House dust mites (HDM) are the most common source of indoor allergens and are associated with allergic diseases worldwide. To benefit allergic patients, safer and non-invasive mucosal routes of oral administration are considered to be the best alternative to conventional allergen-specific immunotherapy. In this study, transgenic rice was developed expressing derivatives of the major HDM allergen Der f 2 with reduced Der f 2-specific IgE reactivity by disrupting intramolecular disulphide bonds in Der f 2. These derivatives were produced specifically as secretory proteins in the endosperm tissue of seeds under the control of the endosperm-specific glutelin GluB-1 promoter. Notably, modified Der f 2 derivatives aggregated in the endoplasmic reticulum (ER) lumen and were deposited in a unique protein body (PB)-like structure tentatively called the Der f 2 body. Der f 2 bodies were characterized by their intracellular localization and physico-chemical properties, and were distinct from ER-derived PBs (PB-Is) and protein storage vacuoles (PB-IIs). Unlike ER-derived organelles such as PB-Is, Der f 2 bodies were rapidly digested in simulated gastric fluid in a manner similar to that of PB-IIs. Oral administration in mice of transgenic rice seeds containing Der f 2 derivatives encapsulated in Der f 2 bodies suppressed Der f 2-specific IgE and IgG production compared with that in mice fed non-transgenic rice seeds, and the effect was dependent on the type of Der f 2 derivative expressed. These results suggest that engineered hypoallergenic Der f 2 derivatives expressed in the rice seed endosperm could serve as a basis for the development of viable strategies for the oral delivery of vaccines against HDM allergy.

Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP 'core'. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions.

Seed storage proteins (SSPs) have been studied for more than 250 years because of their nutritional value and their impact on the use of grain in food processing. More recently, the use of seeds for the production of recombinant proteins has rekindled interest in the behavior of SSPs and the question how they are able to accumulate as stable storage reserves. Seed cells produce vast amounts of SSPs with different subcellular destinations creating an enormous logistic challenge for the endomembrane system. Seed cells contain several different storage organelles including the complex and dynamic protein storage vacuoles (PSVs) and other protein bodies (PBs) derived from the endoplasmic reticulum (ER). Storage proteins destined for the PSV may pass through or bypass the Golgi, using different vesicles that follow different routes through the cell. In addition, trafficking may depend on the plant species, tissue and developmental stage, showing that the endomembrane system is capable of massive reorganization. Some SSPs contain sorting signals or interact with membranes or with other proteins en route in order to reach their destination. The ability of SSPs to form aggregates is particularly important in the formation or ER-derived PBs, a mechanism that occurs naturally in response to overloading with proteins that cannot be transported and that can be used to induce artificial storage bodies in vegetative tissues. In this review, we summarize recent findings that provide insight into the formation, function, and fate of storage organelles and describe tools that can be used to study them.

Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.

In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS(W)) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS(M)-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.

Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz).(Montagnac JA, Davis CR, Tanumihardjo SA.(2009) Compr Rev Food Sci Food Saf 8:181-194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Cereal seeds provide an ideal production platform for high-value products such as pharmaceuticals and industrial materials because seeds have ample and stable space for the deposition of recombinant products without loss of activity at room. Seed storage proteins (SSPs) are predominantly synthesized and stably accumulated in maturing endosperm tissue. Therefore, understanding the molecular mechanisms regulating SSP expression and accumulation is expected to provide valuable information for producing higher amounts of recombinant products. SSP levels are regulated by several steps at the transcriptional (promoters, transcription factors), translational and post-translational levels (modification, processing trafficking, and deposition). Our objective is to develop a seed production platform capable of producing very high yields of recombinant product. Towards this goal, we review here the individual regulatory steps controlling SSP synthesis and accumulation.

