On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

Compositional changes in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L. incubated at 25 C for 3 days in the dark were irradiated with continuous red light to induce spore germination and cell growth during following 7 days. A portion of spores were cultured for 8 days in the dark as non-irradiated control. Rhizoidal and protonemal cells were observed at 3 days after transferring spores to the irradiation conditions. During 10 days of the experimental period, changes in the contents of following cell constituents were investigated: total lipid, total soluble sugar, reducing sugar, insoluble glucan, organic acid, protein, soluble α-amino N, and major free amino acids. A large part of nutrient reserves of spores was found to be lipid, whose content decreased markedly as spores germinated. Soluble and insoluble carbohydrates also provided carbon and energy sources during imbibition and germination. Two main reserve proteins were detected by SDS-polyacrylamide gel electrophoresis. These proteins disappeared mostly during germination. Major free amino acids could be assorted into three groups by their patterns of fluctuation during the germination.     

Protein Synthesis During Rehydration, Germination and Seedling Growth of Naturally and Precociously Matured Soybean Seeds (Glycine max)

Soybean seeds [Glycine max (L.) Merr.] synthesize de novo andaccumulate several non-storage, soluble polypeptides duringnatural and precocious seed maturation. These polypeptides havepreviously been coined ‘maturation polypeptides’.The objective of this study was to determine the fate of maturationpolypeptides in naturally and precociously matured soybean seedsduring rehydration, germination, and seedling growth. Developingsoybean seeds harvested 35 d after flowering (mid-development)were precociously matured through controlled dehydration, whereasnaturally matured soybean seeds were harvested directly fromthe plant. Seeds were rehydrated with water for various timesbetween 5 and 120 h. Total soluble proteins and proteins radio-labelledin vivo were extracted from the cotyledons and embryonic axesof precociously and naturally matured and rehydrated seed tissuesand analyzed by one-dimensional PAGE and fluorography. The resultsindicated that three of the maturation polypeptides (21, 31and 128 kDa) that had accumulated in the maturing seeds (maturationpolypeptides) continued to be synthesized during early stagesof seed rehydration and germination (5–30 h after imbibition).However, the progression from seed germination into seedlinggrowth (between 30 and 72 h after imbibition) was marked bythe cessation of synthesis of the maturation polypeptides followedby the hydrolysis of storage polypeptides that had been synthesizedand accumulated during seed development. This implied a drasticredirection in seed metabolism for the precociously maturedseeds as these seeds, if not matured early, would have continuedto synthesize storage protein reserves. Glycine max (L.) Merr, soybean, cotyledons, maturation, germination/seedling growth     

Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.     

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: III. INSOLUBLE PROTEIN SYNTHETIC PATTERN CHANGES WITHIN THE ENDOSPERM OF Ricinus communis L. SEEDS

Kermode, A. R., Gifford, D. J. and Bewley, J. D. 1985. The roleof maturation drying in the transition from seed developmentto germination. III. Insoluble protein synthetic pattern changeswithin the endosperm of Ricinus communis L. seeds.—J.exp. Bot. 36: 1928–1936. Immature seeds of Ricinus communisL. cv. Hale (castor bean) removed from the capsule at 30 or40 days after pollination (DAP) can be induced to germinateby being subjected to drying. This desiccation–inducedswitch from development to germination is mirrored by a change,upon subsequent rehydration, in the pattern of insoluble proteinsynthesis within the endosperm storage tissue. During normaldevelopment from 25–40 DAP there is rapid synthesis ofthe insoluble (11S) crystalloid storage protein. At later stagesof development (45 and 50 DAP), crystalloid protein synthesisdeclines markedly and synthesis of new insoluble proteins commences.Following premature drying at 30 or 40 DAP, the pattern of insolubleprotein synthesis upon rehydration is virtually identical tothat following imbibition of the mature seed. Proteins synthesizedduring normal late development (at 45 and 50 DAP) are producedup to 48 h after imbibition; a subsequent change in the patternof insoluble protein synthesis occurs between 48 and 72 h. Thus,in contrast to the rapid switch in the pattern of soluble proteinsynthesis induced by drying, insoluble protein syntheses withinthe endosperm are redirected towards those uniquely associatedwith a germination/growth programme only after a considerabledelay following mature seed imbibition, or following rehydrationof the prematurely dried seed. Nevertheless, these results supportour contention that drying plays a role in the suppression ofthe developmental metabolic programme and in the permanent inductionof a germination/growth programme. Key words: Desiccation, crystalloid storage proteins, castor bean, seed development, seed germination     

