Sucrose-supplemented Medium Research Articles

Expression of Sucrose Transporters from Vitis vinifera Confer High Yield and Enhances Drought Resistance in Arabidopsis.

Sucrose is the predominant form of sugar transported from photosynthetic (source) to non-photosynthetic (sink) organs in higher plants relying on the transporting function of sucrose transporters (SUTs or SUCs). Many SUTs have been identified and characterized in both monocots and dicots. However, the function of sucrose transporters (SUTs or SUCs) from Vitis is not clear. As the world’s most planted grape species, Vitis vinifera owns three sucrose transport activity verified SUTs. In this study, we constructed three kinds of VvSUC (Vitis vinifera SUC)-overexpressing transgenic Arabidopsis. VvSUC-overexpressing transgenic Arabidopsis was cultured on sucrose-supplemented medium. VvSUC11- and VvSUC12-overexpressing lines had similar thrived growth phenotypes, whereas the size and number of leaves and roots from VvSUC27-overexpressing lines were reduced compared with that of WT. When plants were cultured in soil, all SUT transgenic seedlings produced more number of leaves and siliques, resulting in higher yield (38.6% for VvSUC12-transformants) than that of WT. Besides, VvSUC27-transformants and VvSUC11-transformants enhanced drought resistance in Arabidopsis, providing a promising target for crop improvement

Toward enhanced hyperforin production in St. John's wort root cultures.

During the past decades, several trials targeted a stable, sustainable and economic production of St. John's wort (Hypericum perforatum) extract. The value of this extract stems from its use to treat depression and skin irritation due to its hyperforin content. Previously, hyperforin-forming in vitro root cultures were established. Here, detailed growth and production kinetics have been analyzed over 40 days of cultivation. In the first 10 days, sucrose was completely hydrolyzed to glucose and fructose. The ammonium consumption supported the increase in the biomass and hyperforin production. When sucrose was replaced with glucose/fructose, the linear growth phase started 6 days earlier and resulted in a higher space-time-yield. The maximum hyperforin production was 0.82mgL-1 day-1, which was 67 % higher than in the sucrose-supplemented standard cultivation. Buffering the sucrose-supplemented medium with phosphate caused a 2.7-fold increase in the product to biomass yield coefficient. However, the combination of monosaccharides and buffering conditions did not cause an appreciable improvements in the production performance of the shake flask approaches. A potential scalability from flask to lab-scale stirred bioreactors has been demonstrated. The results obtained offer a basis for a scalable production of hyperforin and a sustainable source for a tissue culture-based phytomedicine.

Serine-rich Repeat Adhesin Gordonii Surface Protein B is Important for Streptococcus gordonii Biofilm Formation

IntroductionStreptococcus gordonii is a predominant member of the oral microflora and has been isolated from root canals of teeth with refractory apical periodontitis. Biofilm formation is important for various dental diseases, and S. gordonii is involved in dental biofilm formation as an early colonizer. Although serine-rich repeat (SRR) adhesins of S. gordonii such as gordonii surface protein B (GspB) are associated with bacterial colonization, the role of GspB in biofilm formation is not clearly understood. In the present study, we investigated the effect of S. gordonii GspB on biofilm formation using wild-type and GspB-deficient mutant S. gordonii strains. MethodsConfocal microscopy and crystal violet assay were used to determine biofilm formation. Bacterial growth was examined by measuring optical density with spectrometry. Bacterial adherence and biofilm on the culture plate and human dentin slices were visualized with a scanning electron microscope. ResultsThe GspB-deficient S. gordonii mutant strain was less potent than the wild-type strain in biofilm formation. Of note, there was no difference in the bacterial growth rate between the mutant and wild-type strains. Differences in biofilm-forming ability between the wild-type and mutant strains were more distinct in the sucrose-supplemented media. Furthermore, the GspB-deficient mutant exhibited attenuated formation of aggregates on the surface of the culture plate and human dentin slices. ConclusionsThese results suggest that GspB is important for S. gordonii biofilm formation, which may contribute to the development of dental biofilm-associated diseases.

