Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

Beneficial implications of sugar beet proteinase inhibitor BvSTI on plant architecture and salt stress tolerance in Lotus corniculatus L.

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus.

The role of endogenous thiamine produced via THIC in root nodule symbiosis in Lotus japonicus.

Thiamine is a pivotal primary metabolite which is indispensable to all organisms. Although its biosynthetic pathway has been well documented, the mechanism by which thiamine influences the legume-rhizobium symbiosis remains uncertain. Here, we used overexpressing transgenic plants, mutants and grafting experiments to investigate the roles played by thiamine in Lotus japonicus nodulation. ljthic mutants displayed lethal phenotypes and the defect could be overcome by supplementation of thiamine or by overexpression of LjTHIC. Reciprocal grafting between L. japonicus wild-type Gifu B-129 and ljthic showed that the photosynthetic products of the aerial part made a major contribution to overcoming the nodulation defect in ljthic. Overexpression of LjTHIC in Lotus japonicus (OE-LjTHIC) decreased shoot growth and increased the activity of the enzymes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. OE-LjTHIC plants exhibited an increase in the number of infection threads and also developed more nodules, which were of smaller size but unchanged nitrogenase activity compared to the wildtype. Taken together, our results suggest that endogenous thiamine produced via LjTHIC acts as an essential nutrient provided by the host plant for rhizobial infection and nodule growth in the Lotus japonicus - rhizobium interaction.

The acclimatization strategies of kidney vetch (Anthyllis vulneraria L.) to Pb toxicity.

Kidney vetch (Anthyllis vulneraria L.) is a well-known Zn hyperaccumulator. Zn often occurs with Pb in one ore; thus, plants inhabiting waste dumps are exposed not only to Zn but also to Pb toxicity. While the response of kidney vetch to Zn toxicity is relatively well known, the Pb survival strategy of Anthyllis vulneraria has not been the subject of investigations. The aim of presented research was to determine the survival strategy of kidney vetch exposed to high lead concentrations. Shoot explants of a calamine kidney vetch ecotype were placed on agar media containing 0.0, 0.5, 1.0, and 1.5 mM Pb. Morphological, physiological, and biochemical responses, in particular photosynthetic apparatus of plantlets, were examined. The most pronounced changes were observed in plants grown on media supplemented with 1.5 mM Pb after 8 weeks of culture. Increased dry weight and high lead accumulation were observed in roots. Similarly, in shoots, increased dry weight and a decreased number of newly formed shoots were recorded. The accumulation of lead was many times lower in shoots than in roots. In leaf cells' ultra-structure, looser arrangement of chloroplast thylakoid grana was observed. Despite the decrease in chlorophyll a and carotenoid content, the photosynthetic apparatus remained efficient due to the lack of photoinhibition and increased electron transport rate beyond photosystem II (PSII). For the first time, an acclimatization mechanism based on maintaining the high efficiency of photosynthetic apparatus resulting from increasing of electron transport rate was described.

Effects of species evenness and dominant species identity on multiple ecosystem functions in model grassland communities.

Ecosystems provide multiple services upon which humans depend. Understanding the drivers of the ecosystem functions that support these services is therefore important. Much research has investigated how species richness influences functioning, but we lack knowledge of how other community attributes affect ecosystem functioning. Species evenness, species spatial arrangement, and the identity of dominant species are three attributes that could affect ecosystem functioning, by altering the relative abundance of functional traits and the probability of synergistic species interactions such as facilitation and complementary resource use. We tested the effect of these three community attributes and their interactions on ecosystem functions over a growing season, using model grassland communities consisting of three plant species from three functional groups: a grass (Anthoxanthum odoratum), a forb (Plantago lanceolata), and a N-fixing forb (Lotus corniculatus). We measured multiple ecosystem functions that support ecosystem services, including ecosystem gas exchange, water retention, C and N loss in leachates, and plant biomass production. Species evenness and dominant species identity strongly influenced the ecosystem functions measured, but spatial arrangement had few effects. By the end of the growing season, evenness consistently enhanced ecosystem functioning and this effect occurred regardless of dominant species identity. The identity of the dominant species under which the highest level of functioning was attained varied across the growing season. Spatial arrangement had the weakest effect on functioning, but interacted with dominant species identity to affect some functions. Our results highlight the importance of understanding the role of multiple community attributes in driving ecosystem functioning.

Water stress induces a differential and spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus.

Water stress is one of the most severe problems for plant growth and productivity. Using the legume Lotus japonicus exposed to water stress, a comparative analysis of key components in metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were made. After water stress treatment plants accumulated proline 23 and 10-fold in roots and leaves respectively, compared with well-watered plants. Significant changes in metabolism of RNS and ROS were observed, with an increase in both protein tyrosine nitration and lipid peroxidation, which indicate that water stress induces a nitro-oxidative stress. In roots, ·NO content was increased and S-nitrosoglutathione reductase activity was reduced by 23%, wherein a specific protein nitration pattern was observed. As part of this response, activity of NADPH-generating dehydrogenases was also affected in roots resulting in an increase of the NADPH/NADP(+) ratio. Our results suggest that in comparison with leaves, roots are significantly affected by water stress inducing an increase in proline and NO content which could highlight multiple functions for these metabolites in water stress adaptation, recovery and signaling. Thus, it is proposed that water stress generates a spatial distribution of nitro-oxidative stress with the oxidative stress component being higher in leaves whereas the nitrosative stress component is higher in roots.