The seed proteins of white spruce and their mobilization following germination

Buffer-soluble proteins that have subunit molecular weights, in the presence of sodium dodecyl sulphate (SDS), of 47, 31 and 27 kilodaltons (kDa) form the major storage proteins in the mature white spruce [ Picea glauca (Moench) Voss] seed. These proteins were found mainly in the megagametophyte. but smaller amounts were identified in the embryo. Following the completion of germination, this reserve was rapidly hydrolyzed in both tissues and probably plays a major nutritional role in the germinated seed. Buffer-insoluble proteins were also found in megagametophytes and embryos from the mature seed. These proteins were soluble in buffer only if SDS was present. Predominant in this class of proteins were several that have a subunit molecular weight and structure that is characteristic of seed crystalloid storage proteins; the subunits were shown to be heterodimers with polypeptide molecular weights in the 33 kDa to 37 kDa and 23.5 kDa to 25 kDa ranges. This reserve was rapidly hydrolyzed in the germinated seed. Storage protein hydrolysis was accompanied by a significant increase in the soluble amino acid pool in both megagametophytes and embryos. Cell-free extracts of mature seed megagametophytes and embryos contained leucine-naphthylamidase (leuNAase) activity. Following germination. this activity was maintained at a constant level in megagametophytes but increased substantially in embryos.     

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.     

Gene Expression Profile Changes in Germinating Rice

Water absorption is a prerequisite for seed germination. During imbibition, water influx causes the resumption of many physiological and metabolic processes in growing seed. In order to obtain more complete knowledge about the mechanism of seed germination, two‐dimensional gel electrophoresis was applied to investigate the protein profile changes of rice seed during the first 48 h of imbibition. Thirty‐nine differentially expressed proteins were identified, including 19 down‐regulated and 20 up‐regulated proteins. Storage proteins and some seed development‐ and desiccation‐associated proteins were down regulated. The changed patterns of these proteins indicated extensive mobilization of seed reserves. By contrast, catabolism‐associated proteins were up regulated upon imbibition. Semi‐quantitative real time polymerase chain reaction analysis showed that most of the genes encoding the down‐ or up‐regulated proteins were also down or up regulated at mRNA level. The expression of these genes was largely consistent at mRNA and protein levels. In providing additional information concerning gene regulation in early plant life, this study will facilitate understanding of the molecular mechanisms of seed germination.     

Loblolly pine seed dormancy. I. The relationship between protein synthesis and the loss of dormancy

Embryos from the mature unstratified loblolly pine ( Pinus taeda L.) seeds used in this study were nondormant: however, they failed to germinate in situ because of constraints imposed by the surrounding tissues. During a stratification period of 35 days of moist chilling at 2°C, seed germinability increased from 19 to 76%. The total lipid content of the megagametophyte did not change during stratification, whereas the total protein content of both megagametophyte and embryo was more variable. The rate of synthesis of buffer soluble proteins in these two tissues increased and electrophoretic analysis showed that while similar proteins were synthesized during the stratification period, changes in the patterns of synthesis of some proteins did occur. In both the embryo and megagametophyte the synthesis of a set of proteins with molecular masses below 46 kDa decreased markedly after 14 days of chilling (DOC). In the megagametophyte, the synthesis of a more diverse set of proteins with molecular masses ranging from 16 to 78 kDa increased after 14 DOC. It is noteworthy that these changes in the patterns of protein synthesis coincided with the greatest relative increase in seed germinability of 35%.     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated     

Hydrolysis of Crystalloid Storage Protein in Castor Bean Seed Endosperm During and Following Germination

Hydrolysis of the insoluble crystalloid storage proteins ofcastor bean endosperm during germination released buffer-solublepolypeptides with molecular weights in the presence of sodiumdodecyl sulphate of 30000–40000. These polypeptides appearto be dimers since the addition of 2-mercaptoethanol decreasestheir molecular weights to 15000–22000. Hydrolysis ofthe crystalloid proteins was detected 12–18 h after seedimbibition (HAI), which is before the completion of germination;maximum rates were attained at 30 HAI. During this period, parallelincreases in free amino acids were observed. Hydrolysis of thecrystalloid proteins during early germination was insensitiveto cycloheximide treatment and therefore did not require newlysynthesized proteases. Hydrolysis was effected by proteaseswhich were made in an inactive form during seed developmentand activated upon seed imbibition. Key words: Castor bean, crystalloid storage protein hydrolysis, seed germination, endosperm     

萌发中花生胚轴的耐干性与热稳定蛋白

成熟花生种子吸胀１８ ｈ 发芽率达１００ ％ 。在这１８ ｈ 的范围内,胚轴即使经干燥处理,萌发生长率仍保持１００ ％ ,而热稳定蛋白含量变化很小。吸胀２４ ｈ 后,经干燥的花生胚完全丧失萌发生长能力。ＳＤＳＰＡＧＥ和双向电泳表明,花生胚轴的热稳定蛋白主要是贮藏蛋白,该蛋白中的花生球蛋白大亚基,伴花生球蛋白Ｉ和２Ｓ 蛋白的降解与胚轴的耐干性丧失有关。     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

