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Page 1 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Phytic Acid and Inorganic Phosphate Composition in Soybean Lines with Independent IPK1 Mutations Jennifer A. Vincent, Minviluz Stacey, Gary Stacey, Kristin D. Bilyeu* Jennifer A. Vincent, Minviluz Stacey, Gary Stacey, Kristin D. Bilyeu, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211; Kristin D. Bilyeu, USDA-ARS, Plant Genetics Research Unit, Columbia, Missouri 65211. Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA or the University of Missouri and does not imply its approval to the exclusion of other products or vendors that may also be suitable. The US Department of Agriculture, Agricultural Research Service, Midwest Area, is an equal opportunity, affirmative action employer and all agency services are available without discrimination. Received ________. *Corresponding author ([email protected]) Abbreviations: FN, fast neutron; IP4, inositol (1,4,5,6) tetrakisphosphate; IP5, inositol (1,3,4,5,6) pentakisphosphate; IPK1, inositol pentakisphosphate 2-kinase; LPA, low phytic acid; m, mutant; P, phosphorus; PA, phytic acid; PA-P, phytic acid phosphorus; PCR, polymerase chain reaction; Pi, inorganic phosphorus; SAS, statistical analysis software; SNP, single nucleotide polymorphism; SPT, single plant thresh; W, wild type The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Abstract Soybean [Glycine max (L.) Merr] seeds contain a large amount of phosphorus (P), which is stored as phytic acid (PA). PA is indigestible by nonruminant livestock and considered an anti-nutritional factor in soybean meal. Several low PA soybean lines have been discovered, but many of these lines have either minor reductions in PA or inadequate germination and emergence. The reduced PA phenotype of soybean line Gm-lpa-ZC-2 was previously shown to be the result of a mutation in a gene encoding an inositol pentakisphosphate 2-kinase on chromosome 14 (14IPK1). While the 14IPK1 mutation was shown to have no impact on germination and emergence, the reduction in PA was modest (up to 50%). Our objective was to determine the effect on seed P partitioning for a novel mutation of an independent IPK1 gene on chromosome six (06IPK1) on its own and in combination with mutant alleles of the 14IPK1. We developed soybean populations and conducted genotype and phenotype association analyses based on the genotype of the 06IPK1 and 14IPK1 genes and the seed P partitioning profile. The lines with both mutant IPK1 genes had very low PA levels, moderate accumulation of inorganic phosphate, and accumulation of high amounts of P in lower inositols. The developed lines did not have significant reductions in germination or field emergence. In addition, characterization of the lower inositols produced in the mutant lines suggests that IPK1 is a polyphosphate kinase and provides some insight into the PA biosynthesis pathway in soybean seeds. Page 2 of 35 Page 3 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Introduction Inositol hexakisphosphate, otherwise known as phytic acid (PA), is a major component of mature soybean seeds, roughly 2%, and it is about 75% of the total phosphorus (P) found in soybean seeds (Raboy et al., 1984). The rest of the P in soybean seeds is in the form inorganic phosphate (Pi) and cellular components, such as protein and nucleotides (Raboy et al., 2001). PA is an important component of seedling germination because phytase hydrolyzes some of the PA-P (PA phosphorus) in order to release Pi, which is utilized by soybean seedlings for energy and nutrients (Urbano et al., 2000). The PA-P in mature soybean seeds, which are fed to livestock and humans, is unavailable for use by nonruminant livestock because phytase enzymes are not present in the digestive tract for breakdown of PA and release of Pi (reviewed in BrinchPedersen et al., 2002). Research to develop low phytic acid (lpa) and high Pi soybean seeds is an ongoing objective that must deliver improved nutritional quality seed meal without introducing any negative agronomic qualities to the seed such as reduced field emergence. Multiple independent sources of lpa soybean lines were discovered and characterized (Sebastian et al., 2000; Wilcox et al., 2000, Yuan et al., 2007; Maroof and Buss, 2011). Further investigation into the genetics of these lines identified mutations in genes encoding enzymes in the PA biosynthesis pathway and PA transporter genes (Gillman et al., 2013; Gillman et al., 2009; Hitz et al., 2002; Yuan, et al., 2012). Unfortunately, many soybean lpa mutants have significantly reduced germination and emergence (Anderson and Fehr, 2008; Maupin and Rainey, 2011; Maupin et al., 2011; The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Meis et al., 2003; Yuan et al., 2007). However, initial characterization of the soybean mutant, Gm-lpa-ZC-2, with a novel lpa mutation, led to the discovery of no significant emergence or germination issues (Frank et al., 2009b; Yuan et al., 2007; Yuan et al., 2009). Gm-lpa-ZC-2 was determined to have a reduction of about 20-50% of PA as well as a proportional increase of Pi and lower inositols (Frank et al., 2009a; Yuan et al., 2007). Characterization of the metabolite profile of Gm-lpa-ZC-2 indicated that there were no decreases in nutritional value (Frank et al., 2009b). Eventually, it was discovered that a mutation in an inositol (1,3,4,5,6)-pentakisphosphate 2-kinase, or IPK1, gene was responsible for the lpa phenotype in Gm-lpa-ZC-2 (Yuan et al., 2012). IPK1 (E.C. 2.7.1.158, inositol-pentakisphosphate 