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

Short-term nutritional treatments grazing legumes or feeding concentrates increase prolificacy in Corriedale ewes.

We tested whether short periods of increased nutrition will improve ovulation rate and prolificacy, irrespective of the method used to synchronise the cycles of the ewes. In Experiment 1, we used 138 Corriedale ewes to evaluate two factors: synchronization treatment (sponges versus a single injection of prostaglandin) and type of pasture (native versus improved with Lotus corniculatus). Ewes were mated at the end of the grazing period and prolificacy was evaluated at lambing. Grazing Lotus corniculatus for 12 days tended to increase the number of twin lambs born (P=0.09). The percentage of ewes showing oestrus during a 9-day period was similar among synchronization treatments. Animals in Experiments 2 (n=282) and 3 (n=288) were allocated to a control group or a group fed a supplement of corn grain and soybean meal for 7 days. Ewes received 2 prostaglandin injections and the supplement was fed from Days 11 to 17 after the second prostaglandin. Ovulation rate was measured in 65 (Experiment 2) and 61 (Experiment 3) ewes that were confirmed to have consumed the supplement and showed oestrus in a 4-day period. The supplement increased ovulation rate by 14% in both experiments (P<0.05). We conclude that Corriedale ewes can respond with increases in prolificacy to a 12-day period grazing Lotus corniculatus and in ovulation rate to 7 days feeding with a supplement rich in energy and protein. Moreover, in these studies, prostaglandin was as effective as sponges for synchronising oestrus, an important factor in future decisions about hormonal management of fertility.

Arbuscular mycorrhizal fungi and plant symbiosis in a saline-sodic soil.

The seasonality of arbuscular mycorrhizal (AM) fungi-plant symbiosis in Lotus glaber Mill. and Stenotaphrum secundatum (Walt.) O.K. and the association with phosphorus (P) plant nutrition were studied in a saline-sodic soil at the four seasons during a year. Plant roots of both species were densely colonized by AM fungi (90 and 73%, respectively in L. glaber and S. secundatum) at high values of soil pH (9.2) and exchangeable sodium percentage (65%). The percentage of colonized root length differed between species and showed seasonality. The morphology of root colonization had a similar pattern in both species. The arbuscular colonization fraction increased at the beginning of the growing season and was positively associated with increased P concentration in both shoot and root tissue. The vesicular colonization fraction was high in summer when plants suffer from stress imposed by high temperatures and drought periods, and negatively associated with P in plant tissue. Spore and hyphal densities in soil were not associated with AM root colonization and did not show seasonality. Our results suggest that AM fungi can survive and colonize L. glaber and S. secundatum roots adapted to extreme saline-sodic soil condition. The symbiosis responds to seasonality and P uptake by the host altering the morphology of root colonization.

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus.

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.

Functional diversity of plant-pollinator interaction webs enhances the persistence of plant communities.

Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm species. Thus, pollinators provide an essential ecosystem service to humankind. However, the impact of human-induced biodiversity loss on the functioning of plant-pollinator interactions has not been tested experimentally. To understand how plant communities respond to diversity changes in their pollinating fauna, we manipulated the functional diversity of both plants and pollinators under natural conditions. Increasing the functional diversity of both plants and pollinators led to the recruitment of more diverse plant communities. After two years the plant communities pollinated by the most functionally diverse pollinator assemblage contained about 50% more plant species than did plant communities pollinated by less-diverse pollinator assemblages. Moreover, the positive effect of functional diversity was explained by a complementarity between functional groups of pollinators and plants. Thus, the functional diversity of pollination networks may be critical to ecosystem sustainability.

Measuring gene flow from two birdsfoot trefoil (Lotus corniculatus) field trials using transgenes as tracer markers.

Genetic engineering is becoming a useful tool in the improvement of plants but concern has been expressed about the potential environmental risks of releasing genetically modified (GM) organisms into the environment. Attention has focused on pollen dispersal as a major issue in the risk assessment of transgenic crop plants. In this study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. Plants of Lotus corniculatus L. transformed with either the Escherichia coli asparagine synthetase gene asnA or the beta-glucuronidase gene uidA, were used as the pollen donor. Nontransgenic plants belonging to the species L. corniculatus L., L. tenuis Waldst. and Kit. ex Willd, and L. pedunculatus Cav., were utilized as recipients. Two experimental fields were established in two areas of central Italy. Plants carrying the uidA gene were partially sterile, therefore only the asnA gene was used as a tracer marker. No transgene flow between L. corniculatus transformants and the nontransgenic L. tenuis and L. pedunculatus plants was detected. As regards nontransgenic L. corniculatus plants, in one location flow of asnA transgene was detected up to 18 m from the 1.8 m2 donor plot. In the other location, pollen dispersal occurred up to 120 m from the 14 m2 pollinating plot.

[Self-fertility problems in birdsfoot trefoil (Lotus corniculatus L.)]. / Problemy samofertyl'nosti u liadventsia rohatoho (Lotus corniculatus L.).

The problems of Lotus corniculatus self-fertility are analyzed. The self-compatibility of hereditivity character, the efficacy of different methods of self-pollination (autogamy, geitonogamy within one plant and among plants of one clone), and usage of the mixture of self-compatible and self-incompatible pollen were studied.