Lipidomics Analysis of Multilamellar Bodies Produced by Amoeba Acanthamoeba castellanii in Co-Culture with Klebsiella aerogenes.

Multilamellar bodies (MLBs) are membrane-bound cytoplasmic organelles of lysosomal origin. In some protozoa, they were considered as lipid storage secretory organelles and feasible participants in cell-to-cell communication. However, for Acanthamoeba castellanii, similar vesicles were indicated only as possible transmission vectors of several pathogenic bacteria without attributing them biological roles and activities. Since amoebae belonging to the genus Acanthamoeba are not only of environmental but also of clinical significance, it is of great importance to fully understand their physiology. Thus, determination of MLB lipid composition could partly address these questions. Because MLBs are secreted by amoebae as a result of bacteria digestion, the co-culture technique with the use of "edible" Klebsiella aerogenes was used for their production. Lipids obtained from The MLB fraction, previously purified from bacterial debris, were analyzed by high-performance thin-layer chromatography, gas chromatography coupled with mass spectrometry, and high-resolution mass spectrometry. Lipidomic analysis revealed that in MLBs, a very abundant lipid class was a non-phosphorous, polar glycerolipids, diacylglyceryl-O-(N,N,N)-trimethylhomoserine (DGTS). Since DGTSs are regarded as a source of nitrogen and fatty acids, MLBs can be considered as lipid storage organelles produced in stress conditions. Further, the identification of phytoceramides and possible new betaine derivatives indicates that MLBs might have a distinct bioactive potential.

Spikelet movements, anther extrusion and pollen production in wheat cultivars with contrasting tendencies to cleistogamy.

BACKGROUND: Cleistogamic flowers are a main barrier in pollen dispersal for cross-pollination necessary in wheat hybrid breeding. The aim of our study was to gain new knowledge on the biology of wheat flowering, in particular on the differences between the cleisto- and chasmogamic forms which has certainly cognitive significance, but it can also be used in practice when seeking a female and male ideotypes for cross hybridization. RESULTS: We characterized the most significant features defining the flowering specificity in two wheat cultivars with contrasting tendency to cleistogamy: Piko (chasmogamous) and Dacanto (cleistogamous). In the field observations we assessed diurnal pattern of anther extrusion and anther extrusion capacity. For the first time we adapted the time lapse method for measuring kinetics of the spikelet movement and 3-D image correlation technique for the non-invasive measurements of potential deformations of the spikelet lemmas. We found that the two cultivars differ in the potential of pollen dispersion for-cross-pollination and in the spikelet kinetics. We also described some anatomical traits that can have potential functional role in floret opening. None of the cultivars showed any symptoms of lemma surface deformation. CONCLUSIONS: The cleistogamic and chasmogamic wheat cultivars differ significantly in the potential for pollen dispersion for cross-pollination, which is mainly related to anther extrusion capacity. Although none of these features differentiated the cultivars clearly, we assume, based on spikelet kinetics and the lack of lemmas surface deformation, that the water transport and turgor of cells is essential for the floret opening and anther extrusion in wheat. The search for parental ideotype should be supported by marker assisted selection, e.g. based of polymorphisms in genes related to aquaporin biosynthesis.

Glandular trichomes of Robinia viscosa Vent. var. hartwigii (Koehne) Ashe (Faboideae, Fabaceae)-morphology, histochemistry and ultrastructure.

MAIN CONCLUSION: Permanent glandular trichomes of Robinia viscosa var. hartwigii produce viscous secretion containing several secondary metabolites, as lipids, mucilage, flavonoids, proteins and alkaloids. Robinia viscosa var. hartwigii (Hartweg's locust) is an ornamental tree with high apicultural value. It can be planted in urban greenery and in degraded areas. The shoots, leaves, and inflorescences of this plant are equipped with numerous persistent glandular trichomes producing sticky secretion. The distribution, origin, development, morphology, anatomy, and ultrastructure of glandular trichomes of Hartweg's locust flowers as well as the localisation and composition of their secretory products were investigated for the first time. To this end, light, scanning, and transmission electron microscopy combined with histochemical and fluorescence techniques were used. The massive glandular trichomes differing in the distribution, length, and stage of development were built of a multicellular and multiseriate stalk and a multicellular head. The secretory cells in the stalk and head had large nuclei with nucleoli, numerous chloroplasts with thylakoids and starch grains, mitochondria, endoplasmic reticulum profiles, Golgi apparatus, vesicles, and multivesicular bodies. Many vacuoles contained phenolic compounds dissolved or forming various condensed deposits. The secretion components were transported through symplast elements, and the granulocrine and eccrine modes of nectar secretion were observed. The secretion was accumulated in the subcuticular space at the trichome apex and released through a pore in the cuticle. Histochemical and fluorescence assays showed that the trichomes and secretion contained lipophilic and polyphenol compounds, polysaccharides, proteins, and alkaloids. We suggest that these metabolites may serve an important function in protection of plants against biotic stress conditions and may also be a source of phytopharmaceuticals in the future.

Oviposition by the vagrant eriophyoid mite Aculops allotrichus on leaves of black locust tree, Robinia pseudoacacia.

