Immature Soybean Seeds Research Articles

Impact of pod and seed photosynthesis on seed filling and canopy carbon gain in soybean.

There is a limited understanding of the carbon assimilation capacity of nonfoliar green tissues and its impact on yield and seed quality since most photosynthesis research focuses on leaf photosynthesis. In this study, we investigate the photosynthetic efficiency of soybean (Glycine max) pods and seeds in a field setting and evaluate its effect on mature seed weight and composition. We demonstrate that soybean pod and seed photosynthesis contributes 13% to 14% of the mature seed weight. Carbon assimilation by soybean pod and seed photosynthesis can compensate for 81% of carbon loss through the respiration of the same tissues, and our model predicts that soybean pod and seed photosynthesis contributes up to 9% of the total daily carbon gain of the canopy. Chlorophyll fluorescence (CF) shows that the operating efficiency of photosystem II in immature soybean seeds peaks at the 10 to 100 mg seed weight stage, while that of immature pods peaks at the 75 to 100 mg stage. This study provides quantitative information about the efficiency of soybean pod and seed photosynthesis during tissue development and its impact on yield.

Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max ).

Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.

Rapidly evolving phosphoenolpyruvate carboxylase Gmppc1 and Gmppc7 are highly expressed in the external seed coat of immature soybean seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon fixation enzyme which probably plays crucial roles in seed development. A greater number of PEPC isoforms are encoded in the soybean genome, while most of the PEPC isoforms are functionally unknown. In this study, we investigated on soybean PEPC expressed in the external layer of seed coat (ELSC) during seed formation. PEPC activity in ELSC ranged from 0.24 to 1.0U/g F.W., which could be comparable to those in whole seeds at U per dry matter. Public RNA-Seq data in separated soybean seed tissues revealed that six plant-type PEPC isogenes were substantially expressed in ELSC, and Gmppc1 and Gmppc7 were highly expressed in hourglass cells of ELSC. Gene Ontology enrichment of co-expressed genes with Gmppc1 and Gmppc7 implicated a role of these isogenes in assisting energy production and cellulose biosynthesis. Comparison of PEPC sequences from 16 leguminous species hypothesized adaptive evolution of the Gmppc1 and Gmppc7 lineage after divergence from the other plant-type PEPC lineages. Molecular diversification of these plant-type PEPC was possibly accomplished by adaptation to the functions of the soybean seed tissues. This study indicates that energy demand in immature seeds may be a driving force for the molecular evolution of PEPC.

The plant-type phosphoenolpyruvate carboxylase Gmppc2 is developmentally induced in immature soy seeds at the late maturation stage: a potential protein biomarker for seed chemical composition.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon-fixing enzyme with critical roles in seed development. Previously we observed a positive correlation between PEPC activity and protein content in mature seeds among soybean cultivars and varietal differences of PEPC activity in immature seeds, which is concordant with seed protein accumulation. Here, we report a PEPC isoform (Gmppc2) which is preferentially expressed in immature soybean seeds at the late maturation stage. Gmppc2 was co-expressed with enzyme genes involved in starch degradation: α-amylase, hexokinase, and α-glucan phosphorylase. Gmppc2 was developmentally induced in the external seed coats, internal seed coats, hypocotyls, and cotyledons at the late maturation stage. The expression of Gmppc2 protein was negatively regulated by the application of a nitrogen fertilizer, which suppressed nodule formation. These results imply that Gmppc2 is involved in the metabolism of nitrogen originated from nodules into seeds, and Gmppc2 might be applicable as a biomarker of seed protein content.Abbreviations: PEP: phosphoenolpyruvate; PEPC: phosphoenolpyruvate carboxylase; RNA-Seq: RNA sequencing; PCA: principal component analysis; SE: standard error.

