Free and Bound Amino Acids during Germination of Maize Seed with Preincorporated Foreign Prolamines

Wheat prolamines were preincorporated into the endosperm of maize seed, and concentrations of free and bound amino acids were measured in the endosperm and seedlings during seed germination. Incorporation of foreign prolamines lowered the rate of decrease of free proline concentration in both the endosperm (sprouting and shoots) and the embryo (sprouting). The seedlings at the stage of shoots were characterized by higher concentrations of aspartic acid, glutamic acid, and tyrosine. At the stage of transition to autotrophic nutrition, the pool of amino acids that are physiologically important for plant growth was greater in maize seedlings with incorporated wheat prolamines.     

Free and bound amino acids during germination of maize seeds with implemented foreign prolamines

Wheat prolamines were preincorporated into the endosperm of maize seed, and concentrations of free and bound amino acids were measured in the endosperm and seedlings during seed germination. Incorporation of foreign prolamines lowered the decrease rate of free proline concentration in both the endosperm (sprouting and shoots) and the embryo (sprouting). The seedlings at the stage of shoots were characterized by higher concentrations of aspartic acid, glutamic acid, and tyrosine. At the stage of transition to autotrophic nutrition, the pool of amino acids that are physiologically important for the plant growth was greater in maize seedlings with incorporated wheat prolamines.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

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     

Peptide uptake in germinating barley embryos involves a dithiol-dependent transport protein

An active transport system for small peptides occurs in the scutellar membrane of germinating barley and serves to move the products of partial hydrolysis of storage proteins from the endosperm into the growing embryo. Transport of peptides, but not amino acids or glucose, is inhibited by the thiol reagents, N-ethylmaleimide and p-chloromercuribenzene sulphonic acid (PCMBS). Peptide substrates protect against PCMBS inactivation. The dithiol-specific reagent, phenylarsine oxide (PAO) also inhibits. The reducing agent, dithiothreitol, reverses the inactivation caused by PCMBS and PAO. We conclude that the peptide transport system contains a redox-sensitive, dithiol-dependent protein.     

Embryology in Trithuria submersa (Hydatellaceae) and relationships between embryo, endosperm, and perisperm in early-diverging flowering plants

? Premise of the study: Despite their highly reduced morphology, Hydatellaceae bear the unmistakable embryological signature of Nymphaeales, including a starch-rich maternal perisperm and a minute biparental endosperm and embryo. The co-occurrence of perisperm and endosperm in Nymphaeales and other lineages of flowering plants, and their respective functions during the course of seed development and embryo germination, remain enigmatic. ? Methods: Development of the embryo, endosperm, and perisperm was examined histologically from fertilization through germination in flowers and fruits of Trithuria submersa. ? Key results: The embryo of T. submersa initiates two cotyledons prior to seed maturity/dormancy, and their tips remain in contact with the endosperm throughout germination. The endosperm persists as a single layer of cells and serves as the interface between the embryo and the perisperm. The perisperm contains carbohydrates and proteins, and functions as the main storage tissue. The endosperm accumulates proteins and aleurone grains and functions as a transfer cell layer. ? Conclusions: In Nymphaeales, the multiple roles of a more typical endosperm have been separated into two different tissues and genetic entities: a maternal perisperm (nutrient acquisition, storage, mobilization) and a minute biparental endosperm (nutrient transfer to the embryo). The presence of perisperms among several other ancient lineages of angiosperms suggests a modest degree of developmental and functional lability for the nutrient storage tissue (perisperm or endosperm) within seeds during the early evolution of flowering plants. Finally, we examine the evolutionary developmental hypothesis that, contrary to longstanding assumptions, an embryo-nourishing perisperm along with a minute endosperm may represent the plesiomorphic condition for flowering plants.     

