroCel-E coating. Two normal-PA (NPA) varieties served as controls. Both irrigated and non-irrigated plots were planted in Blacksburg and Orange, Virginia, USA in 2014 and 2015. Results revealed that three seed treatments (fungicides Rancona Summit and ApronMaxx, as well as Priming + Rancona) significantly improved field emergence by 6.4–11.6% across all genotypes, compared with untreated seeds. Seed priming was negatively associated with emergence across LPA genotypes. Seed treatments did not increase the yield of any genotype. LPA genotypes containing mips or lpa1/lpa2 mutations, produced satisfactory emergence similar to NPA under certain soil and environmental conditions due to the interaction of genotype and environment. Effective seed treatments applied to LPA soybeans along with the successful development of LPA germplasm by soybean breeding programs, will increase use of LPA varieties by commercial soybean growers, ultimately improving animal nutrition while easing environmental impact.

Quantitative proteomic analyses of two soybean low phytic acid mutants to identify the genes associated with seed field emergence

BackgroundSeed germination is essential to crop growth and development, and ultimately affects its harvest. It is difficult to breed soybeans low in phytic acid with a higher seed field emergence. Although additional management and selection could overcome the phytate reduction, the mechanisms of seed germination remain unknown.ResultsA comparative proteomic analysis was conducted between two low phytic acid (LPA) soybean mutants (TW-1-M and TW-1), both of which had a deletion of 2 bp in the GmMIPS1 gene. However, the TW-1 seeds showed a significantly lower field emergence compared to the TW-1-M. There were 282 differentially accumulated proteins (DAPs) identified between two mutants at the three stages. Among these DAPs, 80 were down-accumulated and 202 were up-accumulated. Bioinformatic analysis showed that the identified proteins were related to functional categories of oxidation reduction, response to stimulus and stress, dormancy and germination processes and catalytic activity. KEGG analysis showed that these DAPs were mainly involved in energy metabolism and anti-stress pathways. Based upon the conjoint analysis of DAPs with the differentially expressed genes (DEGs) previously published among three germination stages in two LPA mutants, 30 shared DAPs/DEGs were identified with different patterns, including plant seed protein, beta-amylase, protein disulfide-isomerase, disease resistance protein, pyrophosphate-fructose 6-phosphate 1-phosphotransferase, cysteine proteinase inhibitor, non-specific lipid-transfer protein, phosphoenolpyruvate carboxylase and acyl-coenzyme A oxidase.ConclusionsSeed germination is a very complex process in LPA soybean mutants. The TW-1-M and TW-1 showed many DAPs involved in seed germination. The differential accumulation of these proteins could result in the difference of seed field emergence between the two mutants. The high germination rate in the TW-1-M might be strongly attributed to reactive oxygen species-related and plant hormone-related genes. All these findings would help us further explore the germination mechanisms in LPA crops.

Development and Evaluation of Low Phytic Acid Soybean by siRNA Triggered Seed Specific Silencing of Inositol Polyphosphate 6-/3-/5-Kinase Gene.

Soybean is one of the leading oilseed crop in the world and is showing a remarkable surge in its utilization in formulating animal feeds and supplements. Its dietary consumption, however, is incongruent with its existing industrial demand due to the presence of anti-nutritional factors in sufficiently large amounts. Phytic acid in particular raises concern as it causes a concomitant loss of indigestible complexed minerals and charged proteins in the waste and results in reduced mineral bioavailability in both livestock and humans. Reducing the seed phytate level thus seems indispensable to overcome the nutritional menace associated with soy grain consumption. In order to conceive our objective we designed and expressed a inositol polyphosphate 6-/3-/5-kinase gene-specific RNAi construct in the seeds of Pusa-16 soybean cultivar. We subsequently conducted a genotypic, phenotypic and biochemical analysis of the developed putative transgenic populations and found very low phytic acid levels, moderate accumulation of inorganic phosphate and elevated mineral content in some lines. These low phytic acid lines did not show any reduction in seedling emergence and displayed an overall good agronomic performance.

Inference of Transcription Regulatory Network in Low Phytic Acid Soybean Seeds.

