Interspecific incompatibility in Populus: inhibition of tube growth and behaviour of the male germ unit inP. deltoides×P. alba cross

Summary In order to better understand the cellular events controlling interspecific incompatibility in the genus Populus, the incompatible cross betweenP. deltoides andP. alba has been investigated both at the light and electron microscopic levels. Stained in decolourized aniline blue and observed by epifluorescence microscopy, most incompatible pollen grains are seen to germinate at the stigma surface. Numerous incompatible pollen tubes reach the base of the style where they are arrested 19 h after pollination. Ultrastructural observations on in vivo growing incompatible pollen tubes confirm these data. Very few cytoplasmic modifications are seen within living pollen tubes reaching the lower end of the style or within arrested ones, except the presence of polymorphic plastids. In this predominantly tricellular system, the male germ unit (MGU) is apparently initiated at pollen maturity as an association between the vegetative nucleus and sperm cells. It is maintained during pollen tube growth within the style and persists within arrested incompatible pollen tubes. The unique observation of an association between a dividing generative cell at metaphase and the vegetative nucleus is also reported. Arrested pollen tubes are characterized by apical deformations and accumulation of callose within their thickened cell walls. These cytological data provide additional information on the cellular events associated with interspecific incompatibility in Populus.Abbreviations DAPI 4,6-diamino-2-phenylindole - FCR fluorochromatic reaction - MGU male germ unit     

Evaluation of heterologous promoters in transgenic Populus tremula × P. alba plants

The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 – 15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between independent transgenic lines for all the constructs tested.     

Postnatal development of β-galactosidase activity in the small intestine of the rat. Effect of adrenalectomy and diet

1. β-Galactosidase activity was studied in homogenates of the proximal and distal thirds of the small intestine from adult and infant rats. o-Nitrophenyl β-d-galactoside served as the substrate. 2. Activity in suckling rats is highest in the distal part of the small intestine. 3. The pH optimum was 3·5 in the distal third of the small intestine in rats aged 5 and 15 days, whereas in the proximal third the maximum was not clearly defined. 4. Activity was higher in both thirds in newborn than in adult rats, expressed per wet wt. or per wt. of protein. In the proximal third activity continually decreases with age, whereas in the distal part there is a rise up to day 15 and then a sudden decrease. Total β-galactosidase activity changes very little in the proximal third during postnatal development; the greatest changes occur in the distal third. 5. Adrenalectomy performed on day 15 postnatally slows down the decrease in β-galactosidase activity, particularly in the distal part. 6. Feeding a lactose diet to infant rats from day 14 postnatally in the presence of the mother rat also slows down the decrease in β-galactosidase activity and this is not found with a diet containing glucose and galactose instead of lactose.     

Glycohydrolases β-hexosaminidase and β-galactosidase are associated with lipid microdomains of Jurkat T-lymphocytes

Growing evidence suggests the presence of active lysosomal enzymes in extra-lysosomal compartments, such as the plasma membrane. Although in the past little attention was paid to glycohydrolases acting on cellular compartments different from lysosomes, there is now increasing interest on plasma membrane-associated glycohydrolases because they should be involved, together with glycosyltransferases, in glycosphingolipids oligosaccharide modification processes regulating cell-to-cell and/or cell-environment interactions in both physiological and pathological conditions. Starting from the previous evidence of the presence of β-hexosaminidase and β-galactosidase at the plasma membrane of cultured fibroblasts, we here investigated the association of these glycohydrolases with lipid microdomains of Jurkat T-lymphocytes. Monosialoganglioside GM3 represents the major glycosphingolipid constituent of T-cell plasma membrane and its amount largely increases after T-cell stimulation. β-hexosaminidase and β-galactosidase cleave specific β-linked terminal residues from a wide range of glycoconjugates and in particular are involved in the stepwise degradation of GM1 to GM3 ganglioside. Here we demonstrated that fully processed plasma membrane-associated β-hexosaminidase and β-galactosidase co-distribute with the lipid microdomain markers and co-immunoprecipitate with the signalling protein lck in Jurkat T-cell. Furthermore, Jurkat cell stimulation up-regulates the expression and activity of lysosomal β-hexosaminidase and β-galactosidase and increases their targeting to lipid microdomains. The non-random distribution of plasma membrane-associated β-hexosaminidase and β-galactosidase and their localization within lipid microdomains, suggest a role of these enzymes in the local reorganization of glycosphingolipid-based signalling units.     

