Crystal structure of hetero hexameric 11S seed storage protein of hazelnut.

Edible plant seeds provide a relatively inexpensive source of protein and make up a large part of nutrients for humans. Plant seeds accumulate storage proteins during seed development. Seed storage proteins act as a reserve of nutrition for seed germination and seedling growth. However, seed storage proteins may be allergenic, and the prevalence of food allergy has increased rapidly in recent years. The 11S globulins account for a significant number of known major food allergens. They are of interest to the public and the agricultural industry because of food safety concerns and the need for crop enhancement. We sought to determine the crystal structure of Cor a 9, the 11 S storage protein of hazelnut and a food allergen. The structure was refined to 1.92 Å, and the R and Rfree for the refined structure are 17.6% and 22.5%, respectively. The structure of Cor a 9 showed a hetero hexamer of an 11S seed storage protein for the first time. The hexamer was two trimers associated back-to-back. Two long alpha helixes at the C-terminal end of the acidic domain of one of the Cor a 9 isoforms lay at the trimer-trimer interface's groove. These data provided much-needed information about the allergenicity of the 11S seed proteins. The information may also facilitate a better understanding of the folding and transportation of 11S seed storage proteins.

Mammalian oocytes store proteins for the early embryo on cytoplasmic lattices.

Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.

Atomic Force Microscopy Study of the Temperature and Storage Duration Dependencies of Horseradish Peroxidase Oligomeric State.

This paper presents an investigation of the temperature dependence of the oligomeric state of the horseradish peroxidase (HRP) enzyme on the temperature of its solution, and on the solution storage time, at the single-molecule level. Atomic force microscopy has been employed to determine how the temperature and the storage time of the HRP solution influence its aggregation upon direct adsorption of the enzyme from the solution onto bare mica substrates. In parallel, spectrophotometric measurements have been performed in order to estimate whether the HRP enzymatic activity changes over time upon the storage of the enzyme solution. The temperature dependence of the HRP oligomeric state has been studied within a broad (15-40 °C) temperature range. It has been demonstrated that the storage of the HRP solution for 14 days does not have any considerable effect on the oligomeric state of the enzyme, neither does it affect its activity. At longer storage times, AFM has allowed us to reveal a tendency of HRP to oligomerization during the storage of its buffered solution, while the enzymatic activity remains virtually unchanged even after a 1-month-long storage. By AFM, it has been revealed that after the incubation of a mica substrate in the HRP solution at various temperatures, the content of the mica-adsorbed oligomers increases insignificantly owing to a high-temperature stability of the enzyme.

Endomembrane-mediated storage protein trafficking in plants: Golgi-dependent or Golgi-independent?

Seed storage proteins (SSPs) accumulated within plant seeds constitute the major protein nutrition sources for human and livestock. SSPs are synthesized on the endoplasmic reticulum and are then deposited in plant-specific protein bodies, including endoplasmic reticulum-derived protein bodies and protein storage vacuoles. Plant seeds have evolved a distinct endomembrane system to accomplish SSP transport. There are two distinct types of trafficking pathways contributing to SSP delivery to protein storage vacuoles: one is Golgi-dependent and the other is Golgi-independent. In recent years, molecular, genetic, and biochemical studies have shed light on the complex network controlling SSP trafficking, to which both evolutionarily conserved molecular machineries and plant-unique regulators contribute. In this review, we discuss current knowledge of protein body biogenesis and endomembrane-mediated SSP transport, focusing on endoplasmic reticulum export and post-Golgi traffic. This knowledge supports a dominant role for the Golgi-dependent pathways in SSP transport in Arabidopsis and rice. In addition, we describe cutting-edge strategies for dissecting the endomembrane trafficking system in plant seeds to advance the field.

Confocal Fluorescence Microscopy Investigation for the Existence of Subdomains within Protein Storage Vacuoles in Soybean Cotyledons.

In legumes, the seed storage proteins accumulate within specialized organelles called protein storage vacuoles (PSVs). In several plant species, PSVs are differentiated into subdomains that accumulate different kinds of proteins. Even though the existence of subdomains is common in cereals and legumes, it has not been reported in soybean PSVs. The two most abundant seed proteins of soybean, 7S and 11S globulins, have different temporal accumulation patterns and exhibit considerable solubility differences that could result in differential accretion of these proteins within the PSVs. Here, we employed confocal fluorescent microscopy to examine the presence or absence of subdomains within the soybean PSVs. Eosin-stained sections of FAA-fixed paraffin embedded soybean seeds, when viewed by confocal fluorescence microscopy, revealed the presence of intricate subdomains within the PSVs. However, fluorescence immunolabeling studies demonstrated that the 7S and 11S globulins were evenly distributed within the PSVs and failed to corroborate the existence of subdomains within the PSVs. Similarly, confocal scanning microscopy examination of free-hand, vibratome and cryostat sections also failed to demonstrate the existence of subdomains within PSVs. The subdomains, which were prominently seen in PSVs of FAA-fixed soybean seeds, were not observed when the seeds were fixed either in glutaraldehyde/paraformaldehyde or glutaraldehyde. Our studies demonstrate that the apparent subdomains observed in FAA-fixed seeds may be a fixation artifact.

