Plant Physiology Preview. Published on May 2, 2017, as DOI:10.1104/pp.16.01929
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Title: Stearoyl-acyl carrier protein desaturase mutations uncover an impact of
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stearic acid in leaf and nodule structure
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Authors: Naoufal Lakhssassi, Vincent Colantonio1, Nicholas D. Flowers1, Zhou Zhou1,
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Jason Henry, Shiming Liu, and Khalid Meksem*
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Author affiliations: Department of Plant, Soil and Agricultural Systems, Southern
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Illinois University, Carbondale, IL 62901, USA (N.L., V.C., Z.Z., S.L., K.M.);
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Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, USA
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(V.C.); Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901,
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USA (N.D.F., J.H.). 1These authors contributed equally to the work.
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Footnotes:
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Author contributions: K.M. designed the research, planned and supervised the work and
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edited the manuscript. N.L. conceived and drafted the manuscript, performed statistical
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analysis, primer design, genotyping analysis, in silico, expression analysis, mutational
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analysis and light microscopy analysis. V.C. conducted protein homology modeling and
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mutational analysis. N.D.F. and J.H. performed leaf and nodule fixation, infiltration,
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sectioning, staining, and light microscopy analysis. N.L., and Z.Z. developed EMS
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mutagenized populations, performed fatty acid analysis, RNA extractions, and PCR gel
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purification. N.L., Z.Z., and S.L. performed DNA extractions. N.L., and S.L. performed
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sequencing analysis. All authors edited the manuscript, reviewed and approved the final
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version.
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Funding: This research was supported in part by the United Soybean Board project
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#1520-532-5607 and 0253 to Khalid Meksem.
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Corresponding author email:[email protected].
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The author responsible for distribution of materials integral to the findings presented in
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this article in accordance with the policy described in the Instructions for Authors
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(www.plantphysiol.org) is: Khalid Meksem ([email protected]).
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Summary: Mutational analysis of SACPD-C reveals an impact of stearic acid
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accumulation in leaf and nodule structure and morphology.
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Abstract
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Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the
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accumulation of seed stearic acid; however, no study has previously reported its
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involvement in leaf stearic acid content and impact on leaf structure and morphology. A
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subset of an EMS mutagenized population of soybean c.v. ‘Forrest’ was screened to
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identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one
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nonsense and four missense Gmsacpd-c mutants were identified to have high levels of
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seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of
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the GmSACPD-C enzyme reveals that most of these mutations were localized near or at
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conserved residues essential for di-iron ion coordination. Soybeans carrying Gmsacpd-c
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mutations at conserved residues showed the highest stearic acid content and were found
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to have deleterious effects on nodule development and function. Interestingly, mutations
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at non-conserved residues show an increase in stearic acid content yet retain healthy
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nodules. Thus, random mutagenesis and mutational analysis allows for the achievement
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of high seed stearic acid content with no associated negative agronomic characteristics.
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Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts
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were significantly more abundant in soybeans with deleterious mutations at conserved
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residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase
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in leaf stearic acid content and an alteration of leaf structure and morphology, in addition
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to differences in nitrogen fixing nodule structure.
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Keywords: Soybean, EMS mutagenesis, fatty acids, stearic acid, SACPD-C, mutational
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analysis, homology modeling, forward genetics, Leghemoglobin.
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Introduction
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Soybean [Glycine max (L.) Merr.] is the most widely consumed legume crop in
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the world, providing 56% of the world’s oilseed production (Soystats, 2014). Soybean oil
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can be found in food products such as margarine, salad dressings, and cooking oils, as
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well as industrial products such as plastics and biodiesel fuel. There are five major fatty
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acids in soybean oil: two saturated fatty acids, palmitic acid (16:0) and stearic acid (18:0),
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and three unsaturated fatty acids, oleic acid (18:1), linoleic acid (18:2), and linolenic acid
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(18:3).
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Oxidatively stable oil with a high melting temperature is necessary for solid fat
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application. Highly-saturated soybean seed oil would be suitable for this end use
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(Clemente and Cahoon, 2009). Palmitic acid not only improves the oxidative stability of
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soybean oil, but also can be used to produce trans-fat-free shortenings, margarines, and
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cosmetic products. However, this saturated short chain fatty acid is undesirable for
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nutrition because its consumption results in an unfavorable lipoprotein profile in blood
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serum (Mensink and Katan, 1990). Stearic acid does not exhibit these cholesterolemic
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effects on human health (Kris-Etherton et al., 1997, Kris-Etherton and Yu, 1997).
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Additionally, stearic acid is less likely to be incorporated into cholesterol esters and has a
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neutral effect on the concentration of blood serum LDL cholesterol (Byfield et al., 2006).
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Soybean oils are typically subjected to hydrogenation to increase stearic acid content.
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When partially hydrogenated, parts of the soybean unsaturated fatty acids are converted
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into unhealthy trans fats (Clemente and Cahoon, 2009). Consumers are increasingly
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avoiding products that contain trans fats, and governments have begun to ban trans fats
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in food products. Because of its high melting temperature and oxidative stability, soybean
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oil with a high stearic acid content may be useful in reducing the need for chemical
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hydrogenation in the production of trans fat free margarines and shortenings (Kok et al.,
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1999, List et al., 1997, Knowlton, 2001). Genetic manipulation of stearic acid is more
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efficient in reducing trans-fats introduced by the hydrogenation process (Clemente and
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Cahoon, 2009). Therefore, the development of soybean germplasm with higher seed
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stearic acid (>20%), oleic acid (>50%), and lower linolenic acid (<3%) content is a main
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objective of the edible oil industry (Ruddle et al., 2013, Ruddle et al., 2014).
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Stearic acid content in soybean seeds is typically between 2-5% of the total fatty
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acids (ILSI, 2010; USDA-ARS, 2012). The enzyme stearoyl-acyl carrier protein
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desaturase (SACPD-C) is responsible for the conversion of stearic acid to oleic acid in
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the developing soybean seed. However, no study has previously reported SACPD-C
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involvement in leaf stearic acid content and impact on leaf structure and morphology.
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Spontaneously occurring mutants of GmSACPD-C, like FAM94-41 (9% stearic acid),
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have been found to have a high seed stearic acid phenotype (Zhang et al., 2008).
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Mutagenesis has been used in some soybean cultivars to reduce the catalytic activity of
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GmSACPD-C and accumulate stearic acid within the seed (Ruddle et al., 2013, Carrero-
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Colon et al., 2014). In this study, soybean lines with high seed stearic acid content were
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isolated using a forward genetic screening, and the correlation of GmSACPD-C mutations
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with an alteration of seed stearic acid content were analyzed. Consequently, five
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Gmsacpd-c mutants were identified by target sequencing with high levels of seed stearic
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acid, all of which will be used as new high stearic soybean germplasm. It has been
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reported that high stearic mutants carrying deletions on GmSACPD-C resulted in nodule
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cell senescence and the formation of necrotic cavities (Gillman et al., 2014).
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Interestingly, in the current study, homology modeling and mutational analysis revealed
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that mutations within non-conserved residues around the iron-binding pocket resulted in
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relatively high stearic acid content, but no cell senescence and necrotic cavities in
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nodules. Finally, the impact of stearic acid accumulation due to GmSACPD-C mutations
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has been identified to affect leaf structure and morphology.
