Generation of New ß-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean ß-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in ß-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean ß-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the ß-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and ß-subunits of soybean ß-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the ß-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and ß-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein ß-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the ß-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

miRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage.

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

Oat AsDA1-2D enhances heat stress tolerance and negatively regulates seed-storage globulin.

The importance of oats has increased because of their high nutritional value and health benefits in the human diet. High-temperature stress during the reproductive growth period has a detrimental effect on grain morphology by changing the structure and concentration of several seed-storage proteins. DA1, a conserved ubiquitin-proteasome pathway component, plays an important role in regulating grain size by controlling cell proliferation in maternal integuments during the grain-filling stage. However, there have been no reports or studies on oat DA1 genes. In this study, we identified three DA1-like genes (AsDA1-2D, AsDA1-5A, and AsDA1-1D) using genome-wide analysis. Among these, AsDA1-2D was found to be responsible for high-temperature stress tolerance via a yeast thermotolerance assay. The physical interaction of AsDA1-2D with oat-storage-globulin (AsGL-4D) and a protease inhibitor (AsPI-4D) was observed using yeast two-hybrid screening. A subcellular localization assay revealed that AsDA1-2D and its interacting proteins are localized in the cytosol and plasma membrane. An in vitro pull-down assay showed that AsDA1-2D forms a complex with both AsPI-4D and AsGL-4D. An in vitro cell-free degradation assay showed that AsGL-4D was degraded by AsDA1-2D under high-temperature conditions and that AsPI-4D inhibited the function of AsDA1-2D. These results suggest that AsDA1-2D acts as a cysteine protease and negatively regulates oat-grain-storage-globulin under heat stress.

Amino acids induce high seed-specific expression driven by a soybean (Glycine max) glycinin seed storage protein promoter.

KEY MESSAGE: We characterize GFP expression driven by a soybean glycinin promoter in transgenic soybean. We demonstrate specific amino acid-mediated induction of this promoter in developing soybean seeds in vitro. In plants, gene expression is primarily regulated by promoter regions which are located upstream of gene coding sequences. Promoters allow transcription in certain tissues and respond to environmental stimuli as well as other inductive phenomena. In soybean, seed storage proteins (SSPs) accumulate during seed development and account for most of the monetary and nutritional value of this crop. To better study the regulatory functions of a SSP promoter, we developed a cotyledon culture system where media and media addenda were evaluated for their effects on cotyledon development and promoter activity. Stably transformed soybean events containing a glycinin SSP promoter regulating the green fluorescent protein (GFP) were generated. Promoter activity, as visualized by GFP expression, was only observed in developing in planta seeds and in vitro-cultured isolated embryos and cotyledons from developing seeds when specific media addenda were included. Asparagine, proline, and especially glutamine induced glycinin promoter activity in cultured cotyledons from developing seeds. Other amino acids did not induce the glycinin promoter. Here, we report, for the first time, induction of a reintroduced glycinin SSP promoter by specific amino acids in cotyledon tissues during seed development.

Characteristics of Seed Vigor in Rice Varieties with Different Globulin Accumulations.

Seed vigor of rice is an important trait for direct seeding. The objective of this study was to reveal the relationship between globulin and seed vigor, and then to explore a method for evaluating seed vigor. Several rice varieties with different levels of 52 kDa globulin accumulation were used to compare seed vigor under normal and aged conditions. Results showed that varieties with high globulin accumulation obtained significantly higher seed vigor, measured by germination percentage and germination index, compared with those varieties with low globulin accumulation under normal and aged conditions. Meanwhile, a significantly higher accumulation of reactive oxygen species (ROS) was observed in the early germinating seeds of varieties with high globulin accumulation compared to those varieties with low globulin accumulation under normal and aged conditions. Collectively, the globulin content could be applied in the evaluation of seed vigor, which contributes to the selection of rice varieties for direct seeding.

Soybean ß-Conglycinin: Structure Characteristic, Allergenicity, Plasma Lipid-Controlling, Prevention of Obesity and Non-alcoholic Fatty Liver Disease.

