c06-11-0691 Israel.indd

Commercialization of soybean [Glycine max (L.) Merr.] varieties with low seed phytic acid will depend on the stability of the trait when grown in soils with a wide range of P availabilities and on the impact of altered P composition on seed protein and oil concentrations. Impacts of defi cient (0.05 mmol L−1) to excessive (0.9 to 1.2 mmol L−1) levels of external P on seed P composition of normal and low phytic acid lines and of altered seed P composition on seed protein and oil synthesis were evaluated. Soybean lines homozygous recessive (pha/pha) at one of two loci with genes that condition the low seed phytic acid trait had the same greater-thanthreefold increase in phytic acid in response to increasing external P as their normal phytic acid parent, ‘AGS Prichard-RR’ (Pha/Pha). This supports the conclusion from previous inheritance studies that the low seed phytic acid trait in CX1834-1-2 is controlled by epistatic interaction between two independent recessive genes. The seed phytic acid concentration in the low phytic acid line G03PHY-443 (derived from CX18341-2) was <2 g phytic acid P kg-1 dry wt. when grown under defi cient to excessive external P. As the P supply increased, seed inorganic P concentrations for this line increased from 0.8 to 4.0 g kg-1 dry wt., compared to an increase of 0.2 to 0.6 g kg-1 dry wt. for the normal phytic acid lines. Seed protein and oil concentrations did not differ signifi cantly between normal and low phytic acid lines. These results support continued development of varieties with low seed phytic acid and high yields. D.W. Israel, USDA-ARS and Dep. of Soil Science, North Carolina State Univ., Raleigh, NC 27695; P. Kwanyuen and J.W. Burton, USDA-ARS, 3127 Ligon St., Raleigh, NC 27607; D.R. Walker, USDA-ARS, 232 National Soybean Research Center, 1101 W. Peabody Dr., Urbana, IL 61801. Received 2 Nov. 2006. *Corresponding author ([email protected]). Abbreviations: HPLC, highperformance liquid chromatography; LG, linkage group; SEPEL, Southeastern Plant Environment Laboratories; SSR, simple sequence repeat. Published in Crop Sci. 47:2036–2046 (2007). doi: 10.2135/cropsci2006.11.0691 © Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 WWW.CROPS.ORG 2037 environmentally friendly has been to add phytase to feed rations. Phytase additions to feed have increased P availability of corn from 15 to 43% in swine (Cromwell et al., 1993) and reduced P excretion by broilers as much as 24%. Phytic acid accumulation in soybean seed begins in early embryogenesis and is linear throughout most of seed development (Raboy and Dickinson, 1987). External P availability infl uences phytic acid P concentrations in soybean seed (Raboy and Dickinson, 1993). For example, phytic acid P concentrations in seed of six soybean cultivars increased an average of four-fold (1.6 to 6.6 g kg−1) when the external P supply to plants was increased from 2 to 50 mg L−1 (Raboy and Dickinson, 1993). Increases in phytic acid P accounted for almost all of the increase in total seed P, as inorganic P and other nonphytic acid P were essentially constant across the range of external P concentrations. Since the mid-1990s genetic and breeding approaches have been used to reduce phytic acid P concentrations and increase bioavailability of P in seeds of grain crops (Larson and Raboy, 1999; Raboy et al., 2000; Shi et al., 2003; Wilcox et al., 2000) used in animal rations, and to ameliorate problems with P excretion in animal manure (Ertl et al., 1998). Genotypes with low seed phytic acid P concentrations have been developed in maize (Larson and Raboy, 1999; Raboy et al., 2000; Shi et al., 2003), rice (Larson et al., 2000), barley (Larson et al., 1998; Dorsch et al., 2003), wheat (Guttieri et al., 2004), and soybean (Wilcox et al., 2000). Chemical mutagenesis has been used to obtain mutants with alterations in diff erent steps of the phytic acid biosynthetic pathway and several classes of phenotypes have been described. Mutations which cause reciprocal eff ects on seed phytic acid and inorganic P concentrations in seed, with no accompanying change in the total P concentration have been designated as low phytic acid 1 (lpa1) mutations (Raboy et al., 2000). Two soybean mutants that fall in the lpa1 class have been identifi ed (Hitz et al., 2002; Wilcox et al., 2000). Other mutations which cause a decrease in phytic acid P concentration and an accumulation of inositol phosphate intermediates have been designated as lpa2 mutations (Raboy et al., 2000; Shi et al., 2003). Recently, a maize mutation that decreases phytic acid P concentration in grain by 66% and causes an accumulation of inositol has been reported (Shi et al., 2005). This mutation was designated