Modification of Growth and Physiological Response of Coastal Dune Species Anthyllis maritima to Sand Burial by Rhizobial Symbiosis and Salinity

The aim of the present study was to establish an experimental system in controlled conditions to study the physiological effect of abiotic/biotic interaction using a rare wild leguminous plant species from coastal sand dunes, Anthyllis maritima. The particular hypothesis tested was that there is an interaction between sand burial, rhizobial symbiosis and salt treatment at the level of physiological responses. Experiment in controlled conditions included 18 treatment combinations of experimental factors, with two intensities of sand burial, rhizobial inoculation and two types of NaCl treatment (soil irrigation and foliar spray). Shoot biomass was significantly affected both by burial and by inoculation, and by interaction between burial and NaCl in the case of shoot dry mass. For plants sprayed with NaCl, burial had a strong significant positive effect on shoot growth irrespective of inoculation. General effect of inoculation with rhizobia on shoot growth of plants without NaCl treatment was negative except for the plants buried 2 cm with sand, where significant stimulation of shoot dry mass by inoculant was found. The positive effect of burial on shoot growth was mainly associated with an increase in leaf petiole height and number of leaves. Performance index significantly increased in buried plants in all treatment combinations, and leaf chlorophyll concentration increased in buried plants independently on burial depth, and only in plants not treated with NaCl. Inoculation led to significant increase of leaf peroxidase activity in all treatment combinations except NaCl-irrigated plants buried for 2 cm by sand. Sand burial stimulated peroxidase activity, mostly in non-inoculated plants, as inoculation itself led to increased enzyme activity. In conclusion, strong interaction between sand burial and NaCl treatment was evident, as the latter significantly affected the effect of burial on growth and physiological indices. Moreover, rhizobial symbiosis had a significant effect on physiological processes through interaction with both sand burial and NaCl treatment, but the effect was rather controversial; it was positive for photosynthesis-related parameters but negative for growth and tissue integrity indices.

Microscopic and transcriptomic analyses of Dalbergoid legume peanut reveal a divergent evolution leading to Nod Factor dependent epidermal crack-entry and terminal bacteroid differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS come from model legumes that exhibit a root-hair mode of bacterial infection in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step towards understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated a comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen scavenging mechanisms in the Papilionoid family. Finally, absence of cysteine-rich motif-1 containing-NCRs, but the recruitment of defensin like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobial symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.

Identification of an Isoflavonoid Transporter Required for the Nodule Establishment of the Rhizobium-Fabaceae Symbiotic Interaction

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Realization of protective and symbiotic properties of soybeans using fungicide seed treatment

Aim. The elaboration of efficient legume-rhizobial symbiosis systems, involving active strains of nodule bacteria, in the combination with fungicide seed treatment may be an alternative method of providing ecologically friendly nitrogen sources to plants and promoting their tolerance to the external factors, which is relevant for preservation and restoration of envi- ronmental quality. Therefore, the aim of our study was to determine the impact of pre-sowing seed treatment with fungi- cides, which differ in the action spectrum of active substances – Standak Top (fipronil, 250 g/l, thiophanate-methyl, 225 g/l, pyraclostrobin, 25 g/l) and Maxim XL (fludioxonyl, 25 g/l, metalaxyl, 10 g/l), on the intensity of the development of lipid peroxidation processes, the activity of antioxidant enzyme ascorbate peroxidase and nitrogen fixation activity in soybeans on the early stages of forming legume-rhizobial symbiosis. Methods. Microbiological (cultivation of a bacterial culture, seed inoculation), physiological (vegetative experiment), biochemical (spectrophotometric determination of the content of lipid peroxidation products and the activity of ascorbate peroxidase; measuring the nitrogen-fixation activity using a gas chro- matography). Results. It was found that pre-sowing fungicide treatment of soybean seeds and subsequent inoculation with active rhizobia of strain 634b did not result in the change in the content of TBA-active products in roots and root nodules (the values of indices were within the experiment deviation). At the same time, after seed inoculation using rhizobia and treat- ment with Maxim XL, there was an increase in the activity of ascorbate peroxidase in the roots from 20.3 to 30.8 %, and with Standak Top – from 20.0 to 29.8 % during the early stages of ontogenesis till the formation of the third ternate leaf. Here, the activity of the enzyme in root nodules increased by 24.7–40.3 % at the fungicidal effect. Our data demonstrate that the combination of fungicide seed treatment and inoculation with active rhizobia does not induce lipid peroxidation processes, but promotes the initiation of protective antioxidant properties in soybeans. It is accompanied with efficient functioning of the symbiotic apparatus, which is manifested in the increase in nitrogen-fixing activity of nodule bacteria, formed by active rhizobia of strain 634b after the seed treatment with Standak Top – by 98.3 and 78.1 % and after Maxim XL – by 78.6 and 196.2 % respectively, during the stages of the second and third ternate leaves. Conclusions. The pre-sowing soybean seed treatment with fungicides Standak Top and Maxim XL and the subsequent inoculation with active rhizobia of strain 634b does not induce the development of lipid peroxidation processes, but increases the activity of the antioxidant enzyme, ascor- bate peroxidase, in the roots and root nodules, which is accompanied with the efficient work of the symbiotic apparatus on the early stages of determining legume-rhizobial symbiosis. This method of seed treatment may be a novel measure, to use in the technologies of cultivating soybeans to enhance the realization of the symbiotic potential and meet the needs of plants in ecologically friendly nitrogen, and to promote the formation of their tolerance to the corresponding cultivation conditions.

Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identified these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might influence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium influx inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1–3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative influence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the fluorophores such as red fluorescent protein (RFP) and yellow fluorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by infiltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modification or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

Identification of an isoflavonoid transporter required for the nodule establishment of the Rhizobium-Fabaceae symbiotic interaction

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by e.g., forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids, that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N2 fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261–357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.