Germanium does not substitute for boron in cross-linking of rhamnogalacturonan II in pumpkin cell walls

Boron (B)-deficient pumpkin (Cucurbita moschata Duchesne) plants exhibit reduced growth, and their tissues are brittle. The leaf cell walls of these plants contain less than one-half the amount of borate cross-linked rhamnogalacturonan II (RG-II) dimer than normal plants. Supplying germanium (Ge), which has been reported to substitute for B, to B-deficient plants does not restore growth or reduce tissue brittleness. Nevertheless, the leaf cell walls of the Ge-treated plants accumulated considerable amounts of Ge. Dimeric RG-II (dRG-II) accounted for between 20% and 35% of the total RG-II in the cell walls of the second to fourth leaves from Ge-treated plants, but only 2% to 7% of the RG-II was cross-linked by germanate (dRG-II-Ge). The ability of RG-II to form a dimer is not reduced by Ge treatment because approximately 95% of the monomeric RG-II generated from the walls of Ge-treated plants is converted to dRG-II-Ge in vitro in the presence of germanium oxide and lead acetate. However, dRG-II-Ge is unstable and is converted to monomeric RG-II when the Ge is removed. Therefore, the content of dRG-II-Ge and dRG-II-B described above may not reflect the actual ratio of these in muro. (10)B-Enriched boric acid and Ge are incorporated into the cell wall within 10 min after their foliar application to B-deficient plants. Foliar application of (10)B but not Ge results in an increase in the proportion of dRG-II in the leaf cell wall. Taken together, our results suggest that Ge does not restore the growth of B-deficient plants.     

Proton and copper adsorption to maize and soybean root cell walls

A surface complexation model which has been used to describe inner-sphere complexation on metal oxide surfaces was applied to the adsorption of Cu by isolated cell walls of 4-day and 28-day-old maize (Zea mays L. cv WF9 × Mo17) and 21-day-old soybean (Glycine max [L.] Merr. cv Dare) roots. Concentration dependence of the titration data prevented the determination of unique pK and capacitance values for the 4-day maize cell walls, though mean values obtained for the intrinsic pK of the titratable carboxyl groups were 3.0 (4-day maize), 3.6 (28-day maize), and 3.0 (21-day soybean) as determined by potentiometric titration with either NaOH or HCl in 20 millimolar NaCl. The constant capacitance model was applied to Cu sorption data from rapid batch equilibrium experiments in an ionic medium of 20 millimolar NaClO₄. Speciation calculations indicated that the formation of a bidentate surface complex was sufficient to describe the experimental data for all three types of plant material, with only one value for the pK and capacitance density. The intrinsic constants of Cu complexation by a neutral site are: log K = −0.3 ± 0.1, −0.2 ± 0.3, and 0.9 ± 0.1 for 4-day and 28-day maize, and 21-day soybean, respectively. The integral capacitance density parameter, which describes the relationship between surface charge density and electrical potential, is several times larger than for crystalline mineral surfaces. This result indicates that the surface electrical potential remains low even when the surface charge density is high. Such behavior is characteristic of gels and porous oxides.     

Tobacco transgenic lines that express fenugreek galactomannan galactosyltransferase constitutively have structurally altered galactomannans in their seed endosperm cell walls

Galactomannans [(1-->6)-alpha-D-galactose (Gal)-substituted (1-->4)-beta-D-mannans] are major cell wall storage polysaccharides in the endosperms of some seeds, notably the legumes. Their biosynthesis in developing legume seeds involves the functional interaction of two membrane-bound glycosyltransferases, mannan synthase (MS) and galactomannan galactosyltransferase (GMGT). MS catalyzes the elongation of the mannan backbone, whereas GMGT action determines the distribution and amount of Gal substitution. Fenugreek (Trigonella foenum-graecum) forms a galactomannan with a very high degree of Gal substitution (Man/Gal = 1.1), and its GMGT has been characterized. We now report that the endosperm cell walls of the tobacco (Nicotiana tabacum) seed are rich in a galactomannan with a very low degree of Gal substitution (Man/Gal about 20) and that its depositional time course is closely correlated with membrane-bound MS and GMGT activities. Furthermore, we demonstrate that seeds from transgenic tobacco lines that express fenugreek GMGT constitutively in membrane-bound form have endosperm galactomannans with increased average degrees of Gal substitution (Man/Gal about 10 in T(1) generation seeds and about 7.5 in T(2) generation seeds). Membrane-bound enzyme systems from transgenic seed endosperms form galactomannans in vitro that are more highly Gal substituted than those formed by controls under identical conditions. To our knowledge, this is the first report of structural manipulation of a plant cell wall polysaccharide in transgenic plants via a biosynthetic membrane-bound glycosyltransferase.     

Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.     

Effect on epidermal cell of soybean protein-degraded products and structural determination of the root hair promoting peptide

Peptide(s) produced from degraded soybean protein by an alkaline protease from Bacillus circulans HA12 (degraded soybean-meal products; DSP) increased the number of both the root hair cells (trichoblasts) and hairless cells (atrichoblasts) of Brassica rapa by about 4.4 times and 1.9 times, respectively. To identify the root hair-promoting peptide(s) in DSP, the origin protein of the root hair-promoting peptide(s) was identified as Kunitz trypsin inhibitor (KTI). The root hair-promoting peptide in the degraded products of KTI was purified and produced a signal of 1,198.2 Da with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) analysis. A search of the amino acid sequence of KTI located the peptide GGIRAAPTGNER, which had a molecular weight identical to 1,198.2 Da. The peptide GGIRAAPTGNER was chemically synthesized, and the synthetic peptide possessed root hair-promoting activity. Thus, it is concluded that this peptide in DSP is the foreign bioactive peptide promoting the differentiation of root hairs.     

A dual-targeted soybean protein is involved in Bradyrhizobium japonicum infection of soybean root hair and cortical cells

The symbiotic interaction between legumes and soil bacteria (e.g., soybean [Glycine max L.] and Bradyrhizobium japonicum]) leads to the development of a new root organ, the nodule, where bacteria differentiate into bacteroids that fix atmospheric nitrogen for assimilation by the plant host. In exchange, the host plant provides a steady carbon supply to the bacteroids. This carbon can be stored within the bacteroids in the form of poly-3-hydroxybutyrate granules. The formation of this symbiosis requires communication between both partners to regulate the balance between nitrogen fixation and carbon utilization. In the present study, we describe the soybean gene GmNMNa that is specifically expressed during the infection of soybean cells by B. japonicum. GmNMNa encodes a protein of unknown function. The GmNMNa protein was localized to the nucleolus and also to the mitochondria. Silencing of GmNMNa expression resulted in reduced nodulation, a reduction in the number of bacteroids per infected cell in the nodule, and a clear reduction in the accumulation of poly-3-hydroxybutyrate in the bacteroids. Our results highlight the role of the soybean GmNMNa gene in regulating symbiotic bacterial infection, potentially through the regulation of the accumulation of carbon reserves.     

Double knockouts of phospholipases Dzeta1 and Dzeta2 in Arabidopsis affect root elongation during phosphate-limited growth but do not affect root hair patterning

Root elongation and root hair formation are important in nutrient absorption. We found that two Arabidopsis (Arabidopsis thaliana) phospholipase Ds (PLDs), PLDzeta1 and PLDzeta2, were involved in root elongation during phosphate limitation. PLDzeta1 and PLDzeta2 are structurally different from the majority of plant PLDs by having phox and pleckstrin homology domains. Both PLDzetas were expressed more in roots than in other tissues. It was reported previously that inducible suppression or inducible overexpression of PLDzeta1 affected root hair patterning. However, gene knockouts of PLDzeta1, PLDzeta2, or the double knockout of PLDzeta1 and PLDzeta2 showed no effect on root hair formation. The expression of PLDzetas increased in response to phosphate limitation. The elongation of primary roots in PLDzeta1 and PLDzeta2 double knockout mutants was slower than that of wild type and single knockout mutants. The loss of PLDzeta2, but not PLDzeta1, led to a decreased accumulation of phosphatidic acid in roots under phosphate-limited conditions. These results indicate that PLDzeta1 and PLDzeta2 play a role in regulating root development in response to nutrient limitation.     

