Incorporating mixed rations and formulated grain mixes into the diet of grazing cows: Effects on milk composition and coagulation properties, and the yield and quality of Cheddar cheese.

Effects of different strategies for feeding supplements to grazing dairy cows on the composition and coagulation properties of milk and the subsequent yield and quality of Cheddar cheese were measured. The experiment used milk from 72 Holstein-Friesian cows, averaging 45d in milk, fed according to 1 of 3 feeding strategies: (1) cows grazed a restricted allowance of perennial ryegrass pasture [approximately 14kg of dry matter (DM)/cow per day, to ground level] supplemented with milled wheat grain fed in the milking parlor and alfalfa hay offered in the paddock (control); (2) same pasture and allowance as control, supplemented with a formulated grain mix containing wheat grain, corn grain, and canola meal fed in the parlor and alfalfa hay fed in the paddock (FGM); or (3) same pasture and allowance as control, supplemented with a partial mixed ration comprising the same formulated grain mix but mixed with alfalfa hay and presented on a feed pad after each milking (PMR). For all strategies, supplements provided the same metabolizable energy and grain:forage ratio (78:22, DM basis). Within each feeding strategy, milk was sampled from cows receiving either 8 or 16kg (DM) of supplement/cow per day. There were 2 replicated groups of 6 cows per supplement amount per dietary strategy; approximately 250L of milk was sampled from each for analyses of composition and coagulation properties and the manufacture of Cheddar cheese. The experiment had a 14-d adaptation period and a 14-d measurement period. For cows fed according to the control strategy, those fed 16kg/cow per day produced milk with lower concentrations of milk fat than cows fed 8kg/cow per day. This effect was not observed for cows fed according to the FGM and PMR strategies. Milk from cows fed 16kg of DM/cow per day according to the control strategy yielded less Cheddar cheese than milk from cows fed according to the PMR strategy, with cheese yields from FGM cows being intermediate. Amount of supplement offered had minor effects on percentages of some fatty acids. We observed few other effects of feeding strategy on milk composition, types of milk protein, milk coagulation properties, or the composition and quality of the resultant Cheddar cheese. These data show that, compared with the traditional control strategy, feeding PMR or FGM may increase milk fat concentration and the subsequent yield of Cheddar cheese without compromising cheese composition or quality.

Milk production responses to different strategies for feeding supplements to grazing dairy cows.

Milk production responses of grazing cows offered supplements in different ways were measured. Holstein-Friesian cows, averaging 45 d in milk, were allocated into 8 groups of 24, with 2 groups randomly assigned to each of 4 feeding strategies. These were control: cows grazed a restricted allowance of perennial ryegrass pasture supplemented with milled wheat grain fed in the milking parlor and alfalfa hay offered in the paddock; FGM: same pasture and allowance as the control supplemented with a formulated grain mix containing wheat grain, corn grain, and canola meal fed in the parlor and alfalfa hay fed in the paddock; PMRL: same pasture and allowance as the control, supplemented with a PMR consisting of the same FGM but mixed with alfalfa hay and presented on a feed pad after each milking; and PMRH: same PMR fed in the same way as PMRL but with a higher pasture allowance. For all strategies, supplements provided the same metabolizable energy and grain:forage ratio [75:25, dry matter (DM) basis]. Each group of 24 cows was further allocated into 4 groups of 6, which were randomly assigned to receive 8, 12, 14, or 16 kg of DM supplement/cow per d. Thus, 2 replicated groups per supplement amount per dietary strategy were used. The experiment had a 14-d adaptation period and a 14-d measurement period. Pasture allowance, measured to ground level, was approximately 14 kg of DM/d for control, FGM, and PMRL cows, and 28 kg of DM/d for the PMRH cows, and was offered in addition to the supplement. Positive linear responses to increasing amounts of supplement were observed for yield of milk, energy-corrected milk, fat, and protein for cows on all 4 supplement feeding strategies. Production of energy-corrected milk was greatest for PMRH cows, intermediate for FGM and PMRL cows, and lowest for control cows. Some of these differences in milk production related to differences in intake of pasture and supplement. Milk fat concentration decreased with increasing amount of supplement for all feeding strategies, but the decline was most marked for the control cows. Milk protein concentration increased for all groups as the amount of supplement increased, but was greater for FGM, PMRL, and PMRH cows than control cows. It is concluded that when supplements are fed to grazing dairy cows, inclusion of corn grain and canola meal can increase milk production even at similar metabolizable energy intakes, and that it does not matter whether these supplements are fed as a PMR or in the parlor and paddock.