Abscisic acid activation of oleosin gene HvOle3 expression prevents the coalescence of protein storage vacuoles in barley aleurone cells.

Protein storage vacuoles (PSVs) in aleurone cells coalesce during germination, and this process is highly coupled with mobilization of PSV reserves, allowing de novo synthesis of various hydrolases in aleurone cells for endosperm degradation. Here we show that in barley (Hordeum vulgare L.) oleosins, the major integral proteins of oleosomes are encoded by four genes (HvOle1 to 4), and the expression of HvOle1 and HvOle3 is strongly up-regulated by abscisic acid (ABA), which shows antagonism to gibberellic acid. In aleurone cells, all HvOLEs were subcellularly targeted to the tonoplast of PSVs. Gain-of-function analyses revealed that HvOLE3 effectively delayed PSV coalescence, whereas HvOLE1 only had a moderate effect, with no notable effect of HvOLE2 and 4. With regard to longevity, HvOLE3 chiefly outperformed other HvOLEs, followed by HvOLE1. Experiments swapping the N- and C-terminal domain between HvOLE3 and other HvOLEs showed that the N-terminal region of HvOLE3 is mainly responsible, with some positive effect by the C-terminal region, for mediating the specific preventive effect of HvOLE3 on PSV coalescence. Three ACGT-core elements and the RY-motif were responsible for ABA induction of HvOle3 promoter activity. Transient expression assays using aleurone protoplasts demonstrated that transcriptional activation of the HvOle3 promoter was mediated by transcription factors HvABI3 and HvABI5, which acted downstream of protein kinase HvPKABA1.

Endomembrane mediated-trafficking of seed storage proteins: from Arabidopsis to cereal crops.

Seed storage proteins (SSPs) are of great importance in plant science and agriculture, particularly in cereal crops, due to their nutritional value and their impact on food properties. During seed maturation, massive amounts of SSPs are synthesized and deposited either within protein bodies derived from the endoplasmic reticulum, or into specialized protein storage vacuoles (PSVs). The processing and trafficking of SSPs vary among plant species, tissues, and even developmental stages, as well as being influenced by SSP composition. The different trafficking routes, which affect the amount of SSPs that seeds accumulate and their composition and modifications, rely on a highly dynamic and functionally specialized endomembrane system. Although the general steps in SSP trafficking have been studied in various plants, including cereals, the detailed underlying molecular and regulatory mechanisms are still elusive. In this review, we discuss the main endomembrane routes involved in SSP trafficking to the PSV in Arabidopsis and other eudicots, and compare and contrast the SSP trafficking pathways in major cereal crops, particularly in rice and maize. In addition, we explore the challenges and strategies for analyzing the endomembrane system in cereal crops.

Wetting of phase-separated droplets on plant vacuole membranes leads to a competition between tonoplast budding and nanotube formation.

Seeds of dicotyledonous plants store proteins in dedicated membrane-bounded organelles called protein storage vacuoles (PSVs). Formed during seed development through morphological and functional reconfiguration of lytic vacuoles in embryos [M. Feeney et al., Plant Physiol. 177, 241-254 (2018)], PSVs undergo division during the later stages of seed maturation. Here, we study the biophysical mechanism of PSV morphogenesis in vivo, discovering that micrometer-sized liquid droplets containing storage proteins form within the vacuolar lumen through phase separation and wet the tonoplast (vacuolar membrane). We identify distinct tonoplast shapes that arise in response to membrane wetting by droplets and derive a simple theoretical model that conceptualizes these geometries. Conditions of low membrane spontaneous curvature and moderate contact angle (i.e., wettability) favor droplet-induced membrane budding, thereby likely serving to generate multiple, physically separated PSVs in seeds. In contrast, high membrane spontaneous curvature and strong wettability promote an intricate and previously unreported membrane nanotube network that forms at the droplet interface, allowing molecule exchange between droplets and the vacuolar interior. Furthermore, our model predicts that with decreasing wettability, this nanotube structure transitions to a regime with bud and nanotube coexistence, which we confirmed in vitro. As such, we identify intracellular wetting [J. Agudo-Canalejo et al., Nature 591, 142-146 (2021)] as the mechanism underlying PSV morphogenesis and provide evidence suggesting that interconvertible membrane wetting morphologies play a role in the organization of liquid phases in cells.

