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In plants, development is a continuing process that takes place under strong fluctuations of the light environment. Here we show that in Arabidopsis thaliana plants grown under intense white light, coupling of the photoreceptor cryptochrome 2 to developmental processes is broader than previously appreciated. Compared to the wild type, the cry2 mutant showed reduced activity of a Lhcb1*2 promoter fused to a reporter, and delayed flowering. The cry2 mutation also reduced the inhibition of hypocotyl growth, the unfolding of the cotyledons, the rate of leaf production during the vegetative phase, and the pace of development after transition to the reproductive stage; but these effects were obvious only in the absence of cryptochrome 1 and in some cases phytochrome A and/or phytochrome B. Complementary, the cry2 mutation uncovered novel roles for cryptochrome 1 and phytochrome A. The activity of the Lhcb1*2 promoter was higher in the cry1 cry2 mutant than in the cry2 mutant, suggesting that cry1 could be involved in blue-light repression of photosynthetic genes. Surprisingly, the phyA cry1 cry2 triple mutant flowered earlier and showed better response to photoperiod than the cry1 cry2 double mutant, indicating that phyA is involved in light repression of flowering. Growth and development were severely impaired in the quadruple phyA phyB cry1 cry2 mutant. We propose that stability and light modulation of development are achieved by simultaneous coupling of phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 to developmental processes, in combination with context-dependent hierarchy of their relative activities.

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Current evidence is inconclusive regarding the point of signaling convergence downstream from different members of the phytochrome family. In transgenic Arabidopsis, the activity of a reporter enzyme under the control of the -453 to +67 fragment of an Lhcb1*2 promoter shows very low fluence responses (VLFRs) and high-irradiance responses (HIRs) mediated by phytochrome A and low-fluence responses (LFRs) mediated by phytochrome B. A 5' deletion of the promoter to -134 abolished the HIR without affecting VLFR or LFR. In transgenic tobacco, VLFR and LFR were observed for the -176 to -31 or -134 to -31 fragments of Lhcb1*2 fused to 35S cauliflower mosaic virus minimal promoters, but only the largest fragment showed HIR. We propose that sustained activation of phytochrome A with far-red light initiates a signaling cascade that deviates from phytochrome B signaling and transient phytochrome A signaling and that this divergence extends as far as the Lhcb1*2 promoter.

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Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.

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The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of beta-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h-1 (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h-1 caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.

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The occurrence of very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR) of phytochrome was investigated for the expression of the gene of beta-glucuronidase (gusA) under the control of the tobacco Lhcb1*2 promoter, in etiolated transgenic tobacco seedlings. The activity of beta-glucuronidase (GUS) showed biphasic responses to the calculated proportion of Pfr provided by light pulses. The first phase (i.e. the VLFR) showed a maximum for Pfr levels characteristic of far-red light. The second phase (i.e. the LFR) was observed at higher Pfr levels and was reversible by far-red light pulses. The strong effect of continuous far-red light (i.e. HIR) was fluence-rate-dependent and could not be replaced either by hourly pulses of the same spectral composition and total fluence or by very low fluences of red light. Deletion of the Lhcb1*2 promoter to -453 caused little loss of GUS activity. The -453 to -31, -270 to -31 and -176 to -31 fragments of the Lhcb1*2 promoter conferred proportionally normal VLFR, LFR and HIR to a truncated (-46 to +8) CaMV 35S minimal promoter. This is the first demonstration of the presence of three phytochrome action modes in the control of the transcriptional activity of a single gene. The cis-acting regulatory elements necessary for VLFR, LFR and HIR are present in a 146 bp fragment of the tobacco Lhcb1*2 promoter.