Effects of partial mixed rations and supplement amounts on milk production and composition, ruminal fermentation, bacterial communities, and ruminal acidosis.

Late-lactation Holstein cows (n=144) that were offered 15kg dry matter (DM)/cow per day of perennial ryegrass to graze were randomized into 24 groups of 6. Each group contained a fistulated cow and groups were allocated to 1 of 3 feeding strategies: (1) control (10 groups): cows were fed crushed wheat grain twice daily in the milking parlor and ryegrass silage at pasture; (2) partial mixed ration (PMR; 10 groups): PMR that was isoenergetic to the control diet and fed twice daily on a feed pad; (3) PMR+canola (4 groups): a proportion of wheat in the PMR was replaced with canola meal to produce more estimated metabolizable protein than other groups. Supplements were fed to the control and PMR cows at 8, 10, 12, 14, or 16kg of DM/d, and to the PMR+canola cows at 14 or 16kg of DM/d. The PMR-fed cows had a lower incidence of ruminal acidosis compared with controls, and ruminal acidosis increased linearly and quadratically with supplement fed. Yield of milk fat was highest in the PMR+canola cows fed 14 or 16kg of total supplement DM/d, followed by the PMR-fed cows, and was lowest in controls fed at these amounts; a similar trend was observed for milk fat percentage. Milk protein yield was higher in the PMR+canola cows fed 14 or 16kg of total supplement DM/d. Milk yield and milk protein percentage were not affected by feeding strategy. Milk, energy-corrected milk, and milk protein yields increased linearly with supplement fed, whereas milk fat percentage decreased. Ruminal butyrate and d-lactate concentrations, acetate-to-propionate ratio, (acetate + butyrate)/propionate, and pH increased in PMR-fed cows compared with controls for all supplement amounts, whereas propionate and valerate concentrations decreased. Ruminal acetate, butyrate, and ammonia concentrations, acetate-to-propionate ratio, (acetate + butyrate)/propionate, and pH linearly decreased with amounts of supplement fed. Ruminal propionate concentration linearly increased and valerate concentration linearly and quadratically increased with supplement feeding amount. The Bacteroidetes and Firmicutes were the dominant bacterial phyla identified. The Prevotellaceae, Ruminococcaceae, and Lachnospiraceae were the dominant bacterial families, regardless of feeding group, and were influenced by feeding strategy, supplement feeding amount, or both. The Veillonellaceae family decreased in relative abundance in PMR-fed cows compared with controls, and the Streptococcaeae and Lactobacillaceae families were present in only minor relative abundances, regardless of feeding group. Despite large among- and within-group variation in bacterial community composition, distinct bacterial communities occurred among feeding strategies, supplement amounts, and sample times and were associated with ruminal fermentation measures. Control cows fed 16kg of DM of total supplement per day had the most distinct ruminal bacterial community composition. Bacterial community composition was most significantly associated with supplement feeding amount and ammonia, butyrate, valerate, and propionate concentrations. Feeding supplements in a PMR reduced the incidence of ruminal acidosis and altered ruminal bacterial communities, regardless of supplement feeding amount, but did not result in increased milk measures compared with isoenergetic control diets component-fed to late-lactation cows.

Effects of different strategies for feeding supplements on milk production responses in cows grazing a restricted pasture allowance.