2-kinase) is the enzyme that adds the last phosphate on the inositol ring to create PA (reviewed in York et al., 1999). Based on in vitro experiments with maize and potato IPK1 recombinant enzymes and work in Arabidopsis, the plant IPK1 gene appears to have the capacity to encode an inositol polyphosphate kinase, with lower inositol phosphates also capable of being substrates in addition to inositol-pentakisphosphate (Caddick et al., 2008; StevensonPaulik et al., 2005; Sun et al., 2007; Sweetman et al., 2006). Soybean is derived from ancient genome duplication events, and the existence of small gene families with members present on different chromosomes provides the opportunity for expressed genes encoding proteins that can be functionally redundant with each contributing to the total enzyme activity for a biochemical reaction step (Schmutz et al., 2010). Alternatively, the independent homologous genes in a gene family can diverge and subfunctionalize by expression or activity. Page 4 of 35 Page 5 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 There are three independent IPK1 genes in the soybean genome on chromosomes 14, 6, and 4 [Glyma14g07880, Glyma06g03310, and Glyma04g03240 in Glycine max v1.1 (Yuan et al., 2012)]. The IPK1 gene on chromosome 14 showed the highest IPK1 gene expression in immature soybean seeds, while the other two genes on chromosome 6 and 4 showed very low expression (Yuan et al., 2012). The soybean line Gm-lpa-ZC-2 contains a splice-site mutation in the chromosome 14 IPK1 gene, resulting in one nonfunctional IPK1 of the three IPK1 genes present (Yuan et al., 2012). Even though the chromosome 14 IPK1 gene was the highest expressed of the three genes in developing seeds, the null alleles of the chromosome 14 IPK1 gene in soybean line Gm-lpa-ZC-2 only reduced the PA content by up to approximately 50% compared to wild-type seeds; therefore, the two remaining IPK1 soybean genes on chromosome 6 and 4 become candidate genes for reverse genetics experiments to either independently reduce PA levels or work in combination with the chromosome 14 IPK1 mutation to create more dramatic reductions in seed PA. In this work, we identified a fast neutron induced soybean line that has a partially deleted IPK1 gene on chromosome 6 (Glyma06g03310). Our goal was to characterize the effect of the chromosome 6 IPK1 mutation on its own and in combination with the Gm-lpa-ZC-2 chromosome 14 IPK1 mutation in terms of P partitioning as well as germination and emergence. In addition, by characterizing the accumulated lower inositol forms, we were able to provide more evidence that IPK1 is an inositol polyphosphate 2-kinase and that soybean seed PA biosynthesis can proceed independently of one proposed route of the lipid-dependent pathway (Stevenson-Paulik et al., 2005). The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Materials and Methods FN irradiation In the fall of 2007, 70,000 Williams 82 seeds were treated with 30 Gy fast neutrons at McClellan Nuclear Radiation Center (University of California Davis, Davis, CA). Seeds were sent to grow in Costa Rica for one generation, and in May 2008, approximately 9,000 single plant thresh (SPT) packets were returned to Columbia, Missouri as M2 seeds. 4096 rows of M2 lines, each representing one M1 plant were grown in the field in 2008. Visual evaluation of M2 plants in the field led to the discovery of one line segregating for a multifoliate phenotype. Two multifoliate plants from the same line were single plant threshed at maturity, and several M3 seeds of one of those multifoliate M2 plants were utilized to characterize the soybean genome. CGH Comparative Genomic Hybridization (CGH) was performed on DNA from a single plant designated FN38 as described by Bolon et al. (2011). NimbleGen soybean CGH microarray, which consists of 696,139 unique oligonucleotide probes (50- to 70mers) were designed from the reference sequence, Williams 82, and spaced at approximately 1.1 kilobase (kb) intervals (platform details can be found in Gene Expression Omnibus accession number GPL11198 at www.ncbi.nlm.nih.gov/geo/). Mutant (Cy3 dye) and reference (Cy5 dye) labeling reactions were performed with 1 µg each of genomic DNA from FN38 (mutant) and Williams 82 (wild-type) leaf tissue samples. Mutant and wild- Page 6 of 35 Page 7 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 type DNA were labeled, quantified, and then hybridized for 72 hours at 42°C on the CGH microarrays. Plant material and segregating population development FN38 characterization of Glyma06g03310 alleles A segregating population was developed by crossing FN38 and ‘Jake’ (Shannon et al., 2007). Jake was used as the female, and it is a mid-group V, registered plant line that was received from Dr. Grover Shannon (Division of Plant Sciences, University of Missouri Columbia-Delta Center). F1 seeds were produced at South Farm in Columbia, Missouri during the summer of 2011. F1 plants were grown in a growth chamber with 14 hour days at 28°C and 10 hour nights at 22°C in the winter of 2011-2012. F2 seeds were subsequently planted at South Farm in the summer of 2012. Individual F2 plants were identified by number and observed for the FN38 multifoliate leaflet phenotype. F2:3 seeds were collected by single plant threshing 20 out of 27 plants; the other 7 plants were not threshed due to death before maturity. Generation and field experiments for the 2-kinase population A segregating population, designated 2-kinase, was developed by crossing FN38 and Gm-lpa-ZC-2, which are homozygous