Viability and integrity of Pinus densiflora seeds stored for 20 years at three different temperatures.

Storage temperature is one of the most important factors determining seed longevity in the genebank. This study aimed to investigate the effect of storage temperature on the seed viability and physiological integrity after a 20-year storage period of Pinus densiflora, a tree species of ecological and economic significance in South Korea. To this end, seeds were collected and stored dry for 20 years at -18°C, 4°C and 25°C. Germination tests were conducted to assess seed viability and vigour, electrolyte leakage analysis was performed to assess cell membrane integrity, and carbohydrate analysis was conducted to assess metabolic integrity during germination. The results revealed that over 20 years, seeds stored at -18°C maintained a high germination percentage (GP; 89%), comparable to initial GP (91%), whilst those stored at 4°C exhibited a decline in GP (44%) along with a decrease in vigour. Seeds stored at 25°C lost their viability entirely. Electrical conductivity of the leachate and leakage of inorganic compounds and soluble sugars were higher with elevated storage temperature, indicating increased imbibition damage. Additionally, changes in carbohydrate content during germination revealed that the loss of viability according to storage temperature is associated with reduced storage reserve utilization and altered carbohydrate metabolism during germination. These results enhance our understanding of the effect of seed storage temperature on longevity and physiological changes of aging in the genebank, serving as a reference for establishing conservation strategies for Pinus densiflora.

Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

Multi-Omics Analysis of a Chromosome Segment Substitution Line Reveals a New Regulation Network for Soybean Seed Storage Profile.

Soybean, a major source of oil and protein, has seen an annual increase in consumption when used in soybean-derived products and the broadening of its cultivation range. The demand for soybean necessitates a better understanding of the regulatory networks driving storage protein accumulation and oil biosynthesis to broaden its positive impact on human health. In this study, we selected a chromosome segment substitution line (CSSL) with high protein and low oil contents to investigate the underlying effect of donor introgression on seed storage through multi-omics analysis. In total, 1479 differentially expressed genes (DEGs), 82 differentially expressed proteins (DEPs), and 34 differentially expressed metabolites (DEMs) were identified in the CSSL compared to the recurrent parent. Based on Gene Ontology (GO) term analysis and the Kyoto Encyclopedia of Genes and Genomes enrichment (KEGG), integrated analysis indicated that 31 DEGs, 24 DEPs, and 13 DEMs were related to seed storage functionality. Integrated analysis further showed a significant decrease in the contents of the seed storage lipids LysoPG 16:0 and LysoPC 18:4 as well as an increase in the contents of organic acids such as L-malic acid. Taken together, these results offer new insights into the molecular mechanisms of seed storage and provide guidance for the molecular breeding of new favorable soybean varieties.

Multi-omic analysis reveals the effects of interspecific hybridization on the synthesis of seed reserve polymers in a Triticum turgidum ssp. durum × Aegilops sharonensis amphidiploid.

BACKGROUND: Wheat grain endosperm is mainly composed of proteins and starch. The contents and the overall composition of seed storage proteins (SSP) markedly affect the processing quality of wheat flour. Polyploidization results in duplicated chromosomes, and the genomes are often unstable and may result in a large number of gene losses and gene rearrangements. However, the instability of the genome itself, as well as the large number of duplicated genes generated during polyploidy, is an important driving force for genetic innovation. In this study, we compared the differences in starch and SSP, and analyzed the transcriptome and metabolome among Aegilops sharonensis (R7), durum wheat (Z636) and amphidiploid (Z636×R7) to reveal the effects of polyploidization on the synthesis of seed reserve polymers. RESULTS: The total starch and amylose content of Z636×R7 was significantly higher than R7 and lower than Z636. The gliadin and glutenin contents of Z636×R7 were higher than those in Z636 and R7. Through transcriptome analysis, there were 21,037, 2197, 15,090 differentially expressed genes (DEGs) in the three comparison groups of R7 vs Z636, Z636 vs Z636×R7, and Z636×R7 vs R7, respectively, which were mainly enriched in carbon metabolism and amino acid biosynthesis pathways. Transcriptome data and qRT-PCR were combined to analyze the expression levels of genes related to storage polymers. It was found that the expression levels of some starch synthase genes, namely AGP-L, AGP-S and GBSSI in Z636×R7 were higher than in R7 and among the 17 DEGs related to storage proteins, the expression levels of 14 genes in R7 were lower than those in Z636 and Z636×R7. According to the classification analysis of all differential metabolites, most belonged to carboxylic acids and derivatives, and fatty acyls were enriched in the biosynthesis of unsaturated fatty acids, niacin and nicotinamide metabolism, one-carbon pool by folate, etc. CONCLUSION: After allopolyploidization, the expression of genes related to starch synthesis was down-regulated in Z636×R7, and the process of starch synthesis was inhibited, resulting in delayed starch accumulation and prolongation of the seed development process. Therefore, at the same development time point, the starch accumulation of Z636×R7 lagged behind that of Z636. In this study, the expression of the GSe2 gene in Z636×R7 was higher than that of the two parents, which was beneficial to protein synthesis, and increased the protein content. These results eventually led to changes in the synthesis of seed reserve polymers. The current study provided a basis for a greater in-depth understanding of the mechanism of wheat allopolyploid formation and its stable preservation, and also promoted the effective exploitation of high-value alleles.

Deciphering the Transcriptional Regulatory Network Governing Starch and Storage Protein Biosynthesis in Wheat for Breeding Improvement.

Starch and seed storage protein (SSP) composition profoundly impact wheat grain yield and quality. To unveil regulatory mechanisms governing their biosynthesis, transcriptome, and epigenome profiling is conducted across key endosperm developmental stages, revealing that chromatin accessibility, H3K27ac, and H3K27me3 collectively regulate SSP and starch genes with varying impact. Population transcriptome and phenotype analyses highlight accessible promoter regions' crucial role as a genetic variation resource, influencing grain yield and quality in a core collection of wheat accessions. Integration of time-serial RNA-seq and ATAC-seq enables the construction of a hierarchical transcriptional regulatory network governing starch and SSP biosynthesis, identifying 42 high-confidence novel candidates. These candidates exhibit overlap with genetic regions associated with grain size and quality traits, and their functional significance is validated through expression-phenotype association analysis among wheat accessions and loss-of-function mutants. Functional analysis of wheat abscisic acid insensitive 3-A1 (TaABI3-A1) with genome editing knock-out lines demonstrates its role in promoting SSP accumulation while repressing starch biosynthesis through transcriptional regulation. Excellent TaABI3-A1Hap1 with enhanced grain weight is selected during the breeding process in China, linked to altered expression levels. This study unveils key regulators, advancing understanding of SSP and starch biosynthesis regulation and contributing to breeding enhancement.

A k-mer-based pangenome approach for cataloging seed-storage-protein genes in wheat to facilitate genotype-to-phenotype prediction and improvement of end-use quality.

Wheat is a staple food for more than 35% of the world's population, with wheat flour used to make hundreds of baked goods. Superior end-use quality is a major breeding target; however, improving it is especially time-consuming and expensive. Furthermore, genes encoding seed-storage proteins (SSPs) form multi-gene families and are repetitive, with gaps commonplace in several genome assemblies. To overcome these barriers and efficiently identify superior wheat SSP alleles, we developed "PanSK" (Pan-SSP k-mer) for genotype-to-phenotype prediction based on an SSP-based pangenome resource. PanSK uses 29-mer sequences that represent each SSP gene at the pangenomic level to reveal untapped diversity across landraces and modern cultivars. Genome-wide association studies with k-mers identified 23 SSP genes associated with end-use quality that represent novel targets for improvement. We evaluated the effect of rye secalin genes on end-use quality and found that removal of ω-secalins from 1BL/1RS wheat translocation lines is associated with enhanced end-use quality. Finally, using machine-learning-based prediction inspired by PanSK, we predicted the quality phenotypes with high accuracy from genotypes alone. This study provides an effective approach for genome design based on SSP genes, enabling the breeding of wheat varieties with superior processing capabilities and improved end-use quality.

The association of protein-bound methionine sulfoxide with proteomic basis for aging in beech seeds.

BACKGROUND: European beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared. RESULTS: Aged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds. CONCLUSIONS: We demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.

Comparative transcriptomic analysis provides insights into the genetic networks regulating oil differential production in oil crops.