Milk production responses of grazing cows offered supplements in different ways were measured. Holstein-Friesian cows, averaging 227 d in milk, were allocated into 6 groups of 36, with 2 groups randomly assigned to each of 3 feeding strategies: (1) cows grazed perennial ryegrass pasture supplemented with milled barley grain fed in the milking parlor and pasture silage offered in the paddock (control); (2) same pasture and allotment supplemented with the same amounts of milled barley grain and pasture silage, but presented as a mixed ration after each milking (PMR 1); and (3) same pasture and allotment, supplemented with a mixed ration of milled barley grain, alfalfa hay, corn silage, and crushed corn grain (PMR 2). For all strategies, supplements provided the same metabolizable energy and grain:forage ratio. [75:25, dry matter (DM) basis]. Each group of 36 cows was further allocated into 4 groups of 9, which were assigned to receive 6, 8, 10, or 12 kg of supplement DM/cow per day. Thus, there were 2 replicated groups per supplement amount per dietary strategy. The experiment had a 14-d adaptation period and an 11-d measurement period. Pasture allotment was approximately 14 kg of DM/d for all cows and was offered in addition to the supplement. Positive quadratic responses to increasing amounts of supplement were observed for yield of milk, energy-corrected milk (ECM), and fat and protein, and positive linear responses for concentrations of fat and protein for cows on all 3 supplement feeding strategies. No difference existed between feeding strategy groups in yield of milk, ECM, or protein at any amount of supplement offered, but yield and concentration of fat was higher in PMR 2 cows compared with control and PMR 1 cows at the highest amounts of supplementation. Responses in marginal ECM production per additional kilogram of supplement were also greater for PMR 2 than control and PMR 1 cows when large amounts of supplement were consumed. For all diets, marked daily variation occurred in ruminal fluid volatile fatty acids and pH, especially in cows fed the largest amounts of supplement. It was concluded that when supplements are fed to grazing dairy cows, a simple mix of grain and pasture silage has no benefit over traditional strategies of feeding grain in the parlor and forage in the paddock. However, yield of milk fat and marginal milk production responses can be greater if the strategy uses an isoenergetic ration that also contains alfalfa hay, corn silage, and corn grain.

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens.

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White embedded tissues to be sectioned at 10 microm providing excellent signal contrast.

A vegetative storage protein homolog is expressed in the growing shoot apex of hybrid poplar.

The ability of poplars (Populus deltoides Bartr. ex Marsh., and Populus trichocarpa Torr. and Gray) to sequester nitrogen in stems in preparation for winter has been associated with the massive accumulation of protein bodies in the bark and xylem ray parenchyma. These protein bodies contain a bark storage protein (BSP) that can account for up to 30% of the total soluble bark protein during the winter months. Perhaps the plant's ability to efficiently cycle nitrogen through BSP is an important aspect of its growth potential. Sequence analysis of BSP led to the identification of a leaf-associated homolog, win4, which was initially isolated because its transcript increased in abundance upon mechanical wounding. The goal of this work was to characterize this putative leaf-associated vegetative storage protein, and determine whether it might perform a storage role in vivo. Antibodies, produced against protein synthesized upon over-expression of the win4 coding region in Escherichia coli, were used to examine the relative abundance of WIN4 protein in response to supplemental nitrogen, and during development. The transcript and protein were most abundant in the youngest leaves and also increased with nitrogen fertilization. Immunolocalization of the protein was performed and showed that WIN4 was associated with cells surrounding the vasculature, and cells of the lower epidermis and stipules of immature leaves. Under moderate nitrogen fertilization regimes, WIN4 accounted for only about 2% of total soluble leaf protein; however, given the cellular specificity and enhancement with nitrogen, the protein is regulated in a manner similar to other vegetative storage proteins. Since poplar is amenable to DNA transformation and regeneration, it is now possible to ask direct questions about the role these proteins play in nitrogen storage in rapidly expanding or in dormant tissue. This type of analysis could determine whether these proteins mainly ameliorate the toxic effects of excess nitrogen, if they are instrumental in controlling nitrogen allocation or if they simply represent an efficient method for sequestering this valuable nutrient.

Methyl jasmonate treatment eliminates cell-specific expression of vegetative storage protein genes in soybean leaves.

Soybean (Glycine max) plants accumulate a vacuolar glycoprotein in the parenchymal cells of leaves, petioles, stems, seed pods, and germinating cotyledons that acts in temporary nitrogen storage during vegetative growth. In situ immunolocalization of this vegetative storage protein (VSP) revealed that it accumulates in those parenchymal cells in close proximity to existing and developing vasculature, as well as in epidermal and cortical cells. The protein was more prevalent in younger, nitrogen-importing tissues before pod and seed development. Removal of actively growing seed pods greatly enhanced VSP accumulation, primarily in bundle sheath and paraveinal mesophyll cells. In situ hybridization of a VSP RNA probe to mRNA in leaf sections demonstrated that cell-specific mRNA accumulation corresponded with the pattern of protein localization. Treatment of leaf explants with 50 micromolar methyl jasmonate resulted in accumulation of VSP mRNA and protein in all cell types.