mutant for Glyma06g03310 and Glyma14g07880, respectively. F1 seeds from the 2012 crosses were sent to our winter nursery in Upala, Costa Rica and planted for advancement. In the subsequent generation, individual F2 plants were tagged and leaflets were sampled for DNA on Whatman FTA (Whatman, Clifton, NJ) cards for genotyping assays conducted in our lab at the University of Missouri, Columbia, MO. Punches of FTA leaf presses were used The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 as templates to determine the IPK1 (chromosome 14 and chromosome 6) genotype. Selected F2 lines for each genotypic class were harvested and single plant threshed (SPT). There were 5 F2 plants for the double mutant class, and two plants each were selected for the other genotypic classes. For the parental lines, five plants each were grown out with the F2 generation, and after harvest, parental lines were SPT. For phenotypic analysis of the Costa Rica seeds, 15 F2:3 seeds were lyophilized and ground per line, including the parental lines. From each line, including the parental lines, 25 mg and 10-15 mg were sampled from the ground samples for PA and Pi analysis, respectively. Seeds of the four selected classes were grown at South Farm in Columbia, Missouri during the summer of 2013 using a complete randomized block design. There were three replicates for the 13 different soybean lines of the various genotypic classes, including parental lines. Each plot consisted of 10 seeds planted per line, and plots with no emergence were replanted 16 days after the initial planting date. The population was bulk harvested by plot, with every line and replication distinguished. Seed samples were prepared for phenotypic analyses by lyophilizing and grinding a 15-seed F2:4 sample of each plot. Two lines in all 3 reps, a 14W/06W and 14m/06m did not germinate or did not live to maturity due to disease, so that the experiment size was reduced by two lines. Genotypic analysis of the sequence around the FN38 deletion FN38 and Williams 82 single seeds were powdered in liquid nitrogen for DNA isolation using the DNeasy Plant Mini Kit (Qiagen Sciences Inc., Germantown, MD). Primers were designed to the region left of the deletion CGH start point and the right of Page 8 of 35 Page 9 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 the deletion CGH end point using Williams 82 as a reference at Phytozome (www.phytozome.net), and the primers were manufactured through IDT (Coralville, IA). Primers were carefully designed to target and amplify various size products on chromosome 6 due to a highly similar region on chromosome 4 of soybean. Primers are listed in Table 1 (pairs for L1, L2, L3, R1, R2, and R3) and were used at a final concentration of 0.5 µM. Reactions were carried out in 20 µL final volume containing 550 ng DNA template, primers, buffer (40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgCl2, 3.75 µg mL‐1 BSA, 200 µM dNTPs), and 0.2X Titanium Taq polymerase (BD Biosciences, Palo Alto, CA) with the following conditions: 95°C for 5 minutes followed by 40 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. An agarose gel, 1.2% concentration, was used to determine the presence of PCR product. IPK1 (Glyma14g07880) molecular marker assay Primer sequences and causative SNP location obtained by Yuan et al. (2012) for the splice site mutation in the IPK1 (Glyma14g07880) gene were utilized in developing a SimpleProbe assay for genotypic discrimination between wild-type, heterozygous, and mutant alleles. The SimpleProbe (Flourescentric, Inc. ,Park City, UT) was designed using Roche Applied Science LightCycler Probe Design software 2.0 (version 1.0, February 2004) to match the wild-type sequence in the 14IPK1 gene. Primers were mixed in a 5:2 asymmetric CTCAGCTTCACCCCTTTC-3’ ratio and (final final concentration concentration 0.5 0.2 µM µM IPK1f: 5’- IPK1r: 5’- CTAACTCAGATTTAATGCC-3’), with the SimpleProbe at a final concentration of 0.2 µM (IPK1: Fluorescein-SPC-GTAGGATGTACCTCTCCTTGAAGCAATTTC-Phosphate). Page 10 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Reactions were carried out in 20 µL final volume containing 5-50 ng DNA template, primers, buffer (40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgCl2, 3.75 µg mL‐1 BSA, 200 µM dNTPs), SimpleProbe, and 0.2X Titanium Taq polymerase. The amplification and melt curve were carried out in a Roche480 Light Cycler according to this reaction: 95°C for 3 minutes followed by 45 cycles of 95°C for 20 seconds, 51°C for 20 seconds, and 72°C for 30 seconds. A melt curve was performed by reading every 0.2°C for 1 second from 50°C to 75°C in order to visualize the properties of the products. Lines containing wild-type 14IPK1 alleles have a peak present at 65°C, while mutant (Gm-lpa-ZC-2 derived splice site) 14IPK lines have a peak present at 59°C; thus, heterozygous lines have two peaks at 59°C and 65°C. IPK1 (Glyma06g03310)-linked molecular assay We sought to identify a SNP tightly linked to the IPK1 (Glyma06g03310) deletion, since a deletion PCR assay could not distinguish plants hemizygous for the deletion from those that were homozygous wild-type for the chromosome 6 region. Based on whole genome resequencing data in various soybean genomes by Lam et al. (2010), we identified a SNP between FN38 and Gm-lpa-ZC-2 positioned in the third exon of Glyma06g03310 at 2,341,942. The SNP causes a V150L missense change in a nonconserved amino acid position in the Gm-lpa-ZC-2 06IPK1. The linked SNP was used to develop a SimpleProbe molecular marker assay. Primers were mixed