Seed Storage Proteins Research Articles

A comparative investigation of seed storage protein fractions: The synergistic impact of molecular properties and composition on anisotropic structuring

Plant protein fractions are complex systems that could represent a versatile platform of building blocks for novel foods. The objective of this work was to evaluate potential common principles leading to anisotropy formation during wet extrusion by comparing different protein concentrates/isolates. Commercially available protein fractions (>63% protein) from different sources, namely pulses (pea, chickpea, fava bean and mung bean), cereals (gluten and rice), and oilseeds (soy and pumpkin), were individually extruded into a thermoplastic mass and comparatively investigated for composition, protein molecular properties, supramolecular interactions, visual anisotropy, rheology, and water mobility. This work shows that the structuring potential of plant protein fractions during thermomechanical processing should be explained by both molecular and colloidal mechanisms acting in concert and involving proteins, polysaccharides, and polyvalent ions. Wet extrusion resulted in the formation of new β-sheet structures in oilseed and cereal proteins through a higher extent of hydrogen bonds (extended β-sheets) and electrostatic interactions, as noted by X-ray photoelectron spectroscopy (XPS). Furthermore, disulfide bonds played a major role in protein-protein interactions. The presence of minerals facilitated the formation of complex coacervates, amenable to elongation during extrusion, enhancing anisotropy. Thus, pumpkin and mung bean extrudates, followed by soy, displayed the highest level of anisotropy, Warner-Bratzler tenderness and storage modulus (E′). These parameters were positively correlated (r > 0.8) with the proportion of newly formed ordered secondary structures (β-sheets and α-helices).

A review of the genetic, physiological, and agronomic factors influencing secondary dormancy levels and seed vigour in Brassica napus L.

Dormancy in canola ( Brassica napus L.) is a complicated process due to many overlapping and interacting factors affecting the absolute dormancy levels. It is unknown if seed dormancy plays a role in the poor stand establishment of planted canola but given that germination and dormancy are two ends of the same continuum, it has been suggested that dormancy may be a culprit of poor establishment. This review synthesizes literature pertaining to secondary dormancy in B. napus and the interaction of genetic, physiological, environmental, and agronomic factors. Seed germination and vigour and the interaction with dormancy are also addressed. The persistence of volunteer canola seed in the soil seedbank is a result of the induction of canola seed into secondary dormancy under adverse environmental conditions such as low temperature or low light. Genetics is a major influencing factor on absolute secondary dormancy (∼50%) in canola. Plant hormones abscisic acid and gibberellic acid and their interactions also influence dormancy with highly dormant genotypes having increased abscisic acid concentration in the seed. Seed sugars, seed storage proteins, glucosinolate content, and growth habit are all additional factors affecting absolute dormancy in B. napus. Furthermore, maternal environmental conditions affect dormancy levels. In addition to genetic, physiological, and environmental factors, farming practices such as harvest timing, and tillage regimes can influence secondary dormancy of canola seed that has entered the seedbank unintentionally. Given the documented high heritability of secondary dormancy, it is feasible to reduce secondary dormancy in canola cultivars; however, consideration of all interacting factors must be given.

A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum.

We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm.

Expression, purification, characterization, and patient IgE reactivity of new macadamia nut iso-allergen

Structural and functional information about food allergens is essential for understanding the allergenicity of food proteins. All allergens belong to a small number of protein families. Various allergens from different families have been successfully produced recombinantly in E. coli for their characterization and applications in allergy diagnosis and treatment. However, recombinant hexameric 11S seed storage protein has not been reported, although numerous 11S legumins are known to be food allergens, including the recently identified macadamia nut allergen Mac i 2. Here we report the production of a macadamia nut legumin by expressing it in E. coli with a substrate site of HRV 3C protease and cleaving the purified protein with HRV 3C protease. The protease divided the protein into two chains and left a native terminus for the C-terminal chain, resulting in a recombinant hexameric 11S allergen for the first time after the residues upstream to the cleavage site flipped out of the way of the trimer-trimer interaction. The 11S allergens are known to have multiple isoforms in many species. The present study removed an obstacle in obtaining homogeneous allergens needed for studying allergens and mitigating allergenicity. Immunoreactivity of the protein with serum IgE confirmed it to be a new isoform of Mac i 2.

A View into Seed Autophagy: From Development to Environmental Responses.

Autophagy is a conserved cellular mechanism involved in the degradation and subsequent recycling of cytoplasmic components. It is also described as a catabolic process implicated in the specific degradation of proteins in response to several stimuli. In eukaryotes, the endoplasmic reticulum accumulates an excess of proteins in response to environmental changes, and is the major cellular organelle at the crossroads of stress responses. Return to proteostasis involves the activation of the Unfolded Protein Response (UPR) and eventually autophagy as a feedback mechanism to relieve protein overaccumulation. Recent publications have focused on the relevance of autophagy in two central processes of seed biology: (i) seed storage protein accumulation upon seed maturation and (ii) reserve mobilization during seed imbibition. Although ER-protein accumulation and the subsequent activation of autophagy resemble the Seed Storage Protein (SSP) deposition during seed maturation, the molecular connection between seed development, autophagy, and seed response to abiotic stresses is still an underexplored field. This mini-review presents current advances in autophagy in seeds, highlighting its participation in the normal course of seed development from embryogenesis to germination. Finally, the function of autophagy in response to the seed environment is also considered, as is its involvement in controlling seed dormancy and germination.

Pea and lentil 7S globulin crystal structures with comparative immunoglobulin epitope mapping

Legumes represent an affordable high protein, nutrient dense food source. However, the vast majority of legume crops contain proteins that are known allergens for susceptible individuals. These include proteins from the 7S globulin family, which comprise a vast majority of seed storage proteins. Here, the crystal structures of 7S globulins from Pisum sativum L. (pea) and Lens culinaris Medicus (lentil) are presented for the first time, including pea vicillin and convicilin, and lentil vicilin. All three structures maintain the expected 7S globulin fold, with trimeric quaternary structure and monomers comprised of β-barrel N- and C-modules. The potential impact of sequence differences on structure and packing in the different crystal space groups is noted, with potential relevance to packing upon seed deposition. Mapping on the obtained crystal structures highlights significant Ig epitope overlap between pea, lentil, peanut and soya bean and significant coverage of the entire seed storage protein, emphasizing the challenge in addressing food allergies. How recently developed biologicals might be refined to be more effective, or how these seed storage proteins might be modified in planta to be less immuno-reactive remain challenges for the future. With legumes representing an affordable, high protein, nutrient dense food source, this work will enable important research in the context of global food security and human health on an ongoing basis.

Characterization of α-gliadin alleles of Japanese wheat cultivars in relation to flour dough extensibility and celiac disease epitopes

Alpha-gliadins are major seed storage proteins of wheat and are involved in dough extensibility, which is related to the end-use properties of flour. However, α-gliadin causes gluten-related diseases such as celiac disease (CD). To characterize the α-gliadin allele types of Japanese cultivars, PCR markers that detect the presence of each α-gliadin gene in Chinese Spring wheat were used to determine allele-types. We surveyed 95 accessions of the Japanese wheat core collection (JWC) and 22 commercial cultivars and found 2, 10, and 4 allele types at the loci Gli-A2, Gli-B2, and Gli-D2, respectively. According to the allele-type frequency, the α-gliadin alleles of JWC showed diversities comparable to those of foreign cultivars. Allele diversities were compared within four populations of JWC, classified by genome-wide single nucleotide polymorphisms. The degree of diversity of alleles reflected the population structures, except for high diversity of the allele-types at Gli-B2 in the classical varieties. The frequencies of alleles related to end-use were higher in newer varieties than in classical varieties and landraces. Allele types that dramatically decreased and increased CD epitopes were not found in JWC, suggesting that selection(s) of α-gliadin genes was not linked to CD epitopes in Japanese cultivars.

Integrated genomic approaches delineate a novel role of ROP1 ENHANCER1 in controlling seed protein content of chickpea.

Utilizing a combinatorial approach of quantitative trait locus (QTL)-Seq and candidate gene-based association mapping, the QTLs and genes responsible for seed protein content (SPC), a major quality trait in chickpea, were identified. Whole genome re-sequencing based QTL-Seq analysis of bulked recombinant inbred lines from a mapping population contrasting for SPC led to the identification of two QTLs [0.94 Mb on Linkage Group (LG)5 and 1.16 Mb on LG6] encompassing three SNPs, displaying the highest ΔSNP index. These highly significant SNPs and their associated genes were validated in 211 chickpea mini-core accessions varying in SPC, revealing a tightly associated marker affecting CaREN1 (ROP1 ENHANCER1) and explaining a phenotypic variation of 23%. This SNP was subsequently converted into a cost effective allele-specific PCR-based marker that could be utilized for rapid screening of SPC during marker assisted breeding. Furthermore, in planta functional validation via knockdown of CaREN1 transcripts led to significant reduction in SPC of chickpea. This decrease in seed protein is likely due to disruption in the formation of CaREN1 protein complexes comprising chaperones, phosphopeptide-binding proteins, and GTPases that mediate folding, transport and accumulation of seed storage proteins, as indicated through affinity purification-mass spectrometry. Taken together, our data will expedite tailoring of chickpea cultivars with augmented SPC.

Comparative proteomic analyses of Tartary buckwheat (Fagopyrum tataricum) seeds at three stages of development

Tartary buckwheat is among the valuable crops, utilized as both food and Chinese herbal medicine. To uncover the accumulation dynamics of the main nutrients and their regulatory mechanism of Tartary buckwheat seeds, microscopic observations and nutrient analysis were conducted which suggested that starch, proteins as well as flavonoid gradually accumulated among seed development. Comparative proteomic analysis of rice Tartary buckwheat at three different developmental stages was performed. A total of 78 protein spots showed differential expression with 74 of them being successfully identified by MALDI-TOF/TOF MS. Among them, granule bound starch synthase (GBSS1) might be the critical enzyme that determines starch biosynthesis, while 11 S seed storage protein and vicilin seemed to be the main globulin and affect seed storage protein accumulation in Tartary buckwheat seeds. Two enzymes, flavanone 3-hydroxylase (F3H) and anthocyanidin reductase (ANR), involved in the flavonoid biosynthesis pathway were identified. Further analysis on the expression profiles of flavonoid biosynthetic genes revealed that F3H might be the key enzyme that promote flavonoid accumulation. This study provides insights into the mechanism of nutrition accumulation at the protein level in Tartary buckwheat seeds and may facilitate in the breeding and enhancement of Tartary buckwheat germplasm.

Transcriptome profiling revealed salt stress-responsive genes in Lilium pumilum bulbs.

Lilium pumilum is an important ornamental, culinary and medicinal bulbous plants with salt tolerance. However, salt tolerance of lily, particularly the bulb, has been studied relatively little, which brings challenges to the cultivation of lily varieties with high salt tolerance. Here, we performed transcriptome sequencing on the bulb organs of L. pumilum under salt stress treatment, analyzed differential gene expressed levels and then identified several key genes associated with salt stress tolerance at genome-wide scale. For the first time, we revealed the obvious response against salt stress for L. pumilum bulb organs, while distinct from those for root organs. Several key genes obtained through transcriptome analysis and DEG screening include NF-YB3 transcription factor, metallothionein type 2 protein, vicilin like seed storage protein and bidirectional sugar transporter SWEET14. Rather than typical ROS scavengers like superoxide dismutase, peroxidase, and glutathione transferase, non-typical ROS scavengers such as the metallothionein type 2 protein, and vicilin like seed storage protein were upregulated in our work. The bidirectional sugar transporter SWEET14 protein and the hormone signaling proteins such as E3-ubiquitin protein ligases, PYL4 and protein phosphatase 2C were also upregulated, suggesting the role of sugars and hormones in the bulb organ responses to salt stress. Co-expression analysis of the DEGs further confirmed that NF-YB3 transcription factor acted as a hub gene, suggesting that salt stress can promote flowering of L. pumilum. Taken together, we identified important candidate genes associated with salt tolerance of the L. pumilum bulb organs, which may provide the excellent basis for further in-depth salt tolerance mechanisms of the lily bulbs.

Loss-of-function mutations of OsbHLH044 transcription factor lead to salinity sensitivity and a greater chalkiness in rice (Oryza sativa L.)

The most hazardous abiotic stress, salinity, restricted the world crop production, and grain chalkiness affected the grain quality to limit consumers' acceptance. The basic helix-loop-helix (bHLH) proteins modulate massive biological processes in plants. Here the CRISPR/Cas9 gene editing mutants were obtained to detect the function of OsbHLH044. The loss-of-function of OsbHLH044 mutants showed numerous altered plant phenotypes. Notably, the osbhlh044 mutants resulted in prominently reduced morphological and physiological parameters under salt stress. Lower antioxidant activities and higher lipid peroxidation and hydrogen peroxide (H2O2) accumulation in the osbhlh044 mutants caused salinity sensitivity due to elevated reactive oxygen species (ROS). Under salt stress, both shoots and roots of the osbhlh044 mutants acquired higher Na+. Moreover, the expression of ion homeostasis-related genes (OsHKTs, OsHAK, OsSOSs, and OsNHX) and ABA-responsive gene (OsLEA3) was significantly altered in the osbhlh044 mutants after salt stress. The expression levels of genes coding for starch (OsAGPL1, OsSSIIa, OsWx, and OsFLO2) and seed storage proteins (GluA1 and Globulin 1) were significantly decreased, indicating that they synthesize less store starch and proteins, resulting in grain chalkiness in the osbhlh044 mutants. Yeast one Hybrid (Y1H) showed that OsbHLH044 could activate salt- (OsHKT1;3, OsHAK7, OsSOS1, OsSOS2, OsNHX2, and OsLEA3 but not OsHKT2;1), and starch-related genes (OsSSIIa, OsWx, and OsFLO2) by binding to the G-boxes of their promoters. Therefore, the OsbHLH044 gene editing mutants revealed multiple functions, specifically a positive regulator of salt stress and grain quality, which might bring new insights into the breeding of rice varieties.

Gene Expression in the Developing Seed of Wild and Domesticated Rice.

The composition and nutritional properties of rice are the product of the expression of genes in the developing seed. RNA-Seq was used to investigate the level of gene expression at different stages of seed development in domesticated rice (Oryza sativa ssp. japonica var. Nipponbare) and two Australian wild taxa from the primary gene pool of rice (Oryza meridionalis and Oryza rufipogon type taxa). Transcriptome profiling of all coding sequences in the genome revealed that genes were significantly differentially expressed at different stages of seed development in both wild and domesticated rice. Differentially expressed genes were associated with metabolism, transcriptional regulation, nucleic acid processing, and signal transduction with the highest number of being linked to protein synthesis and starch/sucrose metabolism. The level of gene expression associated with domestication traits, starch and sucrose metabolism, and seed storage proteins were highest at the early stage (5 days post anthesis (DPA)) to the middle stage (15 DPA) and declined late in seed development in both wild and domesticated rice. However, in contrast, black hull colour (Bh4) gene was significantly expressed throughout seed development. A substantial number of novel transcripts (38) corresponding to domestication genes, starch and sucrose metabolism, and seed storage proteins were identified. The patterns of gene expression revealed in this study define the timing of metabolic processes associated with seed development and may be used to explain differences in rice grain quality and nutritional value.

Predictors of anaphylaxis to peanut and tree nuts in a Mediterranean population.