The 32-Kilodalton Vegetative Storage Protein of Salix microstachya Turz : Characterization and Immunolocalization.

A 32-kilodalton vegetative storage protein, found in Salix microstachya Turz. bark during the overwintering period, was purified and characterized using several polyacrylamide gel electrophoretic procedures. Solubility characteristics and amino acid analyses were also performed. The protein is water soluble, is glycosylated, has no disulfide-bonded subunits, but is composed of a family of isoelectric isomers. The majority of these isomers are basic. Characteristic of storage proteins, the protein is rich in glutamine/glutamate and asparagine/aspartate (28%), the basic nature of the isomers indicating that most of these amino acid residues are in the amide form. The protein was purified using preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and antibodies raised in chickens. Immunoblot analysis suggested an annual cyclic nature of the accumulation and mobilization of this vegetative storage protein. Immunologically, it is related to a similar molecular weight protein found in the bark of Populus deltoides Marsh. but not to any overwintering storage proteins of the other hardwoods tested. Indirect immunolocalization revealed that the protein was sequestered in protein-storage vacuoles in parenchymatous cells of the inner bark tissues of Salix during the winter months.

Seasonally fluctuating bark proteins are a potential form of nitrogen storage in three temperate hardwoods.

The inner bark tissues of three temperate hardwoods contain specific proteins which undergo seasonal fluctuations. Increases in particular proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, occur within the bark of several Acer, Populus and Salix spp. during late summer and early autumn. These proteins are abundant in the bark throughout the winter and their levels decline the following spring. Light and electron microscopy showed that the parenchyma cells of the inner bark are packed with spherical organelles throughout the overwintering period. These organelles are rich in protein and analogous to protein bodies found in cells of mature seeds. The protein bodies of the parenchyma cells are replaced by large central vacuoles during spring and summer, presumably as a result of the mobilization of the storage protein and fusion of the protein bodies. The high levels of specific proteins in inner bark tissues and the presence of protein bodies within the parenchyma cells indicate that the living cells of the bark act as a nitrogen reserve in overwintering temperate hardwoods.

Transport and posttranslational processing of the vacuolar enzyme α-mannosidase in jack-bean cotyledons.

α-Mannosidase (EC 3.2.1.24) is a vacuolar enzyme which occurs abundantly in the cotyledons of the jack-bean (Canavalia ensiformis (L.) DC). The mature enzyme is a tetramer with two polypeptides each of relative molecular mass (Mr) 66000 and Mr 44000. The enzyme has an interesting molecular structure because in its native form, it does not bind to concanavalin A (ConA) in spite of the presence of a high-mannose glycan. α-Mannosidase is synthesized in the developing cotyledons of jack-beans at the same time as the abundant proteins canavalin and ConA. The enzyme is synthesized as a precursor which has an Mr of 110000 and is associated with the endoplasmic reticulum (ER). Antibodies against the deglycosylated subunits cross-react with the Mr-110000 precursor. Processing of the precursor to the constituent polypeptides occurs posttranslationally, probably in the protein bodies. Immunocytochemical evidence shows that α-mannosidase is present in the ER and the Golgi complex of developing cells, and accumulates in the protein bodies.Labeling with [(3)H]glucosamine shows that after processing only the Mr-66000 polypeptide has glucosamine-containing glycans. The synthesis of these glycans is inhibited by tunicamycin, indicating that they are asparagine-linked oligosaccharides. Analysis of the glycans shows that there is a large glycan that is retained by ConA and a small glycan that is not retained by ConA. The large glycan is only partially sensitive to α-mannosidase because of the presence of a terminal glucose residue. Cross-reaction of the large subunit with an antiserum directed against small, complex glycans of plant glycoproteins indicates that this polypeptide probably has a xylose-containing glycan. Pulse-chase experiments carried out in the presence of tunicamycin show that the presence of glycans is not required for transport of α-mannosidase out of the ER-Golgi system.

Phytin is synthesized in the cotyledons of germinated castor-bean seeds in response to exogenously supplied phosphate.