in a 2:5 asymmetric ratio (final concentration 0.2 µM IP52Kf: 5’-GCATGAGAGGTAAGAG -3’ and final concentration 0.5 µM IP52Kr: 5’- GCCAAGGGAATTTCTCG -3’), with the SimpleProbe at a final concentration of 0.1 µM (IP52K: Fluorescein-SPC-GGCATTTCAATACCATGAGTGAAGACTGA-Phosphate). Page 11 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Reaction conditions were as above with thermocycling parameters: 95°C for 3 minutes followed by 45 cycles of 95°C for 20 seconds, 56°C for 20 seconds, and 72°C for 20 seconds. A melt curve was performed by reading every 0.2°C for 1 second from 52°C to 77° The wild-type (FN38) peak was present at 65°C, while the alternate (Gm-lpa-ZC2) peak was at 60°C; thus, the heterozygous peaks were at 60°C and 65°C. Germination and emergence The germination experiment was performed using a version of the rag doll test (http://edis.ifas.ufl.edu/ag182). Three sheets of germination paper were wet with double de-ionized water. Excess water was allowed to drip off before laying two sheets flat and placing 25 F2:4 seeds from each replication grown in Columbia, Missouri on the sheets. The other sheet of germination paper was placed on top of the seeds, and the sheets were then rolled into a tube and placed onto a tray. The tray was placed into an incubator at 28°C and kept moist. The seeds were examined after 3 days, and those seeds with a hypocotyl were identified as germinated seedlings. The emergence experiment was conducted at South Farm in Columbia, Missouri in 2014. Fifty F2:4 seeds from each replication grown in Columbia, Missouri were hand planted into a 48 inch row into prepared seed beds utilizing a complete randomized block design. Emergence was scored seven days after planting. Pi quantification by colorimetric assay Seed inorganic phosphate was quantified as described by Gillman, et al (2009). PA, IP5, and IP4 quantification by HPLC The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Page 12 of 35 An HPLC method was used to quantify seed PA-P (Chen and Li, 2003). A 25 mg lyophilized ground seed sample was combined with 0.5 mL extraction buffer (500 mM HCl) shaken for 1 hour at room temperature, and centrifuged at 20,000 g for 15 minutes. The supernatant was filtered through a 0.22-micron filter, and 75 µL of filtrate was analyzed by ion chromatography using a Dionex CarboPac PA-100 guard column and a CarboPac PA-100 analytical column on a Dionex ICS-5000 chromatography system with post column derivitization (Thermo Scientific Dionex, Waltham, MA). The linear elution gradient was effected by two eluents: deionized water and 0.5 M HCl; time 0 min, 8% 0.5 M HCl; time 30, 100% 0.5 M HCl; time 35, 100% 0.5 M HCl; time 35.1, 100% 0.5 M HCl; time 40, 8% 0.5 M HCl. A post-column derivitization was achieved with a solution of 1 g L-1 Fe(NO3)3 in 0.33 M HClO4 using a 750-mL knitted coil and was followed by detection at 295 nm. Flow rates of eluent and post-column solution were 1.0 and 0.4 mL minute-1, respectively. PA standard (PA dipotassium salt; Sigma), at 1 mM concentration, eluted at 29.8 minutes, and a standard curve was calculated from serial dilutions of 4 mM PA standard. Results were converted to µg PA-P mg seed-1. I(1,3,4,5,6)P5 (inositol pentaphosphate ammonium salt; Cayman Chemical, Ann Arbor, Michigan) and I(1,4,5,6)P4 (inositol tetraphosphate sodium salt; Cayman Chemical) standards, at 1 mM concentration, eluted at 22.5 and 15.2 minutes, respectively, and a standard curve was calculated from serial dilutions of 1 mM. Results were converted to µg IP5-P mg seed-1 and µg IP4-P mg seed-1. Elution times for I(1,3,4,5)P4 and I(1,3,4,6)P4 (A.G. Scientific, San Diego, CA) were 11.6 and 10.7 minutes, respectively. I(3,4,5,6)P4 (A.G. Scientific) eluted with the same retention time as I(1,4,5,6)P4. Page 13 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Statistical analysis All statistical analyses were carried out using the SAS 9.3 software (SAS Institute Inc., Cary, NC). All lines were grouped into 6 categories based on their genotype with the parents separate. For Costa Rica produced seed, it was a completely randomized design, while Missouri was replicated three times in randomized complete block design. Basic statistic parameters for each data set were obtained using the MIXED procedure using either a single- or double-factor ANOVA. For Costa Rica, categories and lines were the classes used, with lines randomized and least square means determined for categories. For, Missouri, another class was used, designated block, due to the replication. For IP5 and IP4, categories with a value of 0 were deleted before analysis; however using the F value, the other categories were able to be identified as statistically different than 0. Page 14 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Results Characterization of deleted IPK1, and other genes, in FN irradiated line A mutant population of fast neutron-treated Williams 82 plants was developed for use in reverse genetics research, but one line was selected for further genomic characterization based on its multifoliate phenotype (data not shown). Several M3 seeds of a multifoliate plant (named FN38) were used as source DNA in Comparative Genomic Hybridization (CGH) for analysis of deletions in the genome. For line FN38, one region on chromosome 6 showed evidence of an almost 90,000 bp deletion (2,252,581 to 2,339,716 Glycine max v1.1). This deletion encompassed eleven genes including at least the 3’ portion of the IPK1 homolog on chromosome 6, Glyma06g03310.9 (Table 2). In the Williams 82 Glycine max v1.1 sequence, the