GmSABP2-1 encodes methyl salicylate esterase and functions in soybean defense against soybean cyst nematode.

KEY MESSAGE: The soybean gene GmSABP2-1 encodes methyl salicylate esterase and its overexpression led to significant reduction in development of pathogenic soybean cyst nematode. Soybean cyst nematode (SCN, Heterodera glycines) is one of the most devastating pests of soybean (Glycine max L. Merr.). In searching for SCN-defense genes, a soybean gene of the methylesterase (MES) family was found to be upregulated in an SCN-resistant soybean line and downregulated in an SCN-susceptible line upon SCN infection. This gene was designated as GmSABP2-1. Here, we report on biochemical and overexpression studies of GmSABP2-1 to examine its possible function in SCN resistance. The protein encoded by GmSABP2-1 is closely related to known methyl salicylate esterases. To determine the biochemical function of GmSABP2-1, a full-length cDNA of GmSABP2-1 was cloned into a protein expression vector and expressed in Escherichia coli. The resulting recombinant GmSABP2-1 was demonstrated to catalyze the demethylation of methyl salicylate. The biochemical properties of GmSABP2-1 were determined. Its apparent Km value was 46.2 ± 2.2 µM for methyl salicylate, comparable to those of the known methyl salicylate esterases. To explore the biological significance of GmSABP2-1 in soybean defense against SCN, we first overexpressed GmSABP2-1 in transgenic hairy roots of an SCN-susceptible soybean line. When infected with SCN, GmSABP2-1-overexpressing hairy roots showed 84.5% reduction in the development of SCN beyond J2 stage. To provide further genetic evidence for the role of GmSABP2-1 in SCN resistance, stable transgenic soybean plants overexpressing GmSABP2-1 were produced. Analysis of the GmSABP2-1-overexpressing lines showed a significant reduction in SCN development compared to non-transgenic plants. In conclusion, we demonstrated that GmSABP2-1 encodes methyl salicylate esterase and functions as a resistance-related gene against SCN.

POWR1 is a domestication gene pleiotropically regulating seed quality and yield in soybean.

Seed protein, oil content and yield are highly correlated agronomically important traits that essentially account for the economic value of soybean. The underlying molecular mechanisms and selection of these correlated seed traits during soybean domestication are, however, less known. Here, we demonstrate that a CCT gene, POWR1, underlies a large-effect protein/oil QTL. A causative TE insertion truncates its CCT domain and substantially increases seed oil content, weight, and yield while decreasing protein content. POWR1 pleiotropically controls these traits likely through regulating seed nutrient transport and lipid metabolism genes. POWR1 is also a domestication gene. We hypothesize that the TE insertion allele is exclusively fixed in cultivated soybean due to selection for larger seeds during domestication, which significantly contributes to shaping soybean with increased yield/seed weight/oil but reduced protein content. This study provides insights into soybean domestication and is significant in improving seed quality and yield in soybean and other crop species.

An (E,E)-α-farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect-induced volatiles.

Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)-α-farnesene. GmAFS is closely related to (E,E)-α-farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN-resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN-susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN-susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two-spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography-mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)-α-farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below-ground and above-ground organs of soybean against nematodes and insects, respectively.

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines.

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production.

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 µM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

Gene expression profiling of resistant and susceptible soybean lines infected with soybean cyst nematode.

Soybean cyst nematode (SCN) is the most devastating pathogen of soybean. Information about the molecular basis of soybean-SCN interactions is needed to assist future development of effective management tools against this pathogen. Toward this end, soybean transcript abundance was measured using the Affymetrix Soybean Genome Array in a susceptible and a resistant reaction of soybean to SCN infection. Two genetically related soybean sister lines TN02-226 and TN02-275, which are resistant and susceptible, respectively, to the SCN race 2 infection were utilized in these experiments. Pairwise comparisons followed by false discovery rate analysis indicated that the expression levels of 162 transcripts changed significantly in the resistant line, of which 84 increased while 78 decreased. However, in the susceptible line, 1,694 transcripts changed significantly, of which 674 increased while 1,020 decreased. Comparative analyses of these transcripts indicated that a total of 51 transcripts were in common between resistance and susceptible responses. In this set, 42 transcripts increased in the resistant line, but decreased in the susceptible line. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of microarray analysis. Of the transcripts to which a function could be assigned, genes were associated with metabolism, cell wall modification, signal transduction, transcription, and defense. Microarray analyses examining two genetically related soybean lines against the same SCN population provided additional insights into the specific changes in gene expression of a susceptible and a resistant reaction beneficial for identification of genes involved in defense.