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Results
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Forward genetic screening identifies high seed stearic acid content mutants
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The distribution of the seed stearic acid of the M3 EMS Forrest mutagenized
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population ranges from 0.41% to 11% (Supplemental Figure S1). Traditionally, the
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average seed stearic acid content of the wild type Forrest, PI 88788, Essex, and Williams
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82 range between 2.4% and 4.1% (Table 1). Interestingly, in the developed Forrest
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mutagenized M3 population, 19 lines presented seed stearic acid content that was twice
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that of the wild type. However, among those 19 M3 lines, only five lines (F605, F620,
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F714, F813, and F869) retained the high stearic acid phenotype in two successive
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generations (Table 1). The average seed stearic acid content in the wild type Forrest was
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3.25%, whereas the five M3 mutants F605, F620, F714, F813, and F869 presented
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5.88%, 6.06%, 6.49%, 6.9%, and 6.52% content, respectively (Supplemental Table 1).
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Interestingly, the seed stearic acid content of the mutants in the M4 generation increased
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to 9.48%, 10.04%, 7.24%, and 6.15%, respectively. Unfortunately, because the F869
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mutant was not advanced to the M4 and M5 generations, no stearic acid content was
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available for these generations.
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Fatty acid analyses show that the mutant lines F714 and F813 presented consistent
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stearic acid content among the three tested generations: M3 (2012), M4 (2013), and M5
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(2015). However, stearic acid content of the mutant lines F605 and F620 were
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significantly different among the M3 and M4 generations (Supplemental Tables 1 and 2).
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In the M3 generation, the stearic acid content of F605 and F620 was 5.88% and 6.06%,
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whereas in the M4 generation, the stearic acid of F605 and F620 increased to 9.48% and
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10.04%, respectively. Surprisingly, in the M5 generation, the mutant line F605 presented
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an extremely high seed stearic acid content of 20.11% (Table 1). This variation within the
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F605 and F620 mutant families may have been due to allelic segregation during these
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three generations. In addition to measuring the stearic acid, the content of palmitic acid,
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oleic acid, linoleic acid, and linolenic acid of each mutant was measured. We observed
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that, between these mutants and the wild type, the contents of the other four types of fatty
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acids only showed a significant difference in oleic acid (a decrease of up to 5% compared
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to the wild type Forrest) (Table 1). Knowing the role of SACPD-C in converting stearic
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acid to oleic acid in the fatty acid biosynthetic pathway, the decrease in oleic acid may be
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due to a reduced activity of SACPD-C.
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Identification of five novel SACPD-C alleles conferring higher stearic acid content in
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soybean seeds
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We analyzed the genomic sequence of GmSACPD-C of the five mutants (F605,
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F620, F714, F813, and F869) and the wild types (Forrest, Essex, and Williams 82).
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Interestingly, F605, F620, F714, F813, and F869 were identified to each carry a mutation
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within GmSACPD-C (C247T, C235T, G229A, C305T, and G340A, respectively) (Figure
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1B). In addition, F620 contains G1777A and G1964T SNPs in the second exon and 3’
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UTR, respectively. These two SNPs are present in the Essex cultivar. Therefore, the F620
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may have crossed with Essex (Figure 1B). These mutations produced five new
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GmSACPD-C alleles including one nonsense mutation (Q83* in F605), and four missense
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mutations (D77N in F714, L79F in F620, P102L in F813, and E114K in F869) (Figure
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1B). PROVEAN predictions were performed on all mutations. Interestingly, four of the
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five missense mutations identified had PROVEAN scores of < -2.5 (Figure 1B). The
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mutations SACPD-CQ83*, SACPD-CD77N (PROVEAN score= -2.19), SACPD-CP102L
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(PROVEAN score= -6.08), and SACPD-CE114K (PROVEAN score= -3.58) were
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predicted to be deleterious to the protein (Figure 1B).
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Identification of conserved GmSACPD-C residues across several dicot species
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In order to study if the new Gmsacpd-c mutations are located in conserved
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positions along the GmSACPD-C predicted protein sequence, an alignment was
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performed between the five Gmsacpd-c screened mutants, the four wild type cultivars
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(Essex, Forrest, Williams 82, and PI88788) and orthologous SACPD-C proteins of
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several plant species obtained from the NCBI database. The alignment of Glycine max
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GmSACPD-C protein sequences with the orthologous SACPD-C proteins from 26 other
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species including SACPD-C from a dicot model (Arabidopsis thaliana), a monocot
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model (Oryza sativa), other monocot and dicot species, the primitive land plant models: a
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lycophyte (Selaginella mollendorffii), a moss (Physcomitrella patens), and a chlorophyte
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(Chlamydomonas reinhardtii) showed striking sequence conservation (Figure 1C). From
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the alignment, the amino acid changes in the four identified missense mutants affected
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residues that were classified within two groups. Group I presented mutations in
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conserved residues: SACPD-CL79F and SACPD-CE114K. However, Group II contained
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mutations
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Interestingly, the seed stearic acid content correlated with both groups. Mutations in
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conserved residues increased to up to 3 times the levels of seed stearic acid content
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(Group I). However, mutations in non-conserved residues resulted in a limited increase in
within
non-conserved
residues:
SACPD-CD77N
and
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SACPD-CP102L.
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seed stearic acid, presenting only 2 times the level contained in the wild type Forrest
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seeds (Group II).
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Mutational analysis of GmSACPD-C
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In order to study the impact of the missense mutations on the catalytic activity of
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the enzyme, homology modeling of GmSACPD-C was carried out. Three missense
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mutants (D77N, L79F, and E114K) were localized directly at the surface of the iron ion
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binding pocket (Figure 2), with one mutation directly altering the negatively charged
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bridging ligand Glu114 into a positively charged lysine (SACPD-CE114K). Because of the
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iron ion pocket localization and alteration of charge in the missense mutants SACPD-
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CD77N and SACPD-CL79F, and the presence of steric hindrance by L79F, these mutations
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are predicted to be affecting iron ion binding kinetics and stability. The missense mutant
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SACPD-CP102L was not localized at the iron ion binding pocket, but was positioned at the
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first residue of the α4 chain, which holds the ligands Glu114 and His117 in place.
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Considering proline’s cyclic conformation in which the secondary amine binds to the α-
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carbon of the protein backbone, a disruption of this conformational rigidity may impact
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the ability of the α4 chain to be in its proper location, disrupting the enzymatic activity of
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GmSACPD-C.
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Analysis of nodule structure reveals morphological differences between Group I and
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Group II Gmsacpd-c mutants
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It has been demonstrated that a homolog of the soybean GmSACPD-C gene was
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also identified as a nodulin gene in yellow lupine (Lupinus luteus) (Swiderski et al.,
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2000). Therefore, the effect of the GmSACPD-C mutation on stearic acid content in the
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nitrogen-fixing nodules was analyzed. Unfortunately, because the F869 mutant was not
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advanced in the M4 and M5 generations, from here on, only four mutants (F605, F620,
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F714, F813) will be further characterized. The average nodule stearic acid content in the
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wild type Forrest was 2.13%. Surprisingly, the four M4 mutants F605, F620, F714, and
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F813 presented a large increase in the nodule stearic acid content, showing 21.09%,
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22.99%, 12.39%, and 15.24%, respectively. These data are in accordance with the two
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groups cited previously, and correlated with stearic acid levels obtained. In fact, members
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belonging to Group I presented the highest nodule stearic acid content (>21%); however,
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Group II members presented a limited stearic acid content (<16%). No significant root
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stearic acid content increase was observed in the mutants (Supplemental Figure S2).