IPK1 gene on chromosome 6 (Glyma06g03310.9) is present as seven exons in the antisense orientation on the chromosome spanning positions 2,338,844 – 2,342,668. Based on detection of PCR amplification products around the putative deletion, we expected that roughly the first three exons of the IPK1 gene (Glyma06g03310.9) were still present in the genome of line FN38. However, the deleted region appeared to abolish the final (seventh) IPK1 exon with the fourth, fifth, and sixth exons in a region of uncertainty (Figure 1). To analyze the effect on P partitioning in seeds for soybeans containing a deletion encompassing part of the chromosome 6 IPK1 gene (Glyma06g03310; Page 15 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 hereafter abbreviated 06IPK1) we analyzed the seed P phenotype and the chromosome 6 deletion genotype in a segregating population developed by crossing FN38 (homozygous for deleted 06IPK1) and Jake (homozygous for presumably functional, wild-type 06IPK1). For field grown FN38, Jake, and the 20 plants in the population, there were no statistically significant differences for PA-P or Pi-P among any of the lines compared to each other or compared to the reference cultivar Williams 82 (data not shown). Combinations of two independent mutant IPK1 alleles A splice-site mutation in the chromosome 14 IPK1 (Glyma14g07880) resulted in altered P partitioning in seeds of the soybean line Gm-lpa-ZC-2 (Yuan et al., 2012). Even though the FN38 deletion of 06IPK1 did not appear to alter P partitioning on its own, we hypothesized that further decreased PA and increased Pi could be obtained for lines containing both the 06IPK1 and chromosome 14 IPK1 (14IPK1) mutations. Thus, a segregating population, designated 2-kinase, was developed by crossing FN38 and Gm-lpa-ZC-2. Soybean lines FN38 and Gm-lpa-ZC-2 each contain homozygous mutant alleles for one IPK1 homolog, 06IPK1/Glyma06g03310 or 14IPK1/Glyma14g07880, respectively. Genotyping assays were developed to accurately score the allele status for Glyma14g07880 and Glyma06g03310 (see Materials and Methods). This second population was developed and assessed in both Columbia, Missouri and Upala, Costa Rica. In Costa Rica, F2 plants were genotyped for the allele status of both of the two IPK1 genes, and genotypic selections were made based on homozygosity at both IPK1 loci to identify at least two plants in the population containing one of the four possible homozygous genotypes for 14IPK1 and 06IPK1. In Columbia, The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Page 16 of 35 Missouri, replicated plots of F2:3 plants were grown, and bulked F2:4 seeds were analyzed for the different genotypic classes. The four selected genotypic classes based on the Glyma14g07880 and Glyma06g03310 allele status were: wild-type/wild-type (14W/06W), wild-type/mutant (14W/06m), mutant/wild-type (14m/06W), and the double mutant (mutant/mutant; 14m/06m). The Gm-lpa-ZC-2 (14m/16W) and FN38 (14W/06m) parents of the population were grown along with the populations. Phosphorus partitioning in seeds of the 2-kinase population produced in two locations For the Costa Rica and Missouri environments, we analyzed Pi as well as PA and lower inositols (IP5 and IP4) for the soybean seeds in the four genotypic classes based on the chromosome 14 and chromosome 6 IPK1 alleles. There were major, biologically significant differences in the accumulation of each P-component for the different genotypic classes in either Missouri or Costa Rica (Table 3). Pi-P is typically low in soybean seeds, and it had been shown to be increased in chromosome 14 IPK1 mutant line Gm-lpa-ZC-2 (Yuan et al., 2012). Our results confirmed the heritability of significantly increased Pi-P in 14m/06W lines compared to lines inheriting all wild-type alleles of IPK1, 14W/06W (approximately a three-fold increase); in addition, we confirmed our results from the first population that showed the chromosome 6 IPK1 mutants have no effect on Pi-P on their own. However, the double mutant genotypic class (14m/06m) showed the highest Pi-P levels, representing approximately a seven to eight-fold increase in Pi-P for the double mutant lines compared to lines inheriting all wild-type alleles of IPK1. Page 17 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 The PA-P results showed a dramatic reduction in PA-P for the double mutant 14m/06m class (Table 3). All of the other genotypic classes were either not statistically different from one another (Costa Rica) or showed a slight but significant reduction in PA-P for the 14m/06W lines (Missouri). The double mutant 14m/06m class contained approximately 22% and 15% of the PA-P as the average of the other genotypic classes in Costa Rica and Missouri, respectively. There were no significant differences for Pi-P or PA-P for the different genotypic classes between the Costa Rica and Missouri environments (data not shown). Since inositol (1,3,4,5,6) pentakisphosphate (IP5) is the substrate for IPK1, we wanted to investigate the accumulation of lower inositols in the different genotypic classes (Table 3). Lower inositols (IP5 and IP4) accumulated in genotypic classes that contained the chromosome 14 IPK1 mutant alleles (14m/16W and 14m/06m). There were significant environment effects for the lower inositols (data not shown). For IP5, the double mutants (14m/06m) accumulated the highest levels of IP5-P in both locations. Lines in the 14m/16W genotypic class produced lower but detectable levels of IP5-P. For inositol-tetrakisphosphate (IP4), the double mutant class 14m/06m