GLYCOPROTEIN MOIETY IN THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA) AS THE BIORECOGNITION SITE FOR THE CRYPTHECODINIUM COHNII-LIKE DINOFLAGELLATE

The Crypthecodinium cohnii -like heterotrophic dinoflagellate preys on the cells of the red microalga Porphyridium sp. UTEX 637, and not on other microalgae. The dinoflagellate contains enzymes that degrade the cell wall complex of this species of alga and not that of other red microalgae. The cells of the red microalgae are encapsulated within a cell wall complex composed of about 10 sugars, sulfate, and proteins. We previously hypothesized that the dinoflagellate recognizes the cell wall of this alga. In this study, we have shown that the biorecognition site is the 66-kDa glycoprotein in the algal cell wall complex. The methodology used in this study was based on changing the algal cell wall composition and examining the prey and chemosensory response of the dinoflagellate. The dinoflagellate was not attracted to the cell wall of other red microalgae, which are similar to that of Porphyridium sp., or to sugars composing its cell wall. However, the dinoflagellate preyed on and was attracted to Porphyridium sp. mutants (DCB resistant) having modified cell wall polysaccharide composition, probably because the 66-kDa cell wall glycoprotein was not changed. The dinoflagellate did not respond chemotactically to enzymatically degraded cell wall complex. Treatment of the cell wall complex with antiserum to the 66-kDa glycoprotein or with the lectin concanavalin A (con A), which binds specifically to α-d-mannosyl and α-d-glucosyl residues, did not affect the chemotactic attraction. However, prey by the dinoflagellate was prevented when the algal cells were blocked with antiserum specific to the 66-kDa glycoprotein or with con A. These latter results provide direct proof that the 66-kDa cell wall glycoprotein isthe recognition site and prey-prevention results from the blocking of this site on the cell wall.     

Identification of a receptor protein in cotton fibers for the herbicide 2,6-dichlorobenzonitrile

The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and apparently specific inhibitor of cellulose synthesis in higher plants. We have synthesized a photoreactive analog of DCB (2,6-dichlorophenylazide [DCPA]) for use as an affinity-labeling probe to identify the DCB receptor in plants. This analog retains herbicide activity and inhibits cellulose synthesis in cotton fibers and tobacco cells in a manner similar to DCB. When cotton fiber extracts are incubated with [³H]DCPA and exposed to ultraviolet light, an 18 kilodalton polypeptide is specifically labeled. About 90% of this polypeptide is found in the 100,000g supernatant, the remainder being membrane-associated. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of this polypeptide indicate that it is an acidic protein which has a similar size in its native or denatured state. The amount of 18 kilodalton polypeptide detectable by [³H]DCPA-labeling increases substantially at the onset of secondary wall cellulose synthesis in the fibers. A similar polypeptide, but of lower molecular weight (12,000), has been detected upon labeling of extracts from tomato or from the cellulosic alga Chara corallina. The specificity of labeling of the 18 kilodalton cotton fiber polypeptide, coupled with its pattern of developmental regulation, implicate a role for this protein in cellulose biosynthesis. Being, at most, only loosely associated with membranes, it is unlikely to be the catalytic polypeptide of the cellulose synthase, and we suggest instead that the DCB receptor may function as a regulatory protein for β-glucan synthesis in plants.     

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes)

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.     

Disruption of cellulose synthesis by 2,6‐dichlorobenzonitrile affects the structure of the cytoskeleton and cell wall construction in Arabidopsis

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6‐dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose‐ and time‐dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N‐ST‐YFP and TGN labelled with VHA‐a1‐GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.     

Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans

Abstract A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

Alterations in light-induced stomatal opening in a starch-deficient mutant of Arabidopsis thaliana L. deficient in chloroplast phosphoglucomutase activity

Stomatal responses to light of Arabidopsis thaliana wild-type plants and mutant plants deficient in starch (phosphoglucomutase deficient) were compared in gas exchange experiments. Stomatal density, size and ultrastructure were identical for the two phenotypes, but no starch was observed in guard cells of the mutant plants whatever the time of day. The overall extent of changes in stomatal conductance during 14 h light–10 h dark cycles was similar for the two phenotypes. However, the slow endogenous stomatal opening occurring in darkness in the wild type was not observed in the mutant plants. Stomata in the mutant plants responded much more slowly to blue light (70 μmol m^?2 s^?1) though the response to red light (250 μmol m^?2 s^?1) was similar to that of wild-type plants. In paradermal sections, stomatal responses to red light (300 μmol m^?2 s^?1) were weak for wild-type plants as well as for mutant plants. Stomatal opening was greater under low blue light (75 μmol m^?2 s^?1) than under red light for the two genotypes. However, in mutant plants, a high chloride concentration (50 mol m^?3) was necessary to achieve the same stomatal aperture as observed for the wild-type plants. These results suggest that starch metabolism, via the synthesis of a counter-ion to potassium (probably malate), is required for full stomatal response to blue light but is not involved in the stomatal response to red light.     

Effect of cellulose synthesis inhibition on growth and the integration of xyloglucan into pea internode cell walls

2,6-Dichlorobenzonitrile (DCB, 100 μm) inhibited by 80 to 85% the incorporation of [³H]glucose into cellulose in stem segments of etiolated pea (Pisum sativum) seedlings. The inhibition lasted for at least 24 h. In the period 1 to 4 h after the excision of the segments, DCB did not influence elongation in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). However, during the period 1 to 24 h after excision, DCB enhanced endogenous and 2,4-D-stimulated elongation by 65 and 34%, respectively. DCB did not affect the incorporation of ³H from [³H]arabinose into xyloglucan, and did not change the ability of the [³H]xyloglucan formed in vivo to bind strongly to the cell wall. Therefore, at least 80 to 85% of newly synthesized cellulose was excess to the requirements for tight wall binding of newly synthesized xyloglucan. This conflicts with the hypothesis that xyloglucan is held in the cell wall solely by direct hydrogen bonding to the surfaces of cellulosic microfibrils.     

A Comparison of the Surface Polysaccharides from Rhizobium leguminosarum 128C53 smrif with the Surface Polysaccharides from Its Exo Mutant

The surface polysaccharides of Rhizobium leguminosarum 128C53 sm^rrif^r (parent) and its exo⁻¹ mutant were isolated and characterized. The parent carries out normal symbiosis with its host, pea, while the exo⁻¹ mutant does not nodulate the pea. The following observations were made. (a) The parent produces lipopolysaccharide (LPS), typical acidic extracellular polysaccharide (EPS), and three additional polysaccharides, PS1, PS2, and PS3. The PS1 and PS2 fractions are likely to be the capsular polysaccharide (CPS) and are identical in composition to the EPS. The PS3 fraction is a small-molecular-weight glucan. (b) The exo⁻¹ mutant produces LPS, EPS, and a PS3 fraction, but does not produce significant amounts of either PS1 or PS2. The LPS from the exo⁻¹ mutant appears to be identical to the parental LPS. Analysis of the EPS from exo⁻¹ shows that it consists of two polysaccharides. One polysaccharide is identical to the LPS and comprises 70% of the exo⁻¹ EPS. The second polysaccharide is identical to the exo⁻¹ PS3 and comprises 30% of the exo⁻¹ EPS. This result shows that the exo⁻¹ mutant does not produce any of the typical acidic parental EPS and that the major polysaccharide released into the media by the exo⁻¹ mutant is intact LPS. The exo⁻¹ mutant PS3 fraction was found to contain two polysaccharides, PS3-1 and PS3-2. The PS3-2 polysaccharide is identical to the parental PS3 described above. The PS3-1 polysaccharide has a composition similar to the polysaccharide portion of the LPS. This result suggests that the exo⁻¹ mutant produces LPS polysaccharide fragments. These LPS polysaccharide fragments are not produced by the parent strain.     