as lpa3. The lpa1, lpa2, and lpa3 mutations can reduce seed phytic acid concentrations by 30% to >90% (Hitz et al., 2002; Larson et al., 1998; Raboy et al., 2000; Shi et al., 2003, 2005). Wilcox et al. (2000) developed a soybean mutant with seed phytic acid P concentrations that are 80% lower than normal. Genes from this material have been crossed into germplasm adapted to several regions of the United States. Inheritance studies have shown that the low seed phytic acid phenotype in CX1834-1–derived lines descended from the soybean mutant of Wilcox et al. (2000) is conditioned by two independently segregating recessive genes designated as pha1 and pha2 (Oltmans et al., 2004). The enzymatic steps in the phytic acid biosynthetic pathway that have been altered in these mutants to generate the low phytic acid phenotype have not been identifi ed, but loci with recessive alleles conditioning low phytic acid in CX1834-1 have been mapped to molecular linkage groups (LGs) N and L (Walker et al., 2006). These genes presumably correspond to pha1 and pha2, and heterozygotes have phytic acid levels (based on inorganic P levels) that are generally similar to those of the wild-type parents (Walker et al., 2006). Data from markers closely linked to the phytic acid loci on LGs L and N have been used to identify backcross-derived lines homozygous for the low phytic acid allele at either or both of the loci (R. Boerma, personal communication, 2006). The response of seed P composition of low phytic acid lines to increased external P availability has not been reported. Since phytic acid synthesis is impaired in these lines, we hypothesized that increased P availability would cause accumulation of high inorganic P concentrations and have little impact on the phytic acid P concentrations in seed of low phytic acid lines. If phosphorylation–dephosphorylation of enzymes such as cytosolic pyruvate kinase regulate the fl ow of C between protein and oil synthesis (Sebastia et al., 2005), concentrations of these constituents may be altered by the accumulation of high inorganic P concentrations in seed of low phytic acid lines. Soils in soybean production areas tend to have high levels of available P, especially those that have received manure applications for a long period of time (Sims et al., 2000). For the low phytic acid trait to be useful in soybean production systems, it must be stable over a range of available soil P levels. The objectives of this study were to assess (i) the stability of the low seed phytic acid trait across levels of external P supply ranging from defi cient to excessive, and (ii) the impact of the low seed phytic acid trait on seed protein and oil concentrations. MATERIALS AND METHODS Plant Materials and Genotypes Experiment 1 A P nutrition experiment was conducted with a pair of low (G03PHY-443) and normal (‘AGS Prichard-RR’, referred to hereafter as Prichard-RR) seed phytic acid lines of soybean developed by Roger Boerma at the University of Georgia. Prichard-RR is a glyphosate-tolerant version of the Maturity Group VII cultivar Prichard, which has a normal seed phytic acid content (Boerma et al., 2001). Line G03PHY443 was derived from a cross between Prichard-RR and CX1834-1-2, a low phytic acid line obtained from J.R. Wilcox (USDA-ARS and Purdue University). Both low phytic acid alleles from CX1834-1-2 (Wilcox et al., 2000) were backcrossed into Prichard-RR using marker-assisted selection. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 2038 WWW.CROPS.ORG CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 supplied nutrient solution (Thomas and Downs, 1991) modifi ed to contain defi cient (0.1 mmol L), suffi cient (0.5 mmol L), or excessive (1.2 mmol L) P and 15.0 mmol L N. Each genotype × P level treatment combination was replicated three times. From emergence until 35 d after planting, pots were fl ushed at 0900 and 1400 h each day with deionized water, with the addition of 0.5 L of appropriate nutrient solution after the 1400-h fl ush. From 35 to 85 d after planting, 0.5 L of the appropriate nutrient solution was applied after both fl ushes with deionized water. From 85 d after planting until harvest, nutrient solution application was discontinued to enhance maturation. At harvest maturity, pod number, seed number, and seed, leaf, and stem dry mass data were collected. A subsample of seed from each plant was dried to constant weight at 60°C, and was ground suffi ciently to pass through a 1-mm screen before being used for measurement of inorganic P, phytic acid P, protein and oil concentrations, and oil composition. Experiment 2 Except for indicated changes, culture conditions were the same as for Experiment 1. Plants were grown in large walk-in