The natural plant stress elicitor cis-jasmone causes cultivar-dependent reduction in growth of the stink bug, Euschistus heros and associated changes in flavonoid concentrations in soybean, Glycine max

To test the hypothesis that the plant stress related elicitor cis-jasmone (cJ) provides protection in soybean pods against the seed-sucking stink bug pest, Euschistus heros, the growth of E. heros on cJ-treated pods was investigated using three soybean cultivars differing in insect susceptibility, i.e. BRS 134 (susceptible), IAC 100 (resistant) and Dowling (resistant). E. heros showed reduced weight gain when fed cJ-treated Dowling, whereas no effect on weight gain was observed when fed other treated cultivars. Using analysis of variance, a three factor (cultivar x treatment x time) interaction was observed with concentrations of the flavonoid glycosides daidzin and genistin, and their corresponding aglycones, daidzein and genistein. There were increases in genistein and genistin concentrations in cJ-treated Dowling at 144 and 120 h post treatment, respectively. Higher concentrations of malonyldaidzin and malonylgenistin in Dowling, compared to BRS 134 and IAC 100, were observed independently of time, the highest concentrations being observed in cJ-treated seeds. Levels of glycitin and malonylglycitin were higher in BRS 134 and IAC 100 compared to Dowling. Canonical variate analysis indicated daidzein (in the first two canonical variates) and genistein (in the first only) as important discriminatory variables. These results suggest that cJ treatment leads to an increase in the levels of potentially defensive isoflavonoids in immature soybean seeds, but the negative effect upon E. heros performance is cultivar-dependent.

ホソヘリカメムシ，アオクサカメムシ，イチモンジカメムシおよびブチヒゲカメムシによるダイズ子実汚斑病（仮称）病原菌Eremothecium coryliの保菌および媒介

Eremothecium coryli has been identified as the causative agent of yeast-spot disease in soybeans. To determine which true bug species might be vectors for yeast-spot disease in Japan, Riptortus clavatus, Nezara antennata, Piezodorus hybneri and Dolycoris baccarum were collected from commercially grown soybean plants in Kyoto and Hiroshima Prefectures, in July to September 2006. The carrying rates of E. coryli were 77.7, 35.7, 20.4 and 43.8% on R. clavatus, N. antennata, P. hybneri and D. baccarum adults, respectively. The carrying rate of E. coryli was 11.5% among R. clavatus nymphs. But E. coryli was not isolated from N. antennata and D. baccarum nymphs. The transmission rates of E. coryli to immature soybean seeds were 81.6, 50.0, 16.7 and 40.0% by R. clavatus, N. antennata, P. hybneri and D. baccarum adults, respectively. The results suggested that these 4 species are capable of carrying and transmitting E. coryli that cause yeast-spot disease in soybeans.

Effects of Nitrogen and Calcium Supply on the Accumulation of Oxalate in Soybean Seeds

Soybean [Glycine max (L.) Merr.] seeds have sufficiently high oxalate concentration that they are not recommended for consumption by individuals prone to formation of kidney stones. One possible precursor of oxalate is glyoxylic acid and this acid is formed in soybean seeds as a product of ureide catabolism. A nodulating soybean variety was compared with its nonnodulating isoline and the lack of nodules resulted in significant decline in ureide supply to developing seeds. However, oxalate concentrations in the seeds of the isoline were not reduced, suggesting that oxalate is not formed via glyoxylate in soybean seeds. Because oxalate and Ca concentrations are often correlated, manipulation of Ca supply to soybean plants was also studied by increasing Ca supply during pod development by 3.5‐fold relative to the level in the rooting medium. Surprisingly, there was no significant effect of Ca supply on Ca concentrations in leaves, pod walls, or seeds, indicating that Ca accumulation is stable in spite of Ca supply. The Ca treatment also did not significantly alter oxalate accumulation in developing seeds. In spite of this lack of treatment effect, the correlation of oxalate and Ca concentrations within sampling times was generally significant. Oxalate concentration was 2‐ to 3‐fold higher in developing seeds than in mature seeds. It is concluded that (i) the correlation between Ca and oxalate in developing soybean seeds is probably related to unknown factors influencing oxalate accumulation and the subsequent binding of Ca by the oxalate accumulated, (ii) lowering oxalate in soybean seeds will most likely be achieved by selection of low‐oxalate genotypes, and (iii) because oxalate concentrations are higher in immature soybean seeds than in mature seeds, those prone to formation of kidney stones should be cautious about consuming immature soybeans.