Developmental Biochemistry of Cottonseed Embryogenesis and Germination: VIII. Free Amino Acid Pool Composition during Cotyledon Development

The composition of the free amino acid pool in embryonic cotton (Gossypium hirsutum) cotyledons is quite distinct from that of endosperm, and that of germinated, greened cotyledons is quite distinct from that of leaves. During germination (including the precocious germination of immature seeds), the pool expands considerably showing a pronounced accumulation of asparagine. The high level of asparagine found in seedling roots and in the cotyledon vascular exudate indicates that this is the major transported amino acid in germination. There is no pool expansion in the presence of abscisic acid. In the presence of actinomycin D, the pool expands, but an enormous accumulation of glutamine takes place. The composition of the pool at any stage is not related to the composition of the isoacceptor transfer RNA pool, nor to the composition of the storage protein. Anaerobiosis leads to an accumulation of aspartate, alanine, and glycine at the expense of asparagine; however, desiccation does not result in an accumulation of proline. Conspicuously high levels of arginine are maintained through embryogenesis and germination. The levels of individual amino acids are presented as nanomol per cotyledon pair and as per cent of total pool.     

Peptide transport by germinating barley embryos: Uptake of physiological di- and oligopeptides

The uptake of a variety of physiological di- and oligopeptides by germinating barley (Hordeum vulgare L.) embryos is described. Peptides as large as pentaalanine can be absorbed. Evidence is presented suggesting the peptides are absorbed intact and subsequently undergo rapid intracellular hydrolysis. Uptake shows stereospecificity. The transport of peptides is generally faster than the transport of amino acids, making it likely that the former could play an important role in the mobilization of the protein storage reserves during germination. The peptide transport system in barley is compared with similar systems from other groups of organisms.Abbreviations Gly-sar glycylsarcosine - Gly-sar-sar glycylsarcosylsarcosine - Gly-sar-sar-sar Glycylsarcosylsarcosylsarcosine     

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S₂ site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1–9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.Abbreviations CMC carboxymethyl cellulose - E-64 transepoxysuccinyl-l-leucylamido-(4-guanidino)butane - EMI N-ethylmaleimide - IEF isoelectricfocusing - Phen 1,10-phenanthroline - PI isoelectric point - PMSF phenylmethylsulfonyl fluoride We thank the American Malting Barley Association for partially funding this work. Germinated barley seeds were kindly prepared by Eddie D. Goplin. Special thanks to Laurie Marinac for her excellent technical assistance.     

ZmEBE genes show a novel,continuous expression pattern in the central cell before fertilization and in specific domains of the resulting endosperm after fertilization

Two novel maize genes expressed specifically in the central cell of the female gametophyte and in two compartments of the endosperm (the basal endosperm transfer layer and the embryo surrounding region) were characterized. The ZmEBE (embryo sac/basal endosperm transfer layer/embryo surrounding region) genes were isolated by a differential display between the upper and the lower half of the kernel at 7 days after pollination (DAP). Sequence analysis revealed ORFs coding for two closely related proteins of 304 amino acids (ZmEBE-1) and 286 amino acids (ZmEBE-2). This size difference was due to differences in the splicing of the two genes. Both protein sequences showed significant similarity to the DUF239 family of Arabidopsis, a group of 22 proteins of unknown function, a small number of which are putative peptidases. ZmEBE genes had a novel cell type-specific expression pattern in the central cell before and the resulting endosperm after fertilization. RT-PCR analysis showed that the expression of both genes started before pollination in the central cell and continued in the kernel up to 20 DAP with a peak at 7 DAP. In situ hybridization revealed that the expression in the kernel was restricted to the basal transfer cell layer and the embryo surrounding region of the endosperm. The expression of ZmEBE-1 was at least 10 times lower than that of ZmEBE-2. Similarly to other genes expressed in the endosperm, ZmEBE-1 expression was subject to a parent-of-origin effect, while no such effect was detected in ZmEBE-2. Sequence analysis of upstream regions revealed a potential cis element of 33 bp repeated 7 times in ZmEBE-1 and ZmEBE-2 between positions -900 and -100. The 1.6 kb ZmEBE-2 upstream sequence containing the seven R7 elements was able to confer expression in the basal endosperm to a Gus reporter gene. These data indicate that ZmEBE is potentially involved in the early development of specialized domains of the endosperm and that this process is possibly already initiated in the central cell, which is at the origin of the endosperm.     