The trafficking machinery of lytic and protein storage vacuoles: how much is shared and how much is distinct?

Plant cells contain two types of vacuoles, the lytic vacuole (LV) and protein storage vacuole (PSV). LVs are present in vegetative cells, whereas PSVs are found in seed cells. The physiological functions of the two types of vacuole differ. Newly synthesized proteins must be transported to these vacuoles via protein trafficking through the endomembrane system for them to function. Recently, significant advances have been made in elucidating the molecular mechanisms of protein trafficking to these organelles. Despite these advances, the relationship between the trafficking mechanisms to the LV and PSV remains unclear. Some aspects of the trafficking mechanisms are common to both types of vacuole, but certain aspects are specific to trafficking to either the LV or PSV. In this review, we summarize recent findings on the components involved in protein trafficking to both the LV and PSV and compare them to examine the extent of overlap in the trafficking mechanisms. In addition, we discuss the interconnection between the LV and PSV provided by the protein trafficking machinery and the implications for the identity of these organelles.

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting.

Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW-1 (1.67 Units g DW-1) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.

Globoids and Phytase: The Mineral Storage and Release System in Seeds.

Phytate and phytases in seeds are the subjects of numerous studies, dating back as far as the early 20th century. Most of these studies concern the anti-nutritional properties of phytate, and the prospect of alleviating the effects of phytate with phytase. As reasonable as this may be, it has led to a fragmentation of knowledge, which hampers the appreciation of the physiological system at hand. In this review, we integrate the existing knowledge on the chemistry and biosynthesis of phytate, the globoid cellular structure, and recent advances on plant phytases. We highlight that these components make up a system that serves to store and-in due time-release the seed's reserves of the mineral nutrients phosphorous, potassium, magnesium, and others, as well as inositol and protein. The central component of the system, the phytate anion, is inherently rich in phosphorous and inositol. The chemical properties of phytate enable it to sequester additional cationic nutrients. Compartmentalization and membrane transport processes regulate the buildup of phytate and its associated nutrients, resulting in globoid storage structures. We suggest, based on the current evidence, that the degradation of the globoid and the mobilization of the nutrients also depend on membrane transport processes, as well as the enzymatic action of phytase.

Effect of Heat Stress on Seed Protein Composition and Ultrastructure of Protein Storage Vacuoles in the Cotyledonary Parenchyma Cells of Soybean Genotypes That Are Either Tolerant or Sensitive to Elevated Temperatures.

High growth temperatures negatively affect soybean (Glycine max (L.) Merr) yields and seed quality. Soybean plants, heat stressed during seed development, produce seed that exhibit wrinkling, discoloration, poor seed germination, and have an increased potential for incidence of pathogen infection and an overall decrease in economic value. Soybean breeders have identified a heat stress tolerant exotic landrace genotype, which has been used in traditional hybridization to generate experimental genotypes, with improved seed yield and heat tolerance. Here, we have investigated the seed protein composition and ultrastructure of cotyledonary parenchyma cells of soybean genotypes that are either susceptible or tolerant to high growth temperatures. Biochemical analyses of seed proteins isolated from heat-tolerant and heat-sensitive genotypes produced under 28/22 °C (control), 36/24 °C (moderate), and 42/26 °C (extreme) day/night temperatures revealed that the accumulation in soybean seeds of lipoxygenase, the ß-subunit of ß-conglycinin, sucrose binding protein and Bowman-Birk protease inhibitor were negatively impacted by extreme heat stress in both genotypes, but these effects were less pronounced in the heat-tolerant genotype. Western blot analysis showed elevated accumulation of heat shock proteins (HSP70 and HSP17.6) in both lines in response to elevated temperatures during seed fill. Transmission electron microscopy showed that heat stress caused dramatic structural changes in the storage parenchyma cells. Extreme heat stress disrupted the structure and the membrane integrity of protein storage vacuoles, organelles that accumulate seed storage proteins. The detachment of the plasma membrane from the cell wall (plasmolysis) was commonly observed in the cells of the sensitive line. In contrast, these structural changes were less pronounced in the tolerant genotype, even under extreme heat stress, cells, for the most part, retained their structural integrity. The results of our study demonstrate the contrasting effects of heat stress on the seed protein composition and ultrastructural alterations that contribute to the tolerant genotype's ability to tolerate high temperatures during seed development.