Molecular cloning and biochemical characterization of an endo-ß-mannanase gene from soybean for soybean meal improvement.

Soybean meal is the most commonly used protein source in animal feeds. Among the undesirable attributes of soybean meal is the high level of ß-mannan, which was determined to be detrimental to the growth performance of animals. ß-Mannan is a type of hemicellulose in the plant cell wall and can be hydrolyzed by endo-ß-mannanase. The goal of this study is to isolate and characterize an endo-ß-mannanase gene from soybean that can be used for genetic improvement of soybean meal. From the sequenced soybean genome, 21 putative endo-ß-mannanase genes were identified. On the basis of their relatedness to known functional plant endo-ß-mannanases, four soybean endo-ß-mannanase genes (GmMAN1 to GmMAN4) were chosen for experimental analysis. GmMAN1 and GmMAN4 showed expression in the soybean tissue examined, and their cDNAs without the sequences for signal peptide were cloned and expressed in Escherichia coli to produce recombinant enzymes. Only GmMAN1 showed endo-ß-mannanase hydrolase activity. Further gene expression analysis showed that GmMAN1 is specifically expressed in cotyledons of seedlings, suggesting a role of GmMAN1 in degrading mannan-rich food reserves during soybean seedling establishment. Purified recombinant GmMAN1 exhibited an apparent K(m) value of 34.9 mg/mL. The catalytic efficiency (k(cat)/K(m)) of GmMAN1 was determined to be 0.7 mL/(mg·s). GmMAN1 was also shown to be active in hydrolyzing the ß-mannan-rich cell wall of soybean seeds.

Measurement of inorganic phosphorus in soybeans with near-infrared spectroscopy.

This study explored the feasibility of near-infrared (NIR) quantitative and qualitative models for soybean inorganic phosphorus (Pi), which is complementary to phytic acid, a component of nutritional and environmental importance. Spectra, consisting of diffuse reflectance (1100-2500 nm) of ground meal and single-bean transmittance (600-1900 nm) of whole seed, were collected on 191 recombinant inbred soybean lines. Partial least-squares regression models were individually developed for soy meal diffuse reflectance, single-bean transmittance, and averaged (24 beans/line) whole seed transmittance data. The best performance was obtained with diffuse reflectance data, in which the standard errors (rmsd) were 263 and 248 mg/kg for cross-validation and validation sets, respectively. Model accuracy was lower for the 24-bean average transmittance spectra and still lower for single beans. Despite the overall poorer modeling ability of Pi with respect to the common macronutrient NIR regressions, such as those for protein and oil, this technique holds promise for use in breeding programs.

Correlations of oil and protein with isoflavone concentration in soybean [Glycine max (L.) Merr.].

Twelve isoflavones were detected by high-performance liquid chromatography in seeds of 17 soybean [Glycine max (L.) Merrill] cultivars grown at three locations. 6' '-O-Malonyldaidzin and 6' '-O-malonylgenistin together constituted 71-81% of total isoflavones, which ranged in concentration from 2038 to 9514 microg/g and averaged 5644 microg/g across locations and cultivars. The total as well as several individual isoflavones had a moderate negative correlation with oil across locations and cultivars. Six cultivars had a moderate or strong negative correlation of total isoflavones with oil. Five cultivars had a moderate or strong positive correlation of total isoflavones with protein. These results suggest that judicious selection of germplasm for soybean breeding may facilitate development of soybean lines with desirable isoflavone concentrations.

A novel approach to determine the glyphosate tolerant trait in soybeans.

The ability of soybean breeders to accurately, economically, and rapidly determine the transfer of the CP4 gene, the gene which confers soybean tolerance to the herbicide glyphosate, to elite soybean lines is essential to development of new glyphosate tolerant soybean (GTS) cultivars. This research focused on a simple greenhouse screening procedure to replace large, costly, and laborious field screening. Non-GTS seed was determined to be susceptible to soaking in a 1% glyphosate solution for 4 h. This process is quicker, more efficient, and as reliable as field screening for determination of glyphosate susceptibility in soybean seed. Furthermore, this research clearly demonstrates that the metabolic pathway of glyphosate activity, the shikimate acid pathway, is active, and the target enzyme of glyphosate, 5-enol-pyruvyl-shikimate-3-phosphate synthase, is present during seed germination.