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

Plants are replete with thousands of proteins and small molecules, many of which are species-specific, poisonous or dangerous. Over time humans have learned to avoid dangerous plants or inactivate many toxic components in food plants, but there is still room for ameliorating food crops (and plants in general) in terms of their allergens and toxins content, especially in their edible parts. Inactivation at the genetic rather than physical or chemical level has many advantages and classical genetic approaches have resulted in significant reduction of toxin content. The capacity, offered by genetic engineering, of turning off (inactivating) specific genes has opened up the possibility of altering the plant content in a far more precise manner than previously available. Different levels of intervention (genes coding for toxins/allergens or for enzymes, transporters or regulators involved in their metabolism) are possible and there are several tools for inactivating genes, both direct (using chemical and physical mutagens, insertion of transposons and other genetic elements) and indirect (antisense RNA, RNA interference, microRNA, eventually leading to gene silencing). Each level/strategy has specific advantages and disadvantages (speed, costs, selectivity, stability, reversibility, frequency of desired genotype and regulatory regime). Paradigmatic examples from classical and transgenic approaches are discussed to emphasize the need to revise the present regulatory process. Reducing the content of natural toxins is a trade-off process: the lesser the content of natural toxins, the higher the susceptibility of a plant to pests and therefore the stronger the need to protect plants. As a consequence, more specific pesticides like Bt are needed to substitute for general pesticides.

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum (ER). Through interactions with chaperones such as the binding protein (BiP) and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein gamma-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.

In spite of a huge number of reports on successful foreign protein production in plastid, in many cases the expression level of other recombinant proteins in the transplastomic plants appears to be very low or even undetectable. We believe that this is mainly due to our limited knowledge of the mechanisms in plastids influencing the maintenance of protein homeostasis, or proteostasis. Plastids retain a whole series of mechanisms for the preservation of their protein balance, including specific proteases, transcriptional and translational control, as well as molecular chaperones and enzymes useful in protein folding. Therefore, it is important to develop basic studies in factors regulating protein synthesis, stability, folding, targeting, and accumulation in plastids, including the protein quality control which contributes to the functional integrity of proteins. Our intention here is not to provide a comprehensive review of the mechanisms that regulate plastid proteostasis, but to discuss some recent insights into this field which might bring beneficial applications in plastid biotechnology for transgene expression and foreign protein accumulation.

Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.

Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.

It is very important for the application of chloroplast engineering to extend the range of species in which this technology can be achieved. Here, we describe the development of a chloroplast transformation system for the sugar beet (Beta vulgaris L. ssp. vulgaris, Sugar Beet Group) by biolistic bombardment of leaf petioles. Homoplasmic plastid-transformed plants of breeding line Z025 were obtained. Transformation was achieved using a vector that targets genes to the rrn16/rps12 intergenic region of the sugar beet plastome, employing the aadA gene as a selectable marker against spectinomycin and the gfp gene for visual screening of plastid transformants. gfp gene transcription and protein expression were shown in transplastomic plants. Detection of GFP in Comassie blue-stained gels suggested high GFP levels. Microscopy revealed GFP fluorescence within the chloroplasts. Our results demonstrate the feasibility of engineering the sugar beet chloroplast genome; this technology provides new opportunities for the genetic improvement of this crop and for social acceptance of genetically modified sugar beet plants.

Several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, it has been shown that a portion of a maize prolamin storage protein, gamma-zein, can be used for the high accumulation of a recombinant protein in novel endoplasmic reticulum (ER)-derived protein bodies of vegetative tissues. In this study, we investigate whether this protein can be expressed in the chloroplast. Our long-term purpose is to use zeolin to produce value-added proteins by fusing these polypeptides with its gamma-zein portion and targeting the recombinant proteins to the ER or to the chloroplast. We show here that zeolin accumulates in the chloroplast to lower levels than in the ER and its stability is compromised by chloroplast proteolytic activity. Co-localization of zeolin and the ER chaperone BiP in the chloroplast does not have a beneficial effect on zeolin accumulation. In this organelle, zeolin is not stored in protein bodies, nor do zeolin polypeptides seem to be linked by inter-chain disulfide bonds, which are usually formed by the six cysteine of the gamma-zein portion, indicating abnormal folding of the recombinant protein. Therefore, it is concluded that to accumulate zeolin in the chloroplast it is necessary to facilitate inter-chain disulfide bond formation.

Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.

Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70-interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture.

Angiosperm stigma supports compatible pollen germination and tube growth, resulting in fertilization and seed production. Stigmas are mainly divided into two types, dry and wet, according to the absence or presence of exudates on their surfaces. Here, we used 2-DE and mass spectrometry to identify proteins specifically and preferentially expressed in the stigmas of maize (Zea Mays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed from the surface of the tobacco stigma. We found that the specifically and preferentially expressed proteins in maize and tobacco stigmas share similar distributions in functional categories. However, these proteins showed important difference between dry and wet stigmas in a few aspects, such as protein homology in "signal transduction" and "lipid metabolism", relative expression levels of proteins containing signal peptides, and proteins in "defense and stress response". These different features might be related to the specific structures and functions of dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed. Our results provide important information on functions of proteins in dry and wet stigmas and reveal aspects of conservation and divergence between dry and wet stigmas at the proteomic level.