Protein and oil concentration of soybean seed cultured in vitro using nutrient solutions of differing glutamine concentration

SummaryOil and protein are the most valuable components of soybean seed. Evidence indicates that growth and composition of soybean seed are controlled by supplies of carbon and nitrogen provided by the maternal plant to the seed, but it is difficult experimentally to control and quantify the precise amount of carbon and nitrogen provided to the seed by the whole plant. To examine whether oil and protein concentrations are affected by the supply of nitrogen to the seed, immature soybean seeds (Glycine max cv. Williams 82) were grown in vitro in nutrient solutions containing 20, 40, 60 or 80 mM of glutamine. The seeds were incubated in Erlenmeyer flasks for 8 days at 25°C. The rate of dry matter accumulation changed from 7.2 to 8.3 mg seed−1 day−1 as the glutamine concentration increased from 20 to 80 mM but the differences were not significant (P 0.05). Seed protein concentration increased as glutamine concentration increased from 294 mg g−1 at 20 mM glutamine to as high as 445 mg g−1 at 80 mM glutamine. Typical in vivo protein concentration of mature soybean seeds is about 400 mg g−1. Oil and protein concentrations were negatively correlated (r2= 0.44), which indicates that oil and protein synthesis are interrelated. Protein synthesis was favoured over oil synthesis when nitrogen became more abundant. The seeds used in this study clearly demonstrated a capacity to respond to nitrogen availability with changes in seed protein concentration.

Effects of temperature on oil and protein concentration in soybean seeds cultured in vitro

SummaryMuch of the economic value of soybean (Glycine max) is based on the amount of protein and oil produced in the seeds. To examine the influence of temperature on seed oil and protein concentration, immature soybean seeds (cv. Williams 82) were grown in vitro at temperatures of 17°C, 21°C, 25°C, 29°C and 33°C. Dry growth rate (DGR) was calculated to be maximal at 23.7°C. Oil and protein concentration and seed growth rate did not show statistical difference (P > 0.05) within the temperature range from 21–29°C. Across all temperatures, however, a quadratic regression on oil concentration (R2 = 0.66) showed a minimum at 24.1°C and a quadratic regression on protein concentration (R2 = 0.59) showed a minimum at 24.3°C. Dilution by increased dry matter accumulation in the seed accounted for much of the variation in oil and protein concentration and the two concentrations were equally affected across temperatures. Consequently, oil and protein concentrations were positively related over the tested range of temperature. It was concluded that under these conditions the rate of dry matter accumulation by soybean seeds was critical in influencing seed oil and protein concentrations.

A soybean plastid‐targeted NADH‐malate dehydrogenase: cloning and expression analyses

A typical soybean (Glycine max) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2-ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyruvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule-enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana, which has a completely different transit peptide. In addition, the soybean nodule-enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of ∼34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene.

Processing and localization of bovine β-casein expressed in transgenic soybean seeds under control of a soybean lectin expression cassette