Following germination of the castor bean (Ricinus communis L.) seed, levels of phytin decline in both the endosperm and the embryo. However, as seedling growth continues, phytin increase in the latter to a level exceeding that present in the mature dry embryo, while phytin declines concomitantly in the endosperm. It is likely that phosphate mobilized from phytin in the endosperm acts as a substrate for phytin synthesis in the embryo. This is supported by the observation that isolated embryos supplied with phosphate accumulate phytin, particularly in the cotyledons. This increase is enhanced whenmyo-inositol is provided concurrently as a carbon source. Phytin synthesis in the cotyledons of the isolated embryos can occur without the attached axis. Whether initially exposed to exogenous phosphate or not, the isolated cotyledons remain competent in their ability to synthesize phytin for an extended post-germinative period, even though the major reserves are being mobilized at this time.

Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum.

In this study we examined the effect of heat stress (up to 6 hours at 43 degrees C) on the biosynthesis and transport of phytohemagglutinin (PHA) in cotyledons of developing seeds of the common bean, Phaseolus vulgaris. Heat stress resulted in a decrease of total protein synthesis and an enhancement of the synthesis of heat shock proteins and PHA. Pulse chase experiments showed that a considerable proportion of the newly synthesized PHA was present in the endoplasmic reticulum (ER)/Golgi fraction and did not readily chase-out. Analysis with endoglycosidase H showed that the oligosaccharide sidechains of PHA were almost entirely in the high mannose configuration, indicating that most of the newly synthesized PHA was in the ER. However, some of the PHA became fucosylated at 43 degrees C, indicating fucosyltransferase activity. That the biosynthesis and secretion of fucosyl-containing cell wall polymers proceeded normally at 43 degrees C provided evidence that certain Golgi functions (i.e. transport to the cell wall) remained unaffected by heat stress. The ER obtained from these heat stress cotyledons had a greater density (1.16 g. cm(-3) at 43 degrees C instead of 1.14 g.cm(-3) at 22 degrees C) in sucrose gradients. Ultrastructural observations showed that the width of the lumen of the ER cisternae had increased from 20 nanometers at 22 degrees C to 60 to 80 nanometers at 43 degrees C; the lumen was filled with electrondense material presumed to be protein. The experiments are interpreted as evidence that heat stress imposes a block in the transport of PHA out of the ER. Whether heat stress affects the ER itself or alters the conformation of PHA, thereby preventing its transport, is not clear.

Urease in jack-bean (Canavalia ensiformis (L.) DC) seeds is a cytosolic protein.

Urease (EC 3.5.1.5) is abundantly present in the seeds of many species of Leguminosae. There is at present conflicting information in the literature about its subcellular location and status as a glycoprotein. We have made a study of the subcellular location of urease in jack-bean cotyledons using an immunocytochemical approach; in addition, we studied the biosynthesis and glycoprotein nature of the enzyme using several biochemical approaches. All the results are in agreement with the interpretation that the seed urease is not a glycoprotein, is synthesized on free polysomes, and is present in the cytosol of the storage parenchyma cells.

Sambucus nigra agglutinin is located in protein bodies in the phloem parenchyma of the bark.

The bark of some young woody stems contains storage proteins which are subject to an annual rhythm: they accumulate in the autumn and are mobilized in the spring. We show here that the bark phoem-parenchyma cells of Sambucus nigra L. contain numerous protein bodies, and that the bark lectin (S. nigra agglutinin) which undergoes an annual rhythm is localized in these protein bodies. The protein bodies in the cotyledons of legume seeds also contain lectin, indicating that lectins may be storage compounds themselves or may have a function in storage and-or mobilization processes.

Correct targeting of the bean storage protein phaseolin in the seeds of transformed tobacco.

The storage protein phaseolin accumulates during seed development in protein bodies in cotyledons of the common bean Phaseolus vulgaris. Hall et al. (In L Van Vloten-Doting, TC Hall, eds, Molecular Form and Function of the Plant Genome, 1985 Plenum Press, In press) recently reported the expression of a gene coding for phaseolin and the accumulation of phaseolin protein in developing seeds of tobacco plants regenerated from transformed callus cells. The protein did not accumulate in other organs of the plants. Mature seeds from normal and transformed tobacco plants were obtained and the subcellular distribution of phaseolin in the seeds was examined using both light and electron microscopic immunocytochemical methods. Phaseolin was found in six of seven transformed tobacco embryos examined, but was present in only one endosperm of five. When present, phaseolin was located exclusively in the protein bodies of the embryonic and endospermic cells. Furthermore, phaseolin was restricted solely to the amorphous matrix of the protein bodies and was excluded from the globoid and proteinaceous crystalloid components of these organelles. The subcellular location of phaseolin in seeds from transformed tobacco plants is similar to that seen in mature seeds of the common bean indicating that in the transformed cells the protein is targeted to the right subcellular compartment.