Nitric oxide accumulation in Arabidopsis is independent of NOA1 in the presence of sucrose

Nitric oxide signals diverse responses in animals and plants. Whereas nitric oxide synthesis mechanisms in animals are well understood, how nitric oxide is synthesized and regulated in plants remains controversial. NOA1 is a circularly permuted GTPase that is important for chloroplast function and is implicated in nitric oxide synthesis. However, the reported consequences of a null mutation in NOA1 are inconsistent. Whereas some studies indicate that the noa1 mutant has severe reductions in nitric oxide accumulation, others report that nitric oxide levels are indistinguishable between noa1 and the wild type. Here, we identify a correlation between the reported ability of noa1 to accumulate nitric oxide with growth on sucrose-supplemented media. We report that noa1 accumulates both basal and salicylic acid-induced nitric oxide only when grown on media containing sucrose. In contrast, nitric oxide accumulation in wild type is largely insensitive to sucrose supplementation. When grown in the absence of sucrose, noa1 has low fumarate, pale green leaves, slow growth and reduced chlorophyll content. These phenotypes are consistent with a defect in chloroplast-derived photosynthate production and are largely rescued by sucrose supplementation. We conclude that NOA1 has a primary role in chloroplast function and that its effects on the accumulation of nitric oxide are likely to be indirect.

Development of vitrified porcine primordial follicles in xenografts

The objective was to cryopreserve porcine primordial follicles by vitrification and to assess the development of these follicles in xenografts. Ovarian tissues containing primordial follicles were collected from neonatal (15-d-old) piglets. They were vitrified in modified tissue culture medium (TCM)-199 containing 15% (v/v) ethylene glycol, 15% (v/v) dimethylsulfoxide, 20% (v/v) fetal calf serum, and 0, 0.25, or 0.5 M sucrose. After 1 wk of storage in liquid nitrogen (LN 2), the tissues were warmed, and the morphology of follicles and oocytes was examined histologically. After vitrification in sucrose-free medium, there were 50 ± 2 (mean ± SEM; n = 10) follicles per tissue, in contrast with 108 ± 10 (n = 10) in fresh tissues. Losses were attributed to puncturing oocytes during the vitrification-warming process, as oocytes were apparently normal after treatment of the sucrose-free vitrification solution without plunging into LN 2. When tissues were vitrified in sucrose-supplemented medium, loss of oocytes decreased (P < 0.05). However, the number of abnormal oocytes having nuclear shrinkage was increased (P < 0.05) by the addition of 0.5 M sucrose; this occurred in a small number of oocytes treated with sucrose-supplemented vitrification solutions without vitrification. After 2 mo of xenografting of vitrified-warmed tissues in SCID (severe combined immune deficiency) mice, primordial follicles developed to the secondary stage (accompanied by oocyte growth), whereas there was development to the antral stage in xenografts of fresh tissues. In conclusion, primordial follicles from neonatal pigs maintained their developmental ability after vitrification and warming, although their developmental rate was slower than that of the fresh control in xenografts.

Effects of probiotic Lactobacillus salivarius W24 on the compositional stability of oral microbial communities

Probiotics are microorganisms beneficial to gastrointestinal health. Although some strains are also known to possess positive effects on oral health, the effects of most intestinal probiotics on the oral microflora remain unknown. We assessed the ability of the intestinal probiotic Lactobacillus salivarius W24 to incorporate into and to affect the compositional stability and cariogenicity of oral microbial communities. Microtiter plates with hydroxyapatite discs were incubated with W24 (“+W24”) or without W24 (“−W24”) and saliva from four individuals in plain (“−sucrose”) or sucrose-supplemented (“+sucrose”) medium. Biofilms were subjected to community profiling by 16S rRNA gene-based Denaturing Gradient Gel Electrophoresis (DGGE) after 72 h growth. Diversity (Shannon–Weaver index) and similarities (Pearson correlation) between biofilm communities were calculated. Microcosms “+sucrose” were less diverse and more acidic than “−sucrose” microcosms ( p < 0.001). The effects of W24 on the community profiles were pH dependent: at pH 4 (“+sucrose”), the respective “+W24” and “−W24” microcosms differed significantly more from each other than if the pH was ∼7 (“−sucrose”). The pH of “+W24/+sucrose” microcosms was lower ( p < 0.05) than the pH of the microcosms supplemented with sucrose alone (“−W24/+sucrose”). Although not able to form a monospecies biofilm, L. salivarius W24 established itself into the oral community if inoculated simultaneously with the microcosm. In the presence of sucrose and low pH, W24 further lowered the pH and changed the community profiles of these microcosms. Screening of probiotics for their effects on oral microbial communities allows selecting strains without a potential for oral health hazards.

Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.