accumulated the highest levels of IP4-P in Costa Rica. In Missouri, the 14m/06m class had a high accumulation of IP4-P, but it was not significantly different than the two 14m/16W classes for IP4-P. Overall, the soybean seeds from the double mutant class 14m/06m produced a characteristic P partitioning profile compared to wild-type and other lpa soybean lines (Figure 2). The main features of P partitioning in 14m/06m lines compared to other soybean lpa lines were very low PA-P levels, moderate accumulation of Pi-P, and the Page 18 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 accumulation of high amounts of P in the lower inositols IP4 and IP5. The severe reduction in PA-P sets the double mutant 14m/06m class apart from the 14m/06W class. The striking accumulation of P in lower inositols rather than Pi-P distinguishes the double mutant 14m/06m class from the soybean lpa1-lpa2 transporter mutant combinations (Gillman et al., 2013; Yuan et al., 2007). Identification of the accumulated IP4 Lines containing the Glyma14g07880 IPK1 mutant alleles had an increase in lower inositols, while lines wild-type for this gene, such as FN38, had no detectable levels of lower inositols. synthesizing PA is Previous research has confirmed that the last step to phosphorylating the 2-position on inositol (1,3,4,5,6) pentakisphosphate by the enzyme inositol-pentakisphosphate 2-kinase, IPK1 (reviewed in Brearley and Hanke, 1996). It is currently unclear whether seed PA is synthesized through a lipid-dependent or lipid-independent pathway (Raboy, 2003; StevensonPaulik et al., 2005). The inositol-tetrakisphosphate intermediate in the lipid-independent pathway is I(3,4,5,6)P4, but for the lipid-dependent pathway the intermediate is I(1,4,5,6)P4 with I(1,3,4,5)P4 and I(1,3,4,6)P4 as possible lipid-dependent interconversion products (Stevenson-Paulik et al., 2005). Since inositol-tetrakisphosphate, along with inositol (1,3,4,5,6) pentakisphosphate, were the dominant lower inositols, 1 mM standards of four inositol tetrakisphosphates [I(1,3,4,6)P4, I(1,3,4,5)P4, I(3,4,5,6)P4, and I(1,4,5,6)P4] were analyzed by high performance liquid chromatography (Figure 3). Based on the retention time of the peak in Gm-lpa-ZC-2 (14m/06W) and the lines with a mutated Page 19 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Glyma14g07880 IPK1 (14m/06W and 14m/06m), it was determined that the accumulated inositol-tetrakisphosphate was I(1,4,5,6)P4 or I(3,4,5,6)P4 (Figure 3). Germination and field emergence For soybeans, alterations in seed PA accumulation have been correlated with reduced germination and emergence. The F2:4 seeds produced in Missouri from the 2kinase population were analyzed for germination and field emergence. The scores for all of the genotypes were above 80% for standard germination, and none of the genotypic classes were statistically different than one another (data not shown). There was significant variation in field emergence per line, but the double mutant class as a whole was not significantly lower emerging than either of the single mutant classes (data not shown). The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Page 20 of 35 Discussion Initial characterization indicated that soybean line FN38 contained a genomic deletion encompassing at least part of a homologous IPK1 (Glyma06g03310), so P partitioning was analyzed to determine if the null allele of the chromosome 6 IPK1 could reduce PA like it’s counterpart mutant IPK1 on chromosome 14 in soybean line Gm-lpaZC-2. The partial deletion of the IPK1 gene in FN38 did not increase seed Pi-P or decrease PA-P compared to the seeds of a registered cultivar line. These results are consistent with expression data reported by Yuan et al. (2012), in which Glyma14g07880 had higher expression in soybean seeds compared to the other two homologous genes (Glyma06g03310 and Glyma04g03240). However, combining the two mutant IPK1 genes produced an enhanced lpa phenotype with a unique P partitioning profile. Our double mutant lines (14m/06m) produced seeds with very low PA and up to 3.2 µg P mg-1 seed in the lower inositols IP5 and IP4. The environment had an effect on the accumulation of lower inositols but not on PA-P or Pi-P, so understanding the variation in the lower inositols in additional environments will be important to further characterizing the lpa trait in these new IPK1 double mutants. In addition, the effect of such high accumulation of IP5 and IP4 will need to be evaluated for nutritional value in soybean meal. Interestingly, a series of studies identified IP5 as a potential anti-cancer compound because it can block the activation of a critical human enzyme which plays a key role in cell survival and proliferation (reviewed in Falasca, 2009). Even though the RNA expression of the 06IPK1 gene was shown to be low in developing seeds (Yuan et al., 2012), this work demonstrated clearly that the encoded Page 21 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 06IPK enzyme is contributing (along with 14IPK1) to the total IPK1 enzyme activity in developing seeds. Functional redundancy due to ancient genome duplication resulting in two- to four- member homologous gene families has been fairly well documented in soybean, particularly in seed composition research. The subtle contributions of additional gene family members have been previously revealed by re-mutagenesis of lines originally found to contain a desirable phenotype (Reinprecht et al., 2009). The genetic redundancy identified here is in contrast to the soybean seed lpa phenotype controlled almost