Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Soybean [Glycine max (L.) Merr] plants were exposed to three temperature regimens during seed development in controlled chambers 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 oC) or a cool temperature (22/18 oC) relative to normal (26/22 oC) 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 oC 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 oC in which of all defense genes, PR10 was uniquely most highly induced. Secondly, we characterized soybean FAD7 and FAD8 gene structure and expression responses to temperature and pathogen stress in order to compare and contrast these features to those reported for higher plant plastidal ω-3 desaturases. We found that the genomic structure and deduced amino acid sequence of soybean FAD7 and FAD8 are similar to other higher plant plastidal omega-3 desaturases: 8 exons and 7 introns, predicted proteins of 453 amino acid residues containing three conserved histidine motifs, amino terminal chloroplast transit peptides, and molecular masses of 51.3 and 51.4 kDa, respectively. GmFAD7 has 76% amino acid sequence identity to GmFAD8. Two complete copies of GmFAD7, one on chromosome 18 and one on chromosome 7 and two complete copies of GmFAD8, one on chromosome 3 and one on chromosome 1 of the Williams 82 soybean genome were found with strong sequence similarity to GmFAD7 and GmFAD8 of cultivar Dare. Dare GmFAD7 transcript expression in leaves remained at a relatively low level, and was unaffected by any of the temperature treatments we imposed, but GmFAD8 transcript accumulation was sharply upregulated by a cool temperature (20/16oC) after a 12 h exposure and total linolenic acid as a percent of total leaf fatty acids increased from 60 to about 68% after 48 h at the cool temperature. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
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. Previous research has also shown that these imposed temperature regimens during seed development alter C:18 fatty acid composition of soybean seeds. Our findings suggest that changes in climate temperature may alter plant-pathogen interactions via altered expression of host defenses and seed fatty acid composition. Inoculation of soybean leaves with the fungal pathogen C. kikuchii differentially upregulated the level of GmFAD7 transcripts to twice that of GmFAD8 by 12 h post inoculation. The response of soybean foliar FAD8 expression and linolenic acid levels to cool temperature was similar to observations in other plants. On the other hand, despite increased soybean FAD7 expression in response to foliar pathogen stress the expected associated increase in foliar linolenic acid was not detected. These findings may contribute to the development of assays to better assess host resistance to soybean seed fungal pathogens.
Publications
- Upchurch, R.G. and Ramirez, M.E. 2011. Soybean plastidal omega-3 fatty acid desaturase genes GmFAD7 and GmFAD8: structure and expression. Crop Sci. 51:1673-1682.
- Upchurch, R.G. and Ramirez, M.E. 2011. Effects of temperature during soybean seed development on defense-related gene expression and fungal pathogen accumulation. Biotechnol. Lett. 33:2397-2404.
- Upchurch, R.G. 2011. Soybean fatty acid desaturation pathway: responses to temperature changes and pathogen infection, in Soybean-Genetics and Novel Techniques for Yield Enhancement. D. Krezhova (ed.) pp.113-128, InTech Press, Vienna
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: We characterized the expression of eight fatty acid metabolism genes in soybean lines expressing the mid-oleic phenotype (50-60% seed oleic acid)in order to determine if the level of gene expression is associated with this phenotype. It is known that seeds of the soybean x-ray mutant line M23 yield mid-oleic oil because of a deletion in the coding region of fatty acid desaturase gene GmFAD2-1A. The five mid-oleic soybean lines we examined did not have mutations or deletions in either the coding regions of GmFAD2-1A or GmFAD2-1B. We found that higher expression of three and/or lower expression of four of the genes was sometimes associated with the mid-oleic trait in these soybean lines. This finding suggests that multiple genes may contribute to the expression of the mid-oleic trait and some of these genes may regulate the expression of the oleic acid biosynthesis genes. A preliminary report of this work was presented at the site visit by the United States Soybean Board of the ARS-Soybean & Nitrogen Fixation Unit, Raleigh on July 21, 2009. This research will be published in the Journal of the American Oil Chemists' Society in 2010. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Agronomically improved M23-derived soybean lines that have the mid-oleic trait have been developed through plant breeding, but sometimes these lines shown trait instability in the field. Since other natural sources, though less well characterized, of the mid-oleic trait are available, these sources need to be analyzed to assist future breeding efforts. We compared the steady-state transcript abundance during seed development of four omega-6 fatty acid desaturase, three delta-9 stearoyl acyl carrier protein desaturase (GmSACPD), and the oleate-ACP thioesterase (GmFATB1a) genes in the five natural mid-oleic varieties and mutant M23 to gene expression data for the conventional non mid-oleic cultivar Dare. We found that higher expression of the three GmSACPD alleles, particularly GmSACPD-C as well as lower expression of GmFAD2-1A, Gm FAD2-1B, GmFAD2-2, and GmFAD2-3 could, in many instances, be associated with the mid-oleic phenotype of the natural mid-oleic varieties and M23. This finding, suggesting that multiple genes are involved in the mid-oleic phenotype, though a challenge for plant breeding, none the less provides options in that there are likely several genetic blueprints for achieving higher seed oleic acid content in soybean.