The AAE14 gene encodes the Arabidopsis o‐succinylbenzoyl‐CoA ligase that is essential for phylloquinone synthesis and photosystem‐I function

Phylloquinone is the one-electron carrier at the A(1) site of photosystem I, and is essential for photosynthesis. Arabidopsis mutants deficient in early steps of phylloquinone synthesis do not become autotrophic and are seedling lethals, even when grown on sucrose-supplemented media. Here, we identify acyl-activating enzyme 14 (AAE14, At1g30520) as the o-succinylbenzoyl-coenzyme A (OSB-CoA) ligase acting in phylloquinone synthesis. Three aae14 mutant alleles, identified by reverse genetics, were found to be seedling lethal, to contain no detectable phylloquinone (< 0.1 pmol mg(-1) fresh weight) compared with 10 pmol mg(-1) fresh weight in wild-type leaves, and to accumulate OSB. AAE14 was able to restore menaquinone biosynthesis when expressed in an Escherichia coli mutant disrupted in the menE gene that encodes the bacterial OSB-CoA ligase. Weak expression of an AAE14 transgene in mutant plants (controlled by the uninduced XVE promoter) resulted in chlorotic, slow-growing plants that accumulated an average of 4.7 pmol mg(-1) fresh weight of phylloquinone. Inducing the XVE promoter in these plants, or expressing an AAE14 transgene under the control of the CaMV 35S promoter, led to full complementation of the mutant phenotype. aae14-mutant plants were also able to synthesize phylloquinone when provided with 1,4-dihydroxy-2-naphthoate, an intermediate in phylloquinone synthesis downstream of the OSB-CoA ligase reaction. Expression of an AAE14:GFP reporter construct indicated that the protein accumulated in discrete foci within the chloroplasts. This and other evidence suggests that the enzymes of phylloquinone synthesis from isochorismate may form a complex in the chloroplast stroma to facilitate the efficient channeling of intermediates through the pathway.

The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp468-Arg497) forms an intermolecular alpha-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.

A VicRK Signal Transduction System in Streptococcus mutans Affects gtfBCD , gbpB , and ftf Expression, Biofilm Formation, and Genetic Competence Development

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans.

Growth of Listeria monocytogenes as influenced by viscosity and water activity

The effects of osmotic stress on Listeria monocytogenes growth parameters was examined in relation to the viscosity of the growth media. In low-viscosity systems, growth of L. monocytogenes in glucose-supplemented media was comparable to growth in sucrose-supplemented media. The relative lag time (RLT: the lag time divided by the generation time) responses were found to increase in the more restrictive water activity conditions. In high-viscosity systems containing polyvinylpyrrolidone (PVP), growth rate was reduced, whereas lag time showed no discernible modification. Osmotic stress in medium- and high-viscosity media supplemented with glucose resulted in approximately exponential increasing of the RLT values. Thus, the biological effects of osmotic stress on L. monocytogenes could be affected by the physical properties of the system, such as viscosity and diffusivity.

Photoautotrophic culture conditions and photosynthetic photon flux influence growth of Lilium bulblets in vitro

Lilium Asiatic hybrid ‘Mona’ bulblets were cultured in vitro for 100 d under photoautotrophic (CO2-enriched conditions and without sucrose in the medium) and heterotrophic (non-enriched CO2 conditions and sucrose-supplemented medium) methods and under various levels of photosynthetic photon flux (PPF). Bulblet growth and net photosynthetic rate (NPR) were analyzed. CO2− and PPF-enriched conditions enhanced the overall growth of bulblets, scale leaves, and roots. Heterotrophic conditions enhanced bulblet growth but higher PPF levels were inhibitory to the development of scale leaves. These results indicate the CO2− and PPF-enriched conditions (photoautotrophic conditions) are beneficial for the production of high-quality bulblets of Asiatic hybrid lilies in vitro

Sugar-induced adventitious roots in Arabidopsis seedlings.

The effects of sugars on root growth and on development of adventitious roots were analyzed in Arabidopsis thaliana. Seeds were sown on agar plates containing 0.0-5.0% sugars and placed vertically in darkness (DD) or under long day (LD, 16 h:8 h) conditions, so that the seedlings were constantly attached to the agar medium. In the sucrose-supplemented medium, seedlings showed sustained growth in both DD and LD. However, only dark-grown seedlings developed adventitious roots from the elongated hypocotyl. The adventitious roots began to develop 5 days after imbibition and increased in number until day 11. They could, however, be initiated at any position along the hypocotyl, near the cotyledon or the primary root. They were initiated in the pericycle in the same manner as ordinary lateral roots. Sucrose, glucose and fructose greatly stimulated the induction of adventitious roots, but mannose or sorbitol did not. Sucrose at concentrations of 0.5-2.0% was most effective in inducing adventitious roots, although 5.0% sucrose suppressed induction. Direct contact of the hypocotyl with the sugar-supplemented agar medium was indispensable for the induction of adventitious roots.

