1m) and day 5 (Fig. 5e). Read from thousands of the leading scholarly journals from SpringerNature, Wiley-Blackwell, Oxford University Press and more. Protein bodies in the endosperm of castor bean seeds (Ricinus communis L.) contain phytin globoids and protein crystalloids embedded in an amorphous proteinaceous matrix.The protein bodies are apparently surrounded by a single membrane. They might represent either the senescing nucellus, which surrounds the embryo sack and collapses as soon as seed maturation is completed, or an integument developing into the seed coat. Nuclear DNA fragmentation in germinating Ricinus endosperm. To get new article updates from a journal on your personalized homepage, please log in first, or sign up for a DeepDyve account if you don’t already have one. Endosperm, the tissue that surrounds and nourishes the embryo in the angiosperm seed. It is the final phase of plant vegetative and reproductive development, preceding the widespread death of cells and organs. Sections were labeled with antibodies against Cys-EP (a) or mitochondrial malate dehydrogenase (b). From days 1 to 5 of germination, glyoxysomes develop rapidly in the endosperm cells and mobilize triglycerides of the oleosomes. Sections from 3-day-old (a–e) and 5-day-old (f–k) germinating endosperm were labeled with α-Cys-EP antibody (a, c, d, f, h, and i), the preimmune serum (b and g), and the α-propeptide antibody (e and k) by using a FITC-labeled secondary antibody. Furthermore, we found a similar cysteine endoprotease-containing organelle in senescing Hemerocallis flowers. Endosperm tissue from Ricinus contains an additional organelle, only slightly smaller than glyoxysomes. (b) Preimmune serum. Ricinosomes were identified in this tissue by electron microscopy and immunocytochemistry. Functionally, PCD differs from degenerative death or necrosis in its nature and biological significance (2). Western blot analysis revealed the 45-kDa proform of Cys-EP in petals, but not in the ovary (data not shown). Endosperm: Onion, castor bean, groundnut, tomato and cereal grains are examples of the endospermic seeds. Production of the propeptide was induced by 1 mM isopropyl β-d-thiogalactopyranoside, and the protein was purified by using Talon metal affinity resin (CLONTECH) according to the manufacturer’s instructions. The protein bodies were isolated by grinding and centrifuging in glycerol. Nuclear DNA was counterstained with 0.1 μg/ml of 4,6-diamidino-2-phenylindole (DAPI; Sigma) for 5 min at room temperature, washed with PBS, and mounted in fluorescence medium. Castor beans store oil and proteins in a living endosperm laterally attached to the cotyledons. This finding parallels the quantification of ricinosomes at day 5 under EM (Table 1). Bo Tian. [Scale bar: (a) 1 cm; (b and c) 0.75 μm; and (d and e) 0.25 μm.]. The incorporation required calcium ions and showed an optimum pH of 7.8 in 2 m m CaCl 2 . (a) Cys-EP is detected (arrows) in the outer part of the endosperm and close to the cotyledons in 3-day-old seedlings. A 315-bp fragment encoding the propeptide of the castor bean endopeptidase (22) was amplified from the cDNA by PCR, changing F21M for the translation start. Arrowheads in c–e and h indicate single ricinosomes. 5 a–c). Most animal cysteine proteases associated with apoptosis cleave after an aspartic acid residue (8) and therefore have been termed caspases. Protein was extracted from developing seeds at stages I–V. Nuclear DNA was not detected by DAPI staining or TUNEL assay in the collapsed endosperm cells near the cotyledons at day 4 (Fig. In some groups (e.g. One mechanism in animals involves leakage of cytochrome c from mitochondria, regulated by BCL-2 and BAX. Search 9. nutritive tissue is endosperm The castor bean (Ricinus) seed has both endosperm and cotyledon. Tissue was fixed, embedded, and stained as described (22), but castor bean endosperm was dehydrated in an ethanol series instead of chemical dehydration with acidified dimethoxypropane. Mitochondria profiles, in contrast, were equally frequent throughout all layers of the endosperm. By the time the nuclear DNA was susceptible to heavy label by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, the ricinosomes had released into the cytoplasm their content of cysteine endoprotease, which became activated because of the cleavage of its propeptide. 