controlled environment chambers with 600 μmol m−2 s−1 of photosynthetically active radiation. This change was needed because chambers used in Experiment 1 were in use by others and would not have been available for 4 mo. Plants were supplied nutrient solution as described in the North Carolina University Phytotron Procedural Manual (Thomas and Downs, 1991). The nutrient solutions were modifi ed to contain defi cient (0.05 mmol L), suffi cient (0.50 mmol L), or excessive (0.90 mmol L) P levels. The defi cient P and excessive P levels were decreased to 0.05 mmol L and 0.90 mmol L, respectively, because less growth potential was expected at the 25% lower light intensity used in this experiment and the 0.1 mmol L P treatment used in Experiment 1 did not decrease seed yield signifi cantly (Tables 1 and 2). Genotype × P level treatment combinations were replicated three times. Chemical Analyses All samples were dried to a constant weight at 60°C before weighing. Phytic acid P content in soybean seed was determined as previously described (Kwanyuen and Burton, 2005) with some modifi cations. Unless otherwise stated, all procedures were performed at room temperature. Soybean seed samples were ground in a ZM 100 centrifugal grinding mill (Retsch, Haan, Germany) equipped with a 24-tooth rotor and 1.0-mm stainless steel ring sieve with the motor speed set at 15,000 rpm. This setting produced ground samples with a uniform particle size of <0.5 mm. For convenience, extractions were performed in 20-mL vials with 0.5 mol L HCl in a ratio of 1:20 (w/v) for 1 h with stirring. Samples (0.5 g) were extracted with 10 mL of 0.5 mol L HCl. Aliquots (2 mL) of crude extract from each sample were centrifuged at 18,000 g for 10 min in a microcentrifuge. A 1-mL aliquot of supernatant containing phytic acid was then fi ltered through a 13-mm/0.22-μm syringe fi lter. Filtered samples could be stored at 4°C for as long as 48 h before high-performance liquid chromatography (HPLC) analysis because the strongly acidic (0.5 mol L HCl) extractant and the low temperature were not favorable for phytase activity. Optimum pH ranges from 4.0 to 7.5 and optimum temperature G03PHY-443 is a BC 4 F 2 –derived line that is homozygous for low phytic acid alleles at loci on LGs L and N (i.e., its genotype is (pha1pha1pha2pha2). Outside the regions surrounding these loci, the genome should be approximately 97% Prichard-RR. Experiment 2 Unique genetic material from the breeding program of Roger Boerma was used to evaluate the impact of individual recessive genes conditioning the low phytic acid trait on diff erences in seed P composition in response to diff erent external P concentrations. The four genotypes (Prichard-RR, G03PHY-443, LG L, and LG N) used in this experiment all had a predominantly Prichard (Boerma et al., 2001) genetic background. Individual low phytic acid alleles from CX1834-1-2 (Wilcox et al., 2000) were backcrossed into Prichard-RR using marker-assisted selection. The LG L and LG N lines are derived from the same BC 4 F 1 parent as GO3PHY-443 but carry low phytic acid alleles at only one of the two mapped phytic acid loci. The LG L line has the pha2/pha2 genotype at the phytic acid locus near Satt561 on LG L, but is homozygous for the Pha1 alleles of the recurrent parent at the other phytic acid locus near Satt237 on LG N (Walker et al., 2006). In contrast, the LG N line has the pha1/ pha1 genotype at the phytic acid locus on LG N but is homozygous for the Prichard-RR Pha2 alleles at the LG L locus. Plants used in this experiment were genotyped at simple sequence repeat (SSR) markers on LGs L and N to confi rm the presence of the expected parental allele at each of the two phytic acid loci. Immature leaves (1–2 cm long) were sampled 25 d after planting and lyophilized. DNA was obtained from this material using a CTAB-based extraction protocol (Keim et al., 1988) and polymerase chain reaction protocols followed those described by Walker et al. (2006). Samples of Prichard-RR and CX1834-12 DNA were included as controls. Satt339 and Satt237, which fl ank the estimated position of the phytic acid locus on LG N, were used to determine the genotype at that locus (Walker et al., 2006). Satt166 and Satt113, which are 4 to 5 cM to one side of the estimated location of the LG L phytic acid locus, respectively, had to be used because SSR markers closer to the locus were monomorphic and therefore uninformative. Plant Culture and Environmental Conditions Experiment 1 Plants were grown in controlled environment chambers (Environmental Growth Chambers Co., Chagrin, OH, as modifi ed by the Southeastern Plant Environment Laboratories [SEPEL]) with 800 μmol m−2 s−1 of photosynthetically active radiation and 