Lotus SHAGGY-like kinase 1 is required to suppress nodulation in Lotus japonicus.

Glycogen synthase kinase/SHAGGY-like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell-differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen-fixing bacterium Mesorhizobium loti. RNAi knock-down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy-root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock-down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON-related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.

Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania.

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

RAPD-inferred genetic variability of some indigenous Rhizobium leguminosarum isolates from red clover (Trifolium pratense L.) nodules.

The application of commercial rhizobial inoculants to legume crops is proving to be an alternative to synthetic fertilizer use. The challenge for sustainable agriculture resides in the compatibility between crop, inoculants and environmental conditions. The evaluation of symbiotic efficiency and genetic diversity of indigenous rhizobial strains could lead to the development of better inoculants and increased crop production. The genetic variability of 32 wild indigenous rhizobial isolates was assessed by RAPD (Random Amplified Polymorphic DNA). The strains were isolated from red clover (Trifolium pratense L.) nodules from two distinct geographical regions of Northern and Eastern Romania. Three decamer primers were used to resolve the phylogenetic relationships between the investigated isolates. Cluster analysis revealed a high diversity; most strains clustered together based on their geographical location.

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus.

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.

Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables.

The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance.

The study of a SPATULA-like bHLH transcription factor expressed during peach (Prunus persica) fruit development.

Extensive studies on the dry fruits of the model plant arabidopsis (Arabidopsis thaliana) have revealed various gene regulators of the development and dehiscence of the siliques. Peach pericarp is analogous to the valve tissues of the arabidopsis siliques. The stone (otherwise called pit) in drupes is formed through lignification of the fruit endocarp. The lignified endocarp in peach can be susceptible to split-pit formation under certain genetic as well as environmental factors. This phenomenon delays processing of the clingstone varieties of peach and causes economical losses for the peach fruit canning industry. The fruitfull (FUL) and shatterproof (SHP) genes are key MADS-box transcription protein coding factors that control fruit development and dehiscence in arabidopsis by promoting the expression of basic helix-loop-helix (bHLH) transcription factors like Spatula (SPT) and Alcatraz (ALC). Results from our previous studies on peach suggested that temporal regulation of PPERFUL and PPERSHP gene expression may be involved in the regulation of endocarp margin development. In the present study a PPERSPATULA-like (PPERSPT) gene was cloned and characterized. Comparative analysis of temporal regulation of PPERSPT gene expression during pit hardening in a resistant and a susceptible to split-pit variety, suggests that this gene adds one more component to the genes network that controls endocarp margins development in peach. Taking into consideration that no ALC-like genes have been identified in any dicot plant species outside the Brassicaceae family, where arabidopsis belongs, PPERSPT may have additional role(s) in peach that are fulfilled in arabidopsis by ALC.

Characterization and expression analysis of AGAMOUS-like, SEEDSTICK-like, and SEPALLATA-like MADS-box genes in peach (Prunus persica) fruit.

MADS-box genes encode transcriptional regulators that are critical for flowering, flower organogenesis and plant development. Although there are extensive reports on genes involved in flower organogenesis in model and economically important plant species, there are few reports on MADS-box genes in woody plants. In this study, we have cloned and characterized AGAMOUS (AG), SEEDSTICK (STK) and SEPALLATA (SEP) homologs from peach tree (Prunus persica L. Batsch) and studied their expression patterns in different tissues as well as in fruit pericarp during pit hardening. AG- STK- and SEP-like homologs, representative of the C-, D-, E-like MADS-box gene lineages, respectively, play key roles in stamen, carpel, ovule and fruit development in Arabidopsis thaliana. Sequence similarities, phylogenetic analysis and structural characteristics were used to provide classification of the isolated genes in type C (PPERAG), type D (PPERSTK) and type E (PPERSEP1, PPERSEP3, PPERFB9) organ identity genes. Expression patterns were determined and in combination with phylogenetic data provided useful indications on the function of these genes. These data suggest the involvement of MADS-box genes in peach flower and fruit development and provide further evidence for the role of these genes in woody perennial trees that is compatible with their function in model plant species.

Molecular and biochemical characterization of the parvulin-type PPIases in Lotus japonicus.

The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.

Tissue-specific down-regulation of LjAMT1;1 compromises nodule function and enhances nodulation in Lotus japonicus.

Plant ammonium transporters of the AMT1 family are involved in N-uptake from the soil and ammonium transport, and recycling within the plant. Although AMT1 genes are known to be expressed in nitrogen-fixing nodules of legumes, their precise roles in this specialized organ remain unknown. We have taken a reverse-genetic approach to decipher the physiological role of LjAMT1;1 in Lotus japonicus nodules. LjAMT1;1 is normally expressed in both the infected zone and the vascular tissue of Lotus nodules. Inhibition of LjAMT1;1 gene expression, using an antisense gene construct driven by a leghemoglobin promoter resulted in a substantial reduction of LjAMT1;1 transcript in the infected tissue but not the vascular bundles of transgenic plants. As a result, the nitrogen-fixing activity of nodules was partially impaired and nodule number increased compared to control plants. Expression of LjAMT1;1-GFP fusion protein in plant cells indicated a plasma-membrane location for the LjAMT1;1 protein. Taken together, the results are consistent with a role of LjAMT1;1 in retaining ammonium derived from symbiotic nitrogen fixation in plant cells prior to its assimilation.

Spatial and temporal organization of sucrose metabolism in Lotus japonicus nitrogen-fixing nodules suggests a role for the elusive alkaline/neutral invertase.

Symbiotic nitrogen fixation (SNF) in legume nodules is a highly energy demanding process, fuelled by plant-supplied carbohydrates mainly in the form of sucrose. In this study, we have combined molecular and biochemical approaches in order to study the spatial and temporal organisation of sucrose metabolism in nitrogen-fixing nodules of the model legume Lotus japonicus, with an emphasis on the neglected role of alkaline/neutral invertase. For this purpose, a full-length cDNA clone coding for an alkaline/neutral invertase isoform, termed LjInv1, was identified in a L. japonicus mature nodule cDNA libraries. Alkaline/neutral invertase activity was also found to be the predominant invertase activity in mature nodules. Real-time reverse-transcription polymerase chain reaction analysis was used in order to study the temporal expression patterns of LjInv1 in parallel with genes encoding acid invertase and sucrose synthase (SuSy) isoforms, and enzymes involved in the subsequent hexose partitioning including hexokinase, phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI). The spatial organisation of sucrose metabolism was studied by in situ localisation of LjInv1 transcripts and alkaline/neutral invertase activity, and SuSy protein during nodule development. Furthermore, the spatial organisation of hexose metabolism was investigated by histochemical localisation of hexokinase, PGM and PGI activities in mature nodules. The results considered together indicate that alkaline/neutral invertase could contribute to both the Glc-1-P and Glc-6-P pools in nodules, fuelling both biosynthetic processes and SNF. Furthermore, transcript profiling analysis revealed that genes coding for hexokinase and putative plastidic PGM and PGI isoforms are upregulated during the early stages of nodule development, while the levels of transcripts corresponding to cytosolic PGM and PGI isoforms remained similar to uninfected roots, indicating a possible role of LjInv1 in producing hexoses for starch production and other biosynthetic processes in developing nodules.