Aquaporin activity of barley tonoplast intrinsic proteins is involved in the delay of the coalescence of protein storage vacuoles in aleurone cells.

The coalescence of protein storage vacuoles (PSVs) is one of the most prominent cellular changes occurring in cereal aleurone cells during germination. This structural change is highly coupled with the functional transition of this organelle from a storage compartment to a lytic section. Gibberellic acid (GA) promotes this process, whereas abscisic acid (ABA) prevents it. Previously, we demonstrated that ABA-inducible HvTIP3;1 plays a decisive role in ABA-mediated prevention of PSV fusion. In this follow-up study, we examined whether the aquaporin activity of tonoplast intrinsic protein (TIP) is related to its preventive effect on PSV fusion using various functional mutants. The defective forms of aquaporin (HvTIP3;1m and HvTIP3;1ΔNPA-GFPs for HvTIP3;1, and HvTIP1;2m for HvTIP1;2) were found to be less effective than the usual form in delaying the PSV fusion process occurring in GA-treated cells. In contrast, overexpression of HvTIP3;1m reduced the preventive effect of ABA on PSV fusion. Upon inhibition of aquaporin activity using mercury, PSV fusion occurred to a greater extent in ABA-treated barley protoplasts. These data suggest that the aquaporin activity of TIP is involved in the deterrent effect of TIP on PSV coalescence. TIP3-GFP barley transgenic seeds showed prolonged expression of the TIP3;1 transcript. Moreover, PSV fusion progressed at a much slower rate compared to wild type. Additionally, the degradation of storage proteins was not as efficient, suggesting that a metamorphic transition of PSVs to lytic organelles is closely correlated with the disappearance of HvTIPs and the PSV fusion process.

Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology.

KEY MESSAGE: Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination.

GmGPA3 is involved in post-Golgi trafficking of storage proteins and cell growth in soybean cotyledons.

As the major nutritional component in soybean seeds storage proteins are initially synthesized on the endoplasmic reticulum as precursors and subsequently delivered to protein storage vacuoles (PSVs) via the Golgi-mediated pathway where they are converted into mature subunits and accumulated. However, the molecular machinery required for storage protein trafficking in soybean remains largely unknown. In this study, we cloned the sole soybean homolog of OsGPA3 that encodes a plant-unique kelch-repeat regulator of post-Golgi vesicular traffic for rice storage protein sorting. A complementation test showed that GmGPA3 could rescue the rice gpa3 mutant. Biochemical assays verified that GmGPA3 physically interacts with GmRab5 and its guanine exchange factor (GEF) GmVPS9. Expression of GmGPA3 had no obvious effect on the GEF activity of GmVPS9 toward GmRab5a. Notably, knock-down of GmGPA3 disrupted the trafficking of mmRFP-CT10 (an artificial cargo destined for PSVs) in developing soybean cotyledons. We identified two putative GmGPA3 interacting partners (GmGMG3 and GmGMG11) by screening a yeast cDNA library. Overexpression of GmGPA3 or GmGMG3 caused shrunken cotyledon cells. Our overall results suggested that GmGPA3 plays an important role in cell growth and development, in addition to its conserved role in mediating storage protein trafficking in soybean cotyledons.

Seeds as Economical Production Platform for Recombinant Proteins.

The cost-effective production of high-quality and biologically active recombinant molecules especially proteins is extremely desirable. Seed-based recombinant protein production platforms are considered as superior choice owing to lack of human/animal pathogenic organisms, lack of cold chain requirements for transportation and long-term storage, easy scalability and development of edible biopharmaceuticals in plants with objective to be used in purified or partially processed form is desirable. This review article summarizes the exceptional features of seed-based biopharming and highlights the needs of exploiting it for commercial purposes. Plant seeds offer a perfect production platform for high-value molecules of industrial as well as therapeutic nature owing to lower water contents, high protein storage capacity, weak protease activity and long-term storage ability at ambient temperature. Exploiting extraordinarily high protein accumulation potential, vaccine antigens, antibodies and other therapeutic proteins can be stored without effecting their stability and functionality up to years in seeds. Moreover, ability of direct oral consumption and post-harvest stabilizing effect of seeds offer unique feature of oral delivery of pharmaceutical proteins and vaccine antigens for immunization and disease treatment through mucosal as well as oral route.

The small GTPase Rab5a and its guanine nucleotide exchange factors are involved in post-Golgi trafficking of storage proteins in developing soybean cotyledon.