THE PRODUCTION OF EXTRACELLULAR POLYSACCHARIDE BY THE UNICELLULAR RED ALGA PORPHYRIDIUM AERUGINEUM1,2

The unicellular red alga Porphyridium aerugineum was shown to be encapsulated by an amorphous, water-soluble, polyanionic polysaccharide of high molecular weight. The encapsulating polysaccharide is qualitatively identical with polysaccharide found dissolved in large quantity in the culture medium. The kinetics of extracellular polysaccharide production as a function of cell age was studied. Rates of production (on a per cell basis) of both encapsulating and dissolved polysaccharides are greatest in stationary phase light-grown cultures. Dissolved polysaccharide was quantitatively isolated by precipitation with cetyl pyridinium chloride, conversion to the calcium salt, and reprecipitation with ethanol. The procedure yields a spectrally pure product, which is composed of glucose, galactose, xylose, and 2 undetermined, sugar components, and has a sulfate content of 7.6% by weight. Electron microscopy of Porphyridium revealed that Golgi vesicles transport, polymerized polysaccharides to and through the cell membrane. Similar vesicles were observed in the multicellular Pseudogloiophloea, indicating that the Golgi complex plays a crucial role in the production of extracellular polysaccharides by the red algae. H¹⁴CO₃^- pulse-label experiments resulted in labeled extracellular polysaccharide in which all the constituent components contained ¹⁴C. Rates of excretion of polysaccharide were found, to follow a cyclic pattern, correlated generally with the division cycle, of the cell.     

The endogenous metabolism ofFibrobacter succinogenes and its relationship to cellobiose transport,viability and cellulose digestion

Fibrobacter succinogenes S85 digested ballmilled cellulose at a rapid rate (0.10 h^–1), but there was a long lag time if the culture was not transferred daily. WhenF. succinogenes was starved for 100h, a large fraction of the cells (>30%) still bound to cellulose, but the lag time was 150h. The lag time was similar for either cellulose- or cellobiose-grown inocula, and lag times were highly correlated (r ² = 0.91) with a decrease in viable cell number. The number of viable cells declined from 10⁸ to 10⁶ in the first 30h of starvation, and by 72h the viable cell number was less than 10³/ml. Cells growing exponentially on cellobiose had a large pool of polysaccharide, and continuous culture experiments indicated that polysaccharide accumulation was not significantly influenced by the growth rate of the culture (approximately 0.7 mg polysaccharide mg^–1 protein). When the cellobiose was depleted, cellular polysaccharide decreased at first order rate of 0.09 h^–1. The rate of endogenous metabolism was initially 0.08mg polysaccharide mg^–1 protein h^–1, and there was little decline in viability until the rate of endogenous metabolism was less than 0.01 mg polysaccharide mg^–1 protein h^–1. When the rate was less than 0.01 mg polysaccharide mg^–1 protein h^–1, the cells could not maintain a sodium gradient, transport cellobiose or grow. The endogenous metabolic rate needed for cell survival was 20 fold less than the maintenance energy of cells growing in continuous culture (0.01 versus 0.232mg carbohydrate mg^–1 protein h^–1).     

Wall architecture in the cellulose-deficientrsw1 mutant ofArabidopsis thaliana: Microfibrils but not microtubules lose their transverse alignment before microfibrils become unrecognizable in the mitotic and elongation zones of roots