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To test the effect of the nodule stearic acid content increase between the two
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groups, light microscopy of nodule cross sections was carried out. Group I exhibited an
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undulate coat phenotype and the presence of a central necrotic cavity. Surprisingly,
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Group II did not show any undulate coat phenotype or central necrotic cavity. To
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elucidate the effect of each mutation on nodule structure, biometric measures were
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collected from the nodule coat (Figure 3F). Interestingly, the nodule average diameter
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showed an increase in coat size across the four Gmsacpd-c mutants analyzed (Figure 3).
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Furthermore, in order to gain insight into changes that caused the central cavity
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formation at a cellular level, nodule cross sections in the wild type and the four identified
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mutants were compared. Microscopy analysis revealed a reduced differentiation of
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vascular bundles observed in Group I (Figure 4). The nodules showed a significant
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increase in size of all cell layers, indicating that all nodule cell types were affected by the
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deleterious GmSACPD-C mutations. In addition, a very strong reduction of bacteroids
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was observed in Group I, with an increased cortex size and an expanded cavity (Figure
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4B). However, no differences in radial organization or defects between the wild type
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Forrest and the Group II mutants were observed. These results indicate that GmSACPD-
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C mutations not only control seed and nodule stearic acid content, but also may
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ultimately impact nodule structure and morphology.
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Analysis of leaf morphology in Gmsacpd-c mutant lines
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Phenotypic analysis of plant development of the Gmsacpd-c mutants revealed a
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common undulate morphology in the leaves (Figure 5A). To investigate the possible
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effect of the GmSACPD-C mutation on the observed leaf phenotype, leaf fatty acid
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content was analyzed. Leaf stearic acid content was significantly increased from 2.48% in
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the Forrest wild type to 6.25%, 5.32%, 6.62%, and 4.97% in the four M4 mutants F605,
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F620, F714, and F813, respectively (Supplemental Figure S2). To gain insight into the
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changes that caused the undulate leaf formation at cellular level, leaf cross-sections in the
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WT and the four identified mutants were analyzed (Figure 5).
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In the wild type cross sections, the leaf anatomy observed beneath the adaxial
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epidermis includes a palisade mesophyll layer that is 1 to 2 cells thick. The cells in this
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region are more uniform in shape, size, and spacing. The spongy mesophyll layer is made
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up of more irregularly shaped cells and contains several air spaces throughout. These air
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spaces often correspond with the stomata found on the abaxial epidermal region of the
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leaf. In the Gmsacpd-c mutants, the palisade layer appears to be 3 to 5 cells thick. In
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addition, the cells are more densely packed and are more elongated than the wild type.
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Interestingly, the air spaces found in the spongy mesophyll layer were reduced in the
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mutants, with the spaces occupied by an increase in either the spongy or palisade
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mesophyll cells. The organization of the cell layers including the upper and lower
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epidermis and vascular bundles were also found to be distorted (Figure 5B). Thus, the
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high leaf stearic acid content of the Gmsacpd-c mutants (F605, F620, F714, and F813)
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may have contributed to the drastic leaf cell disorganization, reflected by a stronger
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undulate phenotype.
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qRT-PCR analysis reveals that Group I Gmsacpd-c mutants have a greater
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expression of nodule leghemoglobins than Group II
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Investigation of the G. max[Williams 82] genome indicates that the leghemoglobin
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(GmLegh) gene family is composed of three members located on chromosomes 10
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(Glyma.10G199000), 20 (Glyma.20G191200), and 11 (Glyma.11G121700), named
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GmLegh-C1, GmLegh-C2, and GmLegh-C3, respectively. First, the expression data of the
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three GmLegh genes was compiled using the public RNAseq data available at Soybase
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(www.soybase.org). Two distinct gene expression patterns were identified for the
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GmLegh gene members (Supplemental Figure S3). Whereas GmLegh-C1 and GmLegh-
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C2 were both highly expressed only in nodules, the GmLegh-C3 had almost no
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expression in most tissues analyzed, exception a minor expression in the roots. Next, the
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expression of the three GmLegh gene family members was analyzed by qRT-PCR. As
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expected, no expression of GmLegh-C3 was detected in any of the soybean lines
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analyzed. Furthermore, both GmLegh-C1 and GmLegh-C2 transcripts were found to be
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significantly more abundant in Group I than in Group II mutants (Figure 6).
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Discussion
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Random mutagenesis as a rheostat for agronomically important traits
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Many of the developed high seed stearic acid soybean germplasm suffer
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agronomic problems (Gillman et al., 2014, Hammond and Fehr, 1983, Lundeen et al.,
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1987, Rahman et al., 1997, Rahman et al., 1995). A sodium azide induced mutant called
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A6 presented extremely high stearic acid content (28%), but was described as non-
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desirable since it was associated with poor germination and low seed yield (Hammond
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and Fehr, 1983, Lundeen et al., 1987, Rahman et al., 1997). An extraordinarily large
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deletion representing 1/8 of chromosome 14 downstream from the GmSACPD-C gene,
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including at least 56 genes, was identified recently, and may explain the mutant A6’s
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extremely poor agronomic characteristics, which has delayed commercialization of high
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seed stearic acid lines (Gillman et al., 2014). Therefore, developing mutants with a large
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genomic deletion is agronomically impractical. Consequently, producing new EMS
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mutants with a single base pair mutation seems to be a better strategy for developing lines
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with agronomically important traits without unintended poor agronomic characteristics.
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Additionally, an increase of certain traits over a threshold of plant tolerance may
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negatively impact its agronomic performance, such as the appearance of a central
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necrotic cavity in the root nodules of high stearic acid soybeans (Gillman et al., 2014).
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Thus, fine-tuning the level of a certain trait is pertinent to the development of improved
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crop plants. This can be reached by studying the impact of mutations on important
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catalytic sites within the enzymatic structure.