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A potato (Solanum tuberosum) genomic library was used to isolate five full-length genes and part of a sixth one, encoding the apoproteins of a light-harvesting complex (LHC). The nucleotide sequences of the five isolated genes showed that they encoded very similar proteins (99.75% to 97.50% identities). The deduced amino acid sequences were homologous to the PSII type I CAB proteins encoded by the Lhcb1 genes. The 5'-flanking regions of the five potato genes shared conserved sequences already detected in other light-responsive genes (62 bp fragment located between the CAAT and TATA boxes containing three GATA motifs). The expression of Lhcb1 genes in potato leaves, stems and roots was analyzed by the primer extension method. At least nine different transcripts can be recognized in leaves. A unique transcript could be detected in stems and not in roots. The regulatory function of the 5'-flanking region of the potato Lhcb1*2 was analyzed in transgenic tobacco plants. The construct used contained the gusA gene which was under the control of the potato Lhcb1*2 promoter. Analysis of transgenic plants revealed that the 5'-flanking region (-1300 to +10, relative to the transcription start site) of the Lhcb1*2 gene was sufficient to confer a phytochrome response as well as organ-specific expression.

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Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.

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Incubation of labeled uridine diphosphate glucose with an enzyme preparation from Rhizobium meliloti or Agrobacterium tumefaciens leads to the formation of a glucan which appears to be identical to the beta 1-2 cyclic glucan described by several workers. This conclusion is based on the molecular size, the formation of sophorose and higher homologs by partial acid hydrolysis, the liberation of only glucose by total acid hydrolysis, and the release of only 3,4,6-tri-O-methylglucose after methylation and hydrolysis. A snail intestinal juice enzyme was found to break down the glucan and its partial hydrolysis products. A beta-glucosidase from sweet almonds degraded sophorose but not the intact glucan.

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A genetic analysis of Agrobacterium tumefaciens chromosomal functions required for virulence was undertaken. Large Tn5-containing cosmid clones were isolated from DNA of avirulent A. tumefaciens mutants having chromosomal Tn5 insertions and exhibiting defective attachment to plant cells. The clones from several different mutants each contained overlapping segments of a 30-kilobase A. tumefaciens chromosomal region, which were physically mapped. All chromosomal Tn5 insertions leading to the avirulent, attachment-defective phenotype were localized within an 11-kilobase portion of this chromosomal virulence region. Transposon Tn3::HoHo1 (Tn3 containing lacZ) was used to simultaneously mutagenize and create lac fusions within the virulence region. This analysis demonstrated the presence of two distinct chromosomal virulence loci, which were 1.5 and 5 kilobases long; transposon insertions into these loci led to avirulence and defective attachment. The beta-galactosidase activity associated with various Tn3::HoHo1-created lac fusions indicated that the loci are transcribed in opposite directions, and complementation studies suggested that each locus consists of a single transcriptional unit. A cosmid clone of the chromosomal virulence region containing a lac fusion in the extreme 3' portion of the 5-kilobase locus was used to demonstrate that expression of this region is dependent on the presence of sequences in the 5' portion of the locus, confirming its operon-like nature.

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The lipid-bound saccharides formed by incubation of uridine diphosphate glucose with a particulate enzyme of Rhizobium meliloti were studied. They behaved like polyprenyl diphosphate saccharides when treated with ammonia or hot phenol, when catalytically hydrogenated, and on DEAE-cellulose chromatography. The saccharide moieties obtained after heating at pH 2 for 10 min at 100 degrees C were separated with a gel filtration column. The following compounds were detected: galactose, glucosyl beta 1-3 galactose (Tolmasky, M. E., Staneloni, R. J., Ugalde, R. A., and Leloir, L. F. (1980) ARch. Biochem. Biophys. 203, 358-364), and some octasaccharides (I). These were compared by paper electrophoresis, thin layer and paper chromatography with an octasaccharide obtained from Alcaligenes faecalis var. myxogenes strain 11 (II). Furthermore, Compounds I and II were compared with the exopolysaccharide of Rhizobium meliloti (III) by partial acid hydrolysis and methylation analysis. The results were consistent with the identity of the repeating unit of Compound III with Compounds I and II except for differences in the substituents (acetyl or succinyl). Studies on the labeling of the lipid-bound saccharides have shown that the sequence is: first, galactose and glucosyl beta 1-3 galactose, then the rest of glucose residues, and finally, the substituents (acetyl and pyruvic acid).

