Identification and characterization of large DNA deletions affecting oil quality traits in soybean seeds through transcriptome sequencing analysis.

KEY MESSAGE: Identification and characterization of a 254-kb genomic deletion on a duplicated chromosome segment that resulted in a low level of palmitic acid in soybean seeds using transcriptome sequencing. A large number of soybean genotypes varying in seed oil composition and content have been identified. Understanding the molecular mechanisms underlying these variations is important for breeders to effectively utilize them as a genetic resource. Through design and application of a bioinformatics approach, we identified nine co-regulated gene clusters by comparing seed transcriptomes of nine soybean genotypes varying in oil composition and content. We demonstrated that four gene clusters in the genotypes M23, Jack and N0304-303-3 coincided with large-scale genome rearrangements. The co-regulated gene clusters in M23 and Jack mapped to a previously described 164-kb deletion and a copy number amplification of the Rhg1 locus, respectively. The coordinately down-regulated gene clusters in N0304-303-3 were caused by a 254-kb deletion containing 19 genes including a fatty acyl-ACP thioesterase B gene (FATB1a). This deletion was associated with reduced palmitic acid content in seeds and was the molecular cause of a previously reported nonfunctional FATB1a allele, fap nc . The M23 and N0304-304-3 deletions were located in duplicated genome segments retained from the Glycine-specific whole genome duplication that occurred 13 million years ago. The homoeologous genes in these duplicated regions shared a strong similarity in both their encoded protein sequences and transcript accumulation levels, suggesting that they may have conserved and important functions in seeds. The functional conservation of homoeologous genes may result in genetic redundancy and gene dosage effects for their associated seed traits, explaining why the large deletion did not cause lethal effects or completely eliminate palmitic acid in N0304-303-3.

Effect of Δ9-stearoyl-ACP-desaturase-C mutants in a high oleic background on soybean seed oil composition.

KEY MESSAGE: Two new sources of elevated seed stearic acid were identified and the feasibility of an elevated stearic acid, high oleic acid germplasm was studied. Soybean [Glycine max (L.) Merr.] oil typically contains 2-4% stearic acid. Oil with at least 20% stearic acid is desirable because of its improved baking properties and health profile. This study identifies two new sources of high stearic acid and evaluates the interaction of high stearic and oleic acid alleles. TCHM08-1087 and TCHM08-755, high stearic acid 'Holladay' mutants, were crossed to FAM94-41-3, a line containing a point mutation in a seed-specific isoform of a Δ9-stearoyl-acyl carrier protein-desaturase (SACPD-C). F2-derived lines were evaluated for fatty acid content in four field environments. Sequencing of SACPDs in TCHM08-1087 and TCHM08-755 revealed distinct deletions of at least one megabase encompassing SACPD-C in both lines. After genotyping, the additive effect for stearic acid was estimated at +1.8% for the SACPD-C point mutation and +4.1% for the SACPD-C deletions. Average stearic acid in lines homozygous for the deletions was 12.2%. A FAM94-41-3-derived line and TCHM08-1087-11, a selection from TCHM08-1087, were crossed to S09-2902-145, a line containing missense mutations in two fatty acid desaturases (FAD2-1A and FAD2-1B). F1 plants were grown in a greenhouse and individual F2 seed were genotyped and phenotyped. No interaction was observed between either FAD2-1A or FAD2-1B and any of the SACPD-C mutant alleles. Seed homozygous mutant for SACPD-C/FAD2-1A/FAD2-1B contained 12.7% stearic acid and 65.5% oleic acid while seed homozygous for the SACPD-C deletion and mutant for FAD2-1A and FAD2-1B averaged 10.4% stearic acid and 75.9% oleic acid.

Effect of a novel mutation in a Δ9-stearoyl-ACP-desaturase on soybean seed oil composition.

Soybean [Glycine max (L.) Merr.] oil typically contains 2-4% stearic acid. Seed oil with 20% stearic acid would be useful for solid fat applications, both for its cooking properties and health benefits. Breeding lines with high stearic acid have been developed, but many suffer from agronomic problems. This study identifies a new source of high stearic acid, determines its relationship with another high stearic locus and presents molecular markers for it is use in breeding. TCJWB03-806-7-19, a 'Holladay' mutant with high stearic acid, was crossed to two FAM94-41-derived lines that contained a point mutation in a seed-specific isoform of a Δ9-stearoyl-acyl carrier protein-desaturase (SACPD-C). Fatty acid analysis was performed over two growing seasons with F(2)-derived lines and transgressive segregation for stearic acid content was observed. Sequencing of SACPD isoforms in TCJWB03-806-7-19 revealed the deletion of an 'A' nucleotide in exon 3 of SACPD-B, which results in a protein whose final 28 amino acids are predicted to differ from Williams 82 SACPD-B. Sorting intolerant from tolerant (SIFT) analysis revealed that this frameshift mutation may affect SACPD-B protein function. Allele-specific genotyping for the SACPD-C point mutation and SACPD-B nucleotide deletion was performed in both populations. Additive effects and R(2) for stearic acid were +3.3 and 0.55 for SACPD-C and +1.9 and 0.19 for SACPD-B. Average stearic acid in lines homozygous for both mutations was 14.6%. This SACPD-B mutation represents a novel high stearic allele.

