Determination of floral initiation in <Emphasis Type="Italic">Malus domestica</Emphasis>: a novel morphogenetic approach

Floral initiation in apple (Malus domestica Borkh) was studied by a novel morphogenetic approach. Developmental stages of apices were evaluated based on the morphology of shoot apical meristem (SAM) from various collection dates. Besides, the frequency of each stage was calculated within apices populations after full blooming (DAFB). Prior to doming of apex, three marked phases were found based on SAM morphology: 1) narrow appearance (vegetative phase), 2) broadened form (transition phase), and 3) prominent shape (commitment phase). A furrow region was formed at the base of leaf primordium during the bract initiation, while significant broadening of SAM was observed. Cell division patterns manifested in modification of anisotropic clusters from isotropic cellular packets, as a result of which profound morphological changes of apices occurred. Based on these findings, we propose that the structural alterations prior to doming may be taken into account for determination of the initial development and reproduction signs in apple trees.     

Differences in the timing of cell death,differentiation and function among three different types of ray parenchyma cells in the hardwood <Emphasis Type="Italic">Populus sieboldii</Emphasis> × <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">grandidentata</Emphasis>

Differences in the timing of cell death, differentiation and function among three different types of ray parenchyma cells in the hardwood Populus sieboldii × P. grandidentata which form uniseriate and homocellular rays were examined and clarified. Ray parenchyma cells died within 5 years, and the disappearance of nuclei from ray parenchyma cells did not occur successively from the pith side, even within individual radial cell lines of a given ray. Cell death occurred earliest in contact cells, which were connected to adjacent vessel elements through pits, in the fourth annual ring from the cambium. Cell death occurred next in intermediate cells, which were located within the same cell lines as contact cells but were not adjacent to vessel elements, in the fourth annual ring from the cambium. Finally, isolation cells, which were located within the other cell lines of a given ray, died in the fifth annual ring from the cambium. Secondary wall thickenings in contact cells and intermediate cells were initiated before those in isolation cells in the current year’s xylem. Most starch grains were localized in intermediate cells, and there were more lipid droplets in contact cells and intermediate cells than in isolation cells. In addition, the largest quantities of protein were found in contact cells. Our results indicate that the position within a ray and neighboring short-lived vessel elements might affect the timing of cell death and differentiation and, thus, the function of long-lived ray parenchyma cells in Populus sieboldii × P. grandidentata.     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

<Emphasis Type="Italic">IXR1</Emphasis> and <Emphasis Type="Italic">HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.     

Ultrastructural changes in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells under stress conditions

Ultrastructural organization of the aerobic yeast Yarrowia lipolytica was studied under conditions of oxidative, heat, and ethanol stresses. It was shown that the following uniform changes in cell ultrastructure did not depend on the type of stress: enlargement of mitochondria, enhanced number and enlargement of peroxisomes, and formation of lipid granules. Similar ultrastructural changes also occurred during the transition of cells to the stationary growth phase. It was shown for the first time that accumulation of polyphosphate granules occurred as a stress response in yeasts. Moreover, numerous globular structures of unknown nature appeared on the cell wall surface under oxidative or heat stress. Under ethanol stress, the cells developed clearly marked deep invaginations of the cytoplasmic membrane. (The same changes in the cytoplasmic membrane were observed in the cells grown on ethanol.) Variations of the cell envelope structure along with the formation of polyphosphate granules were not observed in the stationary growth phase. Ultrastructural changes in the cells under stress conditions are in agreement with the previous data on survival, respiratory activity, and variations of the antioxidant systems.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Expression and Assembly of Recombinant Surface Layer Proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Most bacterial surface layers (SLs) are formed by self-assembly of a single type of protein. Native and recombinant surface layer monomers are capable to self-assemble on solid substrates and in solution to highly regular nanosized arrays which make them attractive for nanobiotechnological applications. In this study, we expressed the surface layer protein SbsC of Bacillus stearothermophilus ATTC 12980, tagged with Enhanced Green Fluorescent Protein, in the yeast Saccharomyces cerevisiae. We observed a network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis, SL structures were formed de novo in the buds. SL assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point.     

