Imaging the ER and Endomembrane System in Cereal Endosperm.

The cereal endosperm is a complex structure comprising distinct cell types, characterized by specialized organelles for the accumulation of storage proteins. Protein trafficking in these cells is complicated by the presence of several different storage organelles including protein bodies (PBs) derived from the endoplasmic reticulum (ER) and dynamic protein storage vacuoles (PSVs). In addition, trafficking may follow a number of different routes depending on developmental stage, showing that the endomembrane system is capable of massive reorganization. Thus, developmental sequences involve progressive changes of the endomembrane system of endosperm tissue and are characterized by a high structural plasticity and endosomal activity.Given the technical dexterity required to access endosperm tissue and study subcellular structures and SSP trafficking in cereal seeds, static images are the state of the art providing a bulk of information concerning the cellular composition of seed tissue. In view of the highly dynamic endomembrane system in cereal endosperm cells, it is reasonable to expect that live cell imaging will help to characterize the spatial and temporal changes of the endomembrane system. The high resolution achieved with electron microscopy perfectly complements the live cell imaging.We therefore established an imaging platform for TEM as well as for live cell imaging. Here, we describe the preparation of different cereal seed tissues for live cell imaging concomitant with immunolocalization studies and ultrastructure.

A bench-top Dark-Root device built with LEGO® bricks enables a non-invasive plant root development analysis in soil conditions mirroring nature.

Roots are the hidden parts of plants, anchoring their above-ground counterparts in the soil. They are responsible for water and nutrient uptake and for interacting with biotic and abiotic factors in the soil. The root system architecture (RSA) and its plasticity are crucial for resource acquisition and consequently correlate with plant performance while being highly dependent on the surrounding environment, such as soil properties and therefore environmental conditions. Thus, especially for crop plants and regarding agricultural challenges, it is essential to perform molecular and phenotypic analyses of the root system under conditions as near as possible to nature (#asnearaspossibletonature). To prevent root illumination during experimental procedures, which would heavily affect root development, Dark-Root (D-Root) devices (DRDs) have been developed. In this article, we describe the construction and different applications of a sustainable, affordable, flexible, and easy to assemble open-hardware bench-top LEGO® DRD, the DRD-BIBLOX (Brick Black Box). The DRD-BIBLOX consists of one or more 3D-printed rhizoboxes, which can be filled with soil while still providing root visibility. The rhizoboxes sit in a scaffold of secondhand LEGO® bricks, which allows root development in the dark and non-invasive root tracking with an infrared (IR) camera and an IR light-emitting diode (LED) cluster. Proteomic analyses confirmed significant effects of root illumination on barley root and shoot proteomes. Additionally, we confirmed the significant effect of root illumination on barley root and shoot phenotypes. Our data therefore reinforces the importance of the application of field conditions in the lab and the value of our novel device, the DRD-BIBLOX. We further provide a DRD-BIBLOX application spectrum, spanning from investigating a variety of plant species and soil conditions and simulating different environmental conditions and stresses, to proteomic and phenotypic analyses, including early root tracking in the dark.

Identification of apple cultivars hypoallergenic for birch pollen-allergic individuals by a multidisciplinary in vitro and in vivo approach.

BACKGROUND: Birch pollen-related apple allergy is the most frequent IgE-mediated food allergy in Central-Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year-round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long-term storage and by clinical challenges of allergic individuals before and after the birch pollen season. METHODS: Proteins were extracted from fruits of 'Santana', 'Golden Delicious' (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1-levels were analysed by mass spectrometry, SDS-PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty-eight allergic individuals underwent single-blinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen-specific IgE-levels were determined. RESULTS: After storage all cultivars except 'Santana' were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen-specific IgE-levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing. CONCLUSIONS: Combined expertise in pomology and allergology identified promising new cultivars for allergic consumers. The evaluation of hypoallergenic apples should incorporate long-term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

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.

ESCRTing in cereals: still a long way to go.