Immunocytochemical localization of phaseolin and phytohemagglutinin in the endoplasmic reticulum and Golgi complex of developing bean cotyledons.

Development of legume seeds is accompanied by the synthesis of storage proteins and lectins, and the deposition of these proteins in protein-storage vacuoles (protein bodies). We examined the subcellular distribution, in developing seeds of the common bean, Phaseolus vulgaris L., of the major storage protein (phaseolin) and the major lectin (phytohemagglutinin, PHA). The proteins were localized using an indirect immunocytochemical method in which ultrathin frozen sections were immunolabeled with rabbit antibodies specific for either PHA or phaseolin. Bound antibodies were then localized using goat-anti-rabbit immunoglobulin G adsorbed onto 4- to 5-nm colloidal gold particles. The sections were post-fixed with OsO4, dehydrated, and embedded in plastic on the grids. Both PHA and phaseolin exhibited a similar distribution in the storage-parenchyma cells, being found primarily in the developing protein bodies. Endoplasmic reticulum and Golgi complexes (cisternal stacks and associated vesicles) also were specifically labeled for both proteins, whereas the cytosol and other organelles, such as mitochondria, were not. We interpret these observations as supporting the hypothesis that the transport of storage proteins and lectins from their site of synthesis, the rough endoplasmic reticulum, to their site of deposition, the protein bodies, is mediated by the Golgi complex.

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling.

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.

Subcellular distribution of phytin in the endosperm of developing castor bean: a possibility for its synthesis in the cytoplasm prior to deposition within protein bodies.

Studies using light and electron microscopy, and energy-dispersive X-ray analysis have allowed us to identify phytin particles within the cytoplasm of the developing endosperm of castor bean (Ricinus communis L.). These particles are present at the time of the formation of globoid particles within the protein bodies, but they are absent from mature tissue with fully formed protein bodies. We suggest that phytin is formed initially in the cytoplasm (perhaps in association with the cisternal endoplasmic reticulum) before being transported to the protein bodies, wherein it condenses to form the globoid.

Deposition of Matrix and Crystalloid Storage Proteins during Protein Body Development in the Endosperm of Ricinus communis L. cv. Hale Seeds.

Protein bodies within the endosperm of castor bean (Ricinus communis L. cv. Hale) seeds arise from numerous small vacuoles which progressively become filled with storage protein, of which the crystalloid proteins make up approximately 70%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the crystalloids are a family of at least four proteins which reduce to two complementary groups after 2-mercaptoethanol treatment. The matrix, which comprises the remainder, has two major components, the soluble albumins and the lectins. The lectins are the only glycoproteins within the mature protein body. Both cytochemical staining and SDS-PAGE indicate that the synthesis of the crystalloid and the majority of matrix proteins begins some 20 days after pollination. Additionally, the crystalloid proteins are synthesized concurrently, whereas there is temporal variation in the synthesis of matrix proteins.

Mineral reserves in castor beans: the dry seed.

Elemental composition and distribution of the mineral reserves in the endosperm and embryo tissues of Ricinus communis cultivars Hale and Zanzibarensis were investigated. Energy dispersive x-ray analysis was used to determine the elemental composition of the globoid crystals, while atomic absorption spectrometry allowed quantification of the elements, particularly Ca, in various seed regions. No major differences were found between the two cultivars with regard to the elemental distribution in globoid crystals. While the majority of globoid crystals contained P, K, and Mg, the occasional one also contained Ca. In extremely rare instances, Fe was detected in globoid crystals. Ca-containing globoid crystals were more common in provascular cell protein bodies in the stem and radicle. Polarized light microscopy, micro-incineration, and acid solubility tests demonstrated the presence of calcium oxalate crystals in the innermost testa which adheres to the endosperm and is often mistakenly identified as endosperm. Atomic absorption spectrometry revealed that most of the calcium present in castor bean seeds is localized in the testa. On a perseed-region basis, the much larger endosperm contains more Ca than does the embryo. However, on a unit-weight basis, the radicle-plus-stem regions contain considerably more Ca than does the cotyledon or endosperm, an observation that is consistent with the observed distribution pattern for Ca-containing globoid crystals.