Publications
- Upchurch, R.G. and Ramirez, M.E. 2010. Gene expression profiles of soybeans with mid-oleic acid seed phenotype. JAOCS (in press)
- Upchurch, R.G. and Ramirez, M.E. 2009. Defense gene expression in soybean seeds inoculated with Cercospora kikuchii and Diaporthe phaseolorum. Annual meeting of the American Phytopathological Society, Portland, Oregon, Pyhytopathology 99:S132
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The compatible interaction of soybean with fungal pathogens Cercospora kikuchii (CK) and Diaporthe phaseolorum (DP)was examined in order to gain a snapshot of the host defense response to these pathogens. Expression of defense genes in pathogen-, mock-, or chemically-treated leaves was measured over a 48 h post-treatment period using soybean gene-specific primers and real time, quantitative RT-PCR. For CK treated leaves, expression as mean fold increase over the mock treated control (FIOC) of pathogenicity-related proteins PR3 and PR10 was strongly upregulated (FIOC ca. 17 and 16) within 24 h as was omega-3 fatty acid desaturase GmFAD7 (FIOC ca.12) within 12 h post-treatment. For DP treated leaves, defense gene upregulation was lower for PR3, PR10 and GmFAD7, but PR1 (FIOC ca. 9) was strongly upregulated within 12 h post-treatment. Soybean defensin (PR12) expression was moderately upregulated by DP treatment (FIOC ca. 5), but not by CK treatment. PR1 was moderately upregulated (FIOC ca. 4) by salicylic acid (SA) and PR3, PR4, and PR10 (FIOCs ca. 6, 6, and 3) by methyl jasmonate (MeJA) treatment. For PR12, no upregulation was detected with either chemical treatment. In contrast, upregulation of GmFAD7 was rapid, within 4 h of treatment with either SA or MeJA, but more strongly upregulated (FIOC ca. 15) by MeJA. Results show that soybean leaf defense mediated through PR gene expression has pathogen-specific differences in upregulation pattern but shares a similar GmFAD7 upregulation response. Mid-oleic soybeans (50-60% oleic acid in seed cotyledons) have sometimes shown increased colonization by Cercospora kikuchii in the field. To examine this phenomenon experimentally, we manipulated the fatty acid content in seeds of two isogenic soybean lines by allowing seed development to occur in three different air temperature environments. Seeds produced in these environments were harvested, individually analyzed for fatty acid composition, and inoculated post harvest with mycelium preparations of Cercospora kikuchii or Diaporthe phaseolorum. Fungal biomass of infected seeds was quantified by measuring in vitro ergosterol content. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
PR genes are generally upregulated in both compatible and incompatible plant-pathogen interactions with appropriate races of the pathogen, but more rapidly and/or to higher levels in plant varieties with R-gene type resistance. PIs with nuclear single dominant gene resistance to both CK and DP have been recently described. Analysis of these PIs inoculated with CK and DP using an expanded defense gene set that we have developed is expected to provide additional insight to our understanding of the basal defense response to fungal pathogens in susceptible and resistant soybean genotypes. Colonization by C. kikuchii was positively correlated with the oleic: linoleic acid ratio (r = 0.55, P < 0.03) and oleic acid content (r = 0.61, P < 0.02), and negatively correlated with linoleic (r = -0.60, P < 0.02) and linolenic (r = -0.58, P < 0.03) acid content. No association was found between the extent of seed colonization by D. phaseolorum and the seed oleic: linoleic acid ratio. Our data suggests that the oleic: linoleic acid ratio can be an important factor for soybean seed colonization by C. kikuchii but may not be a factor for colonization by D. phaseolorum.
