Small Protein Bodies Research Articles

Mecanismos asociados con la modificación del endospermo en maíz de calidad proteínica

Protein quality maize (QPM) combines the protein quality of the opaque-2 (o2) mutant with a vitreous endosperm. These characteristics have allowed breeding programs worldwide to produce QPM genotypes that help alleviate malnutrition of people in developing countries from Africa, Asia and Latin America with a cereal-based diet. However, the development of these materials has been inefficient due to the limited knowledge about the molecular basis of the conversion of the soft o2 endosperm into a vitreous phenotype in QPM. This conversion has been associated with an increase in small protein bodies rich in 27 kDa γ-zein, the synthesis of starch with a higher proportion of amylose and short-intermediate amylopectin chain branches that favors the compaction of the starch granules, as well as alterations in the amyloplast envelope that favors the interaction between starch granules and protein bodies. Additional studies about the mechanisms involved in the modification of the endosperm in QPM will contribute to produce materials with good agronomic characteristics and protein quality.

High molecular weight glutenin subunit (HMW-GS) 1Dx5 is concentrated in small protein bodies when overexpressed in wheat starchy endosperm

The high molecular weight glutenin subunits (HMW-GSs) are key determinants of the processing properties of wheat and over-expression of HMW-GS by transgenesis has therefore been widely studied as a strategy to improve grain quality. Two routes of protein body (PB) formation operate in the developing endosperm, with glutenin-rich PBs being formed by direct accumulation within the lumen of the ER and most gliadins being transported via the Golgi apparatus and deposited within the vacuole. However, nothing is known about the effects of HMW-GS over-expression on the operation of these routes. In this study, light microscopy was used to determine the morphology and distribution of protein bodies in developing and mature grains of transgenic wheat with high over-expression of HMW-GS 1Dx5. Many small ER-derived PBs were observed in the cytoplasm of endosperm cells in the transgenic line, but very few in control grains. Immunofluorescence microscopy showed that these smaller PBs contained mainly HMW-GSs. Image analysis confirmed that the number of the smallest PBs was significantly higher in sub-aleurone cells. The segregation of the HMW-GS in ER-derived PBs may affect grain processing quality as over-expressions of HMW-GS 1Dx5 is known to result in the formation of insoluble polymers and an over-strong dough phenotype.

The dynamics of protein body formation in developing wheat grain

SummaryWheat is a major source of protein in the diets of humans and livestock but we know little about the mechanisms that determine the patterns of protein synthesis in the developing endosperm. We have used a combination of enrichment with 15N glutamine and NanoSIMS imaging to establish that the substrate required for protein synthesis is transported radially from its point of entrance in the endosperm cavity across the starchy endosperm tissues, before becoming concentrated in the cells immediately below the aleurone layer. This transport occurs continuously during grain development but may be slower in the later stages. Although older starchy endosperm cells tend to contain larger protein deposits formed by the fusion of small protein bodies, small highly enriched protein bodies may also be present in the same cells. This shows a continuous process of protein body initiation, in both older and younger starchy endosperm cells and in all regions of the tissue. Immunolabeling with specific antibodies shows that the patterns of enrichment are not related to the contents of gluten proteins in the protein bodies. In addition to providing new information on the dynamics of protein deposition, the study demonstrates the wider utility of NanoSIMS and isotope labelling for studying complex developmental processes in plant tissues.

The influence of nitrogen on the development and accumulation of protein bodies in the developing endosperm of wheat caryopses

The aim of the present work was to reveal the histological changes of protein bodies (PBs) in the developing wheat endosperm under nitrogen (N) treatment. For this purpose, the development and accumulation of PBs in the dorsal and ventral regions of wheat endosperm affected by N application at booting stage were investigated using light microscopy and Image-Pro Plus 6.0 software. The endosperm without N treatment contained many smaller PBs that were scattered in endosperm cells in an unordered pattern, whereas the endosperm with N treatment contained many larger PBs or aggregations that were concentrated in a certain region of endosperm cells. The amount and relative areas of PBs in wheat varieties cvs. Xumai 30 and Yangmai 13 were significantly increased by N application. However, the cultivars differed with the degree of response to N being cv. Xumai 30>cv. Yangmai 13. These differences also varied with position in the endosperm in the order ventral>dorsal region. The initiation of PBs occurred 3days earlier in N-treated endosperm than the control.