The <Emphasis Type="Italic">alp1-1315</Emphasis> mutation of the tubulin-folding cofactor D gene delays the mitosis initiation in <Emphasis Type="Italic">cdc25-22</Emphasis> mutant cells of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Tubulin-folding cofactor D is necessary for the assembly of tubulin heterodimers and, possibly, plays additional roles in the cell. The effects of cofactor D, microtubules, and/or tubulin dimers on the mitosis initiation were studied in Schizosaccharomyces pombe. It was found for the first time that S. pombe cells with the alp1-1315 and cdc25-22 mutations remained highly viable at 36°C for 8 h, in contrast to cells with the alp1-1315 mutation alone. The progression of cdc25-22 alp1-1315 cells through mitosis after a cell division arrest at 36°C was described. When transferred to 25°C, cdc25-22 alp1-1315 cells displayed a lag of approximately 30 min in Plo1-GFP appearance in the spindle pole body (SPB), 1 h in chromosome condensation, and 75 min in spindle formation. Thus, the initiation of mitosis in cdc25-22 alp1-1315 cells was delayed as compared with cdc25-22 cells. Since treatment of cdc25-22 cells with a microtubule-destabilizing drug during an arrest is known to cause a premitotic arrest with low activity of the mitosis-promoting factor (MPF), it was assumed that an impaired integrity of microtubules and/or lack of tubulin dimers in the nucleus were responsible for the delayed mitosis initiation in cdc25-22 alp1-1315 cells and in cdc25-22 cells treated with a microtubule-destabilizing drug. The progression through mitosis after a cdc25-22 arrest was extremely slow in cdc25-22 alp1-1315 cells, which was attributed to the de novo formation of tubulin dimers.     

Multiple alleles at <Emphasis Type="Italic">Early flowering 1</Emphasis> locus making variation in the basic vegetative growth period in rice (<Emphasis Type="Italic">Oryza</Emphasis> <Emphasis Type="Italic">sativa</Emphasis> L.)

A recently established rice breeding program in low latitudes aims to develop varieties with extremely long basic vegetative growth (BVG) periods and weak photoperiod sensitivities. The Taiwanese japonica variety Taichung 65 (T65) harbors a recessive allele ef1 at the Ef1 (Early flowering 1) locus, thereby exhibiting an extremely long BVG period. The previous reported functional allele Ehd1 (Early heading date 1), located on chromosome 10, encodes a B-type response regulator, thereby shortening the BVG period, whereas its nonfunctional allele ehd1 greatly prolongs the BVG period. A conventional analysis using F₂ and F₃ populations and a subsequent CAPS analysis based on the amino acid sequences of Ehd1 and ehd1 showed that Ef1 and Ehd1 were at the same locus. The CAPS analysis also indicated that the Taiwanese japonica varieties with extremely long BVG periods all harbor ef1, but that ef1 does not exist among indica and japonica varieties in the low latitudes. Since ef1 has not been found in any japonica varieties outside Taiwan, this allele might have originated in Taiwan. Sequence analysis revealed that the mutant allele ef1-h, which prolongs the BVG period even more than ef1 does, harbors an mPing insertion in exon 2, which causes the complete loss of gene function. Our results indicate that both ef1 or ef1-h alleles can be used as new gene sources in developing rice varieties with extremely long BVG periods for low latitudes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The isolation and analysis of a soybean <Emphasis Type="Italic">CO</Emphasis> Homologue <Emphasis Type="Italic">GmCOL10</Emphasis>

The CONSTANS (CO) gene is a key regulator of the response to photoperiod in the model plant Arabidopsis thaliana, and its homologues are present in many plant species. We describe here the isolation of the CO homologue for zinc finger protein gene GmCOL10 (Glycine max CONSTANS-Like 10) from the soybean cultivar Kennong18. Sequence comparisons showed that the closest A. thaliana gene to GmCOL10 is COL5. The expression of GmCOL10 was regulated in a circadian manner, especially under short-day conditions. The expression of GmCOL10 was concentrated in vegetative organs, and in particular in the unifoliolates and cotyledons. An analysis of subcellular localization found GmCOL10 in the nucleus. Our data suggested that GmCOL10 was not related to the photoperiodic pathway of floral transition as Arabidopsis CO does.     