RAPD Linkage Mapping in a Populus adenopoda×P. alba F1 Family

Random amplified polymorphic DNAs(RAPDs) were used to construct linkage maps of the parents of a Populus adenopoda Maxim. x P. alba L. Fl family. A set of 620 random oligonucleotide primers were screened and 128 primers were selected to generate RAPD markers within a sample of 80 Fl progenies. A total of 333 segregating loci [ (326( 1:1 ) ,7(3:1 ) ] were identified. Among the 326 1:1 segregating loci (238 loci from P. adenopoda and 88 loci from P. dba),36 loci (26 loci in P. adenopoda and 10 loci in P. dba) were found distorted from the normal 1:1 ratio. Altogether 290 loci segregating 1:1 (testcross configuration) were used to construct parent-specific linkage maps,212 for P. alba and 78 for P. adenopoda. The resulting linkage maps consisted of 189 marker loci in 20 groups (four or more loci per group), 6 triples and 16 pairs for P. dba, which cover the map distance about 2 402.4 cM, and 41 linked marker loci for P. adenopoda which cover map distance about 479.4 cM. Further study is warranted to locate some important quantitative trait loci (QTLs) based on the maps.     

Genetic relationship between β-galactosidase and β-fucosidase in the mouse

Evidence for the identity of β-galactosidase and β-fucosidase enzymes in the house mouse was obtained by examination of the enzyme activities in animals from different crosses between C57BL/Kl mice with high galactosidase and fucosidase activities and DBA/2/Kl mice with low activities. There is a strong correlation between the activities of these two enzymes in different tissues of F₂ animals. A comparison of the fractionation properties of β-galactosidase and β-fucosidase showed that the two activities had a parallel distribution and identical thermostability. These data suggest that the same enzyme catalyzes the hydrolysis of both substrates.     

Enhancement of growth rate and β-galactosidase activity,and variation in organic acid profile of Bifidobacterium animalis subsp. lactis Bb 12

The behavior of Bifidobacterium animalis subsp. lactis Bb 12 under batch cultivation, after continuous culturing for up to 12 d, was monitored in skim milk-based media. Previous continuous culture for longer than 6 d affected the physiology of said microorganism. The minimum inhibitory concentrations of lactic and acetic acids increased from 18 to 26 g/l, whereas the molar ratio of acetic to lactic acid increased from 0.8 to 1.55, when the previous continuous culture increased its duration from 1 to 12 d. The specific lactose consumption rate decreased from 0.94 to 0.77 g_lactose/g_{cell dry mass}/h within the batch culture timeframe; this was concomitant with greater amounts of acetic and formic acids, and lower amounts of lactic acid produced. The β-galactosidase activity increased as continuous culturing time increased, and reached 446 units/ml by 12 d; however, the rate of enzyme synthesis decreased concomitantly. Succinic acid was produced during the exponential growth and stationary phases of the batch culture, but the former at exponential growth phase was higher as the continuous culturing time was longer. For comparison purposes, batch cultivation of samples taken from continuous cultures by 1 and 12 d was done using a semi-synthetic medium with glucose as carbon source; a pattern similar to that observed when using skim milk-based media was observed.     

Influence of lactose and lactate on growth and β-galactosidase activity of potential probiotic Propionibacterium acidipropionici

Dairy propionibacteria are microorganisms of interest for their role as starters in cheese technology and as well as their functions as probiotics. Previous studies have demonstrated that Propionibacterium acidipropionici metabolize lactose by a β-galactosidase that resists the gastrointestinal transit and the manufacture of a Swiss-type cheese, so that could be considered for their inclusion in a probiotic product assigned to intolerant individuals. In the present work we studied the effect of the sequential addition of lactose and lactate as first or second energy sources on the growth and β-galactosidase activity of P. acidipropionici Q4. The highest β-galactosidase activity was observed in a medium containing only lactate whereas higher final biomass was obtained in a medium with lactose. When lactate was used by this strain as a second energy source, a marked increase of the intracellular pyruvate level was observed, followed by lactate consumption and increase of specific β-galactosidase activity whereas lactose consumption became negligible. On the contrary, when lactose was provided as second energy source, lactic acid stopped to be metabolized, a decrease of the intracellular pyruvate concentration was observed and β-galactosidase activity sharply returned to a value that resembled the observed during the growth on lactose alone. Results suggest that the relative concentration of each substrate in the culture medium and the intracellular pyruvate level were decisive for both the choice of the energetic substrate and the β-galactosidase activity in propionibacteria. This information should be useful to decide the most appropriate vehicle to deliver propionibacteria to the host in order to obtain the highest β-galactosidase activity.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Photosynthetic activity and chlorophyll pigment concentration in Medicago x varia T. Martyn leaves treated with the Tytanit growth regulator