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SACPD is a homodimeric enzyme with each homomer containing a di-iron
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coordination complex (Lindqvist et al., 1996). Crystal structure of SACPD in Ricinus
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reveals that the two iron ions of the di-iron complex are liganded by residues on four of
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the α-helices (Lindqvist et al., 1996). The first iron has highly conserved ligands
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Glu105/76 and His146/117 and the second iron has ligands Glu196/167 and His232/203
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with the amino acid positions corresponding to the Ricinus and soybean protein
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sequences, respectively. The two residues, Glu143/114 and Glu229/200, act as bridging
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ligands in the coordination space. From here on, only amino acid soybean positions will
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be noted. By mapping the high stearic acid Gmsacpd-c mutations onto a homology model
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based on the wild type Forrest, we can begin to understand how the mutations affect the
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enzymatic activity of GmSACPD-C. Due to the steric hindrance of the missense L79F on
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the di-iron core, and the nonsense truncated Q83* mutations, a strong reduction of
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enzymatic activity was reflected by the highest seed stearic acid contents of 10% and
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20%, respectively (Group I) (Figure 1 and Figure 2). However, the D77N and P102L
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mutations did not have as much of a deleterious impact on the protein activity, which is
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reflected by the limited stearic acid contents of 7% and 6%, respectively (Group II),
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observed through the M5 generation. In the case of P102L, although the mutation was not
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localized at the iron ion binding pocket, due to a disruption of the conformational rigidity
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on the α4 chain, a decrease in enzymatic activity is observed. Such mutations allow for a
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fine-tuning of the enzymatic kinetics, leading to an increase in the desired trait without
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the drawback of poor agronomic characteristics. This is supported by the observation that
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only mutations localized within conserved residues (Group I) presented unhealthy
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nodules with cell senescence, a necrotic cavity, reduced differentiation of vascular
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bundles, and a drastic decrease in infected cells. However, no differences in radial
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organization between the Forrest wild type nodules and the lines carrying mutations
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localized outside of conserved residues (Group II) were observed.
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Effects of Gmsacpd-c mutations in leaf structure and morphology
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Studies on metabolomic profiling showed a differential accumulation of fatty
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acids (precursors to membrane phospholipids) between roots and nodules and between
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infected and uninfected root hairs (Colebatch et al., 2004). Mutants with high levels of
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saturated fatty acids within the leaves of Arabidopsis have been described. The fad5
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mutant, with an increased palmitic acid content, due to a deficiency in the activity of the
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chloroplast 16:O-MGD desaturase, contains 24% palmitic acid and 1% stearic acid in
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leaves (25% total saturates) (Kunst et al., 1989). A fabl mutant had a reduced activity of
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3-ketoacyl-ACP synthase 11, and contained 22% palmitic acid and 1% stearic acid in
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leaves (23% total saturates) (Wu et al., 1994). Both fabl and fad5 mutants had no visible
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phenotype at normal growth temperatures (Wu et al., 1994). However, a fab2 mutant, due
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to a mutation partially affecting the action of the stromal SACPD, contains 12% palmitic
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acid and 20% stearic acid in leaves (31% total saturates) (Lightner et al., 1994). This
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mutant has a profoundly altered growth and morphology characterized by a reduction in
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the size of almost all organs, including leaves. In this study, the F605 nonsense Gmsacpd-
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c mutant presented similar saturated fatty acid phenotypes (27% total saturates), with
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6.9% and 20.1% contributed by palmitic and stearic acids, respectively (Table I).
344
Interestingly, mutations of GmSACPD-C exhibited different morphological phenotypes
345
of the leaves than the stromal Arabidopsis fab2 mutant. In fact, the Gmsacpd-c mutations
346
in soybeans affected the organization of the leaf cell layers conferring a strong undulate
347
leaf phenotype. This is exhibited by the increased abundance and elongation of the
348
palisade mesophyll layer, the reduction of air spaces in the spongy mesophyll layer, and
349
the abnormal differentiation of the vascular bundles within the mutant leaves.
350
It has been previously reported that fatty acid composition and saturation level
351
affect membrane integrity, therefore, allowing plants to tolerate heat stress (Murakami et
352
al., 2000). In fact, increasing degree of saturation enhance lipids heat stability, positively
353
impacting plant tolerance to high temperature in various species (Raison et al., 1982,
354
Grover et al., 2000). However, double bonds present in unsaturated fatty acid are easily
355
attacked by hydroxyl free radical under stress environment (Murakami et al., 2000). The
356
presence of these free radicals can cause membrane lipid peroxidation, and thus
357
membrane disruption (Smirnoff, 1993, Foyer et al., 1994). Moreover, it has been shown
358
that heat stress associated with cellular membrane damage cause physiological injuries
359
and leaf senescence, leading to cell solute leakage and disruption of cell function (Blum
360
and Ebercon, 1981, Marcum, 1998). Therefore, increasing saturated fat content in leaves,
361
like stearic acid, may positively impact plant tolerance to heat stress, and avoid cellular
362
membrane damage.
363
364
Effects of Gmsacpd-c mutations on leghemoglobin expression
365
It has been shown that products of SACPD, such as oleic acid precursors, were
366
found to increase significantly in root hairs in response to rhizobial infection by
367
Bradyrhizobium japonicum (Brechenmacher et al., 2010). Although the sacpd-c mutants
368
have shown large increases in stearic acid, only the oleic acid was found to have a
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12
369
reduction from the wild type levels. Association between legumes and rhizobia results in
370
the formation of root nodules, where symbiotic nitrogen fixation occurs (Herridge et al.,
371
2008).
372
Leghemoglobin (Legh) is an abundant hemeprotein of legume nodules and plays
373
an essential role as an oxygen carrier. Leghemoglobin is generally produced by legumes
374
in response to the colonization of roots by nitrogen fixing bacteria (O'Brian et al., 1987).
375
However, roots not colonized by rhizobia do not accumulate leghemoglobin.
376
Leghemoglobin buffers the concentration of free oxygen in the infected plant cells to
377
ensure the proper function of root nodules (O'Brian et al., 1987). Expression analysis
378
revealed that Group I exhibiting deleterious Gmsacpd-c mutations had the highest
379
abundance of leghemoglobin transcripts. Deleterious Gmsacpd-c mutations around
380
conserved catalytic residues in Group I may negatively affect iron ion binding, which
381
may result in an increase in iron ion availability within the nodules. Since leghemoglobin
382
also uses an iron cofactor, we speculate that the decrease of nodule GmSACPD-C iron
383
binding may allow for an increase in leghemoglobin iron binding and expression (Brear
384
et al., 2013). This is supported by the greater abundance of both Legh-C1 and Legh-C2
385
transcripts in Group I mutants as compared to the Group II mutants or to the Forrest wild
386
type.
387
In this study, we have shown that independent GmSACPD-C mutations confer
388
high stearic acid content in seeds, nodules, and leaves. These mutations also affected leaf
389
and nodule morphology, cell organization, and vasculature. Using random mutagenesis
390
coupled with homology modeling and mutational analysis, high seed stearic acid content
391
can be achieved while the associated negative agronomic characteristics are
392
circumvented. Although the impact of stearic acid accumulation on leaf and nodule
393
structure and morphology has been revealed, its mechanism of action needs to be further
394
studied and the cross-talk between SACPD and leghemoglobin still needs to be
395
elucidated.
396
397
Materials and Methods
398
Development of an EMS mutagenesis Forrest population
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13
399
The soybean cv. Forrest seed was obtained from Southern Illinois University
400
Carbondale Agricultural Research Center and used to develop an EMS mutagenized
401
population. The wild type Forrest seed was mutagenized with 0.6% EMS as described
402
previously (Meksem et al., 2008), and planted to harvest 1588 M2 families of seed in
403
2011. This population was successively advanced to M3 generation in 2012, M4
404
generation in 2013, and M5 generation in 2014 at the Southern Illinois University
405
Carbondale. In total, more than 1000 mutant families (lines) of M3/M4/M5 were
406
developed.