The multivesicular body (MVB) sorting pathway provides a mechanism for the delivery of cargo destined for degradation to the vacuole or lysosome. The endosomal sorting complex required for transport (ESCRT) is essential for the MVB sorting pathway by driving the cargo sorting to its destination. Many efforts in plant research have identified the ESCRT machinery and functionally characterised the first plant ESCRT proteins. However, most studies have been performed in the model plant Arabidopsis thaliana that is genetically and physiologically different to crops. Cereal crops are important for animal feed and human nutrition and have further been utilized as promising candidates for recombinant protein production. In this review, I summarize the role of plant ESCRT components in cereals that are involved in efficient adaptation to environmental stress and grain development. A special focus is on barley (Hordeum vulgare L.) ESCRT proteins, where recent studies show their quantitative mapping during grain development, e.g. associating HvSNF7.1 with protein trafficking to protein bodies (PBs) in starchy endosperm. Thus, it is indispensable to identify the molecular key-players within the endomembrane system including ESCRT proteins to optimize and possibly enhance tolerance to environmental stress, grain yield and recombinant protein production in cereal grains.

Russell-Like Bodies in Plant Seeds Share Common Features With Prolamin Bodies and Occur Upon Recombinant Protein Production.

Although many recombinant proteins have been produced in seeds at high yields without adverse effects on the plant, endoplasmic reticulum (ER) stress and aberrant localization of endogenous or recombinant proteins have also been reported. The production of murine interleukin-10 (mIL-10) in Arabidopsis thaliana seeds resulted in the de novo formation of ER-derived structures containing a large fraction of the recombinant protein in an insoluble form. These bodies containing mIL-10 were morphologically similar to Russell bodies found in mammalian cells. We confirmed that the compartment containing mIL-10 was enclosed by ER membranes, and 3D electron microscopy revealed that these structures have a spheroidal shape. Another feature shared with Russell bodies is the continued viability of the cells that generate these organelles. To investigate similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds, we crossed plants containing ectopic ER-derived prolamin protein bodies with a line accumulating mIL-10 in Russell-like bodies. This resulted in seeds containing only one population of protein bodies in which mIL-10 inclusions formed a central core surrounded by the prolamin-containing matrix, suggesting that both types of protein aggregates are together removed from the secretory pathway by a common mechanism. We propose that, like mammalian cells, plant cells are able to form Russell-like bodies as a self-protection mechanism, when they are overloaded with a partially transport-incompetent protein, and we discuss the resulting challenges for recombinant protein production.

Microscopic and Proteomic Analysis of Dissected Developing Barley Endosperm Layers Reveals the Starchy Endosperm as Prominent Storage Tissue for ER-Derived Hordeins Alongside the Accumulation of Barley Protein Disulfide Isomerase (HvPDIL1-1).

Barley (Hordeum vulgare) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is characterized by a high accumulation of seed storage proteins (SSPs); and the aleurone, which has a prominent role during seed germination. Prolamins accumulate in distinct, ER-derived protein bodies (PBs) and their trafficking route is spatio-temporally regulated. The protein disulfide isomerase (PDI) has been shown to be involved in PB formation. Here, we unravel the spatio-temporal proteome regulation in barley aleurone, subaleurone, and starchy endosperm for the optimization of end-product quality in barley. We used laser microdissection (LMD) for subsequent nanoLC-MS/MS proteomic analyses in two experiments: in Experiment One, we investigated the proteomes of dissected barley endosperm layers at 12 and at ≥20 days after pollination (DAP). We found a set of 10 proteins that were present in all tissues at both time points. Among these proteins, the relative protein abundance of D-hordein, B3-hordein and HvPDIL1-1 significantly increased in starchy endosperm between 12 and ≥20 DAP, identifying the starchy endosperm as putative major storage tissue. In Experiment Two, we specifically compared the starchy endosperm proteome at 6, 12, and ≥20 DAP. Whereas the relative protein abundance of D-hordein and B3-hordein increased between 6 and ≥20 DAP, HvPDIL1-1 increased between 6 and 12 DAP, but remained constant at ≥20 DAP. Microscopic observations showed that these relative protein abundance alterations were accompanied by additional localization of hordeins at the periphery of starch granules and a partial re-localization of HvPDIL1-1 from PBs to the periphery of starch granules. Our data indicate a spatio-temporal regulation of hordeins and HvPDIL1-1. These results are discussed in relation to the putative role of HvPDIL1-1 in end-product quality in barley.

Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.

Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater.

Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins, and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence of the variable regions using the peptide mass-assisted cloning strategy and expressed a scFv (single-chain variable fragment) format of this antibody in yeast and a chimeric full-size version in leaves of Nicotiana tabacum and Nicotiana benthamiana to facilitate inexpensive and scalable production. The specific antigen-binding activity of the purified antibody was verified by surface plasmon resonance spectroscopy and ELISA, confirming the same binding specificity as its hybridoma-derived counterpart. The plant-derived antibody was used to design a lateral flow immunoassay (dipstick) for the sensitive detection of [Arg4]-microcystins at concentrations of 100-300 ng/L in freshwater samples collected at different sites. Plant-based production will likely reduce the cost of the antibody, currently the most expensive component of the dipstick immunoassay, and will allow the development of further antibody-based analytical devices and water purification adsorbents for the efficient removal of toxic contaminants.

Imaging the ER and Endomembrane System in Cereal Endosperm.

The cereal endosperm is a complex structure comprising distinct cell types, characterized by specialized organelles for the accumulation of storage proteins. Protein trafficking in these cells is complicated by the presence of several different storage organelles including protein bodies (PBs) derived from the endoplasmic reticulum (ER) and dynamic protein storage vacuoles (PSVs). In addition, trafficking may follow a number of different routes depending on developmental stage, showing that the endomembrane system is capable of massive reorganization. Thus, developmental sequences involve progressive changes of the endomembrane system of endosperm tissue and are characterized by a high structural plasticity and endosomal activity.Given the technical dexterity required to access endosperm tissue and study subcellular structures and (seed storage protein) SSP trafficking in cereal seeds, static images are the state of the art providing a bulk of information concerning the cellular composition of seed tissue. In view of the highly dynamic endomembrane system in cereal endosperm cells, it is reasonable to expect that live cell imaging will help to characterize the spatial and temporal changes of the system. The high resolution achieved with electron microscopy perfectly complements the live cell imaging.We therefore established an imaging platform for TEM as well as for live cell imaging. Here, we describe the preparation of different cereal seed tissues for live cell imaging concomitant with immunolocalization studies and ultrastructure.

Cell layer-specific distribution of transiently expressed barley ESCRT-III component HvVPS60 in developing barley endosperm.

The significance of the endosomal sorting complexes required for transport (ESCRT)-III in cereal endosperm has been shown by the identification of the recessive mutant supernumerary aleurone layer1 (SAL1) in maize. ESCRT-III is indispensable in the final membrane fission step during biogenesis of multivesicular bodies (MVBs), responsible for protein sorting to vacuoles and to the cell surface. Here, we annotated barley ESCRT-III members in the (model) crop Hordeum vulgare and show that all identified members are expressed in developing barley endosperm. We used fluorescently tagged core ESCRT-III members HvSNF7a/CHMP4 and HvVPS24/CHMP3 and the associated ESCRT-III component HvVPS60a/CHMP5 for transient localization studies in barley endosperm. In vivo confocal microscopic analyses show that the localization of recombinantly expressed HvSNF7a, HvVPS24 and HvVPS60a differs within barley endosperm. Whereas HvSNF7a induces large agglomerations, HvVPS24 shows mainly cytosolic localization in aleurone and subaleurone. In contrast, HvVPS60a localizes strongly at the plasma membrane in aleurone. In subaleurone, HvVPS60a was found to a lesser extent at the plasma membrane and at vacuolar membranes. These results indicate that the steady-state association of ESCRT-III may be influenced by cell layer-specific protein deposition or trafficking and remodelling of the endomembrane system in endosperm. We show that sorting of an artificially mono-ubiquitinated Arabidopsis plasma membrane protein is inhibited by HvVPS60a in aleurone. The involvement of HvVPS60a in different cell layer-specific trafficking pathways, reflected by localization of HvVPS60a at the plasma membrane in aleurone and at the PSV membrane in subaleurone, is discussed.

The dynamic behavior of storage organelles in developing cereal seeds and its impact on the production of recombinant proteins.

Cereal endosperm is a highly differentiated tissue containing specialized organelles for the accumulation of storage proteins, which are ultimately deposited either within protein bodies derived from the endoplasmic reticulum, or in protein storage vacuoles (PSVs). During seed maturation endosperm cells undergo a rapid sequence of developmental changes, including extensive reorganization and rearrangement of the endomembrane system and protein transport via several developmentally regulated trafficking routes. Storage organelles have been characterized in great detail by the histochemical analysis of fixed immature tissue samples. More recently, in vivo imaging and the use of tonoplast markers and fluorescent organelle tracers have provided further insight into the dynamic morphology of PSVs in different cell layers of the developing endosperm. This is relevant for biotechnological applications in the area of molecular farming because seed storage organelles in different cereal crops offer alternative subcellular destinations for the deposition of recombinant proteins that can reduce proteolytic degradation, allow control over glycan structures and increase the efficacy of oral delivery. We discuss how the specialized architecture and developmental changes of the endomembrane system in endosperm cells may influence the subcellular fate and post-translational modification of recombinant glycoproteins in different cereal species.