A dominant loss of function mutation in myo-inositol phosphate synthase (MIPS) gene and recessive loss of function mutations in two multidrug resistant protein type-ABC transporter genes not only reduce the seed phytic acid levels in soybean, but also affect the pathways associated with seed development, ultimately resulting in low emergence. To understand the regulatory mechanisms and identify key genes that intervene in the seed development process in low phytic acid crops, we performed computational inference of gene regulatory networks in low and normal phytic acid soybeans using a time course transcriptomic data and multiple network inference algorithms. We identified a set of putative candidate transcription factors and their regulatory interactions with genes that have functions in myo-inositol biosynthesis, auxin-ABA signaling, and seed dormancy. We evaluated the performance of our unsupervised network inference method by comparing the predicted regulatory network with published regulatory interactions in Arabidopsis. Some contrasting regulatory interactions were observed in low phytic acid mutants compared to non-mutant lines. These findings provide important hypotheses on expression regulation of myo-inositol metabolism and phytohormone signaling in developing low phytic acid soybeans. The computational pipeline used for unsupervised network learning in this study is provided as open source software and is freely available at https://lilabatvt.github.io/LPANetwork/.

Phytic acid dynamics during seed development and it’s composition in yellow and black Indian soybean (Glycine max L.) genotypes through a modified extraction and HPLC method

Identifying or designing low phytic acid soybean demands an improved understanding of its dynamics and a need for a reliable and accurate phytate quantification method and hence an improved quantitative technique for accurate and rapid quantification of phytic acid (PA) using high-performance liquid chromatography is proposed in this paper. A rapid PA extraction method utilizing sonication of sample in 0.78 M HCl for 3 min followed by mechanical agitation and separation using strong anion exchange column in a vacuum manifold was optimized. The elution of PA was performed using a RP-C18 column with an isocratic mobile phase [Acetonitrile, 35 mM formic acid and tetrabutylammonium hydroxide (4.8: 5.1: 0.1, v/v/v)]. The modified method was rapid, accurate, precise, and reproducible with relative standard deviation of 1.80 and 3.01 % (n = 10, for 1 mg ml−1) for within and between days respectively with linearity (R2 = 0.999, P < 0.05), low limit of detection (LOD = 7.8 μg ml−1) and limit of quantification (LOQ = 31.25 μg ml−1). PA dynamics was found increased in a linear trend from initial to later developing stages until maturity in both yellow (DS-9814) and black (DS-MM-64) genotypes. The PA content ranged from 2.38–4.72 g 100g−1 in the 20 soybean genotypes screened and the variability in PA content was more in black genotypes (P < 0.0001). The black soybean genotypes JS 76-205 (2.38 g 100g−1), Kalitur (2.50 g 100g−1) and UPSL 652 (2.54 g 100g−1), inherently rich in anthocyanin, contains the lowest PA content and hence can serve as potential genotypes with nutraceutical benefits.

Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation

Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

Inositol Metabolism in Developing Seed of Low and Normal Phytic Acid Soybean Lines

While inositol has key roles in phytic acid and raffinosaccharide synthesis, its concentration in developing seed of low phytic acid soybean [Glycine max (L.) Merr.] lines derived from CX1834 (Wilcox et al., 2000) has not been compared to that of normal lines. Concentrations of metabolites in the phytic acid and raffinosaccharide biosynthesis have been measured in mature seed of CX1834‐derived lines but not throughout seed development. Our objective was to compare concentrations of inositol and metabolites associated with phytic acid and raffinosaccharide synthesis in developing seed of CX1834‐derived and normal lines. Plants were cultured with complete nutrient solutions in growth chambers with 650 to 700 μmol m−2 s−1 of photosynthetically active radiation and a 26/22°C day/night temperature. Seed inositol concentrations were high (60 to 90 mmol kg−1 seed dry wt.) at 20 d after flowering (DAF) and decreased 95% by maturity in both normal and low phytic acid lines. In two of three experiments, low phytic acid lines had significantly (p ≤ 0.05) greater seed inositol concentrations than normal lines at the first two sampling dates, but differences at maturity were not significant. Seeds of low phytic acid and normal lines had statistically similar concentrations (p &gt; 0.05) of partially phosphorylated inositol intermediate (inositol triphosphate [IP3]), stachyose, raffinose, and phytase activity throughout development. These results corroborate previous studies that ruled out defects in genes coding myo‐inositol‐1‐P synthase, inositol kinases, and phytase as the basis for the low seed phytic acid trait in CX1834‐derived lines.