Publications
- Upchurch, R.G. and Ramirez, M.E. 2008. Defense gene induction in the compatible interaction of soybean with Cercospora kikuchii and Diaporthe phaseolorum. Fourth International Conference on Legume Genomics and Genetics
- Upchurch, R.G. 2008. Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress. Biotech. Lett. 30:967-977.
- Xue, H.Q., Upchurch, R.G., and Kwanyuen, P. 2008. Relationships between oleic and linoleic acid content and seed colonization by Cercospora kikuchii and Diaporthe phaseolorum. Plant Dis. 92:1038-1042.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Seeds of mid-oleic soybeans have sometimes shown increased colonization by Cercospora kikuchii and other fungi in the field. To examine these phenomena experimentally, we manipulated the fatty acid content in seeds of two isogenic soybean lines by allowing seed development to occur in three different air temperature environments. Seeds produced in these environments were harvested, individually analyzed for fatty acid composition, and inoculated with mycelium preparations of the fungal seed pathogens Cercospora kikuchii (Matsumoto & Tomoy.) or Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. sojae. Fungal biomass of infected seeds was quantified by measuring in vitro ergosterol content. For both soybean lines, colonization by C. kikuchii was positively correlated with the oleic: linoleic acid ratio and oleic acid content, and negatively correlated with linoleic and linolenic acid content. This association was not found between the extent of seed colonization by D.
phaseolorum and the seed oleic: linoleic acid ratio. Our data suggests that the oleic: linoleic acid ratio can be an important factor for soybean seed colonization by C. kikuchii but may not be a factor for colonization by D. phaseolorum.
PARTICIPANTS: Dr. Huiqin Xue, Crop Science Department, NC State University, Raleigh, NC 27695 (Now at Monsanto Corporation, Ames, Iowa) Dr. Prachuab Kwanyuen, USDA-ARS Soybean and Nitrogen Fixation Unit, NC State University, Raleigh, NC 27695
Impacts
Fatty acids are crucial components of cellular membranes in seeds as well as other plant parts that provide a structural barrier to the environment. Fatty acids also contribute to inducible stress resistance mechanisms. Linolenic acid is itself a signaling molecule and the precursor for phyto-oxylipin biosynthesis. Thus, alteration of seed fatty acid content may lead to altered susceptibility of seeds to pathogen attack. Fatty acids play a central role in the interaction of fungi with oilseeds. Evidence suggests that plant fatty acid (and oxylipin) species signal and regulate fungal development, pathogen colonization of oilseeds, and mycotoxin production. Plants commonly encounter the abiotic stresses of low or elevated temperature and biotic pathogen and insect attack, sometimes simultaneously. Even with the best farm land and agricultural practices, the impacts of these stresses can significantly reduce the productivity of agricultural food and fiber crops. Thus, the
current situation of diminishing farm land worldwide and the potential heightened effects of global climate change on environmental, pathogen and insect stresses provide increased impetus to understand stress resistance in crop plants. More complete knowledge of fatty acid unsaturation, mobilization, and signaling processes may significantly aid the development of effective strategies for managing abiotic and biotic stresses for these plants.
Publications
- Byfield, G.E. and Upchurch, R.G. 2007. Effect of temperature on delta-9 stearoyl-ACP and microsomal omega-6 desaturase gene expression and fatty acid content in developing soybean seeds. Crop Sci. 47:1698-1704.
- Byfield, G.E. and Upchurch, R.G. 2007. Effect of temperature on microsomal omega-3 linoleate desaturase gene expression and linolenic acid content in developing soybean seeds. Crop Sci. 47:2445-2452.