Fusion, rupture, and degeneration: the fate of in vivo-labelled PSVs in developing barley endosperm.

Cereal endosperm is a highly differentiated tissue containing specialized organelles for the accumulation of storage proteins. The endosperm of barley contains hordeins, which are ultimately deposited within protein storage vacuoles (PSVs). These organelles have been characterized predominantly by the histochemical analysis of fixed immature tissue samples. However, little is known about the fate of PSVs during barley endosperm development, and in vivo imaging has not been attempted in order to gain further insight. In this report, young seeds were followed through development to characterize the dynamic morphology of PSVs from aleurone, subaleurone, and central starchy endosperm cells. TIP3-GFP was used as a PSV membrane marker and several fluorescent tracers were used to identify membranes and monitor endomembrane organelles in real time. Whereas the spherical appearance of strongly labelled TIP3-GFP PSVs in the aleurone remained constant, those in the subaleurone and central starchy endosperm underwent substantial morphological changes. Fusion and rupture events were observed in the subaleurone, and internal membranes derived from both the tonoplast and endoplasmic reticulum were identified within these PSVs. TIP3-GFP-labelled PSVs in the starchy endosperm cells underwent a dramatic reduction in size, so that finally the protein bodies were tightly enclosed. Potential desiccation-related membrane-altering processes that may be causally linked to these dynamic endomembrane events in the barley endosperm are discussed.

Live Cell Imaging During Germination Reveals Dynamic Tubular Structures Derived from Protein Storage Vacuoles of Barley Aleurone Cells.

The germination of cereal seeds is a rapid developmental process in which the endomembrane system undergoes a series of dynamic morphological changes to mobilize storage compounds. The changing ultrastructure of protein storage vacuoles (PSVs) in the cells of the aleurone layer has been investigated in the past, but generally this involved inferences drawn from static pictures representing different developmental stages. We used live cell imaging in transgenic barley plants expressing a TIP3-GFP fusion protein as a fluorescent PSV marker to follow in real time the spatially and temporally regulated remodeling and reshaping of PSVs during germination. During late-stage germination, we observed thin, tubular structures extending from PSVs in an actin-dependent manner. No extensions were detected following the disruption of actin microfilaments, while microtubules did not appear to be involved in the process. The previously-undetected tubular PSV structures were characterized by complex movements, fusion events and a dynamic morphology. Their function during germination remains unknown, but might be related to the transport of solutes and metabolites.

The formation, function and fate of protein storage compartments in seeds.

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.

Interactome of the plant-specific ESCRT-III component AtVPS2.2 in Arabidopsis thaliana.

The endosomal sorting complexes required for transport (ESCRT) guides transmembrane proteins to domains that bud away from the cytoplasm. The ESCRT machinery consists of four complexes. ESCRT complexes 0-II are important for cargo recognition and concentration via ubiquitin binding. Most of the membrane bending function is mediated by the large multimeric ESCRT-III complex and associated proteins. Here we present the first in vivo proteome analysis of a member of the ESCRT-III complex which is unique to the plant kingdom. We show with LC-MS/MS, yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) that coimmunoprecipitated proteins from Arabidopsis thaliana roots expressing a functional GFP-tagged VACUOLAR PROTEIN SORTING 2.2 (AtVPS2.2) protein are members of the ESCRT-III complex and associated proteins. Therefore we propose that at least in plants the large ESCRT-III membrane scaffolding complex consists of a mixture of SNF7, VPS2 and the associated VPS46 and VPS60 proteins. Apart from transmembrane proteins, numerous membrane-associated but also nuclear and extracellular proteins have been identified, indicating that AtVPS2.2 might be involved in processes beyond the classical ESCRT role. This study is the first in vivo proteome analysis with a tagged ESCRT-III component demonstrating the feasibility of this approach and provides numerous starting points for the investigation of the biological process in which AtVPS2.2 is involved.