407
408
SCN-infection phenotyping
409
All M3 lines were screened with the SCN-infection phenotyping as described
410
previously (Liu et al., 2011). Seedlings were inoculated with infective eggs from PA3
411
population (HG type 0). Briefly, cysts were extracted from Essex infested roots and soil
412
by flotation in water and collected on a 250-μm sieve. Harvested cysts were gently
413
crushed using a drill press and the eggs were collected on a 25-μm sieve (Faghihi and
414
Ferris, 2000). The eggs were further diluted to 1,000 eggs/mL of water. Individual
415
seedlings were inoculated with 1 ml of the egg suspension. Plants were maintained in the
416
growth chamber at 27°C. Cyst counts were performed at 30 days post-inoculation.
417
418
Forward genetic analysis of seed fatty acid
419
All M3, M4, and M5 lines were analyzed for seed fatty acid composition using
420
the two-step procedure as outlined by (Kramer et al., 1997). Five seeds per mutant from
421
the M3, M4, and M5 generations: thirty wild type Forrest seeds, and four Essex, Williams
422
82, and PI 88788 wild type seeds were randomly selected for fatty acid analysis. Briefly,
423
each seed was scarified and then loaded into one 16 mm x 200 mm tube with a Teflon-
424
lined screw cap. Next, 2mL of sodium methoxide was added into each tube and then the
425
tube was incubated in a water bath at 50ºC for 10 min. Afterwards, 3 mL of 5%
426
methanolic HCl was added into each tube after the tube was cooled for 5 min.
427
Subsequently, the tube was incubated in a water bath at 80ºC for 10 min, cooled for 7
428
min, and then 7.5 mL of 6% (w/v) potassium carbonate and 1 mL of hexane were added.
429
Finally, the tubes were centrifuged at 1200 g for 5 min to separate the layers. The
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
14
430
aqueous layer was then transferred into vials and used for the fatty acid analysis using a
431
Shimadzu GC-2010 gas chromatograph equipped with a flame ionization detector and a
432
Supelco 60-m SP-2560 fused silica capillary famewax column (0.25 mm i.d. x 0.25 µm
433
film thickness). The helium carrier gas was maintained at a linear velocity of 23 cm/s.
434
The oven temperature was programmed for 175ºC for 22 min, then increased at 15ºC
435
/min to 225ºC maintained for 3 min. The injector and detector temperatures were set at
436
255ºC. Peaks were identified by comparing the retention times with those of the
437
corresponding standards (Nu-Chek-Prep., Elysian, MN; Supelco, Bellefonte, PA). The
438
contents of palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were
439
calculated using the statistical computing package JMP Pro 12.
440
441
Detection of genotypes and mutations of the mutagenized population
442
Young leaf tissue of the Gmsacpd-c mutants, and four soybean lines (wild type
443
Forrest, Essex, Williams 82, and PI 88788) were used to extract DNA using the CTAB
444
method (Rogers and Bendich, 1994) with small modifications. The specific primers
445
(Supplemental Table S3) were designed to amplify the fragments covering all
446
GmSACPD-C exons from the extracted DNAs with 38 cycles of PCR amplification at
447
94ºC for 30 s, 51ºC for 30 s, and 72ºC for 1 min. The PCR products were purified by
448
enzymatic clean-up or the specific bands were recovered from the agarose gels after
449
electrophoresis using the Qiagen gel extraction kit (QIAquick). Then, the purified PCR
450
fragments were sequenced by GENEWIZ Company (www.genewiz.com). The genotypes
451
of the mutants were determined by comparison of the genomic, known cDNA, and
452
predicted protein sequences of the genes with those of Forrest, Essex, Williams 82, and
453
PI 88788 using DNASTAR Lasergene software. PROVEAN predictions were performed
454
on all mutations. PROVEAN predicts whether an amino acid substitution or indel has an
455
impact and affects the biological protein function based on sequence homology and the
456
physical properties of amino acids (Choi, 2012, Choi et al., 2012, Choi and Chan, 2015).
457
PROVEAN variant predictions with a score equal to or below -2.5 are predicted to be
458
deleterious.
459
460
Multiple sequence alignment of SACPD-C
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15
461
To evaluate the position of substitutions, multiple sequence alignments were
462
performed with the ClustalW program, and mutations were overlaid on the protein
463
structure of the wild type lines. In silico analyses of the five identified Forrest
464
GmSACPD-C mutants; SACPD-CD77N, SACPD-CL79F, SACPD-CQ83*, SACPD-CP102L and
465
SACPD-CE114K, in addition to SACPD-C orthologous from other 26 plant species were
466
performed using the MegAlign (DNASTAR Lasergene 8) software package and the
467
Clustal W algorithm. All parameter values correspond to default definitions.
468
469
Fixation and infiltration of nodules and leaves
470
Soybean root nodules and leaves from the Gmsacpd-c mutants and Forrest wild
471
type were washed with triple distilled nano-pure water to remove soil, then placed in a
472
sodium phosphate buffer (0.05 M, 7.2 pH) for 24 hours. The samples were longitudinally
473
sectioned and transferred to an Eppendorf tube and fixed in a 2% gluteraldehyde solution
474
buffered with a sodium phosphate buffer (0.05 M, 7.2 pH) for 1 hour at 22oC, then
475
overnight at 4oC. The samples were washed 4 times (30 minutes each) in the same
476
phosphate buffer described above. After the fourth buffer rinse, the samples were placed
477
in fresh buffer solution and held overnight at 4oC. The samples were post-fixed in a 2%
478
osmium tetraoxide water solution (triple distilled nano-pure) for 10 minutes. Three
479
consecutive water rinses (30 minutes each) were immediately performed post-fixation.
480
The osmicated specimens were serially dehydrated with water and ethanol (25%-100%)
481
with each dehydration lasting 20 minutes. The ethanol was serially replaced with proplyn
482
oxide (50%-100%). The samples were then serially infiltrated with a proplyn oxide
483
Spurr’s resin (25%-100%) solution for 5 days. The infiltrated nodules and leaves were
484
placed in BEEM capsules with fresh 100% Spurr’s resin and baked for 2 days at 60oC.
485
Samples were sectioned on an ultramicrotome at a 1,000µm thickness. Staining was
486
performed using picrofushin and 1% methylene blue in a 50% ethanol and 50% water
487
solution. For the hand-sectioned nodules, sections were placed on a glass slide and
488
stained with 1% toluidine blue. Prepared slides were viewed with a Leica DM5000B
489
compound light microscope and imaged with a Q-Imaging Retiga 2000R digital camera.
490
491
Homology Modeling of GmSACPD-C proteins
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16
492
Homology modeling of a putative GmSACPD-C protein structure was conducted
493
with Deepview and Swiss Model Workspace software using the protein sequence from
494
Forrest and an available SACPD crystal structure from Ricinus communis as a template;
495
PDB accession 1OQ4 (Moche et al., 2003). All residues were modeled against this
496
template with a sequence identity of 69%. Mutation mapping and visualizations were
497
performed using the UCSF Chimera package (Pettersen et al., 2004).