Storage protein is the most abundant nutritional component in soybean seed. Morphology-based evidence has verified that storage proteins are initially synthesized on the endoplasmic reticulum, and then follow the Golgi-mediated pathway to the protein storage vacuole. However, the molecular mechanisms of storage protein trafficking in soybean remain unknown. Here, we clone the soybean homologs of Rab5 and its guanine nucleotide exchange factor (GEF) VPS9. GEF activity combined with yeast two-hybrid assays demonstrated that GmVPS9a2 might specifically act as the GEF of the canonical Rab5, while GmVPS9b functions as a common activator for all Rab5s. Subcellular localization experiments showed that GmRab5a was dually localized to the trans-Golgi network and pre-vacuolar compartments in developing soybean cotyledon cells. Expression of a dominant negative variant of Rab5a, or RNAi of either Rab5a or GmVPS9s, significantly disrupted trafficking of mRFP-CT10, a cargo marker for storage protein sorting, to protein storage vacuoles in maturing soybean cotyledons. Together, our results systematically revealed the important role of GmRab5a and its GEFs in storage protein trafficking, and verified the transient expression system as an efficient approach for elucidating storage protein trafficking mechanisms in seed.

A Review of Plant Vacuoles: Formation, Located Proteins, and Functions.

Vacuoles, cellular membrane-bound organelles, are the largest compartments of cells, occupying up to 90% of the volume of plant cells. Vacuoles are formed by the biosynthetic and endocytotic pathways. In plants, the vacuole is crucial for growth and development and has a variety of functions, including storage and transport, intracellular environmental stability, and response to injury. Depending on the cell type and growth conditions, the size of vacuoles is highly dynamic. Different types of cell vacuoles store different substances, such as alkaloids, protein enzymes, inorganic salts, sugars, etc., and play important roles in multiple signaling pathways. Here, we summarize vacuole formation, types, vacuole-located proteins, and functions.

[Progress in endosomal Na⁺,K⁺/H⁺ antiporter in Arabidopsis thaliana].

Important progress has been made in the interpretation of subcellular location, ion transport characteristics and biological functions of endosomal Na⁺,K⁺/H⁺ antiporter in Arabidopsis thaliana. The endosomal Na⁺,K⁺/H⁺ antiporter contain two members, AtNHX5 and AtNHX6, whose amino acid sequence similarity is 78.7%. Studies have shown that AtNHX5 and AtNHX6 are functionally redundant, and they are all located in Golgi, trans-Golgi network (TGN), endoplasmic reticulum (ER) and prevacuolar compartment (PVC). AtNHX5 and AtNHX6 are critical for salt tolerance stress and the homeostasis of pH and K⁺. It has been reported that there are conservative acidic amino acid residues that can regulate their ion activity in the endosomal NHXs transmembrane domain, which plays a decisive role in their own functions. The results of the latest research indicate that endosomal NHXs affect vacuolar transport and protein storage, and participate in the growth of auxin-mediated development in A. thaliana. In this paper, the progress of subcellular localization, ion transport, function and application of endosomal NHXs in A. thaliana was summarized.

Impact of co-expression of maize 11 and 18 kDa δ-zeins and 27 kDa γ-zein in transgenic soybeans on protein body structure and sulfur amino acid content.

The methionine-rich seed storage proteins of maize have been expressed in transgenic plants as a means to improve the overall sulfur amino acid content of seed. Previous attempts to increase the sulfur amino acid content of soybean seeds by this approach has met with limited success. It has been shown co-expression of different class of zeins can result in their stable accumulation in transgenic plants. In this study, conventional crosses between transgenic plants individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein were made to obtain plants that simultaneously express both the δ-zein and γ-zein. Transmission electron microscopic observation of thin-sections of transgenic soybean seeds revealed that the zeins accumulated in ER-derived protein bodies (PBs) which were found sparsely scattered in cytoplasm. The size of these PBs varied from 0.2 to 0.6 µm in soybean plants individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein. In contrast, soybeans co-expressing the 18 kDa δ-zein and 27 kDa γ-zein the PBs was 3-4 times larger. Electron microscopic observation also revealed the sequestration of PBs inside the vacuoles where they could be subjected to degradation by vacuolar proteases. Amino acid analysis of transgenic soybean individually expressing 11, 18 kDa δ-zeins and 27 kDa γ-zein revealed only a minimal increase in the overall methionine content compared to the wild-type. In contrast, plants co-expressing 18 kDa δ-zein and 27 kDa γ-zein showed a significant increase (27%) in the methionine content compared to the control seeds.