Accumulation Of Seed Storage Proteins Research Articles

Proteomic profile of the germinating seeds reveals enhanced seedling growth in Arabidopsis rpp1a mutant.

Ribosomal phosphoprotein P1 (RPP1) is an integral component of the P-protein stalk in the 60S subunit of eukaryotic ribosomes and is required for the efficient elongation of translation. Previously, Arabidopsis RPP1A was revealed to be involved in the regulation of seed size and seed storage protein accumulation. In this work, the seedling growth analysis shows that the knockout mutation of Arabidopsis RPP1A significantly promoted seedling growth, particularly in the shoots. The label-free quantitative proteomic analysis demonstrated that a total of 593 proteins were differentially accumulated between the germinating seeds of the wild-type Col-0 and rpp1a mutant. And these proteins were significantly enriched in the intracellular transport, nitrogen compound transport, protein transport, and organophosphate metabolic process. The abundance of proteins involved in the RNA and protein processing processes, including ncRNA processing and protein folding, were significantly increased in the rpp1a mutant. Mutation in RPP1A highlighted the effects on the ribosome, energy metabolism, and nitrogen metabolism. The abundance of enzymes involved in glycolysis and pyruvate mechanism was decreased in the germinating seeds of the rpp1a mutant. Whereas the processes of amino acid biosynthesis, protein processing in endoplasmic reticulum, and biosynthesis of cofactors were enhanced in the germinating seeds of the rpp1a mutant. Taken together, the lack of RPP1A triggered changes in other ribosomal proteins, and the higher amino acid contents in the seedlings of the rpp1a mutant probably contributed to enhanced biosynthesis, processing, and transport of proteins, resulting in accelerated growth. Our results show the novel role of a P-protein and shed new light on the regulatory mechanism of seedling growth.

Transcriptome and Proteome Analysis Revealed the Influence of High-Molecular-Weight Glutenin Subunits (HMW-GSs) Deficiency on Expression of Storage Substances and the Potential Regulatory Mechanism of HMW-GSs.

The processing quality of wheat is affected by seed storage substances, such as protein and starch. High-molecular-weight glutenin subunits (HMW-GSs) are the major components of wheat seed storage proteins (SSPs); they are also key determinators of wheat end-use quality. However, the effects of HMW-GSs absence on the expression of other storage substances and the regulation mechanism of HMW-GSs are still limited. Previously, a wheat transgenic line LH-11 with complete deletions of HMW-GSs was obtained through introducing an exogenous gene Glu-1Ebx to the wild-type cultivar Bobwhite by transgenic approach. In this study, comparative seed transcriptomics and proteomics of transgenic and non-transgenic lines at different seed developmental stages were carried out to explore the changes in genes and proteins and the underlying regulatory mechanism. Results revealed that a number of genes, including genes related to SSPs, carbohydrates metabolism, amino acids metabolism, transcription, translation, and protein process were differentially enriched. Seed storage proteins displayed differential expression patterns between the transgenic and non-transgenic line, a major rise in the expression levels of gliadins were observed at 21 and 28 days post anthesis (DPA) in the transgenic line. Changes in expressions of low-molecular-weight glutenins (LMW-GSs), avenin-like proteins (ALPs), lipid transfer proteins (LTPs), and protease inhibitors (PIs) were also observed. In addition, genes related to carbohydrate metabolism were differentially expressed, which probably leads to a difference in starch component and deposition. A list of gene categories participating in the accumulation of SSPs was proposed according to the transcriptome and proteome data. Six genes from the MYB and eight genes from the NAC transcription families are likely important regulators of HMW-GSs accumulation. This study will provide data support for understanding the regulatory network of wheat storage substances. The screened candidate genes can lay a foundation for further research on the regulation mechanism of HMW-GSs.

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.

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.

Transcription Factor DOF4.1 Regulates Seed Longevity in Arabidopsis via Seed Permeability and Modulation of Seed Storage Protein Accumulation.