BACKGROUND: Plants differ more than threefold in seed oil contents (SOCs). Soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed (Brassica napus), and sesame (Sesamum indicum) are four important oil crops with markedly different SOCs and fatty acid compositions. RESULTS: Compared to grain crops like maize and rice, expanded acyl-lipid metabolism genes and relatively higher expression levels of genes involved in seed oil synthesis (SOS) in the oil crops contributed to the oil accumulation in seeds. Here, we conducted comparative transcriptomics on oil crops with two different SOC materials. In common, DIHYDROLIPOAMIDE DEHYDROGENASE, STEAROYL-ACYL CARRIER PROTEIN DESATURASE, PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE, and oil-body protein genes were both differentially expressed between the high- and low-oil materials of each crop. By comparing functional components of SOS networks, we found that the strong correlations between genes in "glycolysis/gluconeogenesis" and "fatty acid synthesis" were conserved in both grain and oil crops, with PYRUVATE KINASE being the common factor affecting starch and lipid accumulation. Network alignment also found a conserved clique among oil crops affecting seed oil accumulation, which has been validated in Arabidopsis. Differently, secondary and protein metabolism affected oil synthesis to different degrees in different crops, and high SOC was due to less competition of the same precursors. The comparison of Arabidopsis mutants and wild type showed that CINNAMYL ALCOHOL DEHYDROGENASE 9, the conserved regulator we identified, was a factor resulting in different relative contents of lignins to oil in seeds. The interconnection of lipids and proteins was common but in different ways among crops, which partly led to differential oil production. CONCLUSIONS: This study goes beyond the observations made in studies of individual species to provide new insights into which genes and networks may be fundamental to seed oil accumulation from a multispecies perspective.

Spatial Distribution and Characteristics of Protein Content and Composition in Japonica Rice Grains: Implications for Sake Quality.

The quantity and composition of rice proteins play a crucial role in determining taste quality of sake, Japanese rice wine. However, the spatial distribution of proteins within rice grains, especially in endosperm tissue, and the differences between rice varieties remain unclear. Here, we analyzed the crude protein contents and composition ratios of table (Nipponbare and Koshihikari) and genuine sake rice varieties (Yamadanishiki, Gohyakumangoku, Dewasansan, Dewanosato, and Yumenokaori) to elucidate their spatial distribution within the Japonica rice grain endosperm. Seven sake rice varieties were polished over five harvest years using a brewer's rice-polishing machine. We obtained fractions at 90-70% (the outermost endosperm fraction), 70-50%, 50-30%, and 30-0% (the central region of the endosperm fraction). Yamadanishiki and Dewanosato exhibited considerably lower crude protein contents than the other cultivars. After applying SDS-PAGE, the protein composition, comprising glutelin/total protein (G/TP), prolamin/TP (P/TP), and G/P ratios of these fractions was determined. In white rice (at a 90% rice-polishing ratio), the average ratio of the major protein composition was G/TP 41%, P/TP 21%, and G/P ratios of 1.97. Gohyakumangoku and Yamadanishiki had higher G/TP ratio, while Dewanosato had a lower value. Despite having lower crude protein contents, Yamadanishiki and Dewanosato exhibited significantly varying G/TP ratios. The G/TP ratio markedly varied among rice varieties, particularly in the rice grains' central region. The 50-30% fraction had the highest P/TP ratio among all tested rice varieties, suggesting spatial differences in P/TP within rice grains. Koshihikari had the lowest P/TP ratio. In addition, the 50-30% fraction had the lowest G/P ratio among all tested rice varieties, with Gohyakumangoku having the highest G/P ratio. Dewanosato had the lowest G/P value, and this value significantly differed from that of Yamadanishiki in the 30-0% fraction. We found substantial differences in protein composition within distinct spatial regions of rice grains, and larger differences among rice varieties were observed in the rice grain's central region.

MicroRNA164e suppresses NAC100 transcription factor-mediated synthesis of seed storage proteins in chickpea.