Soybean has become an important world commodity because of its low price, good nutritional value and recognized functional health benefits in recent years. ß-conglycinin is one of the major storage proteins in soybean. It has captured a growing interest recently because of its allergenicity and potential health benefits, which continues to drive the research and commercial development of ß-conglycinin-based food products and ingredients. In this review, the structure, the amino acid composition, extraction methods and electrophoretic pattern of ß-conglycinin are briefly summarized. Studies on ß-conglycinin by allergenicity, plasma lipid-controlling, obesity and nonalcoholic fatty liver disease are highlighted, critically discussing their main shortcomings and challenges and identifying the research gaps. Studies to date have demonstrated the cultivation of ß-conglycinin with health benefits as functional ingredients and foodstuffs. The current research focuses on proteins, mainly challenging the mechanisms of subunit/peptide effects interaction and identifying and characterizing the hidden biological activities in the polypeptide chains. There is much scope for further exploration into various aspects of ß-conglycinin, such as the selection of mutant strains and genetic engineering and prospects on targeted ß-conglycinin exploitation in the nutraceutical area. In addition, the safety evaluation of ß-conglycinin and its stabilized emulsions deserve more attention to food-related applications.

A retrospective study of clinical and laboratory features and treatment on cats highly suspected of feline infectious peritonitis in Wuhan, China.

Feline infectious peritonitis (FIP) is a systemic, potentially fatal viral disease. The objectives of this study were to review clinical and laboratory features and treatment of cats highly suspected of FIP in Wuhan, China. The clinical records of 127 cats highly suspected of FIP were reviewed for history, clinical signs, physical findings, and diagnostic test results. Sex, neutering status, breed, age, and month of onset of disease were compared with the characteristics of the clinic population. Age and neutering status were significantly correlated with FIP-suspicion. Sex, breed and onset month were not associated with FIP. There were many more FIP-suspected cases in cats in young cats or male intact cats. Effusion was observed in 85.8% of the FIP-suspected cats. Increased serum amyloid A (SAA) and lymphopenia were common laboratory abnormalities in the FIP cases. Furthermore, 91.7% of the cats highly suspected of FIP had an albumin/globulin (A/G) ratio < 0.6, while 85.3% had an A/G ratio < 0.5. The mortality rate for FIP-suspected cats was 67%, and six submitted cases were confirmed by FIP-specific immunohistochemistry. Of the 30 cats treated with GS-441524 and/or GC376, 29 were clinically cured. The study highlights the diverse range of clinical manifestations by clinicians in diagnosing this potentially fatal disease. A/G ratio and SAA were of higher diagnostic value. GS-441524 and GC376 were efficient for the treatment of FIP-suspected cats.

Genetic determinants of seed protein plasticity in response to the environment in Medicago truncatula.

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M. truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition.

Linkage and association study discovered loci and candidate genes for glycinin and ß-conglycinin in soybean (Glycine max L. Merr.).

KEY MESSAGE: Linkage mapping and GWAS identified 67 QTLs related to soybean glycinin, ß-conglycinin and relevant traits. Polymorphisms of the candidate gene Gy1 promoter were associated with the glycinin content in soybean. The major components of storage proteins in soybean seeds are glycinin and ß-conglycinin, which play important roles in determining protein nutrition and soy food processing properties. Increasing the protein content while improving the ratio of glycinin to ß-conglycinin is substantially important for soybean protein improvement. To investigate the genetic mechanism of storage protein subunits, 184 recombinant inbred lines (RILs) derived from a cross of Kefeng No. 1 and Nannong 1138-2 and 211 diverse soybean cultivars were used to detect loci related to glycinin (11S), ß-conglycinin (7S), the sum of glycinin and ß-conglycinin (SGC), and the ratio of glycinin to ß-conglycinin (RGC). Sixty-seven QTLs and 11 hot genomic regions were identified as affecting the four traits. One genetic region (q10-1) on chromosome 10 was associated with multiple traits by both linkage and association analysis. Eight genes in 11 hot genomic regions might be related to soybean protein subunit. The candidate gene analysis showed that polymorphisms in Gy1 promoters were significantly correlated with the 11S content. The QTLs and candidate genes identified in the present study allow for further understanding the genetic basis of 11S and 7S regulation and provide useful information for marker-assisted selection (MAS) in soybean quality improvement.

eQTL mapping of the 12S globulin cruciferin gene PGCRURSE5 as a novel candidate associated with starch content in potato tubers.