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

BACKGROUND The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles. RESULTS We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer. CONCLUSIONS This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.

Zeins are the major storage proteins in maize (Zea mays) endosperm, and their accumulation in zein bodies derived from the endoplasmic reticulum is well characterized. In contrast, relatively little is known about post-Golgi compartments or the trafficking of vacuolar proteins in maize endosperm, specifically the presence of globulins in structures resembling protein storage vacuoles that appear in early to mid-stage seed development. We investigated this pathway by expressing and analyzing a recombinant reporter glycoprotein during endosperm maturation, using a combination of microscopy and sensitive glycopeptide analysis. Specific N-glycan acceptor sites on the protein were followed through the stages of grain development, revealing a shift from predominantly paucimannosidic vacuolar glycoforms to predominantly trimmed glycan structures lacking fucose. This was accompanied by a change in the main subcellular localization of the protein from large protein storage vacuole-like post-Golgi organelles to the endoplasmic reticulum and zein bodies. The endogenous storage proteins corn alpha-globulin and corn legumin-1 showed a similar spatiotemporal profile both in transgenic plants expressing the reporter glycoprotein and in wild-type plants. This indicates that the shift of the intracellular trafficking route, as observed with our reporter glycoprotein, may be a common strategy in maize seed development.

Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell type differentiation under in vitro conditions (Gruis et al. 2006). We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short co-cultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms interchain disulfide bonds that facilitate the formation of ER-located protein bodies. Wild-type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C-terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Delta 418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild-type protein. Moreover, Delta 418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.

Stable accumulation of storage proteins, lipids and carbohydrates is a hallmark of the plant seed, and is a characteristic that is typically deficient in existing platforms for recombinant protein manufacture. One of the biological sequestration mechanisms that facilitate the folding, assembly and stabilization of plant seed storage proteins involve the de novo formation of unique intracellular organelles, the endoplasmic reticulum (ER)-derived protein bodies (PBs). In cereals, such as maize, PBs are formed directly in the lumen of the ER of endosperm cells and contain zeins, a group of polypeptides, which account for more than half of the total seed protein mass. The 27 kD gamma zein protein localizes to the periphery of the PBs surrounding aggregates of other zeins (including a zein and delta zein). Heterologous expression of gamma zein has been shown to result in the formation of PB-like structures, and the N-terminal proline-rich domain of gamma zein (Zera), containing eight PPPVHL repeats and a Pro-X sequence is by itself capable of directing ER retention and PB formation in non-seed tissues. We present a novel approach to produce recombinant proteins in plants based on the ability of gamma zein-Zera domain to store recombinant proteins inside PBs. Zera domain fused to several proteins, including a enhanced cyan fluorescent protein (ECFP), calcitonin (Ct) and epidermal growth factor (EGF), were cloned into vectors for transient or stable transformation of tobacco plants. In tobacco leaves, we observed the formation of dense, ER-localized structures containing high concentrations of the respective target proteins. The intact synthetic organelles containing Zera fusions were readily isolated from cellular material using density-based separation methods.

ABSTRACT: BACKGROUND: Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein g zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells. RESULTS: Various Zera fusions with fluorescent and therapeutic proteins accumulate in induced PB-like organelles in all eukaryotic systems tested: tobacco leaves, Trichoderma reesei, several mammalian cultured cells and Sf9 insect cells. This accumulation in membranous organelles insulates both recombinant protein and host from undesirable activities of either. Recombinant protein encapsulation in these PBs facilitates stable accumulation of proteins in a protected sub-cellular compartment which results in an enhancement of protein production without affecting the viability and development of stably transformed hosts. The induced PBs also retain the high-density properties of native seed PBs which facilitate the recovery and purification of the recombinant proteins they contain. CONCLUSIONS: The Zera sequence provides an efficient and universal means to produce recombinant proteins by accumulation in ER-derived organelles. The remarkable cross-kingdom conservation of PB formation and their biophysical properties should have broad application in the manufacture of non-secreted recombinant proteins and suggests the existence of universal ER pathways for protein insulation.