In vitro growth and shoot multiplication of Wrightia tomentosa Roem et Schult in a controlled carbon dioxide environment

In vitro growth and multiplication of shoots of a woody tree species Wrightia tomentosa in a controlled carbon dioxide environment was studied. The cultures were grown on BA supplemented MS medium with or without 3% sucrose. A range of CO2 concentrations (0.0, 0.6, 10.0 and 40.0 g m−3) was controlled in small chambers by using solutions of NaHCO3, Na2CO3, KHCO3 and K2CO3. To obtain a CO2-free environment, a saturated solution of KOH was kept in the chambers. It was concluded that the growing shoot cultures required either sucrose in the medium as a carbon source or an ambient CO2 environment. Complete absence of a carbon source caused severe browning of the shoots and death within 30 days. The cultures grew better with 10.0 g m−3 carbon dioxide in the environment than with 3.0% sucrose in the medium. With both CO2 and sucrose being available, the best response was obtained at 0.6 g m−3 CO2 in the chamber. At this concentration the rate of shoot multiplication was nearly double the standard rate obtained when exposed to the natural CO2 level and sucrose-supplemented medium. Total fresh and dry weight, leaf number and area per cluster also showed the best response under this condition.

Binding of oral streptococci to human fibrinogen.

The interaction of oral streptococci with human fibrinogen was investigated. Streptococcus gordonii was chosen as a representative species to study the binding to fibrinogen. S. gordonii DL1 adhered to immobilized fibrinogen and bovine serum albumin. Binding to immobilized fibrinogen was saturable, concentration and temperature dependent. The binding of S. gordonii DL1 to fibrinogen was inhibited by anti-fibrinogen antibody. Heating of the bacteria for 1 h at 95 degrees C resulted in 90% inhibition of the binding. Trypsin treatment of the bacteria resulted in decreased binding. Neither lipoteichoic acid nor culturing of the bacteria in a sucrose-supplemented medium had any effect on the binding. S. gordonii, Streptococcus sanguinis, Streptococcus mitis, and Streptococcus oralis bound to the immobilized fibrinogen, but mutans streptococci did not. None of the oral streptococci tested bound to the fibrinogen in fluid phase. These results suggest that the binding of S. gordonii DL1 to immobilized fibrinogen is mediated through a specific protein adhesin-receptor interaction, and fibrinogen acts as a cryptitope.

Starch Accumulation Is Associated with Adventitious Root Formation in Hypocotyl Cuttings of Pinus radiata

This study of the internal physiology of adventitious root formation in Pinus radiata was performed without the potential complications from microbial contamination and nutritional stress. Hypocotyl cuttings of radiata pine were cultured in half strength MS nutrient medium supplemented with IBA (indole-3-butyric acid), IBA + kinetin, kinetin, or without phytohormones (control). Averages of 8.35 and 0.08 roots per cutting were formed in IBA and in growth regulator-free treatments, respectively. No roots were formed in IBA + kinetin, kinetin, or sucrose-free treatments at day 30 after excision of hypocotyls. Changes in fresh weight, sugar, and starch content were measured at established developmental stages associated with adventitious root formation. Sucrose-supplemented medium was required for higher levels of sugar, starch, and root formation in rooting region of IBA-treated hypocotyls. Starch accumulation, in particular, seems to have potential as a biochemical marker before root primordium emergence in light of the following observations in the IBA treatment. Starch began to build up preferentially in cells involved in or in close proximity to potential sites of new root primordium formation (that is, the cells on the inside of the cortex and the pith) before any visible organized root primordia and then began to disappear during root primordium formation. The substantial starch accumulation associated with IBA treatment was not observed in the IBA + kinetin, kinetin alone, growth regulator-free, sucrose-free treatments (nonrooting treatments) or in the nonrooting region of hypocotyls treated with IBA.

RPE, a plant gene involved in early developmental steps of nematode feeding cells.

Sedentary plant-parasitic nematodes are able to induce the redifferentiation of root cells into multinucleate nematode feeding sites (NFSs). We have isolated by promoter trapping an Arabidopsis thaliana gene that is essential for the early steps of NFS formation induced by the root-knot nematode Meloidogyne incognita. Its pattern of expression is similar to that of key regulators of the cell cycle, but it is not observed with the cyst nematode. Later in NFS development, this gene is induced by both root-knot and cyst nematodes. It encodes a protein similar to the D-ribulose-5-phosphate 3-epimerase (RPE) (EC 5.1.3.1), a key enzyme in the reductive Calvin cycle and the oxidative pentose phosphate pathway (OPPP). Quantitative RT-PCR showed the accumulation of RPE transcripts in potato, as in Arabidopsis NFS. Homozygous rpe plants have a germination mutant phenotype that can be rescued in dwarf plants on sucrose-supplemented medium. During root development, this gene is expressed in the meristems and initiation sites of lateral roots. These results suggest that the genetic control of NFSs and the first stages of meristem formation share common steps and confirms the previous cytological observations which indicate that root cells undergo metabolic reprogramming when they turn into NFSs.