498
499
Rhizobia inoculation assay of the identified high stearic Gmsacpd-c mutants
500
Soybean plants were grown on autoclaved sandy and organic soil mixture (50:50)
501
and watered as needed. USDA Bradyrhizobium japonicum strain 110 was grown in a
502
modified arabinose gluconate (MAG) medium for 6 days at 28ºC until it reached the
503
exponential growth phase. Bacterial inoculation was done at the time of sowing (10%
504
volume by seed weight). Inoculated plants were grown in the greenhouse at 27°C and
505
70% humidity, with 16-hour artificially supplemented light. Root, nodule, and leaf
506
samples were frozen dry for fatty acid analysis as described previously, or macerated in
507
liquid nitrogen for qRT-PCR analysis.
508
509
qRT-PCR analysis of Leghemoglobin gene family in Glycine max
510
Soybean seedlings from the four EMS mutants and the Forrest wild type were
511
grown in the growth chamber for one week, and then inoculated with Bradyrhizobium
512
japonicum strain 110 from the USDA. After five weeks, total RNA was isolated from
513
root samples using a Qiagen RNeasy Plant Mini Kit (cat# 74904). Total RNA was DNase
514
treated and purified using a Turbo DNA-free Kit (QAmbion/life technologies AM1907).
515
RNA was quantified using a Nanodrop 1000 (V3.7), then a total of 400 nanograms of
516
treated RNA was used to generate cDNA using the cDNA synthesis Kit (Thermoscript,
517
life technologies, 11146-025) with random hexamers. One-tenth of a 20 μL RT reaction
518
was used in gene specific quantitative PCR with the Power SYBR® Green PCR Master
519
Mix kit (Applied Biosystems™ #4368706). A list of primers used in this work is found in
520
Supplemental Table 3. For each genotype, RNA from three biological replicates was used
521
for quantification, and then normalized using the delta Cq method with Ubiquitin used as
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
17
522
a reference gene (ΔCq= Cq(TAR) – Cq(REF). Each gene’s expression was exponentially
523
transformed to the expression level using the formula (ΔCq Expression = 2-ΔCq). A
524
negative control (–RT) reaction was also performed in all the samples.
525
526
527
528
Statistical analysis
All the presented results were performed with the analysis of variance comparison
using the statistical computing package JMP Pro 12.
529
530
Accession numbers
531
The SACPD (EC 1.14.19.2) nucleotide and amino acid sequences of the new
532
identified high stearic acid alleles in addition to the Forrest wild type are deposited at
533
NCBI under GenBank accession numbers; SACPD-CQ83*(F605) (KX856376), SACPD-
534
CL79F(F620) (KX856377), SACPD-CD77N(F714) (KX856378), SACPD-CP102L(F813)
535
(KX856379), SACPD-CE114K(F869) (KX856380), SACPD-C_Forrest_WT (KX856375).
536
The new developed germplasm (seeds) are property of Southern Illinois University (SIU).
537
Access to the germplasm is subjected to Transfer Material Agreement Form.
538
539
Figure Legends
540
Figure 1. SACPD-C mutations of the screened five high stearic acid mutants. A:
541
SACPD-C gene model and polymorphisms (G1777A and G1964T) between PI88788,
542
Forrest, Essex and Williams 82. B: Predicted SACPD-C proteins sequences showing
543
amino acid differences between the five mutants identified by forward genetics: four
544
missense mutations (D77N, L79F, P102L, and E114K) and one nonsense mutation
545
(Q83*) within the SACPD-C. Provean predictions of the four missense mutations
546
identified are included. C: Protein sequence alignment of the five SACPD-C screened
547
mutants showing the identified mutations: SACPD-CD77N, SACPD-CL79F, SACPD-CQ83*,
548
SACPD-CP102L and SACPD-CE114K, in addition to SACPD-C orthologous from other 26
549
plant species. SACPD-C proteins identified from the following model plants; S.
550
Mollendorffii (lycophyte), P. patens (moss), C. reinhardtii (algae), O. sativa (monocots),
551
and A. thaliana (eudicots), in addition to other eudicots and monocots were included in
552
the alignment and showed striking sequence conservation. SACPD-C mutations within
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
18
553
residues conserved in all species analyzed are in blue boxes (Group I). Mutations within
554
non-conserved residues are in orange boxes (Group II). Red stars represent the conserved
555
E105/76, E143/114, and H146/117 residues important for iron ion binding. Primers used
556
for genotyping are indicated by arrows.
557
Figure 2. Homology modeling of the GmSACPD-C from Forrest with important
558
catalytic residues and the five identified sacpd-c missense mutations mapped. (A)
559
and (B) One subunit containing a di-iron coordination complex. The first iron has highly
560
conserved ligands Glu76 and His117, the second iron has ligands Glu167 and His203,
561
and the two residues Glu114 and Glu200 act as bridging ligands in the coordination
562
space. Iron ions are highlighted in orange. (C) and (D) One subunit with the five
563
missense mutants highlighted in different colors showing an iron ion pocket localization.
564
(E) The predicted homodimeric GmSACPD-C enzyme.
565
566
Figure 3. Comparison of the cortex and cavity measurements between the Gmsacpd-
567
c mutants and the Forrest WT (A) Nodule Horizontal Outer diameter. (B) Nodule
568
Vertical Outer diameter. (C) Nodule Horizontal Inner diameter. (D) Nodule Vertical
569
Inner diameter. (E) Biometric measures of the nodule coat size using the formula ((ho-
570
hi)+(vo-vi))/2). (F) Dissecting light microscope images of a hand sectioned nodule from
571
the Forrest WT showing details of: (HO) Horizontal outer, (HI) Horizontal inner, (VO)
572
Vertical outer, (VI) Vertical inner. F605 (n=21), F620 (n=22), F714 (n=20), F813 (n=25),
573
and WT (n=20).
574
575
Figure 4. Comparison of nodule structure and morphology of the Forrest-WT and
576
the four sacpd-c mutants. (A) Dissecting light microscope images of hand sectioned
577
nodules showing necrotic central cavity in the deleterious Group I Gmsacpd-c mutants
578
(F605 and F620), and its absence in the case of the Group II mutants (F714 and F813).
579
(B) Nodule cross sections stained with methylene blue and picrofushin showing typical
580
nodule cortex and vascular tissue in the Forrest WT, compared to the central cavity,
581
reduced infected cells, less vascular bundle differentiation, and decreased bacteroids of
582
the Group I mutants (magnification 100X). (NZ) Necrotic zone, (UZ) Undulated zone,
583
(IC) Infected cells, (UC) Uninfected cells, (VB) Vascular Bundles. Dark red arrows
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
19
584
present the undulated phenotype presented by Group I mutants. Black arrows represent a
585
separation between infected cell zones by uninfected cells in Group I mutants. This
586
separation was absent in the wild type Forrest and Group II mutants.
587
588
Figure 5. Comparison of leaf structure and morphology of the Forrest-WT and the
589
four Gmsacpd-c mutants. (A) Leaf images showing typical soybean leaf structure in the
590
Forrest WT, compared with the rigid undulated phenotype presented by the Gmsacpd-c
591
mutants. (B) Leaf cross sections stained with methylene blue showing typical (UE, LE)
592
upper and lower epidermis, (PM, SM) palisade and spongy mesophyll, and (VB)
593
vascular bundle (xylem and phloem) organization. This structure was drastically altered
594
in the four Gmsacpd-c mutants.