Tuber starch content (TSC) is a very important trait in potato (Solanum tuberosum L.). This study is the first to use expression quantitative trait loci (eQTL) mapping of transcript-derived markers for TSC in potato. Thirty-four differentially expressed genes were selected by comparing the RNA-seq data of contrasting bulked segregants. For the 11 candidate genes, we determined their relative expression levels across the segregating diploid potato population using RT-qPCR. We detected 36 eQTL as candidate genes distributed on all twelve potato chromosomes, and nine of them overlapped with QTL for TSC. Peaks for two eQTL, eAGPaseS-a and ePGRCRURSE5, were close to the corresponding loci of the large subunit of ADP-glucose pyrophosphorylase (AGPaseS-a) and the 12S globulin cruciferin gene (PGCRURSE5), respectively. The eQTL peaks for AGPaseS-a and PGRCRURSE5 explained 41.0 and 28.3% of the phenotypic variation at the transcript level. We showed the association of the DNA markers for AGPaseS-a and PGRCRURSE5 with QTL for TSC, and significant correlation between the expression level of PGRCRURSE5 and TSC. We did not observe a significant correlation between the expression level of AGPaseS-a and TSC. We concluded that the cruciferin gene PGRCRURSE5 is a novel candidate involved in the regulation of starch content in potato tubers.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman-Birk protease inhibitor and suppresses the accumulation of the ß-subunit of ß-conglycinin in soybean seeds.

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5'-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the ß-subunit of ß-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman-Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC-MS and GC-MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37-52% and 15-19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

Study on Effect of Extraction Techniques and Seed Coat on Proteomic Distribution and Cheese Production from Soybean Milk.

Soybean-based food products are a major source of protein. In the present study, proteins in soybean milk from seeds of the cultivar Bunya (Glycine max) were extracted using the cheesecloth and the centrifuge methods. The milk was produced through mechanical crushing of both whole and split seeds in water. Following separation by either the cheesecloth or centrifuge, proteins were isolated from the soybean milk by using thiourea/urea solubilisation and then separated them using two-dimensional polyacrylamide gel electrophoresis. The isolated proteins were identified by mass spectrometry. A total of 97 spots were identified including 49 that displayed different abundances. Of the two separation techniques, centrifuge separation gave higher protein extraction and more intense protein spots than cheesecloth separation. Eleven of the ß-subunits of ß-conglycinin, three of the α-subunits of ß-conglycinin, and four of the mutant glycinin showed different levels of abundances between separation techniques, which might be related to subsequent cheese quality. Notably, split-seed soybean milk has less allergenic proteins with four α-subunits of ß-conglycinin compared to whole-seed milk with eight of those proteins. The sensory evaluation showed that the cheese produced from split-soybean milk received higher consumer preferences compared to that of whole seed, which could be explained by their proteomic differences. The demonstrated reference map for whole and split-seed soybean milk could be further utilized in the research related to soybean cheesemaking.

Proteome Analysis of Hordein-Null Barley Lines Reveals Storage Protein Synthesis and Compensation Mechanisms.

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.

Comprehensive Comparison of Clinically Relevant Grain Proteins in Modern and Traditional Bread Wheat Cultivars.

Bread wheat (Triticum aestivum L.) is one of the most valuable cereal crops for human consumption. Its grain storage proteins define bread quality, though they may cause food intolerances or allergies in susceptible individuals. Herein, we discovered a diversity of grain proteins in three Ukrainian wheat cultivars: Sotnytsia, Panna (both modern selection), and Ukrainka (landrace). Firstly, proteins were isolated with a detergent-containing buffer that allowed extraction of various groups of storage proteins (glutenins, gliadins, globulins, and albumins); secondly, the proteome was profiled by the two-dimensional gel electrophoresis. Using multi-enzymatic digestion, we identified 49 differentially accumulated proteins. Parallel ultrahigh-performance liquid chromatography separation followed by direct mass spectrometry quantification complemented the results. Principal component analysis confirmed that differences among genotypes were a major source of variation. Non-gluten fraction better discriminated bread wheat cultivars. Various accumulation of clinically relevant plant proteins highlighted one of the modern genotypes as a promising donor for the breeding of hypoallergenic cereals.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds.

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity.

Identification of antigenic sites destructed by high hydrostatic pressure (HHP) of the ß subunit of ß-conglycinin.