Development of protein-enriched chickpea varieties necessitates an understanding of specific genes and key regulatory circuits that govern the synthesis of seed storage proteins (SSPs). Here, we demonstrated the novel involvement of Ca-miR164e-CaNAC100 in regulating SSP synthesis in chickpea. Ca-miRNA164e was significantly decreased during seed maturation, especially in high-protein accessions. The miRNA was found to directly target the transactivation conferring C-terminal region of a nuclear-localized transcription factor, CaNAC100 as revealed using RNA ligase-mediated-rapid amplification of cDNA ends and target mimic assays. The functional role of CaNAC100 was demonstrated through seed-specific overexpression (NACOE) resulting in significantly augmented seed protein content (SPC) consequential to increased SSP transcription. Further, NACOE lines displayed conspicuously enhanced seed weight but reduced numbers and yield. Conversely, a downregulation of CaNAC100 and SSP transcripts was evident in seed-specific overexpression lines of Ca-miR164e that culminated in significantly lowered SPC. CaNAC100 was additionally demonstrated to transactivate the SSP-encoding genes by directly binding to their promoters as demonstrated using electrophoretic mobility shift and dual-luciferase reporter assays. Taken together, our study for the first time established a distinct role of CaNAC100 in positively influencing SSP synthesis and its critical regulation by CamiR164e, thereby serving as an understanding that can be utilized for developing SPC-rich chickpea varieties.

Autophagy maintains endosperm quality during seed storage to preserve germination ability in Arabidopsis.

To preserve germination ability, plant seeds must be protected from environmental stresses during the storage period. Here, we demonstrate that autophagy, an intracellular degradation system, maintains seed germination ability in Arabidopsis thaliana. The germination ability of long-term (>5 years) stored dry seeds of autophagy-defective (atg) mutant and wild-type (WT) plants was compared. Long-term stored (old) seeds of atg mutants showed lower germination ability than WT seeds, although short-term stored (new) seeds of atg mutants did not show such a phenotype. After removal of the seed coat and endosperm from old atg mutant seeds, the embryos developed into seedlings. Autophagic flux was maintained in endosperm cells during the storage period, and autophagy defect resulted in the accumulation of oxidized proteins and accelerated endosperm cell death. Consistent with these findings, the transcripts of genes, ENDO-ß-MANNANASE 7 and EXPANSIN 2, which are responsible for degradation/remodeling of the endosperm cell wall during germination, were reduced in old atg mutant seeds. We conclude that autophagy maintains endosperm quality during seed storage by suppressing aging-dependent oxidative damage and cell death, which allows the endosperm to perform optimal functions during germination, i.e., cell wall degradation/remodeling, even after long-term storage.

Orchestrating seed storage protein and starch accumulation toward overcoming yield-quality trade-off in cereal crops.

Achieving high yield and good quality in crops is essential for human food security and health. However, there is usually disharmony between yield and quality. Seed storage protein (SSP) and starch, the predominant components in cereal grains, determine yield and quality, and their coupled synthesis causes a yield-quality trade-off. Therefore, dissection of the underlying regulatory mechanism facilitates simultaneous improvement of yield and quality. Here, we summarize current findings about the synergistic molecular machinery underpinning SSP and starch synthesis in the leading staple cereal crops, including maize, rice and wheat. We further evaluate the functional conservation and differentiation of key regulators and specify feasible research approaches to identify additional regulators and expand insights. We also present major strategies to leverage resultant information for simultaneous improvement of yield and quality by molecular breeding. Finally, future perspectives on major challenges are proposed.

Characterization of barley (Horduem vulgare) lys3 mutants identifies genes under the regulation of the prolamin-box binding transcription factor and elucidates its role in endosperm promoter methylation during grain development.

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific ß-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant's phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., ß-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low ß-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

Sulfate transport and metabolism: strategies to improve the seed protein quality.