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Progress 10/01/05 to 09/30/06
Outputs
The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post-translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean microsomal omega-3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega-3 fatty acid desaturases (FAD3s) catalyze the insertion of a third double bond into the linoleic (C18:2) acid to produce linolenic (C18:3) acid. At 35 days after flowering (DAF), transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5-15 fold, GmFAD3B by 4-9 fold, and GmFAD3C by 2-3 fold in seeds that developed in a warm (D/N=30/26oC) versus a cool (D/N=22/18oC) environment. At this stage of seed development, decreased omega-3 desaturase gene expression
levels were positively associated with decreased linolenic acid content in the seed that developed in the warm environment. The reductions in linolenic acid content (39-50 %) measured in seed developing in the warm environment were substantial when compared to the reductions (27-67 %) reported for linolenic acid content measured in the mature seed of omega-3 desaturase soybean mutants.
Impacts
Linolenic acid content in soybean oil is critical since high levels cause the oil to oxidize, become rancid, and lose quality. Previous research has show that linolenic acid content in soybean seeds is a heritable trait and significantly the result of the action of the three omega-3 fatty acid desaturase genes present in soybean. In order to maintain the production of high quality soybean oil, the environmental factor, growth temperature, which interacts with plant genotype to regulate linolenic acid accumulation in seed must be assessed. The present investigation was conducted to determine to what extent omega-3 fatty acid desaturase gene expression changes in developing soybean seeds in response to changes in growth temperature and how these changes are associated with seed linolenic acid content. In the warm environmrnt, the expression of each omega-3 desaturase gene was significantly reduced and this contributed to a reduction in the linolenic acid content of
39-50 %. The warm temperature induced reduction was substantial when compared to the reduction in linolenic acid (27-67 %) measured in the mature seed of omega-3 desaturase soybean mutants.
Publications
- Xue, H.Q., Upchurch, R.G. and Kwanyuen, P. 2006. Ergosterol as a quantifiable biomass marker for Diaporthe phaseolorum and Cercospora kikuchii. Plant Disease 90: 1395-1398.
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Progress 10/01/04 to 09/30/05
Outputs
The ∆9 stearoyl-ACP desaturases (SACPDs) are important enzymes for the acclimation of organisms, including soybean, to low temperatures. Since these enzymes carry out important housekeeping functions such as lipid membrane biosynthesis they are under constitutive control, but may also be supplemented by fine regulation at both the transcriptional and post-transcriptional levels. We measured the transcript accumulation of SACPD-A and -B in developing soybean seeds across cold (22/18oC) normal (26/22oC), and warm (30/26oC) temperatures by real time, reverse transcriptase-PCR. Transcript accumulation of SACPD-A and -B decreased by up to 69% with increasing temperature in cultivars Dare, A6 (a high stearate line), and N01-3544 (a mid-oleic line). Stearic acid content of A6 was inversely related to the level of SACPD-A and -B expression at the warm and cold temperatures, as might be expected, but the stearic acid content of N01-3544 and Dare did not appear to be
affected by temperature despite dynamic changes in the expression levels of SACPD in these lines. This suggests that in these two lines stearate does not accumulate with higher temperature because it may be metabolically reclaimed as it is synthesized. Similarly, but unexpectedly, the oleic acid content of all three lines was inversely related to the levels of SACPD expression at the warm and cold temperatures. This suggests that transcription control of SACPD may not be a crucial factor regulating oleic acid content in soybean.
Impacts
The ∆9 stearoyl-ACP desaturases (SACPDs)convert stearic to oleic acid and are important enzymes for the acclimation of organisms, including soybean, to low temperatures. We measured the transcript accumulation of SACPD-A and -B in developing soybean seeds across cold (22/18oC) normal (26/22oC), and warm (30/26oC) temperatures by real time, reverse transcriptase-PCR. Transcript accumulation of SACPD-A and -B decreased by up to 69% with increasing temperature in cultivars Dare, A6 (a high stearate line), and N01-3544 (a mid-oleic line). Stearic acid content of A6 was inversely related to the level of SACPD-A and -B expression at the warm and cold temperatures, as might be expected, but the stearic acid content of N01-3544 and Dare did not appear to be affected by temperature despite dynamic changes in the expression levels of SACPD in these lines. This suggests that soybean lines and cultivars vary in their response to temperature with respect to stearic and oleic
acid accumulation although temperature effects on SACPD transcript accumulation appear similar across lines and cultivars.