Seed longevity is modulated by multiple genetic factors in Arabidopsis thaliana. A previous genome-wide association study using the Elevated Partial Pressure of Oxygen (EPPO) aging assay pinpointed a genetic locus associated with this trait. Reverse genetics identified the transcription factor DOF4.1 as a novel seed longevity factor. dof4.1 loss-of-function plants generate seeds exhibiting higher germination after accelerated aging assays. DOF4.1 is expressed during seed development and RNAseq data show several putative factors that could contribute to the dof4.1 seed longevity phenotype. dof4.1 has reduced seed permeability and a higher levels of seed storage proteins mRNAs (cruciferins and napins) in developing seeds, as compared to wild-type seeds. It has been reported that mutant lines defective in cruciferins or napins present reduced seed longevity. The improved longevity of dof4.1 is totally lost in the quadruple mutant dof4.1 cra crb crc, but not in a dof4.1 line depleted of napins, suggesting a prominent role for cruciferins in this process. Moreover, a negative regulation of DOF4.1 expression by the transcription factor DOF1.8 is suggested by co-inoculation assays in Nicotiana benthamiana. Indeed, DOF1.8 expression anticorrelates with that of DOF4.1 during seed development. In summary, modulation of DOF4.1 levels during seed development contributes to regulate seed longevity.

A proteomic analysis of Arabidopsis ribosomal phosphoprotein P1A mutant

Ribosomal proteins are involved in the regulation of plant growth and development. However, the regulatory processes of most ribosomal proteins remain unclear. In this study, Arabidopsis plants with the mutation in ribosomal phosphoprotein P1A (RPP1A) produce larger and heavier seeds than wild-type plants. A comparative quantitative label-free proteomic analysis revealed that a total of 215 proteins were differentially accumulated between the young siliques of the wild type and rpp1a mutant. Knockout of RPP1A significantly reduced the abundance of proteins involved in carboxylic acid metabolism and lipid biosynthesis. Consistent with this, a metabolic analysis showed that the organic acids in the tricarboxylic acid cycle and the carbohydrates in the pentose phosphate pathway were severely reduced in the mature rpp1a mutant seeds. In contrast, the abundance of proteins related to seed maturation, especially seed storage proteins, was markedly increased during seed development. Indeed, seed storage proteins were accumulated in the mature rpp1a mutant seeds, and the seed nitrogen and sulfur contents were also increased. These results indicate that more carbon intermediates probably enter the nitrogen flow for the enhanced synthesis of seed storage proteins, which might subsequently contribute to the enlarged seed size in the rpp1a mutant. SignificanceRibosomes are responsible for protein synthesis and are generally perceived as the housekeeping components in the cells. In this study, the knockout of RPP1A leads to an increased seed size through repressing carbon metabolism and lipid biosynthesis, and increasing the synthesis of seed storage proteins. Meanwhile, the abundance of seed storage proteins and the nitrogen and sulfur concentrations were increased in the mature rpp1a mutant seeds. The results provide a novel insight into the genetic regulatory networks for the control of seed size and seed storage protein accumulation, and this knowledge may facilitate the improvement of crop seed size.

Genetic Background Negates Improvements in Rice Flour Characteristics and Food Processing Properties Caused by a Mutant Allele of the PDIL1-1 Seed Storage Protein Gene

Phenotypic differences among breeding lines that introduce the same superior gene allele can be a barrier to effective development of cultivars with desirable traits in some crop species. For example, a deficient mutation of the Protein Disulfide Isomerase Like 1–1 (PDIL1-1) gene can cause accumulation of glutelin seed storage protein precursors in rice endosperm, and improves rice flour characteristics and food processing properties. However, the gene must be expressed to be useful. A deficient mutant allele of PDIL1-1 was introduced into two rice cultivars with different genetic backgrounds (Koshihikari and Oonari). The grain components, agronomic traits, and rice flour and food processing properties of the resulting lines were evaluated. The two breeding lines had similar seed storage protein accumulation, amylose content, and low-molecular-weight metabolites. However, only the Koshihikari breeding line had high flour quality and was highly suitable for rice bread, noodles, and sponge cake, evidence of the formation of high-molecular-weight protein complexes in the endosperm. Transcriptome analysis revealed that mRNA levels of fourteen PDI, Ero1, and BiP genes were increased in the Koshihikari breeding line, whereas this change was not observed in the Oonari breeding line. We elucidated part of the molecular basis of the phenotypic differences between two breeding lines possessing the same mutant allele in different genetic backgrounds. The results suggest that certain genetic backgrounds can negate the beneficial effect of the PDIL1-1 mutant allele. Better understanding of the molecular basis for such interactions may accelerate future breeding of novel rice cultivars to meet the strong demand for gluten-free foods.