The purpose of the research was to determine the effect of the foliar use of a growth regulator with the trade name of Tytanit, containing titanium ascorbate, on photosynthetic activity and chlorophyll content in Medicago × varia T. Martyn leaves. There were two kinds of plots: C – control series; Ti – plants treated with Tytanit, containing 8.5 g of titanium in 1 dm³. The following parameters were determined: maximum photosystem II efficiency (F_v/F_m) in a dark-adapted state, actual photosystem II efficiency (ΔF/F_m’) in a light-adapted state, photochemical quenching factor (QP), non-photochemical quenching factor (QN), and chlorophyll a and b content. The Fisher-Snedecor test was used to determine whether the impact of experimental factors was significant, and the HSD 0.05 value was calculated using Tukey’s test. Compared to control, the photosynthetic apparatus performance of alfalfa was positively affected by the regulator compared to control. Tytanit applied to plant leaves increased their photosynthetic activity as a result of an increase in the content of chlorophyll pigments. It was also found that periods of rainfall deficiency did not affect the beneficial effects of the regulator.     

Ontogenesis of the α-MSH, β-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity

Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.     

Influences of medium consistency and shoot density on in vitro shoot proliferation of Populus alba × P. grandidentata

The in vitro shoot proliferation of Populus alba × P. grandidentata was affected by the medium consistency and shoot density, but not by three sizes of vessels. After 4 weeks of culture, the fresh weight and number of shoots per explant on liquid medium were significantly greater than those on agar-solidified medium. In particular, 3.2 shoots, 7 mm or longer per explant, were produced on liquid medium compared with 1.6 shoots per explant or agar-solidified medium. The fresh weight per explant after 4 weeks of culture on liquid medium and agar-solidified medium were 0.68 and 0.25 g, respectively. Increasing the number of shoots per vessel slowed the growth of the explants as measured by fresh weight and the number of shoots produced. There was little difference in the number of shoots produced between vessels with 1 or 2 shoots per vessel, but there were many fewer shoots produced when 3 shoots were placed in each vessel.Journal Paper No. J-11977 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2210.     

In vitro organogenesis and somatic embryogenesis from punctured leaf of Populus nigra × P. maximowiczii

Various explant sources of Populus nigra × P. maximowiczii were used to examine the effects of growth hormones on morphogenesis in vitro. Initial experiments indicated that punctured leaves were superior to non-punctured ones for both callus growth and formation of shoots and roots on MS medium containing various types and concentrations of growth hormones. After 6 weeks in culture, an average of 178 shoots, 129 roots and 3.1 g fresh weight of callus were directly produced from the abaxial side of each punctured leaf. The best combinations of growth hormones for shoot, root and callus proliferation were 0.88 M BA plus 0.05 M 2,4-D, 0.44 M BA plus 2.69 M NAA and 0.44 M BA plus 2.26 M 2,4-D, respectively. Embryoids were also formed on callus derived from punctured leaves. The number of embryoids varied from 0 to 30 per punctured leaf. Adventitious shoots also developed simultaneously with the embryos. Embryoids were removed with a scalpel at the early developmental stages and placed on MS medium lacking growth regulators. Regenerated plantlets were transferred to pots containing vermiculite for normal growth in the greenhouse.     

An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba?��?P. berolinensis and Populus davidiana?��?P. bolleana

Transgenic technology has been successfully used for gene function analyses and trait improvement in cereal plants. However, its usage is limited in woody plants, especially in the difficult-to-transform but commercially viable hybrid poplar. In this work, an efficient regeneration and transformation system was established for the production of two hybrid aspen clones: Populus alba × P. berolinensis and Populus davidiana × P. bolleana. A plant transformation vector designed to express the reporter gene uidA, encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was used to detect transformation event at early stages of plant regeneration, and to optimize the parameters that may affect poplar transformation efficiency. Bacterium strain and age of leaf explant are two major factors that affect transformation efficiency. Addition of thidiazuron (TDZ) improved both regeneration and transformation efficiency. The transformation efficiency is approximately 9.3% for P. alba × P. berolinensis and 16.4% for P. davidiana × P. bolleana. Using this system, transgenic plants were usually produced in less than 1 month after co-cultivation. The growth characteristics and morphology of transgenic plants were identical to the untransformed wild type plants, and the transgenes could be inherited by vegetative propagation, as confirmed by PCR, Southern blotting, RT–PCR and β-glucuronidase staining analyses. The establishment of this system will help to facilitate the studies of gene functions in tree growth and development at a genome level, and as well as the introduction of some valuable traits in aspen breeding.     