Amino Acid transport in germinating castor bean seedlings

During germination and early growth of the castor bean (Ricinus communis) nitrogenous constituents from the endosperm are transferred via the cotyledons to the growing embryo. Exudate collected from the cut hypocotyl of 4-day seedlings contained 120 millimolar soluble amino nitrogen and glutamine was the predominant amino acid present, comprising 35 to 40% of the total amino nitrogen. To determine the nature of nitrogen transfer, the endosperm and hypocotyl were removed and glutamine uptake by the excised cotyledons was investigated. Uptake was linear for at least 2 hours and the cotyledons actively accumulated glutamine against a concentration gradient. The uptake was sensitive to respiratory inhibitors and uncouplers and efflux of glutamine from the excised cotyledons was negligible. Transport was specific for the l-isomer. Other neutral amino acids were transported at similar rates to glutamine. Except for histidine, the acidic and basic amino acids were transported at lower rates than the neutral amino acids. For glutamine transport, the K(m) was 11 to 12 millimolar and the V(max) was 60 to 70 micromoles per gram fresh weight per hour. Glutamine uptake was diminished in the presence of other amino acids and the extent of inhibition was greatest for those amino acids which were themselves rapidly transported into the cotyledons. The transport of amino acids, on a per seedling basis, was greatest for cotyledons from 4-to 6-day seedlings, when transfer of nitrogen from the endosperm is also maximal. It is concluded that the castor bean cotyledons are highly active absorptive organs transporting both sucrose and amino acids from the surrounding endosperm at high rates.     

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.     

Nitrogen metabolism during synchronous growth of Chlorella. II. Free-, peptide-, and protein-amino acid distribution

The levels of free-, peptide-, and protein-amino acids were measured during the synchronous growth and division cycle of a thermophilic strain of Chlorella pyrenoidosa. Most of the protein amino acids exhibited little periodism (as % of total cellular-N); however, the free- and peptide-amino acids showed a variety of dramatic changes in level during the cell cycle. Fractionation of the acid-soluble peptides by Sephadex gel-filtration showed that an average of only 2.8% of the peptide amino acids were associated with peptides of high molecular weight (> 5000), while approximately 75% of the peptide amino acids were components of low molecular weight peptides (< 700). The low molecular weight peptides were predominately made up of relatively few amino acids (i.e., alanine, glutamate, lysine, glycine and arginine accounted for approximately 92% of the low molecular weight peptide amino acids). Several experiments revealed that nucleotide-peptides do not contribute significantly to the pool of acid-soluble peptides during the cell cycle of this organism.     

Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis.     

Amino acids and quaternary nitrogen compounds in the germinating wheat grain

The tissues of the quiescent wheat grain contained free amino acids and quaternary nitrogen compounds. During germination the amino acid levels increased several fold. In the aleurone tissue and starchy endosperm glutamine was the predominant amino acid. Asparagine was predominant in the seedling tissues. Choline and glycine betaine were the principal quaternary nitrogen compounds present. The aleurone tissue and the embryo/seedling contained large quantities of glycine betaine. The increase in free amino acid levels in the aleurone tissue during the first 2 days of germination occurred independently of the embryo. After the second day, the further increase in levels was dependent upon the presence of the embryo and of gibberellic acid (GA). Estimation of the individual amino acids and quaternary nitrogen compounds released from incubating aleurone layers into aqueous media revealed a selective release of some compounds and retention of others. The process was regulated by GA. Possible mechanisms for the release of amino acid and its control by GA are discussed.