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins.

Sugar levels altered by ectopic expression of a yeast-derived invertase affect cellular differentiation of developing cotyledons of Vicia narbonensis L.

In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in cotyledons of Vicia narbonensis. As a result, sucrose decreased whereas hexoses accumulated. We analysed cell structure and cellular differentiation in cotyledons expressing the yeast-invertase. Transgenic cells contained large and long-persisting vacuoles apparently serving as storage compartments for hexoses and clusters of storage-protein aggregates. In the wild-type, large vacuoles did not persist but were replaced by smaller protein bodies. During maturation and desiccation, the transgenic cells showed plasmolysis and vesiculation of the endo-membrane system. Immunogold-labelling revealed that the storage proteins vicilin and legumin were present within the cytoplasm and the extraprotoplasmic space and were attached to membranes of the endoplasmic reticulum and the nuclei. Protein storage vacuoles in mature seeds appeared heterogeneous and only partially filled. The data suggest that sugars control the subcellular organisation of the vacuolar system. Transcript levels encoding a tonoplast intrinsic protein, a marker for membranes of protein storage vacuoles, remained unchanged whereas mRNA levels of a hexose and a sucrose transporter increased. Generally, transgenic seeds appeared to be physiologically younger than wild-type seeds of the same age. The data underline the important role of sugars in legume seed development.

Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4

Embryogenic units of friable maize callus are formed as globular or oblong packets of tightly associated meristematic cells. These units are surrounded by conspicuous cell walls visible in light microscopy after staining with basic fuchsin. Transmission electron microscopy revealed that embryogenic cells are rich in endoplasmic reticulum, polysomes and small protein bodies, and that the outermost layer of their cell walls is composed of fibrillar material. Electron microscopy has also shown that this material covers the surface of embryogenic cells as a distinct layer which we denote as extracellular matrix surface network (ECMSN). Employing histochemical staining with β-glucosyl Yariv phenylglycoside, we localized arabinogalactan-proteins (AGPs) to the outer cell walls of embryogenic units including ECMSN. The most prominent staining was found in cell-cell junction domains. Large non-embryogenic callus cells were not stained with this AGP-specific dye. Immunofluorescence and silver-enhanced immunogold labelling using monoclonal antibody JIM4 has shown that the ECMSN of embryogenic cells is equipped with JIM4 epitope, while non-embryogenic callus cells are devoid of this epitope. We propose that some specific AGPs of the ECMSN might be relevant for cell-cell adhesion and recognition of embryogenic cells during early embryogenic stages, and that the JIM4 antibody can serve as an early marker of embryogenic competence in maize callus culture.

Alcohol‐Soluble Proteins and Protein Body Formation in the Endosperm of Tall Fescue

Alcohol‐soluble proteins (prolamins) are the predominant storage proteins in the endosperm of most of the cereals. Prolamins are deposited within membrane bound structures called protein bodies. Our objective was to determine if there is some commonality in protein body formation between agronomically important cereals and grasses. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) of alcohol‐soluble proteins from tall rescue seeds revealed abundant proteins ranging in size from 73 to 23 kDa. Immunoblot analysis using wheat (Triticum aestivum L.) prolamin antiserum revealed limited antigenic homology between wheat and tall rescue (Festuca arundinaceae Schreb.) prolamins. The amino acid composition of tall rescue alcohol‐soluble proteins showed a high concentration of glutamine and proline residues, similar to those found in several cereal prolamins. Uitrastructural studies of the endosperm cells of tall rescue seeds at 15 d after anthesis revealed the presence of numerous vacuoles that contained protein inclusions. Extensive rough endoplasmic reticulum was seen during this developmental stage. The endoplasmic reticulum, which was often dilated, gave rise to numerous, small vacuole‐like structures. At 20 d after anthesis, the rough endoplasmic reticulum contained flocculent material within the lumen of the dilated areas. Protein aggregates were surrounded by numerous vesicles whose membranes were studded with ribosomes. Organized membrane lattices were seen at the surface of protein bodies and at the junctions where small protein bodies were fusing with larger ones. Protein A‐gold immunocytochemistry employing wheat prolamin antiserum specifically localized the alcohol‐soluble proteins within the protein bodies.