Comparative Analysis of Growth and Photosynthetic Characteristics of (Populus simonii × P. nigra) × (P. nigra × P. simonii) Hybrid Clones of Different Ploidides

To evaluate differences among poplar clones of various ploidies, 12 hybrid poplar clones (P. simonii × P. nigra) × (P. nigra × P. simonii) with different ploidies were used to study phenotypic variation in growth traits and photosynthetic characteristics. Analysis of variance showed remarkable differences for each of the investigated traits among these clones (P < 0.01). Coefficients of phenotypic variation (PCV) ranged from 2.38% to 56.71%, and repeatability ranged from 0.656 to 0.987. The Pn (photosynthetic rate) photosynthetic photon flux density (PPFD) curves of the 12 clones were S-shaped, but the Pn-ambient CO₂ (Ca) curves were shaped like an inverted “V”. The stomatal conductance (Gs)-PPFD and transpiration rate (Tr)-PPFD curves had an upward tendency; however, with increasing PFFD, the intercellular CO₂ concentration (Ci)-PPFD curves had a downward tendency in all of the clones. The Pn-PPFD and Pn-Ca curves followed the pattern of a quadratic equation. The average light saturation point and light compensation point of the triploid clones were the highest and lowest, respectively, among the three types of clones. For Pn-Ca curves, diploid clones had a higher average CO₂ saturation point and average CO₂ compensation point compared with triploid and tetraploid clones. Correlation analyses indicated that all investigated traits were strongly correlated with each other. In future studies, molecular methods should be used to analyze poplar clones of different ploidies to improve our understanding of the growth and development mechanisms of polyploidy.     

Genetic and morphological differentiation in Populus nigra L.: isolation by colonization or isolation by adaptation?

Identifying processes underlying the genetic and morphological differences among populations is a central question of evolutionary biology. Forest trees typically contain high levels of neutral genetic variation, and genetic differences are often correlated with geographic distance between populations [isolation by distance (IBD)] or are due to historic vicariance events [isolation by colonization (IBC)]. In contrast, morphological differences are largely due to local adaptation. Here, we examined genetic (microsatellite) and morphological (from a common garden experiment) variation in Populus nigra L., European black poplar, collected from 13 sites across western Europe and grown in a common garden in Belgium. Significant genetic differentiation was observed, with populations from France displaying greater admixture than the distinct Spanish and central European gene pools, consistent with previously described glacial refugia (IBC). Many quantitative traits displayed a bimodal distribution, approximately corresponding to small‐leaf and large‐leaf ecotypes. Examination of nine climatic variables revealed the sampling locations to have diverse climates, and although the correlation between morphological and climatic differences was significant, the pattern was not consistent with strict local adaptation. Partial Mantel tests based on multivariate summary statistics identified significant residual correlation in comparisons of small‐leaf to large‐leaf ecotypes, and within the small‐leaf samples, but not within large‐leaf ecotypes, indicating that variation within the small‐leaf morphotype in particular may be adaptive. Some small‐leaf populations experience climates very similar to those in large‐leaf sites. We conclude that adaptive differentiation and persistent IBC acted in combination to produce the genetic and morphological patterns observed in P. nigra.     

Illuminating the binding interactions of galactonoamidines during the inhibition of β-galactosidase (E. coli)

Several galactonoamidines were previously identified as very potent competitive inhibitors that exhibit stabilizing hydrophobic interactions of the aglycon in the active site of β-galactosidase (Aspergillus oryzae). To elucidate the contributions of the glycon to the overall inhibition ability of the compounds, three glyconoamidine derivatives with alteration in the glycon at C-2 and C-4 were synthesized and evaluated herein. All amidines are competitive inhibitors of β-galactosidase (Escherichia coli) and show significantly reduced inhibition ability when compared to the parent. The results highlight strong hydrogen-bonding interactions between the hydroxyl group at C-2 of the amidine glycon and the active site of the enzyme. Slightly weaker H-bonds are promoted through the hydroxyl group at C-4. The inhibition constants were determined to be picomolar for the parent galactonoamidine, and nanomolar for the designed derivatives rendering all glyconoamidines very potent inhibitors of glycosidases albeit the derivatized amidines show up to 700-fold lower inhibition activity than the parent.