Sulfur-containing amino acids (SAA), namely methionine, and cysteine are crucial essential amino acids (EAA) considering the dietary requirements of humans and animals. However, a few crop plants, especially legumes, are characterized with suboptimal levels of these EAA thereby limiting their nutritive value. Hence, improved comprehension of the mechanistic perspective of sulfur transport and assimilation into storage reserve, seed storage protein (SSP), is imperative. Efforts to augment the level of SAA in seed storage protein form an integral component of strategies to balance nutritive quality and quantity. In this review, we highlight the emerging trends in the sulfur biofortification approaches namely transgenics, genetic and molecular breeding, and proteomic rebalancing with sulfur nutrition. The transgenic 'push and pull strategy' could enhance sulfur capture and storage by expressing genes that function as efficient transporters, sulfate assimilatory enzymes, sulfur-rich foreign protein sinks, or by suppressing catabolic enzymes. Modern molecular breeding approaches that adopt high throughput screening strategies and machine learning algorithms are invaluable in identifying candidate genes and alleles associated with SAA content and developing improved crop varieties. Sulfur is an essential plant nutrient and its optimal uptake is crucial for seed sulfur metabolism, thereby affecting seed quality and yields through proteomic rebalance between sulfur-rich and sulfur-poor seed storage proteins.

Spatio-temporal transcriptome and storage compound profiles of developing faba bean (Vicia faba) seed tissues.

Faba bean (Vicia faba) is a legume grown in diverse climate zones with a high potential for increased cultivation and use in food due to its nutritional seeds. In this study, we characterized seed tissue development in faba bean to identify key developmental processes; from embryo expansion at the expense of the endosperm to the maturing storage stages of the bean seed. A spatio-temporal transcriptome profiling analysis, combined with chemical nutrient analysis of protein, starch, and lipid, of endosperm and embryo tissues at different developmental stages, revealed gene expression patterns, transcriptional networks, and biochemical pathways in faba bean. We identified key players in the LAFL (LEC1, ABI3, FUS3, and LEC2) transcription factor network as well as their major repressors VAL1 and ASIL1. Our results showed that proteins accumulated not only in the embryo but also in the endosperm. Starch accumulated throughout seed development and oil content increased during seed development but at very low levels. The patterns of differentially expressed transcripts encoding proteins with functions in the corresponding metabolic pathways for the synthesis of these storage compounds, to a high extent, aligned with these findings. However, the early expression of transcripts encoding WRI1 combined with the late expression of oil body proteins indicated a not manifested high potential for lipid biosynthesis and oil storage. Altogether, this study contributes to increased knowledge regarding seed developmental processes applicable to future breeding methods and seed quality improvement for faba bean.

Seed Collection in Temperate Trees-Clean, Fast, and Effective Extraction of Populus Seeds for Laboratory Use and Long-term Storage.

Seeds ensure the growth of a new generation of plants and are thus central to maintaining plant populations and ecosystem processes. Nevertheless, much remains to be learned about seed biology and responses of germinated seedlings to environmental challenges. Experiments aiming to close these knowledge gaps critically depend on the availability of healthy, viable seeds. Here, we report a protocol for the collection of seeds from plants in the genus Populus. This genus comprises trees with a wide distribution in temperate forests and with economic relevance, used as scientific models for perennial plants. As seed characteristics can vary drastically between taxonomic groups, protocols need to be tailored carefully. Our protocol takes the delicate nature of Populus seeds into account. It uses P. deltoides as an example and provides a template to optimize bulk seed extraction for other Populus species and plants with similar seed characteristics. The protocol is designed to only use items available in most labs and households and that can be sterilized easily. The unique characteristics of this protocol allow for the fast and effective extraction of high-quality seeds. Here, we report on seed collection, extraction, cleaning, storage, and viability tests. Moreover, extracted seeds are well suited for tissue culture and experiments under sterile conditions. Seed material obtained with this protocol can be used to further our understanding of tree seed biology, seedling performance under climate change, or diversity of forest genetic resources. Key features • Populus species produce seeds that are small, delicate, non-dormant, with plenty of seed hair. Collection of seed material needs to be timed properly. • Processing, seed extraction, seed cleaning, and storage using simple, sterilizable laboratory and household items only. Obtained seeds are pure, high quality, close to 100% viability. • Seeds work well in tissue culture and in experiments under sterile conditions. • Extractability, speed, and seed germination were studied and confirmed for Populus deltoides as an example. • Can also serve as template for bulk seed collection from other Populus species and plant groups that produce delicate seeds (with no or little modifications). Graphical overview.

Comprehensive amino acid composition analysis of seed storage proteins of cereals and legumes: identification and understanding of intrinsically disordered and allergenic peptides.