Summary Thersw1 mutant ofArabidopsis thaliana has a single amino acid substitution in a putative glycosyl transferase that causes a temperature-dependent reduction in cellulose production. We used recently described methods to examine root growth by surface marker particles, cell wall structure by field emission scanning electron microscopy and microtubule alignment by immunofluorescence after the mutant is transferred to its restrictive temperature. We find that raising the temperature quickly accelerates root elongation in both wild type and mutant, presumably as a result of general metabolic stimulation, but that in the mutant, the rate declines within 7–8 h and elongation almost ceases after 24 h. Radial swelling begins at about 6 h in the mutant and root diameter continues to increase until about 24 h. The normal transverse alignment of microfibrils is severely impaired in the mutant after 8 h, and chemical inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile causes a similar loss of orientation. After 24 h, microfibrils are not clearly visible in the walls of cells that would have been in the mitotic and early-elongation zone of wild-type roots. Changes in older cells are less marked; loss of transverse microfibril orientation occurs without disruption to the transverse orientation of cortical microtubules. The wild type shows none of the changes except for acceleration of elongation, which in its case is sustained. We conclude that microfibril alignment requires the normal functioning of RSW1 and that, in view of the effects of dichlorobenzonitrile, there may be a more general linkage between the rate of cellulose production and its proper alignment.Abbreviations DCB 2,6-dichlorobenzonitrile - REGR relative elemental growth rate Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

A mutant of Candida albicans deficient in beta-N-acetylglucosaminidase (chitobiase)

A mutant of Candida albicans ATCC 10261 was isolated that was defective in the production of beta-N-acetylglucosaminidase (chitobiase). The mutant grew normally in minimal medium supplemented with either glucose or N-acetyl-D-glucosamine (GlcNAc) as carbon and energy source, and the cells formed germ-tubes at 37 degrees C when induced to do so with GlcNAc. However, unlike the wild-type parent strain, the mutant strain did not utilize N,N'-diacetylchitobiose for growth. The mutant and parent strains had similar growth rates on glucose or GlcNAc, similar rates of uptake of these sugars and similar rates of 14C-labelled amino acid incorporation. The chitobiase mutant did, however, contain 53-85% more chitin than the wild-type strain. No reversion of the mutant phenotype was observed following induction of mitotic recombination with UV light, suggesting that the mutant allele (chi) was carried homozygously in the chitobiase-deficient mutant. Although the chitobiase-deficient mutant was pathogenic, it was not as virulent as the wild-type strain.     

Use of hybridoma-resistant variant cell lines to study H-2D b/FMR-specific cytotoxic T cells

An H-2D ^b b heterozygous tumor cell line and a variant subclone bearing a mutant gene product were used to analyze the H-2D^b specificity of cytotoxic T lymphocytes (CTL) generated during a Moloney murine sarcoma virus (MSV) infection. When the mutant cells were used as targets for MSV-specific CTL, the amount of cell lysis, compared with that seen with the nonmutant parental cells, was drastically decreased. However, cells of the mutant clones remained susceptible to allogeneic CTL specific for the nonmutant H-2D^b molecule. The mutant cells also did not differ from the parent cells in their level of viral antigen expression. Biochemically the parental and mutant molecules were similar but not identical. The data indicate that minor alterations of the H-2 antigens caused by somatic mutation may prevent virus-infected cells from being recognized as targets by CTL.     

Growth and Chromatic Adaptation of Nostoc sp. Strain MAC and the Pigment Mutant R-MAC

A spontaneous, stable, pigmentation mutant of Nostoc sp. strain MAC was isolated. Under various growth conditions, this mutant, R-MAC, had similar phycoerythrin contents (relative to allophycocyanin) but significantly lower phycocyanin contents (relative to allophycocyanin) than the parent strain. In saturating white light, the mutant grew more slowly than the parent strain. In nonsaturating red light, MAC grew with a shorter generation time than the mutant; however, R-MAC grew more quickly in nonsaturating green light.

When the parental and mutant strains were grown in green light, the phycoerythrin contents, relative to allophycocyanin, were significantly higher than the phycoerythrin contents of cells grown in red light. For both strains, the relative phycocyanin contents were only slightly higher for cells grown in red light than for cells grown in green light. These changes characterize both MAC and R-MAC as belonging to chromatic adaptation group II: phycoerythrin synthesis alone photocontrolled.