entirely by the MIPS1 gene, despite the fact that three additional closely related MIPS genes are present in the soybean genome (Chappell et al., 2006; Hitz et al., 2002; Yuan et al., 2007). In the present case, the mutation of the 14IPK1 gene alone has a desirable phenotypic effect, while mutation of the 06IPK1 gene only reveals a phenotypic effect when combined with the 14IPK1 mutation. However, the double mutant lines produced an improved P partitioning profile with significantly less PA and more available P than the 14IPK mutation alone. The germination and emergence of the 14m/06m seeds produced in our temperate environment did not reveal any obvious defects. We attempted to assess the emergence capacity of seeds produced in the Costa Rica environment, but uneven destruction of germinated seedlings by pests prevented the analysis. Further research will be necessary to identify and more fully characterize any negative effects of the altered P partitioning profile in the IPK1 double mutants on field emergence. Besides soybean, IPK1 mutations have been characterized in maize and Arabidopsis. In Arabidopsis, multiple putative IPK1 genes were identified in the genome, but only one (atipk1; At5g42810) appeared to contribute to seed IPK1 enzyme Page 22 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 activity (Kim and Tai, 2011; Stevenson-Paulik et al., 2005). However, mutation of At5g42810 only reduced seed PA levels by about 50%; when the atipk1 mutation was combined with an I(1,3,4)P3 6-/3-/5- kinase (AtIPK2-β; At5g61760) mutation, seed PA was reduced to near undetectable levels (Stevenson-Paulik et al., 2005). Along with a large accumulation of I(1,3,4,5,6)P5, the atipk1-1 mutant accumulated a mixture of IP4s, including I(3,4,5,6)P4, I(1,3,4,6)P4, and I(1,4,5,6)P4 in seeds. In maize, a reverse genetics method was used to target mutation in the maize IPK1 gene, one of two genes in the maize genome sharing 98% sequence identity in the coding regions (Shukla et al., 2009; Sun et al., 2007). Maize IPK1 mutants demonstrated a distribution of lpa phenotypes consistent with at least a partial reduction of the total IPK1 enzyme activity in developing seeds (Shukla et al., 2009). Characterization of the lower inositols in the IPK1 mutant maize seeds was not presented (Shukla et al., 2009). The biochemical pathway resulting in the production of PA in plant seeds still has many steps that have eluded characterization. However, both in vitro characterization of recombinant IPK1 enzymes and the analysis of an increasing collection of IPK1 mutants suggest that plant IPK1 genes encode a polyphosphate kinase that can utilize potentially different IP3 and IP4 species as well as I(1,3,4,5,6)P5 (Caddick et al., 2008; Stevenson-Paulik et al., 2005; Sun et al., 2007; Sweetman et al., 2006). Since I(1,3,4,5,6)P5 and IP4 species accumulated in the soybean IPK1 mutants, our results are consistent with soybean IPK1s acting as polyphosphate kinases. Two lipid-dependent routes and a lipid-independent route for PA synthesis in seeds have been proposed (Stevenson-Paulik et al., 2005). Our results narrow down the possible pathways to one lipid-dependent [through I(1,4,5,6)P4] and one lipid-independent [through I(3,4,5,6)P4], Page 23 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 while ruling out the second lipid-dependent pathway [through I(1,3,4,5)P4 or I(1,3,4,6)P4]. The identification of new lpa crops may lead to a better understanding of both the importance of PA to seeds and provide crops with enhanced nutritional qualities for foods and feeds. Acknowledgements We would like to thank the Paul Little and Christi Cole for their superb technical assistance. This research was supported by the United Soybean Board. We would also like to thank Pengyin Chen (University of Arkansas) for his generous sharing of Gm-lpa-ZC-2 seed. Page 24 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 References Anderson B.P., and W.R. Fehr. 2008. Seed source affects field emergence of lowphytate soybean lines. Crop Sci 48:929-932 Bolon Y.T., W.J. Haun, W.W. Xu, D. Grant, M.G. Stacey, R.T. Nelson, D.J. Gerhardt, J.A. Jeddeloh, G. Stacey, G.J. Muehlbauer, J.H. Orf, S.L. Naeve, R.M. Stupar, and C.P. Vance. 2011. Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean. 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Schematic representation for the deletion region and IPK1 gene on FN38 chromosome 6. A. A portion of chromosome 6 present in soybean line FN38 is represented by the solid black horizontal line interrupted by gray lines representing deleted regions. CGH detected probes were deleted from position 2,252,581 to 2,339,716, represented here as solid gray ovals on solid gray chromosome lines (condensed relative distance with hash marks). Connecting the known present chromosome region to the deleted are solid and dashed gray lines representing deletion of the chromosome confirmed by PCR, and uncertain chromosome regions, respectively. L1, L2, L3, R1, R2, and R3 represent the approximate positions of the PCR amplicons, with black font representing amplicons positive for PCR and gray font representing no PCR amplification for FN38 DNA. A scale is given under the right side of the deletion indicating chromosome positions from 2,339,000 to 2,342,000, which also is the approximate position of IPK1 (Glyma06g03310) in the antisense orientation on the chromosome. B. Representation of the sense orientation of the IPK1 (Glyma06g03310) gene on chromosome 6. Exons are represented as boxes connected by lines representing