400 μL L−1 CO 2. A day/night temperature of 26/22°C (±0.50°C of set point in day and ±0.25°C of set point at night) was used throughout the experimental period. A 9-h photosynthetic period and a 15-h dark period interrupted with photomorphogenic irradiance for 3 h (to prevent fl owering) were used daily until the second trifoliolate leaf began to unroll. At this stage, the night interruption was discontinued to induce fl owering. Three seeds of the soybean lines described above were planted into thoroughly moistened peatlite–gravel mix (SUNGRO Horticulture, Bellevue, WA) in 25-cm (6-L) pots and placed into the controlled environment chambers. After emergence, pots were thinned to one healthy seedling. Plants were R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . CROP SCIENCE, VOL. 47, SEPTEMBER–OCTOBER 2007 WWW.CROPS.ORG 2039 ranges from 35 to 70°C for phytases from diff erent organisms (Phillippy, 2003). Chromatography was performed on a binary HPLC system with a 4 by 250 mm IonPac AS7 analytical column (Dionex Corp., Sunnyvale, CA) equipped with a 4 by 50 mm IonPac AG7 guard column. Elution of phytic acid was achieved with a 15-min linear gradient of 0.01 mol L 1methylpiperazine (pH 4.0) to 0.5 mol L NaNO 3 in 0.01 mol L 1-methylpiperazine (pH 4.0) at a fl ow rate of 1 mL min. Wade’s color reagent (Wade and Morgan, 1955) consisting of 0.015% (w/v) FeCl 3 and 0.15% (w/v) 5-sulfosalicylic acid (also at fl ow rate of 1 mL min) and phytic acid that eluted from the column were mixed in a mixing tee, with inline check valves installed before the mixing tee to prevent back fl ow. The postcolumn reaction was allowed to take place in a 250-μL sample loop at the combined fl ow rate of 2 mL min. The absorbance was monitored at 500 nm while the detector signals and/or phytic acid peaks were processed and integrated by the chromatographic data acquisition system. Inorganic P in seed was determined by the microtitre plate assay method described by Larson et al. (2000). This involved extraction of 100 mg of dry seed, ground to pass through a 1-mm screen, with 3.0 mL of 12.5% w/v trichloroacetic acid containing 25 mmol L MgCl 2. Aliquots (10 μL) of extracts were diluted with 90 μL of deionized water and reacted with 100 μL of Chen’s reagent (1 vol. 0.02 mol L ammonium molybdate, 1 vol. 10% w/v ascorbic acid, 1 vol. 3.0 mol L sulfuric acid, and 2 vol. distilled water) (Chen et al., 1956) in wells of microtitre plates. Two standard curves for inorganic P (0–1.5 μg per well) were established on each plate by adding appropriate volumes of 1.0 mmol L K 2 HPO 4 to diff erent wells along with the extractant and deionized water. One hour after adding Chen’s reagent, absorbance at 882 nm was determined for each well using a microplate reader (Model MQX200, BIO-TEK Instruments, Winooski, VT). The absorption maximum for the phospho-molybdenum blue complex that forms on addition of Chen’s reagent is 882 nm (Murphy and Riley, 1962). Absorbance readings for samples were corrected by subtracting absorbance of reagent blanks. Total P concentration in seeds and leaves was measured with inductively coupled plasma emission spectroscopy (Novozamsky et al., 1986). Total protein was measured by the Dumas reductive combustion method coupled with thermal conductivity detection (AOCS, 1995a). Total oil concentration was determined with pulsed NMR on whole seeds (AOCS, 1995b). Fatty acid composition of oil was measured by the method of Wilson et al. (2001). Statistics In both experiments, genotype × P treatment combinations were replicated three times and arranged in a randomized block design (two blocks in one chamber and one block in a second chamber set for the same environmental conditions). The SAS GLM procedure (SAS Institute, 1999) was used for the statistical analysis. Appropriate Fisher Protected LSD values for dependent variables were calculated for comparison of treatment means when either main eff ects or genotype × P treatment interaction eff ects were signifi cant at the 0.05 probability level. RESULTS AND DISCUSSION Two of fi ve P nutrition experiments that we conducted with low and normal seed phytic acid genotypes are presented in this report. Response of seed P composition to increasing P supply for the three unpublished experiments was the same as for the two experiments presented in this report. These genotypes (CX1834-A-1-1, normal and CX1834-A-1-4, low) were early selections from Dr. Wilcox’s mutagenesis program (Wilcox et al., 2000) and were not as genetically Table 1. Signifi cant differences for genotype and P level main effects and genotype (G) × P level interactions on seed attributes measured in Experiment 1. Trait Genotype P level G × P level Coeffi cient variation