Role of a positive regulator of root hair development,CAPRICE, in Arabidopsis root epidermal cell differentiation

In Arabidopsis, root hairs are formed only from a set of epidermal cells named trichoblasts or hair-forming cells. Previous studies showed CAPRICE (CPC) promotes differentiation of hair-forming cells by controlling a negative regulator, GLABRA2 (GL2), which is preferentially expressed in hairless cells. Here, we show that CPC is also predominantly expressed in the hairless cells, but not in the neighboring hair-forming cells, and that CPC protein moves to the hair-forming cells and represses the GL2 expression. We also show that the N terminus of bHLH protein interacts with CPC and is responsible for the GL2 expression. We propose a model in which CPC plays a key role in the fate-determination of hair-forming cells.     

CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination

A chronic demyelinating disease results from murine infection with the neurotropic strain JHM of mouse hepatitis virus (MHV-JHM). Demyelination is largely immune mediated. In this study, the individual roles of CD4 and CD8 T cells in MHV-induced demyelination were investigated using recombination-activating gene 1-/- (RAG1-/-) mice infected with an attenuated strain of MHV-JHM. These animals develop demyelination only after adoptive transfer of splenocytes from mice previously immunized to MHV. In this study, we show that, following adoptive transfer, virus-specific CD4 and CD8 T cells rapidly infiltrate the CNS of MHV-JHM-infected RAG1-/- mice. Adoptive transfer of CD4 T cell-enriched donors resulted in more severe clinical disease accompanied by less demyelination than was detected in the recipients of undepleted cells. Macrophage infiltration into the gray matter of CD4 T cell-enriched recipients was greater than that observed in mice receiving undepleted splenocytes. In contrast, CD8 T cell-enriched recipients developed delayed disease with extensive demyelination of the spinal cord. MHV-JHM-infected RAG1-/- mice receiving donors depleted of both CD4 and CD8 T cells did not develop demyelination. These results demonstrate that the development of demyelination following MHV infection may be initiated by either CD4 or CD8 T cells. Furthermore, they show that CD4 T cells contribute more prominently than CD8 T cells to the severity of clinical disease, and that this correlates with increased macrophage infiltration into the gray matter.     

Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.     

Adsorption and exchange of Ca,Mg and K-ions on the root cell walls of clover and rye-grass

Summary The ion exchange properties of clover and rye-grass root cell walls were studied quantitatively. Three sets of experiments were performed: adsorption of Ca, Mg and K ions versus pH, adsorption versus cation concentration and exchange isotherms Ca–Mg and Mg–K. A model has been developed. It allows the satisfactory prediction of results (except for pH curves) with the adjustment of a minimum of parameters. The total charge (RT), on its own, accounts for the difference between the ion exchange properties of the clover and rye grass cell walls. The selectivities observed on root material are very different from those observed on soil exchange complexes. The decreasing affinities of cell walls for cations follow the sequence: Ca>MgK for cell walls. These differences of selectivity are much larger than those usually observed for soil exchange complexes.     

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.     

Sex ratio in flea infrapopulations: number of fleas, host gender and host age do not have an effect

This study set out to determine whether the sex ratio of fleas collected from host bodies is a reliable indicator of sex ratio in the entire flea population. To answer this question, previously published data on 18 flea species was used and it was tested to see whether a correlation exists between the sex ratio of fleas collected from host bodies and the sex ratio of fleas collected from host burrows. Across species, the female:male ratio of fleas on hosts correlated strongly with the female:male ratio of fleas in their burrows, with the slope of the regression overlapping 1. Controlling for flea phylogeny by independent contrasts produced similar results. It was also ascertained whether a host individual is a proportional random sampler of male and female fleas and whether the sex ratio in flea infrapopulations depends on the size of infrapopulations and on the gender and age of a host. Using field data, the sex ratio in infrapopulations of 7 flea species parasitic on 4 rodent species was analysed. Populations of 3 species (Nosopsyllus iranus, Parapulex chephrenis and Xenopsylla conformis) were significantly female-biased, whereas male bias was found in 1 species (Synosternus cleopatrae). In general, the sex ratio of fleas collected from an individual rodent did not differ significantly from the sex ratio in the entire flea population. Neither host gender, and age nor number of fleas co-occurring on a host affected (a) the sex ratio in flea infrapopulations and (b) the probability of an infrapopulation to be either female- or male-biased.     