Morphological and molecular characterization of <Emphasis Type="Italic">Calothrix</Emphasis> isolates obtained from diverse environments in India

Thirty cyanobacterial strains of Calothrix (family Rivulariaceae) isolated from diverse geographical regions of India were analyzed using morphological and molecular approaches. Most of the isolates were planktonic while some grew benthically. Significant differences were observed with regard to the shape and size of the vegetative cells, heterocysts, and akinetes. Analyses of molecular polymorphisms using Restriction Fragment Length Polymorphisms (RPLP) of 16S rRNA genes with the reference strain led to unambiguous differentiation of the isolates as well as understanding of their genetic relationships.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Lactobacillus plantarum (LP) has previously been used for the treatment and prevention of intestinal disorders and disease. However, the role of the LP surface layer adhesive protein (SLAP) in inhibition of epithelial cell disruption is not fully understood. The aim of the present study was to investigate the protective effects of purified SLAP on Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). The role of ERK in LP-mediated inhibition of tight junction (TJ) injury was also evaluated in order to determine the molecular mechanisms underlying the protective effects of LP in epithelial cells. SLAP was extracted and purified from LP cells using a porcine stomach mucin-Sepharose 4B column. SLAP-mediated inhibition of bacterial adhesion was measured using a competition-based adhesion assay. Expression of TJ-associated proteins, maintenance of TJ structure, and levels of extracellular signal regulated kinase (ERK) and ERK phosphorylation were assessed in SLAP-treated cells by a combination of real-time PCR, western blotting, and immunofluorescence microscopy. Cell permeability was analyzed by measurement of trans-epithelial electrical resistance (TER) and dextran permeability. The effect of SLAP on levels of apoptosis in epithelial cells was assessed by flow cytometry. Results from these experiments revealed that treatment with SLAP decreased the level of adhesion of EPEC to Caco-2 cells. SLAP treatment also enhanced expression of TJ proteins at both the mRNA and protein levels and affected F-actin distribution. Although ERK levels remained unchanged, ERK phosphorylation was increased by SLAP treatment. Caco-2 cells treated with SLAP exhibited increased TER and decreased macromolecular permeability, which was accompanied by a decrease in the level of apoptosis. Together, these results suggest that LP-produced SLAP protects intestinal epithelial cells from EPEC-induced injury, likely through a mechanism involving ERK activation.     

Cellular Interactions and the Secondary Response <Emphasis Type="Italic">in vitro</Emphasis>

ALTHOUGH the role of cellular cooperation in the induction of the immune response has become firmly established only recently¹, morphological evidence suggesting that such cooperation takes place is quite old. Reports² of the aggregation of lymphoid cells around macrophages³ have been confirmed: “islets”, “rosettes” or “clusters” in cultures of cells (derived from humans⁴, guinea-pigs⁵, rabbits⁶ or mice⁷) stimulated with antigen or PHA⁸ were reported. We have investigated cluster formation to ascertain its relationship, if any, to the antigen-induced stimulation of sensitized cells⁹. We used peripheral blood leucocytes from rabbits immunized to bovine serum albumin (BSA) or to human red blood cells (HRBC). The BSA was given in complete Freund's adjuvant (three intramuscular injections of 7.5 mg BSA each, into the hind legs at weekly intervals). HRBC (1 ml.) was given once into the ear vein, as a 20% suspension in saline. Cell cultures and ³H-thymidine incorporation were measured as before¹⁰. To prepare cell smears, cells were washed three times and suspended in one drop of normal rabbit serum and 1 µl. of the suspension was spread on a microscope slide. This ensured a reasonably constant number of cells per slide and made possible comparisons between different experiments. Smears were fixed with methanol and stained with Giemsa.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of ten threatened species of <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae)

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.     