We have examined the processing and subcellular localization of a chimeric gene consisting of the bovine milk protein, β-casein, under the control of a soybean seed lectin promoter and its 32 amino acid signal sequence in the seeds of transgenic soybean plants. The β-casein expressed in developing soybean seeds is a doublet with apparent molecular weight slightly smaller than the bovine β-casein and expression of the protein was highest in immature cotyledons. The casein proteins were purified from the immature soybean seeds by immunoaffinity chromatography and were analyzed by two-dimensional gel electrophoresis, blotting, and amino terminal sequencing. The N-terminal sequences of both of the doublet soybean casein polypeptides were identical to the N-terminal sequence of the bovine β-casein indicating that the 32 amino acid lectin signal sequence was cleaved precisely from the chimeric protein in developing soybean seeds. Analysis of the purified soybean β-casein polypeptides by mass spectrometry (MALDI-MS) showed that they are not phosphorylated. Absence of added phosphate groups is the cause of the size difference between the soybean β-casein and native bovine β-casein protein. Immunolocalization experiments showed that the casein protein was found in the protein storage vacuoles (PSV) in developing and mature soybean seeds. The precise removal of the 32 amino acid lectin amino terminal sequence from the chimeric lectin–casein fusion suggests that the lectin expression cassette can be used for production of pharmaceutical or other recombinant proteins of added value in the developing soybean seed.

Immature soybean seeds as a vegetable or snack food: acceptability by American consumers

Using American panelists, we examined the palatability of frozen immature soybean seeds (R 6) and freeze-dried soybean snacks (FDSS) prepared from selected immature soybean genotypes ( Hutcheson, NTCPR93-286, NTCPR93-1557 and Honey Brown). The frozen immature soybean seeds were evaluated in comparison with frozen peas [ Pisum sativum (L.) sativum] and lima beans [ Phaseolus lunatus (L.) lunatus], whereas the FDSS prototypes were evaluated based on their appearance and eating quality. Frozen immature soybean seeds were rated favorably compared to other legume products. The overall rank sum indexes (ORSI) for frozen peas, NTCPR93-286, Hutcheson, and lima beans were 4, 10, 10, and 16, respectively. Small ORSI values reflected higher consumer preference. The ORSI of the FDSS products were 13 for Hutcheson, 23 for NTCPR93-286, 24 for NTCPR93-1557, and 26 for Honey Brown. Although Hutcheson and NTCPR93-286 were among the top rated varieties, all FDSS used in this study showed a favorable rating in all attributes of eating quality. The result suggests that American consumers could accept the immature frozen soybean as a vegetable or FDSS.

Retention and changes of soy isoflavones and carotenoids in immature soybean seeds (Edamame) during processing.

Isoflavones and carotenoids in four experimental genotypes and Hutcheson cultivar soybeans were evaluated as a function of processing treatments and maturity. Total isoflavone and carotenoid contents were affected by genotypes and maturity stages (p < 0.0001). Total isoflavones ranged from 472 microg/g (in NTCPR93-40) to 2280 microg/g (in Hutcheson). Lutein contents ranged from 895 (in NTCPR93-286) to 2119 (in Honey Brown), and beta-carotene ranged from 291 (in Hutcheson) to 491 (in NICPR92-40) microg/100 g. Mean total isoflavone retention percentages in immature Hutcheson soybeans were 46% (boiling), 53% (freezing), and 40% (freeze-drying). Mean retentions of lutein and beta-carotene, respectively, were 92 and 73% in frozen, 62 and 62% in boiled, and 34 and 27% in freeze-dried soybeans. Boiling caused a substantial increase in daidzin, genistin, and genistein. The results show that post-harvest changes in total isoflavones and carotenoids in soybeans are influenced by processing methods, but genotype has an effect on isoflavone and carotenoid profiles during seed development.

Color Classifier for Symptomatic Soybean Seeds Using Image Processing.

Symptoms associated with fungal damage, viral diseases, and immature soybean (Glycine max) seeds were characterized using image processing techniques. A Red, Green, Blue (RGB) color feature-based multivariate decision model discriminated between asymptomatic and symptomatic seeds for inspection and grading. The color analysis showed distinct color differences between the asymptomatic and symptomatic seeds. A model comprising six color features including averages, minimums, and variances for RGB pixel values was developed for describing the seed symptoms. The color analysis showed that color alone did not adequately describe some of the differences among symptoms. Overall classification accuracy of 88% was achieved using a linear discriminant function with unequal priors for asymptomatic and symptomatic seeds with highest probability of occurrence. Individual classification accuracies were asymptomatic 97%, Alternaria spp. 30%, Cercospora spp. 83%, Fusarium spp. 62%, green immature seeds 91%, Phomopsis spp. 45%, soybean mosaic potyvirus (black) 81%, and soybean mosaic potyvirus (brown) 87%. The classifier performance was independent of the year the seed was sampled. The study was successful in developing a color classifier and a knowledge domain based on color for future development of intelligent automated grain grading systems.