Melanophores in the stripes of adult zebrafish do not have the nature to gather,but disperse when they have the space to move

Animal skin pattern is one of the good model systems used to study the mechanism of pattern formation. Molecular genetic studies with zebrafish have shown that pigment cells play a major role in the mechanism of stripe formation. Among the variety of cellular events that may be involved in the mechanism, aggregation of melanophores has been suggested as an important factor for pattern formation. However, only a few experimental studies detected the migration ability of melanophores in vivo. Here, we tried to determine whether melanophores really have the ability to aggregate in the skin of zebrafish. Melanophores in the adult stripes are packed densely and they rarely move. However, when the neighboring pigment cells are killed, they move and regenerate the stripe pattern, suggesting that melanophores retain the migration ability. To analyze the migration, we ablated a part of the melanophores by laser to give free space to the remaining cells; we then traced the migration. Contrary to our expectation, we found that melanophores repulsed one another and dispersed from the aggregated condition in the absence of xanthophores. Apparent aggregation may be forced by the stronger repulsive effect against the xanthophores, which excludes melanophores from the yellow stripe region.     

Chlorogenic acid participates in the regulation of shoot,root and root hair development in Hypericum perforatum

Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, is one of the most widespread soluble phenolic compounds in the plant kingdom. Although CGA is known to have important roles in plant function, its relevance in plant de novo organogenesis is not yet understood. With a series of experiments, here we show that CGA has a potential role in shoot, root and root hair development. In the first phase of our investigation, we developed an efficient and novel thin cell layer (TCL) regeneration protocol for Hypericum perforatum which could bridge all the in vitro morphogenetic stages between single cell and complete plant. Tissues at different morphogenetic states were analysed for their phenolic profile which revealed that shoot differentiation from callus tissues of H. perforatum is accompanied by the onset of CGA production. Further, the relevance of CGA in de novo organogenesis was deciphered by culturing highly organogenic root explants on media augmented with various concentrations of CGA. Results of this experiment showed that CGA concentrations lower than 10.0 mg l^?1 did not affect shoot organogenesis, whereas, higher concentrations significantly reduced this process in a concentration-dependent manner. In spite of the differential concentration-dependent effects of CGA on shoot regeneration, supplementation of CGA did not have any effect on the production of lateral roots and root hairs. Interestingly, CGA showed a concentration-dependent positive correlation with lateral roots and root hairs production in the presence of α-naphthaleneacetic acid (NAA). When the culture medium was augmented with 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), induction of shoots, lateral roots and root hairs from the explants was significantly affected. Addition of an optimum concentration of CGA in these cultures partially restored all these organogenic processes.     

Co-occurring graminoid and forb species do not differ in their root morphological response to soil heterogeneity

We measured the proliferation of roots into experimental nutrient patches in a grassland community, distinguishing roots of graminoids and forbs. Biomass, length, and specific length were estimated for roots of each of the two functional groups, collected from patches differing in nutrient concentration, and established at four different times during a season. The ratio of graminoid and forb roots was compared with the graminoid-forb ratio in the above-ground biomass. Plant roots proliferated more intensively into patches with higher nutrient concentration, but the roots of the two functional groups had a similar ability to target richer patches. Relative proportion of graminoids was higher below-ground than above-ground and changed during the season, being lowest after mowing. Specific root length was higher for graminoid species, but did not respond to nutrient concentration in patches for either functional group. This is the first study to provide comparative information about root morphological response for graminoids and forbs, measured in a real, semi-natural plant community. The study shows no significant overall difference in the ability of these two functional types to place roots into nutrient-rich patches, but indicates other important differences among the two functional groups.