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmotic stress, created by high agarose or carbohydrate concentrations, on the regeneration of fertile plants from protoplast-derived colonies of several indies (IR43, Jaya, Pusa Basmati 1) and japonica (Taipei 309) rice varieties. Observations of the cultures developed on media containing one of these carbohydrates (cellobiose, fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose), each at 88 mM, indicated that maltose was the preferential carbon source for the proliferation of embryogenic callus and shoot regeneration. Maltose-containing medium induced shoot formation in 24-66% of the protoplast-derived tissues, depending upon the rice variety, compared to shoot regeneration from 4-32% of the tissues in sucrose-supplemented medium. Media containing 288 mM maltose or an equimolar combination of 88 mM maltose and 200 mM mannitol, caused water loss from calli and promoted the growth of embryogenic calli. These calli formed shoots with greater frequencies when subsequently transferred to shoot regeneration medium with 88 mM maltose. A medium containing 88 mM maltose and semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarose had a similar beneficial effect on the growth of embryogenic calli and simultaneously supported high-frequency (48-55%) shoot formation. The optimum shoot regeneration frequencies (60-78%) were obtained when protoplast-derived colonies were serially cultured on to shoot regeneration medium containing 1.0% (w/v) agarose for 4 weeks, followed by a 2-week culture period on the same medium with 0.5% (w/v) agarose. Plants regenerated on medium containing maltose and/or 1.0% (w/v) agarose were phenotypically normal and fertile.

Isolation of high-chlorophyll-fluorescence mutants of Arabidopsis thaliana and their characterisation by spectroscopy, immunoblotting and northern hybridisation.

Thirty-four recessive photosynthetic mutants of the high-chlorophyll-fluorescence (hcf) phenotype have been isolated by screening 7700 M2 progenies of ethyl methane sulfonate-treated seeds of Arabidopsis thaliana. Most of the mutants isolated were found to be seedling-lethal, but could be grown on sucrose-supplemented media. Chlorophyll (Chl) fluorescence induction, absorption changes in the reaction-centre chlorophyll of PS I (P700) at 830 nm and Chl a/Chl b ratios were recorded in order to probe the photosynthetic functions and to define the mutational lesion. These studies were complemented by immunoblot and Northern analyses which finally led to the classification of the mutants into six different groups. Four classes of mutants were affected in PS I, PS II (two different classes) or the intersystem electron-transport chain, respectively. A fifth mutant class was of pleiotropic nature and the sixth class comprised a Chl b-deficient mutant. Several of the mutants showed severe deficiencies in the levels of subunits of PS I, PS II or the cytochrome b6/f complex. Thus the mutational lesions could be located precisely. Only one mutant was defective in the transcript patterns of some plastid-encoded photosynthesis genes. Hence most of the mutants isolated appear to be affected in translational and post-translational regulatory processes of thylakoid membrane biogenesis or in structural genes encoding constituent subunits of the thylakoid protein complexes.

Modulation of the anti- candida activity of apo-lactoferrin by dietary sucrose and tunicamycin in vitro

The sensitivity to apo-lactoferrin (apo-LF) of two oral isolates each of Candida albicans and C. krusei preincubated in either sucrose- or tunicamycin-supplemented media was investigated. Known apo-LF-sensitive isolates of both C. albicans and C. krusei demonstrated significantly increased resistance to apo-LF when preincubated in sucrose-supplemented media, but such preincubation had no effect on an apo-LF-resistant isolate of C. albicans, irrespective of the sugar concentration in the medium. Surprisingly, an apo-LF-resistant isolate of C. krusei demonstrated increased susceptibility to apo-LF when preincubated in sucrose. Further, when preincubated with tunicamycin, a chemical that interferes with candidal cell-wall formation, the susceptibility to apo-LF of the resistant isolates increased significantly. These results, when extrapolated in vivo, imply that dietary sucrose may diminish the anticandidal potency of salivary lactoferrin. However, the aberrant behaviour of a single isolate of C. krusei under these circumstances is possibly due to the different cell-wall structure of this yeast and needs to be further investigated.