Primary characterization of dominant cell surface proteins of halotolerant methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Cell surface-associated proteins with molecular masses of 27 and 80 kDa found in Methylomicrobium alcaliphilum were studied. The MEALZv2_1030034 and MEALZv2_1030035 genes encoding these proteins were identified in the partially annotated genome of the methanotroph. Polypeptides MEALZv2_1030034 and MEALZv2_1030035 showed high homology (>50% identity) with the surface proteins CorA and CorB of Methylomicrobium album BG8 expressed in conditions of low copper content in the growth medium. Electron microscopic analysis with antibodies revealed localization of the 27-kDa protein in the base of the cup-shaped structures of S-layers. The mutant with an insertion in the MEALZv2_1030034 gene lost the ability to grow on the medium with methane, but grew in the presence of 0.2% methanol. In this case, the cup-shaped structures of S-layers were not bound to the cell wall but occurred as regular aggregates in the intercellular space. The 80-kDa protein was found mainly in the periplasm, had a peroxide-degrading activity, and was classified as a di-heme cytochrome c peroxidase. The mutant deficient in the MEALZv2_1030035 gene grew on methane at a high rate and had higher activities of C₁ compound oxidation enzymes than did the parent strain. The role of the proteins MEALZv2_1030034 and MEALZv2_1030035 and distribution of their homologues in other methanotrophs are discussed.     

Influence of <Emphasis Type="Italic">Daphnia</Emphasis> infochemicals on functional traits of <Emphasis Type="Italic">Microcystis</Emphasis> strains (Cyanobacteria)

We conducted a laboratory experiment to investigate the influence of Daphnia infochemicals on growth rate, microcystin production, colony formation and cell size of eight Microcystis strains isolated from two lakes. The strains were characterized genetically by their 16S-23S rDNA ITS sequence. The experiment was composed of four treatments: (1) a control using filtered WC medium, (2) addition of Scenedesmus obliquus culture medium filtrate, (3) addition of Daphnia magna culture medium filtrate and (4) addition of sodium octyl sulphate, a commercially available Daphnia infochemical. Our results showed that sympatric strains differed strongly for the measured functional traits, while no correlations between traits were found. Between-strain differences in growth rate, microcystin production, colony formation and cell size were generally larger than the differences in phenotypes observed between treatments. Despite this, several strains reacted to the infochemicals by changing functional trait values. Daphnia culture medium filtrate and, to a lesser extent, sodium octyl sulphate had a negative influence on the growth rate of half of the strains and stimulated microcystin production in one strain, but the latter effect was not Daphnia-specific as Scenedesmus culture medium filtrate had the same effect. Daphnia culture medium filtrate also induced colony formation in one strain. Our data suggest that Daphnia infochemicals generally have a weak influence on growth rate, microcystin production and colony formation of Microcystis strains as compared to the inter-strain variability, while existing inducible effects are highly strain-specific.     

Identification and Characterization of an Autolysin Gene, <Emphasis Type="Italic">atlh</Emphasis>, from <Emphasis Type="Italic">Streptococcus downei</Emphasis>

An autolysin gene, atlh, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. Atlh protein encoded by atlh is composed of 879 amino acids, with a molecular weight of 95,902.26. Atlh possesses four 15-amino-acid residue repeats in the putative cell-wall-binding domain and has a catalytic domain in the C-terminus. The deduced amino acid sequence of atlh showed homology to S. mutans autolysin AtlA (68.4% similarity). Inactivation of atlh resulted in elongated chain formation compared to the parent strain. Recombinant proteins Atlh and its derivatives were constructed and analyzed by zymography. Zymographic analysis revealed that the Asp-771 residue of Atlh was essential for lytic activity and that lytic activity was not diminished by the deletion of repetitive regions in the putative cell-wall-binding domain of Atlh. Biofilm assay showed that the wild-type strain formed glucose- and sucrose-dependent biofilms, the atlh mutant diminished this ability. These results suggest that Atlh is associated with cell separation and biofilm formation.