The scutellum of Avena: a structure to maximize exploitation of endosperm reserves

NEGBI, M. & SARGENT, J. A., 1986. The scutellum of Avena : a structure to maximize exploitation of endosperm reserves . The scutella of graminaceous embryos possesses a well developed vascular system and a surface epithelium of closely packed cells. The epithelial cells typically elongate during germination and separate to form papillae, thereby increasing the interfacial area for exchange between the endosperm and embryo. Avena and a dozen non-crop genera are distinguishable by a scutellum which grows into and through the endosperm. Thus diffusion pathways for mobilized reserves in Avena are kept short. The course of elongation of the scutellum and of its epithelial cells in A. Jatua was studied during germination and seedling establishment. A comparison was made with with the fully developed scutellum of Secale cereale which does not elongate but only forms papillae. It is proposed that the competitive success of A.Jatua as a weed is attributable in part to an ability, conferred by its scutellum, to exploit its seed reserves steadily and fully during germination and seedling development.     

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane

Thiol-affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devised to label substrate protectable protein with the thiol specific reagent [14C] N-ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol-affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

Fruit Structure and Development in Dinochloa and Ochlandra (Gramineae-Bambusoideae)

Fruit structure and development is examined in species of Dinochloaand Ochlandra, in the context of bamboo systematics. In bothgenera there is a thick pericarp, and at maturity the endospermhas become compressed by the massive scutellum, although bothembryo and endosperm initially follow the normal pattern fordevelopment for grasses. The scutellum, endosperm and pericarpcontain food reserves in the form of starch granules. Thereare some differences between the two genera, notably the structureof the mature pericarp and the position of the embryo, and theseare discussed in relation to vivipary and the lack of a dormancyor dispersal phase in these genera Gramineae, Bambusoideac, Dinochloa, Ochlandra, Melocanna, fleshy fruits, fruit wall     

Expression profile of maize (Zea mays) scutellar epithelium during imbibition

The scutellum is a shield-shaped structure surrounding the embryo axis in grass species. The scutellar epithelium (Sep) is a monolayer of cells in contact with the endosperm. The Sep plays an important role during seed germination in the secretion of gibberellins and hydrolytic enzymes and in the transport of the hydrolized products to the growing embryo. We identified 30 genes predominantly expressed after imbibition in the Sep as compared to other parts of the scutellum. A high proportion of these genes is involved in metabolic processes. Some other identified genes are involved in the synthesis or modification of cell walls, which may be reflected in the changes of cell shape and cell wall composition that can be observed during imbibition. One of the genes encodes a proteinase that belongs to a proteinase family typical of carnivorous plants. Almost nothing is known about their role in other plants or organs, but the scutellar presence may point to a "digestive" function during germination. Genes involved in the production of energy and the transport of peptides were also identified.     

Patterns of proteolytic enzyme activities in different tissues of germinating corn (Zea mays L.)

Profiles of pH dependence and activities of live proteolytic enzymes, amino- and carboxypeptidase and endopeptidases active at pH 3.8, 5.4 and 7.5, with casein as substrate, were determined in crude extracts from the various organs of corn seedlings during germination and early development (30°C, dark, 8 d). With respect to the endopeptidases, caseolytic activities at pH 3.8, 5.4 and 7.5 in extracts from endosperm increased concurrently with loss of endosperm N during germination; however, the relative amounts of the pH 7.5 activity were very small. In scutellum extracts, caseolytic activities at both pH 5.4 and 7.5 increased during the initial stages of development but only the increase at pH 5.4 was concurrent with loss of scutellar N. In shoot extracts, caseolytic activities at pH 5.4 and 7.5 were very low and remained relatively constant. There was a progressive increase in shoot N with time. In root extracts, caseolytic activities at pH 5.4 and 7.5 were higher (3-fold) than in shoot extracts. The activity at pH 5.4 remained constant while the activity at pH 7.5 increased during germination. The rate of accumulation of N by the root was low after day 5. The pattern and ratio but not the amounts of the pH 5.4 and 7.5 caseolytic activities of the root were similar to those observed in senescing leaves of field-grown corn. Addition of mercaptoethanol increased (several-fold) the caseolytic activities at pH 3.8 and 5.4, especially the latter, but not the pH 7.5 activity in endosperm extracts and increased the pH 5.4 activity in extracts from scutellum (30%) and roots (30%) while the effect in shoot extracts was negligible. Carboxypeptidase activity was relatively low in young tissue (root tip, 3-d-old shoots) and increased with development of the various organs except the roots (whole) where the activity remained relatively constant. The increases in carboxypeptidase activities were concurrent with decreases in N from endosperm and scutellum; this result indicates that this enzyme in these tissues may be involved (cooperatively with endopeptidases) in the mobilization of reserve protein.Of all the enzymes tested, only carboxypeptidase activity was markedly (in excess of 50%) inhibited by phenylmethylsulfonylfluoride. Only aminopeptidase activity was found in appreciable amounts in endosperm and scutellum of dry kernels. Aminopeptidase activity was highest in organs with high metabolic activity (scutella, shoot, root tips) and decreased in plant parts undergoing rapid loss of nitrogen (endosperm, senescing leaves).Abbreviations AP aminopeptidase - CA caseolytic activity - CP carboxypeptidase - ME mercaptoethanol     