595
596
Figure 6. qRT-PCR analysis of Leghemoglobin gene family in the four Gmsacpd-c
597
mutant soybean lines. Differences in expression of the Legh-C1 (A) and LeghC2 (B)
598
genes between Group I and Group II mutants. LeghC3 gene was not expressed.
599
Expression values were normalized using Ubiquitin as reference. The gene-specific
600
primers designed for qRT-PCR analysis are detailed in Supplementary Table S3. Results
601
were analyzed with the analysis of variance (ANOVA), P<0.0001.
602
603
Supplemental materials
604
Supplemental Figure S1. The distribution of stearic acid contents in the seed oil of the
605
Forrest M3 population.
606
Supplemental Figure S2. Levels of the stearic acid of the screened high stearic acid
607
sacpd-c mutants and the wild type Forrest in the M4 (2013) generation.
608
Supplemental Figure S3. Expression levels of soybean Leghemoglobin genes in planta
609
based
610
(http://www.soybase.org/soyseq).
611
Supplemental Table S1. Level of stearic acid in seed oil of the five screened high stearic
612
mutants and the Forrest wild type were averaged for n M3 (2012) lines for each mutation.
613
Supplemental Table S2. Level of stearic acid in seed oil of the four screened high stearic
614
mutants and the Forrest wild type were averaged for n M4 (2013) lines for each mutation.
on
Soyseq
resource
available
from
RNAsequencing
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
data
20
615
Supplemental Table S3: The primers used for genotyping, sequencing and qRT-PCR
616
analysis.
617
618
619
Supplemental Figure S1. The distribution of stearic acid contents in the seed oil of the
620
Forrest M3 population. The dark color in the middle of each graph shows the wild type
621
Forrest distribution, n >1000. Top of the graph represents a box plot representing the
622
quartiles.
623
Supplemental Figure S2. Levels of the stearic acid of the screened high stearic acid
624
sacpd-c mutants and the wild type Forrest in the M4 (2013) generation. Content of stearic
625
acid of the mutants were measured in (A) nodules, (B) leaves, (C) roots, and (D) nodules.
626
Averages and standard deviations are shown. Results were performed with the analysis of
627
variance (ANOVA) test comparison, P < 0.1.
628
Supplemental Figure S3. Expression levels of soybean Leghemoglobin genes in planta
629
based
630
(http://www.soybase.org/soyseq). The figure shows that GmLb-C1 and GmLb-C2
631
encodes the nodule specific genes, whereas GmLb-C3 had almost no expression in most
632
tissues analysed, with the exception of a very small trace of expression in the roots.
633
Supplemental Table S1. Level of stearic acid in seed oil of the five screened high stearic
634
mutants and the Forrest wild type were averaged for n M3 (2012) lines for each mutation.
635
Averages and standard deviations are shown.
636
Supplemental Table S2. Level of stearic acid in seed oil of the four screened high stearic
637
mutants and the Forrest wild type were averaged for n M4 (2013) lines for each mutation.
638
Averages and standard deviations are shown.
639
Supplemental Table S3: The primers used for genotyping, sequencing and qRT-PCR
640
analysis.
on
Soyseq
resource
available
from
RNAsequencing
data
641
642
Acknowledgments
643
The authors wish to acknowledge the excellent technical assistance with GC
644
analysis provided by Darcie Hastings. We also would like to thank Dr. Judy Davie for
645
providing the qRT-PCR machine, to Abhinav Adhikari for his assistance, and to all the
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
21
646
student workers who contributed to the EMS mutagenized Forrest population
647
development.
648
649
References
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24
Table 1. Stearic acid content in the seed oil of the 5 screened high stearic acid
mutants was averaged for n homozygous M3 (2012), M4 (2013) or M5 (2014) lines.
The five fatty acids contents in seed oil of the four screened Forrest, Essex, Williams
82 and PI 88788 wild types were also averaged for n lines. Averages and standard
deviations are shown.
Lines
Palmitic Acid
(16:0)
Stearic Acid
(18:0)
Oleic Acid
(18:1)
Linoleic Acid
(18:2)
Linolenic Acid
(18:3)
Stearic acid change %
n
Forrest
10.93±0.59
3.25±0.15
18.89±2.34
53.71±3.28
6.89±0.79
_
33
F-605
6.93±1.08
20.11±1.68
20.77±7.70
31.39±4.99
4.32±0.50
636.63
4
F-620
9.60±0.27
10.04±2.08
12.91±1.50
53.03±0.94
7.52±0.36
267.76
5
F-714
10.09±0.53
7.24±1.89
14.88±1.57
53.73±2.41
8.23±1.09
165.20
7
F-813
9.78±1.33
6.90±1.27
14.7±3.49
42.91±3.63
6.7±1.81
152.74
5
F-869
8.50±1.62
6.52±2.07
11.79±1.76
41.44±4.33
8.29±1.55
138.82
3
Essex
10.91±0.42
4.11±0.41
18.27±1.61
52.8±0.9
54.91±0.64
50.54
4
Williams 82
9.96±0.7
3.74±0.36
16.29±5.18
50.78±1.88
7.08±0.96
36.99
4
PI 88788
9.05±0.71
2.42±0.33
15.59±6.44
41.05±2.96
8.36±1.75
-11.35
4
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Glyma14g27990-SACPD-C
Amplicon1
Amplicon2
Rv1
Fw1
Fw2
Rv2
ATG
TGA
847
570
229
235
247
305
340
1
B
GroupI
GroupII
C
G.maxF605
G.maxF620
G.maxF714
G.maxF813
G.maxF869
G.maxF-WT
A.thaliana
L.luteus
P.vulgaris
T.cacao
G.arboreum
C.cajan
R.communis
V.vinifera
P.trichocarpa
C.arie>num
P.vulgaris
E.grandis
M.truncatula
S.pennelliii
A.duranensis
A.ipaensis
N.tabacum
V.Angularis
O.sa>va
S.bicolor
Z.mays
S.italica
H.vulgare
S.mollendorffii
P.patens
C.reinharoI
PI,F,WI82
Essex
Mutantlineno.
F605
F620
F869
F714
F813
ForrestWT
GCC C G
GCC C G
GCT C G
GTC C G
GCC C A
ACC C G
GCC T G
GCC C G
Stearic%
20.11±1.68
10.04±2.08
6.52±2.07
7.24±1.89
6.90±1.27
2.73±0.95
1861
1964
444
SACPD-C
1777
A
G A G
A
G
G
G G
G
T
G
T
G
G
G
G
Effect PROVEAN PredicCons
Q83*
-
Deleterious
L79F
-3.737
Deleterious
E114K
-3.588
Deleterious
D77N
-2.195
Neutral
P102L
-6.085
Deleterious
-
-
-
Figure1.SACPD-CmutaConsofthescreenedfivehighstearicacidmutants.A:SACPD-Cgenemodelandpolymorphisms(G1777AandG1964T)
between PI88788, Forrest, Essex and Williams 82. B: Predicted SACPD-C proteins sequences showing amino acid differences between the five
mutantsiden[fiedbyforwardgene[cs:fourmissensemuta[ons(D77N,L79F,P102L,andE114K)andonenonsensemuta[on(Q83*)withinthe
SACPD-C.Proveanpredic[onsofthefourmissensemuta[onsiden[fiedareincluded.C:ProteinsequencealignmentofthefiveSACPD-Cscreened
mutants showing the iden[fied muta[ons: SACPD-CD77N, SACPD-CL79F, SACPD-CQ83*, SACPD-CP102L and SACPD-CE114K, in addi[on to SACPD-C
orthologous from other 26 plant species. SACPD-C proteins iden[fied from the following model plants; S. Mollendorffii (lycophyte), P. patens
(moss),C.reinhard7i(algae),O.sa7va(monocots),andA.thaliana(eudicots),inaddi[ontoothereudicotsandmonocotswereincludedinthe
alignment and showed striking sequence conserva[on. SACPD-C muta[ons within residues conserved in all species analyzed are in blue boxes
(GroupI).Muta[onswithinnon-conservedresiduesareinorangeboxes(GroupII).RedstarsrepresenttheconservedE105/76,E143/114,and
H146/117residuesimportantforironionbinding.Primersusedforgenotypingareindicatedbyarrows.