Leaf-dwelling mites often prefer to feed on young leaves and also are more likely to inhabit the abaxial leaf side. The aim of our study was to examine whether leaf age may affect production and distribution of eggs on black locust leaves by females of Aculops allotrichus. The eriophyoids were tested for 2.5 days on 'trimmed' compound leaves (with only two opposite leaflets left), which were maintained in vials filled with water. For the experiments we used leaves of three categories: (1) the 'youngest', in which both halves of the adaxial side of leaflets still adhered to each other (and usually remained folded for the next few hours), (2) 'young' with already unfolded leaflets, and (3) 'mature' with fully expanded leaflets. The tested females laid significantly more eggs on developing leaves than on 'mature' ones, although they deposited the highest number of eggs on the 'young' leaves. The distribution of eggs on adaxial or abaxial leaf sides also depended on leaf age. On the 'youngest' leaves, eriophyoids placed similar numbers of eggs on both sides of a blade. However, the older the leaf, the more willingly females deposited eggs on the abaxial side. Our biochemical and morphometrical analyses of black locust leaves indicated significant changes in the contents of nutrients and phenols within leaf tissue, and in the density of trichomes and thickness of the outer epidermal cell walls, correlated with leaf age. Their possible effects on the production and distribution of eggs on leaves by A. allotrichus are discussed.

Transcriptomic Changes in Medicago truncatula and Lotus japonicus Root Nodules during Drought Stress.

Drought is one of the major environmental factors limiting biomass and seed yield production in agriculture. In this research, we focused on plants from the Fabaceae family, which has a unique ability for the establishment of symbiosis with nitrogen-fixing bacteria, and are relatively susceptible to water limitation. We have presented the changes in nitrogenase activity and global gene expression occurring in Medicago truncatula and Lotus japonicus root nodules during water deficit. Our results proved a decrease in the efficiency of nitrogen fixation, as well as extensive changes in plant and bacterial transcriptomes, shortly after watering cessation. We showed for the first time that not only symbiotic plant components but also Sinorhizobium meliloti and Mesorhizobium loti bacteria residing in the root nodules of M. truncatula and L. japonicus, respectively, adjust their gene expression in response to water shortage. Although our results demonstrated that both M. truncatula and L. japonicus root nodules were susceptible to water deprivation, they indicated significant differences in plant and bacterial response to drought between the tested species, which might be related to the various types of root nodules formed by these species.

Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus.

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical ß-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root's stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula.

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA(-)). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1-MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.

Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99-100% in the case of nodAC and nifH genes, and 98-99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar "glycyphyllae", based on nodA and nodC genes sequences.

Review article: The meristem in indeterminate root nodules of Faboideae.

In this review, the anatomy of indeterminate legume root nodule is briefly summarized. Next, the indeterminate nodule meristem activity, organization and cell ultrastructure are described in species with a distinct nodule meristem zonation. Finally, the putative primary endogenous factors controlling nodule meristem maintenance are discussed in the context of the well-studied root apical meristem (RAM) of Arabidopsis thaliana.

Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan.

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99-100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97-98% for nodC, and 97-100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.

The incomplete substitution of lipopolysaccharide with O-chain prevents the establishment of effective symbiosis between Mesorhizobium loti NZP2213.1 and Lotus corniculatus.

Mesorhizobium loti NZP2213.1 mutant obtained after random Tn5 mutagenesis of M. loti NZP2213 was inefficient in nitrogen fixation on Lotus corniculatus. The transposon insertion was located within an ORF with a sequence similarity to a putative glycosyl transferase from Caulobacter crescentus. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the mutant produced LPS of the same O-chain length but only half of the entire smooth LPS, compared to that of the parental strain. A greater diversity of the anomeric region as determined by NMR spectroscopy, reflected structural differences in the mutant repeating units represented by 6-deoxytalose, 2-OAc-6-deoxytalose, and 2-OMe-6-deoxytalose. In contrast to the completely O-acetylated 6-deoxytalose in wild-type OPS only partial O-acetylation was found in the mutant. The decrease of the LPS species with O-chains seems to be correlated with 6-deoxytalose deficiency. Microscopic examination of the nodules induced by the mutant revealed disturbances in infection thread development and premature senescence of symbiosomes. The impairment of mutant-induced symbiosomes to sustain latter stages of symbiosis could be a consequence of the decreased ratio of the hydrophobic to the hydrophilic LPSs.

Ferritins and nodulation in Lupinus luteus: iron management in indeterminate type nodules.

An ability to form symbiotic associations with rhizobia and to utilize atmospheric nitrogen makes legumes ecologically successful. High iron content in legume grains, partially relocated from root nodules, is another-nutritional-advantage of this group of plants. The ferritin complex is the major cell iron storage and detoxification unit and has been recognized as a marker of many stress-induced responses. The possible participation of ferritin in nodule formation and functioning was investigated here. Correlation of increased accumulation of both ferritin polypeptide and mRNA with actual in situ localization of ferritin allowed ferritin synthesis in the developing, indeterminate-type root nodules to be related to differentiating bacteroid tissue. This kind of tissue, in contrast to the determinate-type nodules, is present in lupin nodules at almost all stages of their development. Interestingly, it was found that, in this type of nodule, senescence starting in the decaying zones induces ferritin accumulation in younger, still active, tissues. Based on the presented data, and in correlation with previous results, some aspects of the regulation of expression of lupin ferritin genes are also discussed.

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia.

Pairwise comparisons of Genista tinctoria (dyer's weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer's weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.