1i). Cotyledons are formed from a) embryos b) seeds c) ovaries d) ovules 7. The specificity of the anti-propeptide antibody was checked by Western blot analysis (data not shown). Developing castor bean (Ricinus communis L.) seeds accumulate lipids and storage proteins in their endosperm. Castor bean (Ricinus communis), unlike Arabidopsis (Arabidopsis thaliana), endosperm is persistent throughout seed development, providing an excellent model in which to dissect the mechanism of endosperm genomic hypomethylation in dicots. In particular, DNA is degraded and cysteine proteases are expressed or activated (5, 6). Preimmune serum was used for the negative control. discover and read the research Ricin is found exclusively in the endosperm of castor seeds and is classified as a type 2 ribosome-inactivating protein. In plants, PCD is essential and occurs during pathogenesis, xylogenesis, reproduction, and senescence (4). Members of this subgroup were identified in senescing flowers of daylily (Hemerocallis) (12) and Christmas bells (Sandersonia aurantiaca; AF133839). Coat protein II-coated vesicles, which transport proteins from the ER to the cis-Golgi, bud off from the ER (31), and a similar process gives rise to oil bodies (32). The protein was further purified by SDS/PAGE, and immunization of rabbits was carried out by using the antigen in acrylamide (Eurogentec, Brussels). Nuclear DNA fragmentation during PCD can be detected by reagents that react with the exposed 3′ hydroxyls on the nucleosomal units (Fig. On day 6, when the oil reserves are depleted, the endosperm becomes increasingly slimy and detaches from the cotyledons. You can change your cookie settings through your browser. Ricinosomes were detected in the outer layer of the endosperm at day 3 (Table 1 and Fig. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. They accumulate later than the cysteine endoproteases involved in mobilization of storage proteins. Image credit: Aurora Fernández Durán (photographer). At day 3 only the 45-kDa proform was detected, but by day 4 the mature form started to appear in the inner layer. The reserve food material in castor seed mostly occurs in the form of (a) carbohydrates (b) proteins (c) oil (d) sugar. In germinating seedlings, the radicula emerges on day 2 (Fig. The detection of ricinosomes is concomitant with the appearance of TUNEL-positive nuclei in senescing endosperm and parallels their progression across the tissue. Type 2 ribosome-inactivating proteins such as ricin from castor oil are lectins, which irreversibly inactivate ribosomes, thus stopping protein synthesis and eventually leading to cell death. At day 2 no nuclear DNA fragmentation was detected; the labeling near the cotyledons was the result of unspecific incorporation of labeled nucleotides, because it also was found in the negative control (Fig. Our findings not only demonstrate that lncRNAs play potential roles in regulating the development of castor bean endosperm and embryo, but also provide novel insights into the parental effects, … Developing castor oil seeds were harvested 32 to 40 days after anthesis. Counterstaining of nucleic DNA with DAPI demonstrates that nuclei are present in all cells except the collapsed endosperm cells (Fig. A comparison of PCD in plants and animals reveals obvious similarities. 1 d, i, o, q, u, and w). Castor seeds have persistent endosperm. Answer: (c) oil. We now have shown that senescing Hemerocallis petals contain a ricinosome-like organelle that is labeled by the antibody to Ricinus Cys-EP. After electroblotting on nitrocellulose, blots were blocked with 1% (wt/vol) BSA in Tris-buffered saline (TBS; 150 mM NaCl) for 1 h at room temperature and incubated with primary antibody raised against the purified 35-kDa Cys-EP (diluted 1:1,000 in blocking solution) for an additional hour. Castor bean oil was used by the Egyptians as a laxative; a paste made from the seeds was also used as a treatment for toothache. Nonspecific sites were blocked with 2% BSA in PBS containing 0.02% sodium azide and 0.05% Tween-20 for 1 h at room temperature. A similar endoprotease (EP-C1) is found in late stages of fruit maturation of Phaseolus vulgaris in ripening pods and is thought to be responsible for the degradation of proteins to be translocated as amino acids to developing seeds (14). A dramatic increase of ricinosomes took place at day 3 with 11.7 ricinosomes per cell section in the inner layer of the endosperm, when the fragmentation of the nuclear DNA began (Fig. There may be also other FAD2 homologs in castor that were not detectable in our EST analyses since we used mRNA from a specific stage of endosperm development. Endosperm with attached cotyledons was fixed in PBS-buffered formaldehyde [4% formaldehyde (wt/vol)] at 4°C overnight and dehydrated in a graded ethanol series.