The seed storage proteins of cereal and legumes are the primary source of amino acids which are required for sustaining the nitrogen and carbon demands during germination and growth. Humans derive most of their dietary proteins from storage proteins in form of a wide variety of foods, for consumption. The amino acid content of most of these proteins is biased and the need for this biasness is not understood. The high abundance of proline, glutamine, and cysteine in cereals makes the gluten fraction viscoelastic. The cereal proteins have less charge and legume proteins have more charge on them. Their non-polar amino acid distribution has large variations. These characteristics are strongly responsible for the partial and complete unfolding of several domains of the storage proteins. Many of the storage proteins share a highly conserved structural feature within the cupin superfamily spread across all kingdoms of life. The intrinsically disordered viscoelastic proteins help in making dough which is vital for the quality of bread. Unfolded regions harbor more immunogenic sequences and cause food-related allergies and intolerance. We have discussed these properties in terms of comparison of cereal and legume storage protein sequences and allergy. Our study supports the findings that large disordered regions contain allergen-representative peptides. Interestingly, a high number of allergen-representative peptides were cleavable by digestive enzymes. Furthermore, unfolded storage proteins mimic microbial immunogens to induce a memory immune response. Results findings can be used to guide the understanding of immunological characteristics of storage proteins and may assist in treatment decisions for food allergy.Communicated by Ramaswamy H. Sarma.

Proteomic profile of tepary bean seed storage proteins in germination with low water potential.

BACKGROUND: Tepary bean (Phaseolus acutifolius A. Gray) is one of the five species domesticated from the genus Phaseolus with genetic resistance to biotic and abiotic stress. To understand the mechanisms underlying drought responses in seed storage proteins germinated on water and polyethylene glycol (PEG-6000) at -0.49 MPa, we used a proteomics approach to identify potential molecular target proteins associated with the low water potential stress response. METHODS: Storage proteins from cotyledons of Tepary bean seeds germinated at 24, 48 and 72 h on water and PEG-6000 at -0.49 MPa were analyzed by one-dimensional electrophoresis (DE) with 2-DE analysis and shotgun mass spectrometry. Using computational database searching and bioinformatics analyses, we performed Gene Ontology (GO) and protein interactome (functional protein association network) String analyses. RESULTS: Comparative analysis showed that the effect of PEG-6000 on root growth was parallel to that on germination. Based on the SDS‒PAGE protein banding patterns and 2-DE analysis, ten differentially abundant seed storage proteins showed changes in storage proteins, principally in the phaseolin and lectin fractions. We found many proteins that are recognized as drought stress-responsive proteins, and several of them are predicted to be intrinsically related to abiotic stress. The shotgun analysis searched against UniProt's legume database, and Gene Ontology (GO) analysis indicated that most of the seed proteins were cytosolic, with catalytic activity and associated with carbohydrate metabolism. The protein‒protein interaction networks from functional enrichment analysis showed that phytohemagglutinin interacts with proteins associated with the degradation of storage proteins in the cotyledons of common bean during germination. CONCLUSION: These findings suggest that Tepary bean seed proteins provide valuable information with the potential to be used in genetic improvement and are part of the drought stress response, making our approach a potentially useful strategy for discovering novel drought-responsive proteins in other plant models.

Editing the 19 kDa alpha-zein gene family generates non-opaque2-based quality protein maize.

Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19 kDa and 22 kDa alpha-zeins causes a floury kernel, unsuitable for agricultural use. Quality protein maize (QPM) overcame the undesirable kernel texture through the introgression of modifying alleles. However, QPM still lacks a functional o2 transcription factor, which has a penalty on non-lysine amino acids due to the o2 mutation. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this paper, gene editing was used to specifically target the 19 kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha-zein knockout. The results showed that editing some, but not all, of the 19 kDa zeins resulted in up to 30% more lysine. An edited line displayed an increase of 30% over the wild type. While not quite the 55% lysine increase displayed by QPM, the line had little collateral impact on other amino acid levels compared to QPM. Additionally, the edited line containing a partially reduced 19 kDa showed an advantage in kernel texture that had a complete 19 kDa knockout. These results serve as proof of concept that editing the 19 kDa alpha-zein family alone can enhance lysine while retaining vitreous endosperm and a functional O2 transcription factor.