Effects of low-phytic acid corn, low-phytic acid soybean meal, and phytase on nutrient digestibility and excretion in growing pigs1

Forty-eight grower pigs were used to evaluate the effects of feeding low phytic acid (LPA) corn, LPA soybean meal, normal corn (NC), normal soybean meal (NSBM), and phytase on nutrient digestibility and excretion. Barrows were blocked by BW (initial BW=45.3+/-1.6 kg) and randomly assigned to 1 of 8 dietary treatments in a 2 x 2 x 2 factorial arrangement (6 pigs/treatment). Pigs were fed twice daily (0700 and 1700 h) at 3 times the ME requirement for maintenance. Phytase was added to the diet at 510 phytase units/kg of feed (where 1 phytase unit is the quantity of enzyme that liberates 1 mumol of inorganic P/min from 0.005 mol/L of sodium phytate at pH 5.5 and 37 degrees C), at the expense of corn starch, and all diets were formulated to provide 0.39% total P, 0.50% Ca, and 1.0% lysine with no supplemental inorganic P. Pigs were adapted to metabolism crates and dietary treatments for 7 d, followed by a 3-d total collection of urine and feces. Total fecal DM excreted, percentage of DM of feces, and percentage of DM digested were not different (P>0.53) among treatments. Fecal P excretion was reduced for pigs fed LPA corn vs. NC (2.85 vs. 3.24+/-0.119 g/d; P=0.024), for pigs fed LPA soybean meal vs. NSBM (2.79 vs. 3.30+/-0.119 g/d; P=0.007), for pigs fed phytase vs. nonphytase diets (2.80 vs. 3.29+/-0.119 g/d; P=0.009), and for pigs fed LPA corn, LPA soybean meal, and phytase vs. NC and NSBM without phytase (2.16 vs. 3.70+/-0.237 g/d; P<0.001). Phosphorus digestibility was increased for pigs fed diets containing LPA corn vs. NC (48.4 vs. 39.9+/-2.27%; P=0.012), for pigs fed phytase vs. nonphytase diets (48.4 vs. 39.9+/-2.27%; P=0.019), and for pigs fed the LPA corn, LPA soybean meal, and phytase diet vs. the NC and soybean meal diet (60.1 vs. 34.1+/-4.5%; P<0.001) and tended to be increased for pigs fed LPA soybean meal vs. NSBM (47.2 vs. 41.1+/-2.27%; P=0.075). Corn type and soybean meal type had no effect (P>0.11) on water-soluble P excretion. However, pigs fed diets containing phytase tended to excrete less total water-soluble P than those without phytase inclusion (1.99 vs. 2.27+/-0.099 g/d; P<0.066). This study demonstrates that feeding any combination of LPA corn, LPA soybean meal, and phytase was additive, significantly improving P digestibility and dramatically decreasing P excretion to reduce the potential impacts of P from pig manure on the environment.

A Modified Colorimetric Method for Phytic Acid Analysis in Soybean

A quantitative, reproducible, and efficient phytic acid assay procedure is needed to screen breeding populations and support genetic studies in soybeans [Glycine max (L.) Merr.]. The objective of this study was to modify the colorimetric Wade reagent method and compare the accuracy and applicability of this new method in determining seed phytic acid content in soybean with three well‐established phytic acid assay methods: anion exchange column (AEC), high‐performance liquid chromatography (HPLC), and 31P nuclear magnetic resonance (NMR). The CV for repeated measurements of a low phytic acid soybean mutant, CX1834‐1‐6, ranged from 1.8 to 4.2% (n = 5), indicating the results were precise and reproducible. Phytic acid content of 42 soybean genotypes as determined by this method showed a correlation of 93.7 to 96.6% with the measurements by AEC, HPLC, and NMR. According to analysis of covariance, using inorganic P content as a predictor, phytic acid P content in a given sample analyzed by the four assay methods can be estimated with four linear regression models at the α = 0.01 level. Compared with HPLC, AEC, and 31P NMR, this modified colorimetric method is simpler and less expensive for assaying a large number of samples, allowing its effective application in breeding and genetic studies of low phytic acid soybean.

Characterization of the MIPS gene family in Glycine max

AbstractPhytic acid (myo‐inositol‐1,2,3,4,5,6‐hexakisphosphate) is the primary storage component of phosphorus in plant seeds. The first step in phytic acid biosynthesis is the de novo synthesis of myo‐inositol, which is catalyzed by the enzyme D‐myo‐inositol 3‐phosphate synthase (MIPS EC 5.5.1.4). Previous work detected four MIPS genes in soybean (Glycine max). However, only a limited amount of data were available for the MIPS gene family and some of the data were conflicting. The work described here clears up these data and characterizes the MIPS gene family for the purposes of reverse genetic technologies. The complete genomic sequence of all four genes was determined and their expression profile was examined by quantitative real‐time reverse transcription‐polymerase chain reaction. Our results indicate that the four MIPS genes are highly conserved and temporally and spatially expressed. The MIPS gene family in the low phytic acid soybean line, CX1834, was also characterized since this line displays a phenotype similar to previously characterized MIPS mutants. These data demonstrate that mutations in MIPS genes are not the cause of the low phytic acid phenotype.