Background: Peanuts (PN) and tree nuts (TN) are major causes of anaphylaxis worldwide. We aimed to determine the clinical and demographic characteristics associated with anaphylaxis in patients sensitized to PN and/or TN in a Mediterranean population. Methods: We conducted a retrospective study, which included 198 patients allergic to PN and/or TN (allergy symptoms plus specific immunoglobulin E [sIgE] sensitization), evaluated in consultations from January 2015 to December 2020. Univariate analysis and multivariate logistic regression models were developed, including demographic, clinical, and laboratory data as independent variables, and anaphylaxis to each PN and/or TN as a dependent variables. Results: Anaphylaxis was associated with an earlier age of onset of allergy to PN, cashew and/or pistachio, and pine nut allergy but not to other TN allergies. Gender, atopic comorbidities, and cofactors were not associated with PN and/or TN anaphylaxis. Anaphylaxis to PN, cashew and/or pistachio, and pine nut were associated with reactivity to a fewer number of PN and/or TN foods. Although sIgE sensitization to lipid transfer proteins (LTP) was highly prevalent in our population, only seed storage protein (SSP) positivity was associated with anaphylaxis in PN allergy. The absence of pathogenesis-related protein family 10 sensitization correlated with PN and hazelnut anaphylaxis. A higher level of sIgE to almond extract predicted anaphylaxis but the level of sIgE to other PN and/or TN extracts did not predict it. Conclusion: The high prevalence of sensitization to the pan-allergen LTP did not seem to have a significant impact in PN and/or TN allergy severity in our study. Instead, other factors, such as early age of onset and positivity for SSPs, seem to strongly associate with anaphylaxis to specific PN and/or TN. These findings may contribute to individual risk assessment in these populations.

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

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.

Genetic Diversity of Seed Storage Proteins in Pigmented and Non-pigmented Indigenous Paddy (Oryza sativa L.) Landraces of Assam, India

Background: Pigmented rice particularly, red and black rice have come into prominence due to their promising antioxidant property. In Assam, all major ecotypes of paddy viz., Ahu, Sali, Bao, Boro have large number of indigenous landraces with red kernel. Although Assam is recognised as a secondary centre of origin and diversity yet gene erosion is assuming serious proportion. It is acknowledged that many indigenous cultivars are facing extinction and there is apprehension that many may be lost even before they are evaluated and characterised. Among different methods, seed protein profile is recognised as fingerprint for a race by International Seed Testing Association (ISTA) as it can resolve interspecific and intraspecific variation without ambiguity. Methods: The present study involves, molecular characterisation and genetic diversity analysis of pigmented and non-pigmented landraces. Seed protein profiles for the paddy landraces were worked out by SDS-PAGE. SDS-PAGE was carried out with two phase gradient buffer system with upper stacking gel and lower separating gel to ensure denaturation of protein while migrating through it. Result: Among the twenty landraces a total of 15 proteins bands were observed in the size range of 12.1 Kd to greater than 122.0 Kd. Highest protein bands were observed in cultivar Ranjit Amon, Bismuthi and Boro (ecotype) all with 15 protein bands. On the other hand, lowest protein bands were recorded in Amona with only 9 bands. Among the protein bands six with molecular weight greater than 122.0 Kd; 112.0 Kd; 97.4 Kd, 90.0 Kd and 15.0 Kd were consistently found in all the landraces. The dendrogram revealed a total of four clusters. The landrace Amona (red) did not belong to the any cluster and most distantly related to others. On the other hand, some are genetically identical. Agnisali, Suhagmani and Tengeri (all white) had no genetic distance between them and hence identical at molecular level. Within the same cluster both red and white rice landraces were observed. Our study suggested that several proteins bands were detected only on specific rice and it could be as biochemical markers for further research.

Association Between the Seed Storage Proteins 2S Albumin and 11S Globulin and Severe Allergic Reaction After Flaxseed Intake.

Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about the allergens implicated in hypersensitivity reactions to flaxseed. The present study aimed to identify the allergens involved in IgE-mediated reactions in 5 patients with a clinical history of severe systemic symptoms after flaxseed consumption. Proteins that were potential allergens with IgE-binding capacity were purified from flaxseed extract using chromatography and identified via MALDI-TOF mass spectrometry. Immunoassays were performed using the 5 allergic patients' sera tested individually and as a pool. Immunoblotting of the flaxseed extract revealed a low-molecular-mass protein (around 13 kDa) in 4 of the 5 patients, while a protein of approximately 55 kDa was detected in 2 patients. The proteins were identified by mass spectrometry as flaxseed 2S albumin, which is included in the WHO/IUIS allergen nomenclature as Lin u 1, and 11S globulin. Inhibition assays revealed in vitro IgE-mediated cross-reactivity between Lin u 1 and peanut and cashew nut proteins, while IgE-mediated recognition of 11S globulin by patients' sera was partially inhibited by several plant-derived sources. Seed storage proteins from flaxseed were involved in the development of severe symptoms in the 5 patients studied and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, this is the first time that 11S globulin has been identified as a potential allergen. Taking these data into account should ensure a more accurate diagnosis.

Allelic state of prolamin-coding loci in the new oat cultivar ‘Tobolyak’

Background. In order to fully reveal the advantages of a cultivar and use it effectively in production, it is necessary to ensure high sowing and varietal qualities of seeds, which requires improvement in breeding and seed production. Biotechnological methods are increasingly used in initial seed production. High efficiency was shown by the method of electrophoresis of seed storage proteins – prolamins. The aim of this study was to assess the biotype composition and allelic state of the prolamin-coding loci in the new spring oat cultivar ‘Tobolyak’ for its further use in initial seed production.Materials and methods. In 2021, 1519 families of cv. ‘Tobolyak’ were studied using the method of native electrophoresis. Three grains from each family were selected for analyses by random sampling. Electrophoresis was carried out in vertical plates of 13.2% polyacrylamide gel at a constant voltage of 500 V for 4.5–5.0 hours.Results and conclusion. The study of cv. ‘Tobolyak’ families resulted in identifying 13 types of avenin patterns with a frequency of occurrence from 96.36 to 0.07%. A new block of avenin components controlled by the allele of the Avn B locus was described. Number 8 was assigned to this block. It was established that cv. ‘Tobolyak’ had been developed by crossing a minor biotype of cv. ‘Taezhnik’ (Avn 4.4.2) with the first biotype of cv. ‘Orion’ (Avn 2.8.2). The basis of cv. ‘Tobolyak’ was one genotype with the Avn 4.8.2 avenin formula. The remaining biotypes arose as a result of crossing the biotypes of cvs. ‘Taezhnik’ and ‘Orion’ as well as through mechanical contamination. To maintain the genetic stability of cv. ‘Tobolyak’, it is recommended to use only families of the first biotype in the process of original seed production.

Characterization of seed storage protein of Moringa oleifera and analyses of their bioactive peptide’s nutraceutical properties [pdf

Characterization of seed storage protein of Moringa oleifera and analyses of their bioactive peptide’s nutraceutical properties [pdf

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

A new demethylase gene, OsDML4, is involved in high temperature-increased grain chalkiness in rice.

High temperature (HT) can affect the accumulation of seed storage materials and cause adverse effects on the yield and quality of rice. DNA methylation plays an important role in plant growth and development. Here, we identified a new demethylase gene OsDML4 and discovered its function in cytosine demethylation to affect endosperm formation. Loss of function of OsDML4 induced chalky endosperm only under HT and dramatically reduced the transcription and accumulation of glutelins and 16 kDa prolamin. The expression of two transcription factor genes RISBZ1 and RPBF was significantly decreased in the osdml4 mutants, which caused adverse effects on the formation of protein bodies (PBs) with greatly decreased PB-II number, and incomplete and abnormally shaped PB-IIs. Whole-genome bisulfite sequencing analysis of seeds at 15 d after pollination revealed much higher global methylation levels of CG, CHG, and CHH contexts in the osdml4 mutants compared with the wild type. Moreover, the RISBZ1 promoter was hypermethylated but the RPBF promoter was almost unchanged under HT. No significant difference was detected between the wild type and osdml4 mutants under normal temperature. Our study demonstrated a novel OsDML4-mediated DNA methylation involved in the formation of chalky endosperm only under HT and provided a new perspective in regulating endosperm development and the accumulation of seed storage proteins in rice.