Publications
- Byfield, G.E., Xue, H.Q., Upchurch, R.G. 2006. Two genes from soybean encoding soluble delta 9 stearoyl-ACP desaturases. Crop Science (accepted September 2005)
- Xue, H.Q., Upchurch, R.G. 2005. Quantification of fungal infection of soybean seed using ergosterol as an indicator of fungal biomass. Phytopathology 95:S114 (Abstract).
- Upchurch, R.G., Rose, M.S., Eweida, M., Zuo, W. 2005. Expression of the cercosporin transporter, CFP, in tobacco reduces frog-eye lesion size. Biotechnology Lett. 27:1543-1550.
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Progress 10/01/03 to 09/30/04
Outputs
The A9 stearoyl acyl-carrier protein desaturase (SACPD) gene of soybean [Glycine max (L.) Merrill] encodes a soluble delta-9 desaturase enzyme that converts stearic to oleic acid. Understanding the regulation of SACPD expression and enzyme activity are thus important steps towards developing soybean lines with altered stearic or oleic acid content. Using primers designed to a G. max SACPD EST sequence, a 3391 bp product was cloned and sequenced from the genome of cultivar Dare. Comparison of the third SACPD exon protein sequence with other Glycine exon 3 sequences revealed unique amino acid variability at positions 309, 312, 355, and 373. Sequence-specific primers were designed for Real-time RT-PCR (reverse transcriptase-polymerase chain reaction) for this region of exon 3. Diagnostic and specific products were recovered with these primers using Dare cDNA template and Dare genomic DNA. Sequencing of a second genomic clone from Dare confirmed that there were two SACPD
genes, designated A and B. Survey of the genomes of 51 soybean lines and cultivars with PCR and the gene-specific primers indicated that all 51 had both genes. Differences between SACPDA and B transcript abundance in soybean tissues while quantifiable were not dramatic. SACPDA and B transcript accumulation at four seed developmental stages between R5 and R6 was essentially parallel, although at stage 1, 3, and 4, SACPDA transcript accumulation was 10 to 15% higher than B. Biochemical analysis of the proteins encoded by these two SACPD genes may reveal if the unique amino acid variability uncovered in this study has any relation to enzyme activity.
Impacts
The gene specific primers developed for the three fatty acid desaturases (SACPD, 2 genes; FAD2-1, 2 genes; and FAD3, 3 genes) will enable us to determine if temperature, which is known to affect fatty acid composition of the seed, also affects the transcript expression of these genes during seed development. This knowledge will contribute to our efforts to produce soybeans lines that stably express desirable seed oil quality traits under a variety of field environments.
Publications
- Byfield, G., Xue, H. , and Upchurch, R.G. 2004. Effects of temperature on desaturase gene expression at various stages of seed development in soybean. Soy2004 Conference, Columbia, MO (Abstract)
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Progress 10/01/02 to 09/30/03
Outputs
The complete genomic sequence of the the stearyl ACP desaturase gene was determined for two soybean cultivars. These sequences along with other incomplete SACPD sequences in the NCBI data base indicate that the soybean SACPD gene has at least two isoforms. Sequence analysis for the FAD 2-1 and FAD 3 desaturase genes indicate that these genes also have at least two isoforms each. Using sequence information for these genes, we developed isoform-specific PCR primers suitable for the reverse transcription, real time PCR procedure. Five soybean cultivars varying in seed fatty acid profile were grown under three temperature regimes in the environmental growth facility (phytotron) at NC State University. Seeds at four developmental stages between R5 and R6 were collected and quick frozen for later RNA isolation. A reverse transcription, real time PCR procedure is being optimized and standard curves and copy number threshold values are being established for the soybean actin
gene. Mature seeds will be collected for fatty acid profile analysis.
Impacts
The isoform specific primers developed for the three fatty acid desaturase genes (SACPD, FAD2-1, and FAD3) will enable us to determine if temperature, which is known to affect fatty acid composition of the seed, also affects the transcript expression of these genes during seed development. This knowledge will contribute to our efforts to produce soybeans lines that stably express desirable seed oil quality traits under a variety of field environments.
Publications
- Upchurch, R.G., Rose, M.S., Eweida, M., Zuo, W. Expression of the cercosporin facilitator protein transporter CFP in tobacco limits infection by Cercospora nicotianae. (2003) Revised and resubmitted to Plant Biotechnology.
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