Figure 2. Homology
modeling of the GmSACPDC from Forrest with important
catalytic residues and the five
identified sacpd-c missense
mutations mapped. (A) and
(B) One subunit containing a
di-iron coordination complex.
The first iron has highly
conserved ligands Glu76 and
His117, the second iron has
ligands Glu167 and His203,
and the two residues Glu114
and Glu200 act as bridging
ligands in the coordination
space. Iron ions are
highlighted in orange. (C) and
(D) One subunit with the five
missense mutants highlighted
in different colors showing an
iron ion pocket localization.
(E) The predicted
homodimeric GmSACPD-C
enzyme.
A
HOCM vs. Lines
0.50
0.50
B
B
0.40
A
HOCM
C
D
0.25
0.25
0.15
0.15
0.20
0.20
F-605
F-620
F-714
Lines
HICM vs. Lines
0.45
0.45
F-813
F-WT
0.35
0.35
0.35
0.30
0.30
A
A
0.20
0.20
B
0.20
0.15
VICM vs. Lines
A
A
B
B
B
0.10
0.10
0.15
F-605
0.12
F-620
A
0.12
F-714
Lines
F-813
F-WT
CCM vs. Lines
F
0.05
F-605
F-620
F-605
F-714
F-620
B
Lines
F-714
VO
C
0.08 0.08
C
D
CCM
0.06 0.06
0.04 0.04
0.02
F-WT
0.15
0.15
0.20
0.10 0.10
F-813
Lines
VICM
HICM
B
0.25
E
F-714
0.25
0.25
0.30
0.10
F-620
0.40
0.40
0.40
0.35
F-605
D
A
0.40
0.25
C
C
0.20
0.20
0.25
0.25
0.30
C
0.30
0.30
D
0.30
0.30
0.15
B
0.35
0.35
VOCM
0.40
0.40
C
A
0.40
0.45
0.45
0.35
0.35
VOCM vs. Lines
0.45
0.45
F-605
F-620
F-605
F-620
F-714
Lines
F-714
F-813
F-813
F-WT
F-WT
HO
HI
VI
F-813
F-WT
F-813
F-WT
Figure 3. Comparison of
the cortex and cavity
measurements between the
Gmsacpd-c mutants and
the Forrest WT (A) Nodule
Horizontal Outer diameter.
(B) Nodule Vertical Outer
d i a m e t e r. ( C ) N o d u l e
Horizontal Inner diameter.
(D) Nodule Vertical Inner
diameter. (E) Biometric
measures of the nodule coat
size using the formula ((hohi)+(vo-vi))/2). (F)
Dissecting light microscope
images of a hand sectioned
nodule from the Forrest WT
showing details of: (HO)
H o r i z o n t a l o u t e r, ( H I )
Horizontal inner, (VO)
Vertical outer, (VI) Vertical
inner. F605 (n=21), F620
(n=22), F714 (n=20), F813
(n=25), and WT (n=20).
A
F-WT
Lines
F-605
Nodule
phenotype
F-620
F-714
F-813
NZ
UZ
StearicA.
content
2.13%±0.48
21.09%±0.98
22.99%±4.06
12.39%±0.94
15.24%±0.9
IC
IC
B
VB
Transversal
secBons
IC
IC
UC
VB
UC
IC
VB
VB
VB
Figure4.ComparisonofnodulestructureandmorphologyoftheForrest-WTandthefoursacpd-cmutants.(A)DissecBnglightmicroscope
images of hand secBoned nodules showing necroBc central cavity in the deleterious Group I Gmsacpd-c mutants (F605 and F620), and its
absenceinthecaseoftheGroupIImutants(F714andF813).(B)NodulecrosssecBonsstainedwithmethyleneblueandpicrofushinshowing
typical nodule cortex and vascular Bssue in the Forrest WT, compared to the central cavity, reduced infected cells, less vascular bundle
differenBaBon,anddecreasedbacteroidsoftheGroupImutants(magnificaBon100X).(NZ)NecroBczone,(UZ)Undulatedzone,(IC)Infected
cells, (UC) Uninfected cells, (VB) Vascular Bundles. Dark red arrows present the undulated phenotype presented by Group I mutants. Black
arrowsrepresentaseparaBonbetweeninfectedcellzonesbyuninfectedcellsinGroupImutants.ThisseparaBonwasabsentinthewildtype
ForrestandGroupIImutants.
A
F-WT
Lines
F-605
F-620
F-714
F-813
Leave
phenotype
StearicA.
content
2.48%±0.42
6.25%±1.35
5.32%±0.12
6.62%±1.85
4.97%±0.49
B
LeafCross
sec<on
100X
UE
LeafCross
sec<on
200X
PM
PM
SM
LE
PM
PM
PM
VB
Figure5.ComparisonofleafstructureandmorphologyoftheForrest-WTandthefourGmsacpd-cmutants.(A)Leafimagesshowingtypical
soybeanleafstructureintheForrestWT,comparedwiththerigidundulatedphenotypepresentedbytheGmsacpd-cmutants.(B)Leafcross
sec<onsstainedwithmethyleneblueshowingtypical(UE,LE)upperandlowerepidermis,(PM,SM)palisadeandspongymesophyll,and(VB)
vascularbundle(xylemandphloem)organiza<on.Thisstructurewasdras<callyalteredinthefourGmsacpd-cmutants.
A
B
20
16
A
100
A
80
A
12
A
60
40
8
4
B
C
B
20
B
C
C
F813-1
F-WT
0
0
F605-1
F620-1
GroupI
F714-1
F813-1
GroupII
F-WT
F605-1
F620-1
GroupI
F714-1
GroupII
Figure6.qRT-PCRanalysisofLeghemoglobingenefamilyinthefourGmsacpd-cmutantsoybeanlines.Differencesinexpressionof
theLegh-C1(A)andLeghC2(B)genesbetweenGroupIandGroupIImutants.LeghC3genewasnotexpressed.Expressionvalueswere
[email protected]ficprimersdesignedforqRT-PCRanalysisaredetailedinSupplementaryTable
S3.Resultswereanalyzedwiththeanalysisofvariance(ANOVA),P<0.0001.
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