ß-conglycinin is one of the most allergenic proteins, and its constituent subunits α', α, and ß are all potential allergens to humans. In the present study, we concentrated on the destructed antigenic sites of ß subunit of ß-conglycinin after high hydrostatic pressure (HHP) treatment. In this paper, the overlapping gene fragments of the ß subunit of ß-conglycinin were amplified by polymerase chain reaction (PCR) and cloned into T7 phage vectors. After being packaged in vitro, the recombinant T7 phage was constructed, and the overlapping fragments of the ß subunit were displayed on the phage surface. The recombinant phages that expressed the overlapping fragments of the ß subunit were used to react with specific antiserum by indirect ELISA to identify the HHP destructed antigenic sites. After three rounds of expression and identification, we used synthetic peptide technology to identify that the obtained fragment was a conformational epitope. We further confirmed that HHP treatment changed the conformational structure of ß-conglycinin, which reduced the antigenicity of the protein.

CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.

BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.

The maize α-zein promoter can be utilized as a strong inducer of cellulase enzyme expression in maize kernels.

Expression of recombinant proteins in plants is a technology for producing vaccines, pharmaceuticals and industrial enzymes. For the past several years, we have produced recombinant proteins in maize kernels using only the embryo, primarily driving expression of foreign genes with the maize globulin-1 promoter. Although strong expression is obtained, these lines use only 10-12% of the seed tissue. If strong embryo expression could be combined with strong endosperm expression, much more recombinant protein could be recovered from a set amount of seed biomass. In this study, we tested three endosperm promoters for expression of a cellulase gene. Promoters tested were rice globulin and glutelin promoters and a maize 19 kDa α-zein promoter. The rice promoters were used in two tandem expression constructs as well. Although the rice promoters were active in producing stable amounts of cellulase, the α-zein promoter was by far the most effective: as much as 9% of total soluble protein was recovered from seed of several independent events and plants. One or two inserts were detected by Southern blot in several lines, indicating that copy number did not appear to be responsible for the differences in protein accumulation. Tissue print analysis indicated that expression was primarily in the endosperm.

Water stress combined with sulfur deficiency in pea affects yield components but mitigates the effect of deficiency on seed globulin composition.

Water stress and sulfur (S) deficiency are two constraints increasingly faced by crops due to climate change and low-input agricultural practices. To investigate their interaction in the grain legume pea (Pisum sativum), sulfate was depleted at the mid-vegetative stage and a moderate 9-d water stress period was imposed during the early reproductive phase. The combination of the stresses impeded reproductive processes in a synergistic manner, reducing seed weight and seed number, and inducing seed abortion, which highlighted the paramount importance of sulfur for maintaining seed yield components under water stress. On the other hand, the moderate water stress mitigated the negative effect of sulfur deficiency on the accumulation of S-rich globulins (11S) in seeds, probably due to a lower seed sink strength for nitrogen, enabling a readjustment of the ratio of S-poor (7S) to 11S globulins. Transcriptome analysis of developing seeds at the end of the combined stress period indicated that similar biological processes were regulated in response to sulfur deficiency and to the combined stress, but that the extent of the transcriptional regulation was greater under sulfur deficiency. Seeds from plants subjected to the combined stresses showed a specific up-regulation of a set of transcription factor and SUMO ligase genes, indicating the establishment of unique regulatory processes when sulfur deficiency is combined with water stress.

Changes to the core and flanking sequences of G-box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters.

Cis-regulatory elements in promoters are major determinants of binding specificity of transcription factors (TFs) for transcriptional regulation. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of both classical (CACGTG) and variant G-box core sequences along with different flanking sequences derived from the promoters of three different highly expressing soybean genes, were constructed and used to regulate a green fluorescent protein (gfp) gene. Use of the classical 6-bp G-box provided information on the base level of GFP expression while modifications to the 2-4 flanking bases on either side of the G-box influenced the intensity of gene expression in both transiently transformed lima bean cotyledons and stably transformed soybean hairy roots. The proximal 2-bp sequences on either flank of the G-box significantly affected G-box activity, while the distal 2-bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Manipulation of the upstream 2- to 4-bp flanking sequence of a G-box variant (GACGTG), found in the proximal region of a relatively weak soybean glycinin promoter, significantly enhanced promoter activity using both transient and stable expression assays, if the G-box variant was first converted into a classical G-box (CACGTG). In addition to increasing our understanding of regulatory element composition and structure, this study shows that minimal targeted changes in native promoter sequences can lead to enhanced gene expression, and suggests that genome editing of the promoter region can result in useful and predictable changes in native gene expression.