Identification and molecular characterization of miRNAs and their target genes associated with seed development through small RNA sequencing in chickpea.

Multiple studies have attempted to dissect the molecular mechanism underlying seed development in chickpea (Cicer arietinum L.). These studies highlight the need to focus on the role of miRNAs in regulating storage protein accumulation in seeds. Therefore, a total of 8,856,691 short-read sequences were generated from a small RNA library of developing chickpea seeds and were analyzed using miRDeep-P to identify 74 known and 26 novel miRNA sequences. Known miRNAs were classified into 22 miRNA families with miRNA156 family being most abundant. Of the 26 putative novel miRNAs identified, only 22 could be experimentally validated using stem loop end point PCR. Differential expression analyses led to the identification of known as well as novel miRNAs that could regulate various stages of chickpea seed development. In silico target prediction revealed several important target genes and transcription factors like SPL, mediator of RNA Polymerase II transcription subunit 12, aspartic proteinase and NACs, which were further validated by real-time PCR analysis. A comparative expression analysis in chickpea genotypes with contrasting seed protein content revealed one known (Car-miR156h) and two novel miRNA (Car-novmiR7 and Car-novmiR23) candidates to be highly expressed in the LPC (low protein content) chickpea genotypes, targets of which are known to regulate seed storage protein accumulation. Therefore, this study provides a useful resource in the form of miRNA and their targets which can be further utilized to understand and manipulate various regulatory mechanisms involved in seed development with the overall aim of improving yield and nutrition attributes in chickpea.

The gibberellin signaling negative regulator RGA-LIKE3 promotes seed storage protein accumulation

Seed storage protein (SSP) acts as one of the main components of seed storage reserves, of which accumulation is tightly mediated by a sophisticated regulatory network. However, whether and how gibberellin (GA) signaling is involved in this important biological event is not fully understood. Here, we show that SSP content in Arabidopsis (Arabidopsis thaliana) is significantly reduced by GA and increased in the GA biosynthesis triple mutant ga3ox1/3/4. Further investigation shows that the DELLA protein RGA-LIKE3 (RGL3), a negative regulator of GA signaling, is important for SSP accumulation. In rgl3 and 35S:RGL3-HA, the expression of SSP genes is down- and upregulated, respectively, compared with that in the wild-type. RGL3 interacts with ABSCISIC ACID INSENSITIVE3 (ABI3), a critical transcription factor for seed developmental processes governing SSP accumulation, both in vivo and in vitro, thus greatly promoting the transcriptional activating ability of ABI3 on SSP genes. In addition, genetic evidence shows that RGL3 and ABI3 regulate SSP accumulation in an interdependent manner. Therefore, we reveal a function of RGL3, a little studied DELLA member, as a coactivator of ABI3 to promote SSP biosynthesis during seed maturation stage. This finding advances the understanding of mechanisms in GA-mediated seed storage reserve accumulation.

Dissecting the nutrient partitioning mechanism in rice grain using spatially resolved gene expression profiling

Rice, a staple food worldwide, contains varying amount of nutrients in different grain tissues. The underlying molecular mechanism of such distinct nutrient partitioning remains poorly-investigated. Here, an optimized rapid Laser Capture Microdissection (LCM) approach was used to individually collect pericarp, aleurone, embryo and endosperm from 10 Days After Fertilization (DAF) old grains. Subsequent RNA-Seq analysis in these tissues have identified 7760 differentially expressed genes (DEGs). Analysis of promoter sequences of tissue specific genes identified many known and novel cis-elements important for grain filling and seed development. Using identified DEGs, comprehensive spatial gene expression pathways were built for spatial accumulation of starch, proteins, lipids and iron. The extensive transcriptomic analysis has provided many novel insights about nutrient partitioning mechanisms, for instance, it reveals a gradient in Seed Storage Protein accumulation across the analysed four tissue-types. It further reveals that partitioning of various minerals, such as iron, is most likely regulated through transcriptional control of their transporters. In addition, the extensive analysis of this study is presented as an interactive online tool (https://biogeek.shinyapps.io/DEGs/) that provides a much-needed resource for future functional genomics studies aimed to improve grain quality and seed development.

