Characterization of somatic embryogenesis in Pelargonium××hortorum mediated by a bacterium

Several cultivars of hybrid seed geranium (Pelargonium×hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos at high frequency when explants were co-cultivated with a morphogenesis promoting bacterium. This bacterium was isolated as an in vitro contaminant from cultures of geranium seedling explants and identified as belonging to the genus Bacillus and species circulans. Co-cultivation of hypocotyl explants with the bacterium promoted somatic embryo formation and improved both the frequency and quality of somatic embryos. In the cultivar Ringo Rose, the least responsive among the cultivars screened, the embryogenic response was more than four times that of axenic cultures. Nearly 70% of these embryos converted into plantlets, while the somatic embryos induced under axenic conditions developed poorly and plantlet formation was inconsistent. Among the different treatments of bacterial culture tested (autoclaved culture, culture filtrate, sonicated bacterial culture, sonication of bacterial culture followed by filtration, HPLC fractionation of crude bacterial lysate), only two HPLC fractions promoted embryogenesis to a marginal degree. Co-cultivation of the explants with bacterium during the first week of induction was crucial for obtaining high-frequency embryogenesis, indicating the role of bacterial stimuli during the induction process. Received: 23 June 1998 / Revision received: 20 August 1998 / Accepted: 27 October 1998     

Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases.

We used the N-terminal amino acid sequence of dihydrolipoamide dehydrogenase from Haloferax volcanii, to design and synthesize two oligonucleotide probes that were used to identify and clone a 4.3 kilobase pair (kbp) fragment from MboI restriction endonuclease digestion of Hf. volcanii genomic DNA. The nucleotide sequence of a 1.5-kbp region of this clone was determined and this revealed an open reading frame that translated into a protein with good homology to dihydrolipoamide dehydrogenase from other sources. The first 48 amino acids were identical with the N-terminal sequence data obtained from the purified protein. The complete primary structure of the halophilic dihydrolipoamide dehydrogenase was analyzed in terms of its homologies to dihydrolipoamide dehydrogenases from other sources and its molecular adaptations to high intracellular ionic strength.     

A Crucial Role for Gibberellins in Stress Protection of Plants

The hypothesis that there is an intimate relationship betweengibberellin levels and plant stress protection has been testedusing near-isogenic lines of a normal and dwarf barley (Hordeumvulgare L.). Application of paclobutrazol (a triazole), inducedstress protection in the normal line and application of GA₃to the dwarf reversed the inherent stress tolerance. Reversalof the dwarf phenotype by specific gibberellins (GAs) suggeststhat the conversion of GA₂₀ to GA₁ and GA₉ to GA₄ has been compromised.These observations indicate that modulation of specific GAsplays a key role in stress protection. The preferential useof non-chemical technologies including enzyme regulation, phytochromeA overexpression, and breeding for induction of stress tolerancein plants are discussed. (Received November 26, 1998; Accepted March 11, 1999)     

Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate.

The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.     

Ca(2+) and Zn(2+) binding properties of peptide substrates of vertebrate collagenase, MMP-1.

To understand the role of Ca(2+) in vertebrate in the structure and action of collagenase, we have examined peptides that interact with recombinant human fibroblast collagenase for their affinities towards Ca(2+) and Zn(2+) in a non-polar solvent. Two of the peptides, GPQGIAGQ and GNVGLAGA, had sequences in collagen which are, respectively, cleaved and not cleaved by collagenase. A third peptide, PSYFLNAG, had a collagenase-cleaved sequence in ovostatin, a globular protein substrate. Peptides TVGCEECTV and CLPREPGL were derived from TIMP-1; the former competitively inhibits collagenase while the latter does not. The relative rates of hydrolysis of the peptides by collagenase had the order GPQGIAGQ>PSYFLNAG>GNVGLAGA. Circular dichroism spectral data in trifluoroethanol showed that while the TIMP control peptide, CLPREPGL, bound only Zn(2+), the other four peptides bound both Ca(2+) and Zn(2+) with definite stoichiometries. Ca(2+) could displace Zn(2+) in the substrate peptides while Zn(2+) displaced Ca(2+) in the TIMP peptide. GPQGIAGQ, PSYFLNAG and TVGCEECTV formed peptide:Ca(2+):Zn(2+) ternary complexes. Our results suggest that both collagen and globular protein substrates of collagenase may bind Ca(2+) and Zn(2+) in the enzyme's active site. This, in turn, may account for the known importance of the non-catalytic Ca(2+) and Zn(2+) in collagenase activity.     

Dihydrolipoamide dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.

Dihydrolipoamide dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.