Changes in the zein composition of protein bodies during maize endosperm development.

Zeins, the seed storage proteins of maize, are synthesized during endosperm development by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum, where they assemble into protein bodies. To better understand the distribution of the various zeins throughout the endosperm, and within protein bodies, we used immunolocalization techniques with light and electron microscopy to study endosperm tissue at 14 days and 18 days after pollination. Protein bodies increase in size with distance from the aleurone layer of the developing endosperm; this reflects a process of cell maturation. The protein bodies within the subaleurone cell layer are the smallest and contain little or no alpha-zein; beta-zein and gamma-zein are distributed throughout these small protein bodies. The protein bodies in cells farther away from the aleurone layer are progressively larger, and immunostaining for alpha-zein occurs over locules in the central region of these protein bodies. In the interior of the largest protein bodies, the locules of alpha-zein are fused. Concomitant with the appearance of alpha-zein in the central regions of the protein bodies, most of the beta- and gamma-zeins become peripheral. These observations are consistent with a model in which specific zeins interact to assemble the storage proteins into a protein body.

Anatomy and composition ofZostera MarinaL.: A potential new crop

Chemical composition of Zostera marina L. seed and shoots was determined. Morphology and histochemistry of mature seeds were studied by fluorescence, brightfield and scanning electron microscopy to locate storage constituents in the seed. Starch content in the mature seed was 51% and was the major storage reserve in the embryo and a minor component in the testa. Starch in the shoots ranged from 0.3 to 2.3%. Protein, located in the embryo as small protein bodies, comprised about 9.0% of the seed. Protein in shoots ranged from 6–15%. Protein quality of both seeds and shoots resembled corn in composition, and the first limiting amino acid was lysine. Shoots were high in minerals, fiber and ash, while seeds were lower in these constituents. Fat was low (0.3–1.7%) in both shoots and seeds.

Early Stages in Wheat Endosperm Formation and Protein Body Initiation

The early stages of endosperm formation and protein body initiation are described for hard red winter wheat using light and transmission electron microscopy. Two days after flowering (DAF) the endosperm was a thin layer of coenocytic cytoplasm lining the embryo sac. By 4 DAF the endosperm had cellularized and completely filled the embryo sac. Enough differentiation had occurred by 6 DAF to distinguish cells destined to become the aleurone layer, sub-aleurone region and central endosperm. Protein bodies were initiated at about 6-7 DAF and were first found near the Golgi apparatus. Wheat was ready for combine harvest at 34 DAF. Enlargement of the small protein bodies near the Golgi apparatus occurred by several mechanisms: (1) fusion with one or more of the dense Golgi vesicles or fusion with other protein bodies, (2) fusion with small electron-lucent Golgi-deri ved vesicles, (3) pinocytosis of a portion of the adjacent cytoplasm into the developing protein body and (4) fusion of large protein bodies with one another at later stages of grain development. Of the four mechanisms described, the pinocytotic vesicles and fusion of protein bodies were the most frequent and consistent processes observed. Direct connections between rough endoplasmic reticulum (RER) and protein bodies were not observed. The results suggest a role for the Golgi apparatus in the initiation of protein bodies. Also, the lack of RER derived vesicles suggests a soluble mode of secretion of storage proteins involved in the enlargement of protein bodies.