Effects of 1Dy12 subunit silencing on seed storage protein accumulation and flour-processing quality in a common wheat somatic variation line

Dissecting the functions of high molecular weight glutenin subunits (HMW-GSs) is helpful for improving wheat quality via breeding. In this study, we used a wheat mutant AS273 in which HMW-GS 1Dy12 was silenced to investigate the silencing mechanism of 1Dy12 and its effects on gluten accumulation and flour-processing quality. Results suggested that the expression of 1Dy12 in AS273 was decreased by one fifth during grain development; a stop codon produced by a base mutation (C/T) led to truncated translation; the absence of 1Dy12 stimulated the accumulation of low molecular weight glutenin subunits (LMW-GSs), gliadins, and glutenin macropolymers, and was resulted in larger protein bodies; AS273 had an inferior flour-processing performance. Based on the outputs achieved in this study it is concluded that 1Dy12 makes important contributions to bread, sponge cake and biscuit-processing quality.

Transcriptome wide identification and characterization of regulatory genes involved in EAA metabolism and validation through expression analysis in different developmental stages of finger millet spikes.

Finger millet is a rich source of seed storage proteins (SSPs). Various regulatory genes play an important role to maintain the quality and accumulation of SSPs in crop seeds. In the present study, nine regulatory genes of EAAs metabolic pathway, i.e., aspartate kinase, homoserine dehydrogenase, threonine synthase, threonine dehydratase, dihydrodipicolinate synthase, cystathionine γ synthase, anthranilate synthase, acetolactate synthase and lysine 2-oxoglutarato reductase/saccharopine dehydrogenase (LOR/SD) were identified from the transcriptomic data of developing spikes of two finger millet genotypes, i.e., GP-45 and GP-1. Results of sequence alignment search and motif/domain analysis showed high similarity of nucleotide sequences of identified regulatory genes with their respective homologs in rice. Results of promoter analysis revealed the presence of various cis-regulatory elements, like nitrogen responsive cis-elements (O2-site and GCN4), light responsive cis-elements, and stress responsive cis-elements. The presence of nine regulatory genes identified from the transcriptomic data of GP-45 and GP-1 was further confirmed by real time expression analysis in high and low protein containing genotypes, i.e., GE-3885 and GE-1437. Results of real time expression analysis showed significantly higher expression (p ≤ 0.01) of regulatory genes in GE-3885 rather than GE-1437 under control and treatment condition. Crude protein content of GE-3885 was found to be significantly higher (p ≤ 0.01) in comparison to GE-1437 under control condition, while under treatment condition GE-1437 was found to be more responsive to KNO3 treatment rather than GE-3885.

Cysteine and Methionine Biosynthetic Enzymes Have Distinct Effects on Seed Nutritional Quality and on Molecular Phenotypes Associated With Accumulation of a Methionine-Rich Seed Storage Protein in Rice.

Staple crops in human and livestock diets suffer from deficiencies in certain “essential” amino acids including methionine. With the goal of increasing methionine in rice seed, we generated a pair of “Push × Pull” double transgenic lines, each containing a methionine-dense seed storage protein (2S albumin from sunflower, HaSSA) and an exogenous enzyme for either methionine (feedback desensitized cystathionine gamma synthase from Arabidopsis, AtD-CGS) or cysteine (serine acetyltransferase from E. coli, EcSAT) biosynthesis. In both double transgenic lines, the total seed methionine content was approximately 50% higher than in their untransformed parental line, Oryza sativa ssp. japonica cv. Taipei 309. HaSSA-containing rice seeds were reported to display an altered seed protein profile, speculatively due to insufficient sulfur amino acid content. However, here we present data suggesting that this may result from an overloaded protein folding machinery in the endoplasmic reticulum rather than primarily from redistribution of limited methionine from endogenous seed proteins to HaSSA. We hypothesize that HaSSA-associated endoplasmic reticulum stress results in redox perturbations that negatively impact sulfate reduction to cysteine, and we speculate that this is mitigated by EcSAT-associated increased sulfur import into the seed, which facilitates additional synthesis of cysteine and glutathione. The data presented here reveal challenges associated with increasing the methionine content in rice seed, including what may be relatively low protein folding capacity in the endoplasmic reticulum and an insufficient pool of sulfate available for additional cysteine and methionine synthesis. We propose that future approaches to further improve the methionine content in rice should focus on increasing seed sulfur loading and avoiding the accumulation of unfolded proteins in the endoplasmic reticulum.Oryza sativa ssp. japonica: urn:lsid:ipni.org:names:60471378-2.

Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s

Cereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues.

An integrated omics analysis reveals molecular mechanisms that are associated with differences in seed oil content between Glycine max and Brassica napus

BackgroundRapeseed (Brassica napus L.) and soybean (Glycine max L.) seeds are rich in both protein and oil, which are major sources of biofuels and nutrition. Although the difference in seed oil content between soybean (~ 20%) and rapeseed (~ 40%) exists, little is known about its underlying molecular mechanism.ResultsAn integrated omics analysis was performed in soybean, rapeseed, Arabidopsis (Arabidopsis thaliana L. Heynh), and sesame (Sesamum indicum L.), based on Arabidopsis acyl-lipid metabolism- and carbon metabolism-related genes. As a result, candidate genes and their transcription factors and microRNAs, along with phylogenetic analysis and co-expression network analysis of the PEPC gene family, were found to be largely associated with the difference between the two species. First, three soybean genes (Glyma.13G148600, Glyma.13G207900 and Glyma.12G122900) co-expressed with GmPEPC1 are specifically enriched during seed storage protein accumulation stages, while the expression of BnPEPC1 is putatively inhibited by bna-miR169, and two genes BnSTKA and BnCKII are co-expressed with BnPEPC1 and are specifically associated with plant circadian rhythm, which are related to seed oil biosynthesis. Then, in de novo fatty acid synthesis there are rapeseed-specific genes encoding subunits β-CT (BnaC05g37990D) and BCCP1 (BnaA03g06000D) of heterogeneous ACCase, which could interfere with synthesis rate, and β-CT is positively regulated by four transcription factors (BnaA01g37250D, BnaA02g26190D, BnaC01g01040D and BnaC07g21470D). In triglyceride synthesis, GmLPAAT2 is putatively inhibited by three miRNAs (gma-miR171, gma-miR1516 and gma-miR5775). Finally, in rapeseed there was evidence for the expansion of gene families, CALO, OBO and STERO, related to lipid storage, and the contraction of gene families, LOX, LAH and HSI2, related to oil degradation.ConclusionsThe molecular mechanisms associated with differences in seed oil content provide the basis for future breeding efforts to improve seed oil content.

Meta-analysis and transcriptome profiling reveal hub genes for soybean seed storage composition during seed development.

Soybean is an important crop providing edible oil and protein source. Soybean oil and protein contents are quantitatively inherited and significantly affected by environmental factors. In this study, meta-analysis was conducted based on soybean physical maps to integrate quantitative trait loci (QTLs) from multiple experiments in different environments. Meta-QTLs for seed oil, fatty acid composition, and protein were identified. Of them, 11 meta-QTLs were located on hot regions for both seed oil and protein. Next, we selected 4 chromosome segment substitution lines with different seed oil and protein contents to characterize their 3years of phenotype selection in the field. Using strand-specific RNA-sequencing analysis, we profile the time-course transcriptome patterns of soybean seeds at early maturity, middle maturity, and dry seed stages. Pairwise comparison and K-means clustering analysis revealed 7,482 differentially expressed genes and 45 expression patterns clusters. Weighted gene coexpression network analysis uncovered 46 modules of gene expression patterns. The 2 most significant coexpression networks were visualized, and 7 hub genes were identified that were involved in soybean oil and seed storage protein accumulation processes. Our results provided a transcriptome dataset for soybean seed development, and the candidate hub genes represent a foundation for further research.

Where do Protein Bodies of Cereal Seeds Come From?

Protein bodies of cereal seeds consist of ordered, largely insoluble heteropolymers formed by prolamin storage proteins within the endoplasmic reticulum (ER) of developing endosperm cells. Often these structures are permanently unable to traffic along the secretory pathway, thus representing a unique example for the use of the ER as a protein storage compartment. In recent years, marked progress has been made in understanding what is needed to make a protein body and in formulating hypotheses on how protein body formation might have evolved as an efficient mechanism to store large amounts of protein during seed development, as opposed to the much more common system of seed storage protein accumulation in vacuoles. The major key evolutionary events that have generated prolamins appear to have been insertions or deletions that have disrupted the conformation of the eight-cysteine motif, a protein folding motif common to many proteins with different functions and locations along the secretory pathway, and, alternatively, the fusion between the eight-cysteine motif and domains containing additional cysteine residues.

