Rabbit bone collagenase inhibitor blocks the activity of other neutral metalloproteinases.

Rabbit bone explants produce a collagenase inhibitor during the first few days in culture and later produce neutral metallo-proteinases, one of which is collagenase. We present evidence that the collagenase inhibitor also blocks the activity of the other neutral metalloproteinases (after activation by 4-amino-phenylmercuric acetate), indicating that one inhibitor may function as a single regulator for all the metalloproteinases involved in connective tisue catabolism.     

Arylamidase activity of neutral proteinase from Saccharomonospora canescens. Comparison with other Zn-proteinases that exhibit the same activity

The arylamidase activity of Zn-proteinase from Saccharomonospora canescens (NPS) was studied with series of peptide nitroanilides of varying amino acid sequence and N-acyl blocking groups. The partial mapping of the enzyme S(1), S(2), S(3), S(4) subsites shows that variations in all positions P(1) to P(4) in the substrate structure affect the catalytic efficiency. The importance of P(4)-S(4) and P(1)-S(1) interactions, which is a characteristic feature of the serine proteinases, is evidenced for the studied Zn-proteinases NPS and serralysin too. The presence of arylamidase activity in the case of Zn-proteinases-astacin EC 3.4.24.21 and serralysin EC 3.4.24.40 is correlated with some specific characteristics of their active site structure: penta-coordinated Zn(2+) and a tyrosyl residue as a fifth ligand to the Zn(2+). It is assumed that this tyrosyl residue plays a role in the productive binding and stabilization of the tetrahedral adduct formed during the reaction of enzyme-catalysed hydrolysis of peptide arylamides of corresponding length and sequence.     

Investigations into seed dormancy in Grevillea linearifolia, G. buxifolia and G. sericea: anatomy and histochemistry of the seed coat

BACKGROUND AND AIMS: Seeds of east Australian Grevillea species generally recruit post-fire; previous work showed that the seed coat was the controller of dormancy in Grevillea linearifolia. Former studies on seed development in Grevillea have concentrated on embryology, with little information that would allow testing of hypotheses about the breaking of dormancy by fire-related cues. Our aim was to investigate structural and chemical characteristics of the seed coat that may be related to dormancy for three Grevillea species. METHODS: Seeds of Grevillea linearifolia, Grevillea buxifolia and Grevillea sericea were investigated using gross dissection, thin sectioning and histochemical staining. Water movement across the seed coat was tested for by determining the water content of embryos from imbibed and dry seeds of G. sericea. Penetration of intact seeds by Lucifer Yellow was used to test for internal barriers to diffusion of high-molecular-weight compounds. KEY RESULTS: Two integuments were present in the seed coat: an outer testa, with exo-, meso- and endotestal (palisade) layers, and an inner tegmen of unlignified sclerenchyma. A hypostase at the chalazal end was a region of structural difference in the seed coat, and differed slightly among the three species. An internal cuticle was found on each side of the sclerenchyma layer. The embryos of imbibed seeds had a water content six times that of dry seeds. Barriers to diffusion of Lucifer Yellow existed at the exotestal and the endotestal/hypostase layers. CONCLUSIONS: Several potential mechanisms of seed coat dormancy were identified. The embryo appeared to be completely surrounded by outer and inner barriers to diffusion of high-molecular-weight compounds. Phenolic compounds present in the exotesta could interfere with gas exchange. The sclerenchyma layer, together with strengthening in the endotestal and exotestal cells, could act as a mechanical constraint.     

BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T-DNA-transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls ( ban ). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT ( Transparent Testa ) genes to two groups according to their epistatic relationship to ban . The flavonoid content of germinated ban and wild-type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild-type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis .     

Aflatoxin contamination in soybeans: role of proteinase inhibitors, zinc availability, and seed coat integrity.