Differential gene expression during seed germination in barley (Hordeum vulgare L.)

A barley cDNA macroarray comprising 1,440 unique genes was used to analyze the spatial and temporal patterns of gene expression in embryo, scutellum and endosperm tissue during different stages of germination. Among the set of expressed genes, 69 displayed the highest mRNA level in endosperm tissue, 58 were up-regulated in both embryo and scutellum, 11 were specifically expressed in the embryo and 16 in scutellum tissue. Based on Blast X analyses, 70% of the differentially expressed genes could be assigned a putative function. One set of genes, expressed in both embryo and scutellum tissue, included functions in cell division, protein translation, nucleotide metabolism, carbohydrate metabolism and some transporters. The other set of genes expressed in endosperm encodes several metabolic pathways including carbohydrate and amino acid metabolism as well as protease inhibitors and storage proteins. As shown for a storage protein and a trypsin inhibitor, the endosperm of the germinating barley grain contains a considerable amount of residual mRNA which was produced during seed development and which is degraded during early stages of germination. Based on similar expression patterns in the endosperm tissue, we identified 29 genes which may undergo the same degradation process. Electronic Publication     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Changes in Levels of alpha-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18°C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for α-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same α-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar α-amylase was excreted into the endosperm compared to aleurone synthesized α-amylase. Aleurone cells appeared to synthesize appreciably more α-amylase than did scutellar tissue.     

The Peptide pools of germinating barley grains: relation to hydrolysis and transport of storage proteins

A quantitative procedure for purifying small peptides from plant tissues, involving both ion-exchange and gel-exclusion chromatography, is described. Peptides were quantified and characterized by using the fluorescence reagents dansyl chloride and fluorescamine. Large pools of small peptides and amino acids have been identified in both the endosperm and embryo of germinating barley grains. The peptide pool of the endosperm increases during the first 3 days of germination, subsequently decreasing, an observation compatible with a role for peptides as intermediates in the breakdown of the storage proteins and their transfer to the embryo. The amino acid composition of these peptides indicates that all the major classes of storage protein contribute to the pool. The concentration of peptides produced in the endosperm during germination is sufficient for the efficient operation of the peptide transport system of the scutellar membrane characterized previously (Higgins and Payne, Planta 136: 71-76, 1977; Planta 138: 211-215 and 217-221, 1978). Data presented here indicate that peptides play at least as important a role as amino acids in the transfer of stored nitrogen from the endosperm to the embryo during germination.     

Endosperm sterol phenotype and germination in wheat

Free and conjugated sterols of endosperm, coats, scutellum, coleoptile and roots have been analysed at different germination stages in two wheat cultivars with different endosperm sterol phenotypes. It seems that sterol metabolism of the developing tissues, namely coleoptile and roots, is not affected by the sterol conjugation profile of the endosperm. Enough sterol is present in the mature embryo to supply the germinating axis during the observation period (144 hr at 16°). The data suggest that sterol is transferred from scutellum to coleoptile and roots during germination.     

Peptide transport by germinating barley embryos

Glycylsarcosine, a dipeptide which is resistant to peptidase activity, was accumulated intact against a concentration gradient by germinating embryos of Hordeum vulgare L., var. Maris Otter, Winter. This is the first clear evidence for the presence of a dipeptide transport system involved in the movement of protein reserves across the scutellum from the endosperm to the embryo during germination.Abbreviations gly-sar glycylsarcosine - TCA trichloroacetic acid     

Secretion of basic and acidic chitinases from salt-adapted and -unadapted winged bean cells

Northern hybridizations were used to study the site of synthesis of three carboxypeptidases (Cpases I-III) which occur in the starchy endosperm of germinating barley grain ( Hordeum vulgare L.). Further evidence was obtained by studying secretion of these enzymes from scutella or aleurone layers separated from germinating grains. Messenger RNA for Cpase II was detected only in developing grain, and the bulk of the mRNA was localized in the starchy endosperm. This suggests that Cpase II is synthesized at the site of its accumulation, the starchy endosperm. In contrast, Cpase I is expressed during germination and the predominant site of synthesis is the scutellum, from which it is secreted into the starchy endosperm. Cpase III is also synthesized during germination, but the bulk of it is synthesized in and secreted from the aleurone layer. Thus, the three carboxypeptidases, all of which seem to play a role in hydrolysis of the reserve proteins in the starchy endosperm during germination, have different sites of synthesis.     