Identification of Inositol 1,3,4-Trisphosphate 5-Kinase and Inositol 1,3,4,5-Tetrakisphosphate 6-Kinase in Immature Soybean Seeds

Abstract In extracts of immature soybean (Glycine max [L.] Merr.) seeds inositol tetrakisphosphate was formed from [3H]inositol 1,3,4-trisphosphate but not from [3H]inositol 1,4,5-trisphosphate. Inositol 1,3,4-trisphosphate kinase was purified to a specific activity of 3.55 min−1 mg−1 by polyethylenimine clarification and anion-exchange chromatography. The partially purified enzyme converted [3H]inositol 1,3,4-trisphosphate to inositol 1,3,4,5-tetrakisphosphate as the major product and inositol 1,3,4,6- and/or 1,2,3,4-tetrakisphosphate as the minor product. Subsequent experiments revealed a separate inositol 1,3,4,5-tetrakisphosphate 6-kinase activity, which could link these enzymes to inositol hexakisphosphate synthesis via the previously reported inositol 1,3,4,5,6-pentakisphosphate 2-kinase. The apparent Km values for inositol 1,3,4-trisphosphate kinase were 200 ± 0 nm for inositol 1,3,4-trisphosphate and 171 ± 4 μm for ATP, and the reaction was not reversible. The kinetics were such that no activity could be detected using unlabeled inositol 1,3,4-trisphosphate and [γ-32P]ATP, which suggested that other kinases may have been observed when less purified fractions were incubated with radiolabeled ATP. Inositol 1,3,4-trisphosphate kinase was nonspecifically inhibited more than 80% by various inositol polyphosphates at a concentration of 100 μm.

Changes in Free Amino Acid and Kjeldahl N Concentrations in Seeds from Vegetable-Type and Grain-Type Soybean Cultivars during the Cropping Season

Immature soybean seeds of the vegetable-type (edamame) cultivars that are harvested 30−40 DAF and consumed as a vegetable are preferred by consumers over those of the grain-type cultivars. This study was carried out mainly to examine differences in the concentrations of free amino acids in seeds of the edamame and grain-type cultivars. Asparagine, alanine, and glutamic acid were the major components in immature seeds of the edamame and grain-type cultivars. The concentrations of these amino acids increased until 30−40 DAF and then decreased in seeds of the edamame cultivars, while in seeds of the grain-type cultivars, they decreased continuously starting 20−30 DAF toward mturity. The differences in free amino acid concentrations between immature seeds of the edamame and grain-type cultivars may indicate that these two types of soybean cultivars belong to genetically different ecotypes. Keywords: Glycine max; soybean; edamame; amino acid; water-soluble nitrogen

Germination of Immature Soybean Seeds to Shorten Reproductive Cycle Duration

The soybean [Glycine max (L.) Merrill] reproductive cycle represents more than 50% of the length of the total growth cycle. Immature embryo culture could be used to shorten this cycle. This study was conducted to develop an efficient technique to shorten the soybean reproductive cycle and to make it possible to develop recombinant lines from populations more rapidly. The importance of a pod pretreatment before embryo extraction, the effects of embryo age on germination rate, embryo position in the pod, and the composition of the germination medium (supplemented with sucrose or not) were analyzed to define an optimal technique. Plant recovery rate and genotype effects on germination were evaluated with 22 genotypes representing a broad genetic background. It was necessary to pretreat immature seed by exposing pods to 26°C for 4 d to induce germination. Best results (up to 100% germination) were obtained from embryos sampled at the end of lag phase of seed development (approximately 18 d after flowering). For three‐seeded pods, the central embryos generally had a higher germination rate than distal and proximal embryos (25 and 44 percentage points, respectively). Whatever the embryo position in the pod, adding sucrose significantly increased the germination of embryos sampled 14 to 18 d after flowering. Plantlets were obtained from 73% of the germinated embryos and the length of cycle was reduced from 130 to 140 d to 65 to 70 d. Average germination of the 22 genotypes was 80%; thus, this technique, paired with the single seed descent method, provides soybean breeders with a tool to develop lines quickly.

