A Secreted Factor Inducs Cell Expansion and Formation of Metaxylem-Like Tracheary Elements in Xylogenic Suspension Cultures of Zinnia

Conditioned medium from mesophyll cell-suspension cultures of Zinnia elegans L. has striking effects on cell expansion and tracheary element differentiation when applied to cultures of freshly isolated mesophyll cells. These effects include (a) induction of early cell expansion, (b) delay in differentiation by 48 h or more, (c) reduction in the synchrony of differentiation, and (d) early formation of very large, metaxylem-like tracheary elements. Like reduced osmotic potential and buffering at pH 5.5, conditioned medium appears to have its primary effect on cell expansion. Partial characterization of the expansion-inducing factor indicates that it is heat stable, of low molecular mass, and is resistant to protease. It also binds reversibly to concanavalin A but is not adsorbed by charcoal. We suggest that the secreted factor may be an oligosaccharide involved in the coordination of cell expansion and differentiation and the regulation of the protoxylem-like to metaxylem-like transition in xylogenic suspension cultures.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.     

The molecular evolution of sperm bindin in six species of sea urchins (Echinoida: Strongylocentrotidae)

The acrosomal protein bindin attaches sperm to eggs during sea urchin fertilization. Complementary to ongoing functional biochemical studies, I take a comparative approach to explore the molecular evolution of bindin in a group of closely related free-spawning echinoid species. Two alleles of the mature bindin gene were sequenced for each of six species in the sea urchin family Strongylocentrotidae. The nucleotide sequences diverged by at least 1% per Myr at both silent and replacement sites. Two short sections flanking the conserved block show an excess of nonsynonymous substitutions. Each is homologous to a region that had been identified as a target of selection in other sea urchin comparisons. A large proportion of the bindin-coding sequence consists of a highly variable repeat region. Bindin sequences, even including the large intron, could not resolve the branching order among five of the species.      

Seed morphology and anatomy of Austrotaxus spicata (Taxaceae) and its systematic position

The anatomy and ultrastructure of seed envelopes of a New Caledonian endemic Austrotaxus spicata were examined for the first time. The systematic position and phylogenetic relations of Austrotaxus were analysed in light of these data. The structure of aril and spermoderm were investigated to demonstrate the similarities with Phyllocladus as well as with Taxus and Pseudotaxus . On the basis of all female reproductive organ characters, Austrotaxus appeared to be fairly isolated and its placing in the independent family Austrotaxaceae was confirmed from the standpoint of comparative anatomy of the seed coat. Taking into consideration that the heterobathmy of features can be the most distinctively traced in the structure of reproductive organs, evaluating the extent of evolutionary advancement of Austrotaxus seems to be rather difficult. However, it is evident that the relationship of Austrotaxus either with Taxaceae or with Podocarpaceae s.l . is considerably remote.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 145 , 437–443.     

Biotechnological improvement of cotton fibre maturity

This mini-review focuses on the prospects and tools for controlling cotton fibre secondary wall thickness. Cotton fibre secondary walls are composed of almost 100% cellulose, and are responsible for fibre maturity and a large component of fibre yield. Improved fibre yield and maturity would result from the ability to control secondary wall cellulose deposition quantitatively, including making the process less sensitive to environmental stress. Both genetic engineering and marker-assisted breeding are possible avenues for effecting such improvements, but first key genes that participate in the regulation and control of secondary wall cellulose biogenesis must be identified. Recent advances towards understanding and manipulating cotton fibre secondary wall deposition that are discussed here include: (i) experimental approaches to identify metabolic participants in cellulose biogenesis; (ii) isolation and characterization of promoters to drive foreign gene expression preferentially during secondary wall deposition; and (iii) a novel set of cDNA sequences representing genes that are differentially expressed during cotton fibre secondary wall deposition compared with primary wall deposition.     

Structure and organization of the bovine beta-globin genes

Genomic clones spanning the entire cow beta-globin gene locus have been isolated and characterized. These clones demonstrate that the linkage of embryonic-like (epsilon) genes and pseudogenes (psi) to the previously described fetal (gamma) and adult (beta) genes is as follows: 5'-epsilon 3-epsilon 4-psi 3-beta-epsilon 1-epsilon 2-psi 1- psi 2-gamma-3'. Present data indicate that, like that of the goat, the fetal and adult genes arose via block duplication of an ancestral four- gene set: epsilon-epsilon-psi-beta. This duplication event preceded the divergence of cows and goats, which occurred greater than or equal to 18-20 Myr ago. However, cows do not have the additional four-gene block containing a preadult/stress globin gene (beta C). Furthermore, the cow fetal cluster contains an extra beta-like pseudogene, which apparently arose by a small-scale duplication. The fixation of this duplication may indicate a possible evolutionary role for pseudogenes.      

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors

Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.     

Abscisic Acid Control of Lectin Accumulation in Wheat Seedlings and Callus Cultures : Effects of Exogenous ABA and Fluridone

Wheat germ agglutinin is found in wheat embryos and a similar lectin is present in the roots of older plants. We report here that 10 micromolar abscisic acid (ABA) produces an average two to three-fold enhancement in the amount of lectin in the shoot base and the terminal portion of the root system of hydroponically grown wheat seedlings. Although ABA stunts seedling growth, a similar growth inhibition produced by ancymidol is not accompanied by elevated lectin levels. To further clarify the role of ABA, wheat callus cultures were employed. Callus derived from immature embryos was grown on growth medium containing various combinations of ABA and 2,4-dichlorophenoxyacetic acid. Those grown in the presence of 10 micromolar ABA exhibit the largest increases in lectin compared to material grown on other regimes. The involvement of ABA in lectin accumulation was further probed with fluridone, an inhibitor of carotenoid synthesis which has also been linked to depressed levels of endogenous ABA. Wheat seedlings grown in the presence of 1 or 10 milligrams per liter fluridone have few or no carotenoids, and wheat germ agglutinin levels in the shoot base and roots are lower compared to controls. The greatest effect (a 39% reduction in the shoot base) is produced at an herbicide concentration of 10 milligrams per liter. Exogenous 10 micromolar ABA greatly stimulates lectin accumulation in the presence of fluridone, but the levels are not as high as those produced by ABA alone. These results indicate that lectin synthesis is under ABA control in both wheat embryos and adult plants.