Soybean trypsin inhibitors are thought to ward off pathogens. Studies with aflatoxigenic strains of Aspergillus flavus and A. parasiticus, frequent soybean contaminants, revealed that trypsin inhibitors do not affect the growth of these fungi and aflatoxin production. Further, the availability of zinc, an essential mineral for aflatoxin synthesis that was thought to explain increased aflatoxin accumulation in cooked compared with raw soybeans, was shown to decrease upon cooking. Seed coat integrity, ensuring limited access and a low moisture content, is responsible for the slow colonization of the seed by A. flavus.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

Systematic relevance of seed coat anatomy in the European heathers (Ericeae, Ericaceae)

The anatomy of the seed coat of the European species of tribe Ericeae (Calluna, Daboecia and Erica) of the Ericaceae family was studied, and the taxonomic importance of their characters was analyzed. The seed coat is mostly formed by a one-cell layer with thick, pitted inner walls and thin outer walls that collapse at maturity over the inner walls. The cell junctions are either raised with anticlinal walls up to four times the height of the periclinal walls or are not raised with similar values for the height of both the anticlinal and periclinal walls. Three main cell junction types were found and described. The thickness of the inner walls is variable, but there is a large overlap among the results for different species. Calluna vulgaris is the only species with no pits, and E. multiflora has a pitted pattern on its inner walls, which is distinctive from the rest of the species. Our main results agree with the external seed morphology, and valuable new data were obtained for certain groups such as the E. cinerea-E. terminalis or the E. scoparia complex. The similarities that are found in seed coat characters are not in accordance with the classical taxonomic delimitation of infrageneric groups within Erica.     

In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage

A comprehensive analysis was carried out of the composition of seed coat mucilage from Arabidopsis thaliana using the Columbia-0 accession. Pectinaceous mucilage is released from myxospermous seeds upon imbibition, and in Arabidopsis consists of a water-soluble, outer layer and an adherent, inner layer. Analysis of monosaccharide composition in conjunction with digestion with pectolytic enzymes conclusively demonstrated that the principal pectic domain of both layers was rhamnogalacturonan I, and that in the outer layer this was unbranched. The macromolecular characteristics of the water-soluble mucilage indicated that the rhamnogalacturonan molecules in the outer layer were in a slightly expanded random-coil conformation. The inner, adherent layer remained attached to the seed, even after extraction with acid and alkali, suggesting that its integrity was maintained by covalent bonds. Confocal microscopy and monosaccharide composition analyses showed that the inner layer can be separated into two domains. The internal domain contained cellulose microfibrils, which could form a matrix with RGI and bind it to the seed. In effect, in the mum5-1 mutant where most of the inner and outer mucilage layers were water soluble, cellulose remained attached to the seed coat. Immunolabeling with anti-pectin antibodies indicated the presence of galactan and arabinan in the inner layer, with the latter only present in the non-cellulose-containing external domain. In addition, JIM5 and JIM7 antibodies labeled different domains of the inner layer, suggesting the presence of stretches of homogalacturonan with different levels of methyl esterification.     

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.     

Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading

Uptake of ¹⁴C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)^?1·min^?1·M^?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)^?1·min ^?1·M ^?1) and mannitol (k = 5.1 μmol·(g FW)^?1·min^?1·M^?1). For glucose an additional saturable system was found (K_m = 0.26 mM, V _max = 4.2 nmol·(g FW)^?1·min^?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10^?7 cm·s^?1) is more than 1 × 10⁶-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo.     

Systematic and phylogenetic significance of the seed coat in the shrubby African Iridaceae, Nivenia, Klattia and Witsenia

MANNINGJ. C. & GOLDBLATT, P., 1991. Systematic and phylogenetic significance of the seed coat in the shrubby African Iridaceae, Nivenia, Klattia and Witsenia. The seeds of Nivenia, Klattia and Witsenia, a natural alliance within Nivenioideae, are among their most distinctive features, and seed number and shape constitute at least two synapomorphies for this monophyletic lineage. In this paper we survey the structure of the seed surface of five species of Nivenia and one each of Klattia and Witsenia by scanning electron microsopy (SEM), and study the development of the seed coat in a representative species of each of the three genera. Outgroups for comparison were selected from genera inside and outside the subfamily. The six genera of Nivenioideae are united in having a 3 (– 2) seriate outer integument but the shrubby genera are unique in the family in their tangentially flattened ovules which mature into depressed, flat, scutiform seeds. They display a range of variation in seed coat characters but are specialized in the subfamily and family in having a transparent outer integument. Other genera have pigmented deposits in the outer epidermis of the outer integument. Nivenia and Witsenia are further specialized in having a partially exfoliating outer integument. Klattia is unique among the shrubby genera in having deposits in the second layer of the tegmen, in retaining an intact outer epidermis of the tegmen, and in the fusiform shape of the epidermal cells and striate surface of the seed. Our results are incorporated in a cladogram that expresses our current understanding of the phylogenetic relationships of Nivenioideae.