Evaluation of some cereals, plants and tubers through protein composition

Wild and cultivated maize, sorghum, rice, amaranth, soybean, and cassava were screened for variability in seed storage proteins. Total seed proteins, albumin (Alb-1 and Alb-2), globulin, alcohol-soluble (A1 and A2), and glutelin (G1 and G2) fractions were investigated by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The comparison was done by the obtained protein patterns and their relative amounts. Using quantitative analysis of the protein composition and the electrophoretic patterns, the relationships between total proteins and amount of individual proteins were determined. Electrophoretic patterns of extracted proteins from investigated samples showed that the main protein subunits were concentrated between 10 and 45 kDa. Variation was found in major fractions and minor bands. Electrophoretic patterns of the protein fractions are directly related to the genetic background of the protein and can be identified and used to certify the genetic makeup of wild, cultivated, or newly derived cereals and plants.     

Disappearance of protein supplements and their fractions in sacco

The rumen degradability of animal and plant protein supplements (eight of each) was assessed in sacco under two feeding regimens. The supplements as such and those obtained after in sacco degradability were fractionated into soluble (albumin and globulin) and insoluble (prolamin and glutehn) proteins. Skimmed milk powder had the highest ratio of soluble to insoluble fractions, followed by cotton seed cake, deoiled mustard cake and blood meal. The casein and soybean meal had a similar but relatively low ratio compared with the above protein supplements. However, these were completely degraded. Corn gluten meal had the lowest ratio of soluble to insoluble fractions, which was reflected in the lowest dry matter and crude protein degradability. Similarly, bone meal and meatcum-bone meal, having low ratios, showed poor degradation of dry matter, crude protein and their fractions. The feeding regimens of the animals had significant (P < 0.05) influence on the degradation of some of the protein supplements. Further, irrespective of the feeding system and the source of protein, albumin was degraded the most and prolamin the least. It was concluded, therefore, that the solubility of protein supplements is an important factor for determining the susceptibility or resistance of protein supplements to rumen degradation.     

A C-terminal sequence of soybean beta-conglycinin alpha' subunit acts as a vacuolar sorting determinant in seed cells

In maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension-cultured cells apart from seed cells. In this study, we examined the VSD of the alpha' subunit of beta-conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild-type alpha' was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid-like structures. Some of the wild-type alpha' was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C-terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The C-terminal region of alpha' was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that alpha' contains two VSDs: one is present in the C-terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C-terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.     

Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance

Development of yellow cedar seeds is completed by about 17-21 months after pollination. Following dispersal from the parent plant, the seeds exhibit a low capacity for germination and typically require an additional year to meet their moist chilling requirements and break dormancy. Biochemical analyses were undertaken in order to address whether seed dormancy is imposed and maintained because the embryo or megagametophyte is immature at the time of seed shedding and hence requires time to complete developmental events before dormancy can be terminated. Major protein reserves of the embryo and megagametophyte are the buffer-insoluble crystalloid (legumin) storage proteins and the water-soluble albumin proteins. SDS-PAGE, fluorography of in vivo synthesized proteins and Western blot analyses showed that the greatest increase in protein reserve synthesis and accumulation occurred between the first and second years of development; deposition of soluble and insoluble storage protein was largely completed in seeds of second-year cones by August, 2-3 months prior to seed dispersal. The period associated with greatest accumulation of storage proteins was accompanied by an increased accumulation of two ER-resident proteins associated with post-translational maturation of storage proteins (binding protein and protein disulphide isomerase). Accumulation of proteins implicated in the acquisition of desiccation tolerance (dehydrins and the tonoplast intrinsic protein, -TiP) occurred between the first and second years of development. Several heat-stable proteins and some of the proteins associated with late development continued to be synthesized after seed shedding and in 13 d moist-chilled mature seeds. However, this did not include the major dehydrin-like protein of yellow cedar seeds. Further, the continued synthesis of heat-stable proteins does not appear to be a factor preventing the germination of yellow cedar seeds following dispersal from the parent plant; rather, the mechanism of dormancy is primarily coat-imposed.     

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover

In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS–PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS–PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.     

Changes in growth,proteins and free amino acids of developing seed and pod of fenugreek

The growth and development under field conditions of the pods and seeds of two cvs of Trigonella foenum graecum are described. Samples were harvested at different stages of ripeness for the determination of dry matter, protein and free amino acid content. During maturation, reserves of solutes are established in the pod wall before the seeds begin their exponential phase of growth. Later, these reserves disappear, providing about 20% of the seed's requirements for nitrogen. SDS-electrophoresis was used to follow the formation of proteins and it was shown that the synthesis of storage proteins takes place prior to dehydration of the seed. Production soluble nitrogenous compounds precedes protein accumulation. Free amino acids follow the same pattern. 4-Hydroxyisoleucine represents nearly 80% of free amino acid of dry seeds. The concentration does not decrease in the later stages of maturation of the seed but this unusual amino acid is absent in the storage proteins of the seeds.