Effects of Ageing on Amylase Activity and Scutellar Cell Structure during Imbibition in Wheat Seed

In wheat seed the scutellum plays an important role in the hydrolysisof stored substrate during germination. This layer is activatedfirst, whilst the aleurone becomes activated later. A good correlationexists between the initiation of visible germination and theappearance of enzyme activity in the scutellum. Enzyme activityin the aleurone becomes apparent only when the germinating seedlingreaches the rapid growth phase. Electron microscopic observationsshow that during the later stages of germination the scutellarcells develop finger like projections. These may serve to absorbendospermic reserves hydrolysed by aleurone amylase. The scutellumof aged non-germinating seeds showed no amylase activity andno finger like projections were produced even after prolongedimbibition.Copyright 1993, 1999 Academic Press Wheat (Triticum aestivum L.), deteriorated, germination, scutellum, scanning electron microscopy, aleurone     

Thioredoxin and germinating barley: targets and protein redox changes

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition. Reduction reached a peak 1 day earlier in the embryo than in the endosperm, day 1 vs. day 2. The profile in both cases resembled those observed with wheat and rice, i.e., the reduction of the storage proteins increased initially and then declined during the period of seedling growth. The extent of the increase in reduction observed with barley endosperm was, however, less pronounced than with the other cereals. Also, unlike wheat and rice, the storage proteins of the endosperm were highly reduced in the dry seed and the sulfhydryl content of glutelins showed no appreciable change during this period. The relative abundance of thioredoxin h during germination and early seedling growth differed in the embryo and endosperm: a progressive decrease in the endosperm (as seen with wheat) vs. an increase in the embryo. Thioredoxin h was found in the major seed tissues in characteristic forms. Three forms were found in the scutellum and aleurone, whereas two, which may represent isoforms, were identified in the root and the shoot. Using a recently developed strategy based on two-dimensional gel electrophoresis, several proteins were identified as specific targets for thioredoxin in the embryo following oxidation with H(2)O(2), among them barley embryo globulin 1, peroxiredoxin and acidic ribosomal protein P(3). The results confirm earlier findings with the endosperm of other cereals and extend the importance of thioredoxin-linked redox change to the germinating embryo for functions that potentially include dormancy, protection against reactive oxygen species, translation and the mobilization of storage proteins.     

Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1

Arabidopsis thaliana is used as a model system to study triacylglycerol (TAG) accumulation and seed germination in oilseeds. Here, we consider the partitioning of these lipid reserves between embryo and endosperm tissues in the mature seed. The Arabidopsis endosperm accumulates significant quantities of storage lipid, and this is effectively catabolized upon germination. This lipid differs in composition from that in the embryo and has a specific function during germination. Removing the endosperm from the wild-type seeds resulted in a reduction in hypocotyl elongation in the dark, demonstrating a role for endospermic TAG reserves in fueling skotomorphogenesis. Seedlings of two allelic gluconeogenically compromised phosphoenolpyruvate carboxykinase1 (pck1) mutants show a reduction in hypocotyl length in the dark compared with the wild type, but this is not further reduced by removing the endosperm. The short hypocotyl phenotypes were completely reversed by the provision of an exogenous supply of sucrose. The PCK1 gene is expressed in both embryo and endosperm, and the induction of PCK1:beta-glucuronidase at radicle emergence occurs in a robust, wave-like manner around the embryo suggestive of the action of a diffusing signal. Strikingly, the induction of PCK1 promoter reporter constructs and measurements of lipid breakdown demonstrate that whereas lipid mobilization in the embryo is inhibited by abscisic acid (ABA), no effect is seen in the endosperm. This insensitivity of endosperm tissues is not specific to lipid breakdown because hydrolysis of the seed coat cell walls also proceeded in the presence of concentrations of ABA that effectively inhibit radicle emergence. Both processes still required gibberellins, however. These results suggest a model whereby the breakdown of seed carbon reserves is regulated in a tissue-specific manner and shed new light on phytohormonal regulation of the germination process.