Genetic and physiological approaches toward understanding the mechanisms underlying the sulfur-regulated expression of β-conglycinin genes

sAccumulation of the β subunit of β-conglycinin, a major seed storage protein of soybean, is known to be upregulated by sulfate deficiency and repressed by exogenous application of methionine in the in vitro culture of immature cotyledons. Accumulation of the β subunit mRNA increased in seeds of soybean plants as the concentration of sulfate in the media was decreased. The promoter of the β subunit was capable of upregulation, suggesting that the regulation mainly occurs at the level of transcription. The level of free sulfate in seeds of soybean grown under sulfur deficiency was dramatically reduced. Immature soybean seeds cultured in vitro in a sulfate deficient medium accumulated an elevated level of the β subunit. These results suggest that the level of sulfate concentration in seeds is a main trigger for the induction of the β subunit accumulation in plants exposed to sulfur deficiency. Possible application of genetical approaches for better understanding of the regulation mechanism is discussed.

Sugar and Cyclitol Determination in Vegetables by HPLC Using Postcolumn Fluorescent Derivatization

ABSTRACTNonreducing sugars such as sucrose, raffinose, myo‐inositol, pinitol and galactinol were separated on a polyamine‐bonded polymer gel (NH2 type) column using acetonitrile‐water as mobile phase. Sugars in the eluate were detected by periodate oxidation and the subsequent derivation of guanidine to fluorescent compounds. Detection limits were: sucrose 2.5, D‐glucose 2.5 and pinitol 4 ng. This highly sensitive method was more accurate and rapid for determining sugar contents in immature soybean seeds, sweet corn kernels and chinese mustard leaves than the conventional method using refractive index detection. Also, this method reduced the injection volumes of extracts to the column and allowed to lengthen the column life.

Positive and negative cis-regulatory regions in the soybean glycinin promoter identified by quantitative transient gene expression

The 5' and 3' flanking regions of the soybean glycinin gene, Gy1, responsible for expression in seeds, were analyzed by quantitative transient expression assay. The construct containing the β-glucuronidase (uidA) reporter gene under the control of the 1.12 kb Gy1 promoter and 0.74 kb Gy1 terminator was introduced into immature soybean seeds and leaves by particle bombardment. To normalize the variability of introduction efficiency, a second reporter gene, firefly luciferase, was cobombarded as an internal standard, and relative activities (GUS/luciferase) were measured. There was a seed-specific β-glucuronidase (GUS) expression, as observed by X-Gluc staining. Compared with the nopaline synthase gene (nos) terminator, the Gy1 terminator enhanced the level of expression in immature seeds, indicating that the terminator region of the glycinin gene is involved in the activation of the gene expression in these seeds. To identify cis-regulatory elements in the glycinin gene upstream sequence, deleted derivatives of the promoter were fused to the luciferase reporter gene. The expression could be measured with a higher accuracy, and constructs were introduced with the internal reporter uidA gene into immature seeds. The results suggest the presence of a positive regulatory element in the -620 to --380 region of the Gy1 promoter. A deletion which eliminates the legumin box with its RY element led to increased relative activity, suggesting that this box is negatively regulating expression of the seed storage protein gene. Analysis of mutant promoters also suggest that the RY element involves negative regulation in seeds. This quantitative transient expression assay using particle bombardment provides a reliable system for the study of seed-specific gene expression in soybeans.