MYC2, MYC3, and MYC4 function redundantly in seed storage protein accumulation in Arabidopsis

Basic helix-loop-helix transcription factors (TFs), namely MYC2, MYC3, and MYC4, interact with Jasmonate Zim-domain proteins and are their direct targets. These TFs have been shown to function synergistically to control Arabidopsis growth and development. Our results showed similar MYC2, MYC3, and MYC4 expression patterns during Arabidopsis seed development, which remained relatively high during seed mid-maturation. MYC2, MYC3, and MYC4 acted redundantly in seed size, weight control, and in regulating seed storage protein accumulation. Triple mutants produced the largest seeds and single and double mutants’ seeds were much larger than those of wild type. The weight of triple mutants’ seeds was significantly higher than that of wild-type seeds, which was accompanied by an increase in seed storage protein contents. Triple mutants’ seeds presented a marked decrease in 2S amounts relative to those in wild-type seeds. Liquid chromatography tandem mass spectra sequencing results indicated that both the relative abundance and the peptide number of CRA1 and CRU3 were greatly increased in triple mutants compared to wild type. The expression of 2S1–2S5 decreased and that of CRA1 and CRU3 increased in triple mutants relative to those in wild types during seed development, which might have contributed to the low 2S and high 12S contents in triple mutants. Our results contribute to understanding the function of MYC2, MYC3, and MYC4 on seed development, and provide promising targets for genetic manipulations of protein-producing crops to improve the quantity and quality of seed storage proteins.

Lack of Globulin Synthesis during Seed Development Alters Accumulation of Seed Storage Proteins in Rice.

The major seed storage proteins (SSPs) in rice seeds have been classified into three types, glutelins, prolamins, and globulin, and the proportion of each SSP varies. It has been shown in rice mutants that when either glutelins or prolamins are defective, the expression of another type of SSP is promoted to counterbalance the deficit. However, we observed reduced abundances of glutelins and prolamins in dry seeds of a globulin-deficient rice mutant (Glb-RNAi), which was generated with RNA interference (RNAi)-induced suppression of globulin expression. The expression of the prolamin and glutelin subfamily genes was reduced in the immature seeds of Glb-RNAi lines compared with those in wild type. A proteomic analysis of Glb-RNAi seeds showed that the reductions in glutelin and prolamin were conserved at the protein level. The decreased pattern in glutelin was also significant in the presence of a reductant, suggesting that the polymerization of the glutelin proteins via intramolecular disulfide bonds could be interrupted in Glb-RNAi seeds. We also observed aberrant and loosely packed structures in the storage organelles of Glb-RNAi seeds, which may be attributable to the reductions in SSPs. In this study, we evaluated the role of rice globulin in seed development, showing that a deficiency in globulin could comprehensively reduce the expression of other SSPs.

Chromodomain, Helicase and DNA-binding CHD1 protein, CHR5, are involved in establishing active chromatin state of seed maturation genes.

Chromatin modification and remodelling are the basis for epigenetic regulation of gene expression. LEAFY COTYLEDON 1 (LEC1), LEAFY COTYLEDON 2 (LEC2), ABSCISIC ACID-INSENSITIVE 3 (ABI3) and FUSCA3 (FUS3) are key regulators of embryo development and are repressed after seed maturation. The chromatin remodelling CHD3 protein PICKLE (PKL) is involved in the epigenetic silencing of the genes. However, the chromatin mechanism that establishes the active state of these genes during early embryo development is not clear. We show that the Arabidopsis CHD1-related gene, CHR5, is activated during embryo development. Mutation of the gene reduced expression of LEC1, ABI3 and FUS3 in developing embryo and accumulation of seed storage proteins. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL in embryo gene expression and seed storage protein accumulation, which likely acted on the promoter region of the genes. CHR5 was shown to be associated with the promoters of ABI3 and FUS3 and to be required to reduce nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in establishing the active state of embryo regulatory genes by reducing nucleosomal barrier, which may be exploited to enhance seed protein production.