introns. Black lines indicate the presence of the region on the chromosome, dashed gray lines represent uncertain chromosome regions, and solid gray lines indicate deletion of the chromosome confirmed by PCR. The chromosome position of the start codon and stop codon are indicated above the gene structure. The approximate position of the R1, R2, and R3 amplicons is indicated below the gene structure, with black font representing amplicons positive for PCR and gray font representing no PCR amplification for FN38 DNA. The positive sign above the third exon represents the position of the SNP used for linkage of the deletion between FN38 and Gm-lpa-ZC-2. Figure 2. Comparison of relative seed phosphorus partitioning in a selection of low phytic acid soybean lines. Each stack totaling 100% of the measured P component data (Pi-P, PA-P, IP5-P, and IP4-P) is from different genotypic classes from this work (from the Missouri environment) as well as three lpa1/lpa2 combinations from Gillman et al., also from a Missouri environment (Gillman et al., 2013). 14W/06W is equivalent to typical wild-type soybean lines. 14m/06W is the genotypic class representing the mutation in Gm-lpa-ZC-2. lpa1a2a indicates lines with null alleles of lpa1 and missense alleles of lpa2; lpa1b2b indicates lines with mis-splicing alleles of lpa1 and null alleles of lpa2; and lpa1a2b indicates lines with null alleles of both lpa1 and lpa2 (Gillman et al., 2013). Page 29 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Figure 3. Separation of inositol phosphates and identification of I(1,4,5,6) or I(3,4,5,6) tetrakisphosphate in soybean seeds with mutations in the chromosome 14 IPK1 (Glyma14g07880). Peaks are identified by retention time as PA, I(1,2,3,4,5,6)P6; IP5, I(1,3,4,5,6,)P5; and several IP4s. A. The output from an ion chromatography run with an injection of 1 mM I(1,3,4,5)P4. B. The output from an ion chromatography run with an injection of 1 mM I(1,3,4,6)P4. C. The output from an ion chromatography run with an injection of 1 mM I(1,4,5,6)P4. I(3,4,5,6)P4 had the same retention time as I(1,4,5,6)P4 (15.2 minutes) and the IP4 present in seed samples. D. The output from an ion chromatography run of Missouri-produced Gm-lpa-ZC-2 (14m/06W) seed. E. The output from an ion chromatography run of Missouri-produced double mutant (14m/06m) seed. Page 30 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Table 1. Primers used to characterize FN38 deletion. Primer Set L1 L2 L3 R1 R2 R3 Forward (5’-3’) CATGATGAGCTTACCCTACTCC AACCTAACTCCACTGTATATCGGAA GGTCGGATTTTTCATCTTGA CAACTACATATGGTGCCGGTT GGAAGGTATAAGAGTGAGAAGC GTAACTGTAACAAATCCTGCTAAAAC Primers are from 5' to 3'. 5’ Position 2250527 2251670 2253292 2341585 2339740 2338825 Reverse (5’-3’) CAAGTCATTCTGGTCTCAGTGTT ATGTTACGTATTCTCGAATAGCAA TCAGTATGCTTGCTGGTTGC ATTATGAATCTCTTACCTCTCATG GTTCAGCTTCTGCTGGTAGG TGCTCTGGAATGAGTGGACA 5’ Position 2250707 2252012 2253547 2341796 2340141 2339173 Product Size 181 343 256 212 402 349 Page 31 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Table 2. Gene identification located within the FN38 deletion identified by comparative genomic hybridization. CGH ID FN38 FN38 FN38 FN38 FN38 FN38 FN38 FN38 FN38 FN38 FN38 Chromosome Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Glyma06 Start Codon 2252754 2259294 2262571 2270034 2274777 2286289 2301058 2305620 2316535 2330907 2342668 Gene Orientation + + + + + + + - Glyma ID Glyma06g03200 Glyma06g03210 Glyma06g03220 Glyma06g03230 Glyma06g03240 Glyma06g03250 Glyma06g03270 Glyma06g03280 Glyma06g03290 Glyma06g03300 Glyma06g03310 Best Arabidopsis TAIR10 hit define BEL1-like homeodomain 3 BEL1-like homeodomain 11 UDP-Glycosyltransferase superfamily protein regulatory particle triple-A ATPase 4A RING/U-box superfamily protein basic leucine-zipper 44 mitogen-activated protein kinase 1 Clathrin adaptor complexes medium subunit family protein HXXXD-type acyl-transferase family protein Sec14p-like phosphatidylinositol transfer family protein Inositol-pentakisphosphate 2-kinase family protein Page 32 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 Table 3. Phosphorus partitioning means and statistical letter groupings for populations grown in Costa Rica and Columbia, MO. Location Costa Rica Columbia, MO Genotype (Glyma14g07880/Glyma06g03310) PA-P FN38 (14W/06m) Gm-lpa-ZC-2 (14m/06W) 14W/06W 14W/06m 14m/06W 14m/06m FN38 (14W/06m) Gm-lpa-ZC-2 (14m/06W) 14W/06W 14W/06m 14m/06W 14m/06m 4.91b* 3.50b 4.84b 5.28b 3.93b 1.00a 5.05b,c 2.67b 5.55c 6.02c 3.22b 0.66a Pi-P IP5-P -1 µg P mg seed 0.15c 0b 0.74a,b 0.27a,b 0.13c 0b 0.09c 0b 0.41b,c 0.16b 1.04a 0.95a 0.10c 0c 0.71b 0.56b 0.15c 0c 0.15c 0c 0.49b 0.39b 1.02a 1.70a IP4-P 0c 1.15b 0c 0c 0.98b 1.96a 0b 1.51a 0b 0b 1.22a 1.50a *Means were calculated using LSMeans in SAS based on each genotype including lines and replicates, if applicable. Means with the same letter are not statistically different using the MIXED procedure with a Bonferroni Adjustment. PA-P, Pi-P, IP5-P, and IP4-P are abbreviations for phytic acid-P, inorganic phosphate-P, I(1,3,4,5,6)P5-P and I(1,4,5,6)P4-P/I(3,4,5,6)P4-P, respectively. Page 33 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 A. L1 L2 R3 R2 L3 339K B. + IPK1 gene (Glyma06g03310.9) R1 R2 R3 340K R1 341K 342K Page 34 of 35 The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077 100% 90% IP4-P 80% 70% 60% IP4-P IP5-P 50% IP5-P PA-P 40% Pi-P 30% PA-P 20% Pi-P 10% 0% 14W/06W 14m/06W 14m/06m lpa1a2a lpa1b2b lpa1a2b Page 35 of 35 A B C D E The Plant Genome Accepted paper, posted 12/04/2014. doi:10.3835/plantgenome2014.10.0077