STARCH GRANULE FORMATION IN DEVELOPING SEEDS OF PISUM SATIVUM L.: EFFECT OF GENOTYPE

Cell development and starch granule formation in seeds of three pea (Pisum sativum L.) genotypes, R/R Rb/Rb, r/r Rb/Rb, and R/R rb/rb, affecting cotyledon starch were compared. Cotyledon cells at 10 days after flowering were highly vacuolated and contained small protein bodies in the vacuoles and small oval starch granules in the cytoplasm in all three genotypes. Gradients of cell development from the center to the periphery of the cotyledon and toward the cotyledon‐hypocotyl axis persisted through the cell enlargement, reserve synthesis, and into the maturation stages of cotyledon development. By 14 days after flowering, many small vacuoles lined with protein deposits had been formed. Vacuoles were only observed in peripheral and basal cells by 18 days after flowering. Starch granules were oval and birefringent in all three genotypes at 10 days. Starch granules in R/R Rb/Rb and R/R rb/rb cotyledons expanded regularly remaining nearly oval and birefringent throughout development. In contrast, starch granules from r/r Rb/Rb cotyledons began to fragment by 14 days after flowering. This process began as a single fissure, followed by a second fissure usually at or near right angles. Finally, because of the fragmentation, the granules appeared compound, and only a portion of the granule was birefringent. All genotypes contained nearly equal volumes of liquid endosperm and embryo at 10 days after flowering. In addition, a layer of parenchyma tissue (ovular and/or endospermic) inside the seed coat was observed. Although, thin walled and poorly defined cytologically, the parenchyma cells contained large numbers of starch granules. These granules were a mixture of simple and compound types in all genotypes. By 18 days after flowering, the parenchyma tissue was reduced to a small layer of cell walls and all starch granules had been mobilized.

Welwitschia mirabilis: Fine Structure of the Germinating Seed V. The Quiescent and Imbibed Gametophyte Interface

Summary Gametophyte cells in the gametophyte-embryo interface zone contain less densely packed reserves and smaller protein bodies (mean diameter 1.2 ,µm) as compared with contiguous and deeper-lying cells. They have more residual cytoplasm, numerous amyloplasts, mitochondria, few microbodies, fragments of ER, but no dictyosomes. Upon imbibition the protein bodies degrade rapidly, starch reserves decline and disappear within 24 h, microbodies increase in number and the ER develops longer single profiles. After 3 days protein bodies start to fuse, microbodies multiply, the ER becomes extensive and sheetlike, and dictyosomes are observed. Between days 4 and 6 ER-derived minivacuoles are autophagically active, accumulating membrane-bound fibrillar material and engulfing lipid bodies; the cell walls have thickened; and mitochondria exhibit a close spatial relationship with lipid bodies. The outer cells of the mature feeder are crushed against the gametophyte. With imbibition physical wetting of outer gametophyte cells precedes that of interface cells, yet nuclear activation and subsequent cellular activity is accelerated in the latter and stand in sharp contrast to the slower metabolism of the former.

Endosperm Structure of High Lysine Sorghum1

Scanning electron micrographs showed that high lysine sorghum [Sorghum bicolor (L.) Moench] endosperm contains fewer and smaller protein bodies than normal sorghum endosperm. This Indicates that an alteration in protein synthesis had occurred in the endosperm of higher lysine sorghums. Improved lysine content resulted because these protein bodies contain mainly alcohol soluble proteins, which are very deficient in basic amino acid content. The marked reduction in protein bodies suggests the change in endosperm protein synthesis was significant and that it may be possible to isolate plump kernels with fewer protein bodies and higher lysine content. The potential use of scanning electron microscopy as a nondestructive tool to select sorghums with plump kernels and high lysine content should be explored.

The Rest Period ofRhododendronFlower Buds

Rhododendron flower development occurs in three easily defined stages: a pre-rest stage, during which petal growth is mainly by cell elongation; an indeterminate rest period; and an after-rest stage, that begins when the flowers resume growth and ends at anthesis. Early in the pre-rest stage of development, protein bodies and amyloplasts accumulate in the petals. The epidermal cells accumulate only protein bodies and the mesophyll cells accumulate amyloplasts that have a few small protein bodies around the periphery. The subepidermal cells and the cells around the vascular bundles accumulate both large protein bodies and amyloplasts. During the rest period there is a cessation of cell elongation and the reserve protein bodies and amyloplasts remain intact. The protein bodies in all of the cells including those around the amyloplasts are proteolized early in the after-rest stage of development. Digestion of the starch granules occurs when the petals are about one-half their final size. Epidermal-cell expansion during after-rest is relatively uniform; the walls between adjacent epidermal cells remain attached to each other. The mesophyll cells elongate irregularly and the walls of adjacent cells separate giving rise to large intercellular spaces. At anthesis the petal cells consist of a cell wall, a parietal cytoplasm, and a large central vacuole.

Studies on the cytophysiology of the fat body of the American silkmoth.

A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.