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This review deals with the structure and addition of the different types of oligosaccharides to asparagine residues in proteins. This process occurs in several steps, first an oligosaccharide which contains N-acetylglucosamine mannose and glucose is built up joined to dolichyl diphosphate. The oligosaccharide is then transferred to a polypeptide chain, loses its glucose, and is modified by removal of some monosaccharides and addition of others giving rise to a variety of saccharides.

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A lipid-bound oligosaccharide was isolated from pea (Pisum sativum) cotyledons incubated with [(14)C]mannose. The oligosaccharide moiety appeared to be identical with the one obtained from rat liver, known to contain three glucoses, nine mannoses, and two N-acetylglucosamines, and to be involved in protein glycosylation.Enzymes obtained from soya (Glycine max) roots and developing pea cotyledons were found to catalyze the transfer of oligosaccharide from the lipid intermediate to endogenous protein. The enzymes require Mn(2+) and detergent for activity. Evidence is presented indicating that the lipid-bound oligosaccharide with three glucoses is transferred faster than that with less. Some of the peripheral mannoses could be removed without affecting the rate of transfer.The protein-bound oligosaccharide, formed by incubation of whole cotyledons or by transfer with the enzyme preparation, could be released by protease and endo-beta-N-acetylglucosaminidase treatment, as expected for an asparagine-bound high mannose oligosaccharide.

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Further work on microsomal glucosidases of rat liver has confirmed that at least two enzymes are involved in the removal of glucose from the glucose-containing oligosaccharide. One acts on the oligosaccharide containing three glucose residues and another on the oligosaccharide which has one or two glucoses. The glucosidase which acts on (Glc)2(Man)9(GlcNAc)2 could be purified with a Concanavalin-A--Sepharose column following by electrofocusing. This purified preparation was active on the oligosaccharide containing one or two glucoses. Heat inactivation and inhibition by disaccharides was parallel for both activities. Inhibition of the glucosidase active on (Glc)3(Man)9(GlcNAC)2 was obtained with kojibiose which has an alpha 1-2 linkage, while the glucosidase acting on (Glc)1-2(Man)9-(GlcNAc)2 was inhibited by nigerose (alpha 1-3 linkage), maltose (alpha 1-4 linkage) and glucose at a higher concentration. None of the beta anomers inhibited. These results are consistent with an alpha configuration of the three glucoses of the dolichyl-diphosphate-linked oligosaccharide. Kojibiose was found to inhibit glucosidase action not only on the free oligosaccharide but also on protein-bound one.

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A compound with properties identical with the glucose-containing dolichyl diphosphate oligosaccharide present in animal tissues was detected in alfalfa roots incubated with [14C]glucose. The products of mild acid hydrolysis behaved the same on paper chromatography, on treatment with specific glucosidases and on N-deacetylation.

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The glycosylation of asparagine residues in proteins is known to occur by transfer from a dolichyl diphosphate oligosaccharide containing glucose. Paper chromatography allowed the separation of oligosaccharides (obtained by acid hydrolysis of the dolichyl diphosphate derivative) containing 1, 2 and 3 glucose residues. Using this procedure it was found that the addition of all three glucoses to the dolichyl diphosphate oligosaccharide occur with dolichyl phosphate glucose as donor. Furthermore only the compound with three glucoses was used as donor in the transfer to protein. The addition of glucose to exogenous dolichyldiphosphate oligosaccharide labelled by transfer from radioactive guanosine diphosphate mannose was detected.

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The glucose-containing oligosaccharides formed by calf thyroid slices incubated with radioactive glucose were studied. A compound soluble in chloroform/methanol/water, 1:1:0.3 (vol/vol), was found that was indistinguishable from the previously described glucose-containing dolichyl diphosphate oligosaccharide formed by liver microsomes. Glycopeptides were prepared by treating the glycoproteins with pronase, the amino acids were removed with alkaline borohydride, and the products were examined by paper electrophoresis and chromatography. A saccharide equal to that which occurs in the glucose-containing dolichyl diphosphate oligosaccharide could not be detected but glucose was found in oligosaccharides that seemed to be smaller by about three to five monosaccharide residues. The same results were obtained by direct treatment of the glycoproteins with alkaline borohydride.

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