Effects of temperature during soybean seed development on defense-related gene expression and fungal pathogen accumulation.

Soybean [Glycine max (L.) Merr] plants were exposed to three temperature regimens during seed development to investigate the effect of temperature on the expression of eight defense-related genes and the accumulation of two fungal pathogens in inoculated seeds. In seeds prior to inoculation, either a day/night warm (34/26 °C) or a cool temperature (22/18 °C) relative to normal (26/22 °C) resulted in altered patterns of gene expression including substantially lower expression of PR1, PR3 and PR10. After seed inoculation with Cercospora kikuchii, pathogen accumulation was lowest in seeds produced at 22/18 °C in which of all defense genes, MMP2 was uniquely most highly induced. For seeds inoculated with Diaporthe phaseolorum, pathogen accumulation was lowest in seeds produced at 34/26 °C in which of all defense genes, PR10 was uniquely most highly induced. Our detached seed assays clearly demonstrated that the temperature regimens we applied during seed development produced significant changes in seed defense-related gene expression both pre- and post inoculation and our findings support the hypothesis that global climate change may alter plant-pathogen interactions and thereby potentially crop productivity.

Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress.

Stress acclimating plants respond to abiotic and biotic stress by remodeling membrane fluidity and by releasing alpha-linolenic (18:3) from membrane lipids. The modification of membrane fluidity is mediated by changes in unsaturated fatty acid levels, a function provided in part by the regulated activity of fatty acid desaturases. Adjustment of membrane fluidity maintains an environment suitable for the function of critical integral proteins during stress. alpha-Linolenic acid, released from membrane lipid by regulated lipase activity, is the precursor molecule for phyto-oxylipin biosynthesis. The modulation of chloroplast oleic acid (18:1) levels is central to the normal expression of defense responses to pathogens in Arabidopsis. Oleic (18:1) and linolenic (18:2) acid levels, in part, regulate development, seed colonization, and mycotoxin production by Aspergillus spp.

Expression of the cercosporin transporter, CFP, in tobacco reduces frog-eye lesion size.

The cercosporin Major Facilitator Superfamily (MFS) transporter, CFP, under the control of the CaMV 35S promoter, was introduced into the Xanthi cultivar of tobacco by Agrobacterium-mediated transformation. CFP(+) transgenic plants were physically indistinguishable from non-transgenic Xanthi and progressed normally through growth to seed set. Accumulation of CFP in the leaf membrane fraction of CFP(+ )transgenic plants was associated with decreased cercosporin phytotoxicity. Frog-eye leaf lesions on CFP(+ )transgenic plants infected with Cercospora nicotianae conidia were smaller but were similar in number to those on non-transgenic plants. We conclude that transgenic expression of CFP may have relevance for a disease control strategy in Cercospora-plant pathosystems where cercosporin is implicated in pathogen virulence.

Expression in sugar beet of the introduced cercosporin toxin export (CFP) gene from Cercospora kikuchii, the causative organism of purple seed stain in soybean.

The Cercospora kikuchii cercosporin export gene, CFP, introduced into Beta vulgaris L. by conjugation with Rhizobium radiobacter, was stably maintained during vegetative propagation as verified by PCR using primers specific for the CFP gene. Transcriptional expression of the CFP gene in leaves was determined by RT-PCR using CFP-specific primers. CFP protein was detected using Western analysis with an affinity-purified polypeptide-specifc antibody. Analysis of the relative susceptibility of CFP-transgenic and non-transgenic sugar beet plants is planned but will probably take several years to complete.

A comparison of the insulin-sensitive transport of chromium in healthy and model diabetic rats.

In response to insulin, chromium(III) is moved from the blood to the tissues where it is ultimately lost in the urine, apparently as the oligopeptide chromodulin; this transfer of chromium is mediated by the protein transferrin. To examine the effects of type 1 and type 2 diabetes on the transport of chromium, the fate of chromium from intravenously introduced (51)Cr(2)-labelled transferrin was monitored after 2 h in healthy and diabetic model rats; the effects of insulin on the transport of chromium in these groups were also examined. Diabetic rats had greater urinary chromium loss, greater movement of chromium from the blood to the tissues, most notably to the skeletal muscle, and an alteration of the distribution of chromium in the blood plasma.

Transgenic assessment of CFP-mediated cercosporin export and resistance in a cercosporin-sensitive fungus.

Cercosporin is a toxic polyketide produced by many phytopathogenic members of the fungal genus Cercospora. Cercospora species, themselves, exhibit the highest level of self-resistance to this almost universally toxic photosensitizer. Although the mechanism of cercosporin self-resistance is multi-faceted, partial resistance does appear to be provided by the encoded product of CFP ( cercosporin facilitator protein), a gene recently isolated from the pathogen of soybean, C. kikuchii. CFP has significant similarity to the major facilitator superfamily of integral membrane transport proteins. We expressed CFP in the cercosporin non-producing, cercosporin-sensitive fungus, Cochliobolus heterostrophus, in order to assess the transport activity of CFP and the contribution of CFP to cercosporin resistance in a fungal species free of endogenous toxin production. Expression of the CFP transgene in this fungus results in increased resistance to cercosporin due, apparently, to its export out of the fungus.