A comparative analysis of the phycobilisomes, isolated from cultures of MAC and R-MAC grown in both red and green light, was performed by polyacrylamide gel electrophoresis in the presence of 8.0 molar urea or sodium dodecyl sulfate. Consistent with the assignment of MAC and R-MAC to chromatic adaptation group II, no evidence for the synthesis of red light-inducible phycocyanin subunits was found in either strain. Phycobilisomes isolated from MAC and R-MAC contained linker polypeptides with relative molecular masses of 95, 34.5, 34, 32, and 29 kilodaltons. When grown in red light, phycobilisomes of the mutant R-MAC appeared to contain a slightly higher amount of the 32-kilodalton linker polypeptide than did the phycobilisomes isolated from the parental strain under the same conditions. The 34.5-kilodalton linker polypeptide was totally absent from phycobilisomes isolated from cells of either MAC or R-MAC grown in green light.

     

Investigations of the turnover of the putative cellulose-synthesizing particle “rosettes” within the plasma membrane ofFunaria hygrometrica protonema cells

Summary In youngFunaria protonemata the influence of various inhibitors and treatments on cell elongation, fine-structure, and particle rosettes within the plasma membrane, putative parts of cellulose synthase complexes, was investigated. Cycloheximide (3×10^–5M) inhibited growth, reduced the number of rosettes and evened the gradient of rosette distribution at the beginning of treatment. The cell fine-structure was unaffected. Actinomycin D (10^–5M and 10^–4) caused an initial but transient decrease in rosette number. Alterations in cell elongation and fine-structure have not been observed. Application of 2.6-dichlorobenzonitrile (10^–5 M) for some minutes reduced the number of rosettes remarkably, while cell elongation seemed to be normal after the filaments had been transferred back to normal medium. An incubation of 2 h or longer stopped growth and caused cells to burst. The number of rosettes then rose to about 50% of the control values. When applied for 7 h biofluor (5×10^–4 M) promoted growth slightly, but generally it retarded it when used for a longer time. It did not markedly affect the number of rosettes. A short heat stock stopped elongation, caused the disappearance of rosettes and affected the structure of the mitochondria and of the Golgi apparatus. Plasmolysed cells did not grow and, initially, did not have rosettes. At reduced turgor, wider cells are formed. Freeze fracturing under UHV conditions and shadowing at very low specimen temperature revealed a small, central depression in the 8 nm rosette particles, suggesting that they are composed of subunits. Our results provide further evidence that the rosettes are parts of the cellulose synthase complexes. Their existence clearly depends on protein synthesis and on the constitution of the plasma membrane, but not on cellulose crystallization.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Dispersed lignin in tracheary elements treated with cellulose synthesis inhibitors provides evidence that molecules of the secondary cell wall mediate wall patterning

Mesophyll cells of Zinnia elegans var. Envy that had been induced to differentiate into tracheary elements (TEs) in suspension culture were treated with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). The deposition of cellulose into the patterned secondary cell wall thickenings typical of TEs was inhibited as demonstrated by reduced incorporation of [¹⁴C]glucose into acetic/nitric insoluble material and absence of cellulose detectable by fluorescence after staining with Tinopal LPW, polarization optics, or labeling with a specific cellulase. Respiration as indicated by release of ¹⁴CO₂ was inhibited to a much lesser extent, supporting a selective mechanism of action of DCB on the cellulose biosynthetic pathway. Patterned secondary cell wall thickenings were deposited in DCB-treated TEs, but these were smaller and less regularly shaped than those of control TEs. These cellulose-depleted thickenings lacked another abundant component of normal thickenings, the hemicellulose xylan, as indicated by absence of labeling with a specific xylanase or an antibody to xylan. DCB-treated TEs also showed dispersed lignin after staining with phloroglucinol, whereas control TEs contained lignin specifically localized to the secondary cell wall thickenings. Isoxaben, another recently described inhibitor of synthesis of acetic/nitric insoluble cell wall material (putatively cellulose), caused the same absence of detectable cellulose and xylan in the thickenings and dispersed lignin. These data suggest that: (i) the localization of lignin is ultimately dependent on the localization of cellulose; (ii) normal patterned wall assembly in TEs occurs in a self-perpetuating cascade in which some molecules of the secondary cell wall mediate patterning of others.     

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M⁻¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid