Analysis of the expression of polyamine biosynthesis genes in nodules of the garden pea (Pisum sativum L.) and the effect of exogenous treatment with polyamines on their development

BACKGROUND: Polyamines are acting as signaling molecules during adaptation to stressful environment and as regulators of plant development. In plants, polyamines are represented mainly by putrescine, spermidine and spermine. The concentration of polyamines in symbiotic nodules of some legumes is 510 times higher than in the other organs, which indicates their important role in the formation and functioning of symbiotic nodules. MATERIALS AND METHODS: We analyzed the expression of genes encoding polyamine biosynthesis enzymes in symbiotic nodules, as well as the effect of exogenous polyamines on the nodule number and the average nodule weight in wild-type SGE plants and symbiotic pea mutants SGEFix-1 (sym40-1) and SGEFix-2 (sym33-3). RESULTS: The comparable expression level of arginine decarboxylase gene (PsADC) was observed in all analyzed nodules, whereas the expression level of ornithine decarboxylase gene (PsODC), was highly increased in nodules of SGEFix-2 (sym33-3) mutant. Treatment of the root system with a 0.1 mM solution of polyamines mixture led to an increase in the average weight of the nodule in wild-type plants and in the SGEFix-2 (sym33-3) mutant plants. CONCLUSIONS: It was shown that the main pathway of putrescine synthesis in wild-type pea symbiotic nodules is the arginine pathway, while the ornithine pathway is probably associated with activation of plant defense reactions. Polyamines acting, apparently, through ethylene, affect the functioning of the nodule meristem.

LysM Receptor-Like Kinase LYK9 of Pisum Sativum L. May Regulate Plant Responses to Chitooligosaccharides Differing in Structure

This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA–CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.

Genetic characterization of pea (Pisum sativum L.) mutants P59 and P60, defective in nitrogen fixation

Several genes involved in development of symbiosis between pea and rhizobia haven’t yet been characterized in detail. Here, the first results of genetic analysis of pea mutants in the symbiotic genes Sym23 and Sym24 are presented.

Shoot Extracts from Two Low Nodulation Mutants Significantly Reduce Nodule Number in Pea

E107 and E132 are pea mutants that nodulate poorly. Because they have a shoot-controlled nodulation phenotype, we asked if their mutated genes were implicated in the autoregulation of nodulation (AON), a mechanism which consists of two systemic circuits, the positive CEP/CRA2 and the negative CLE/SUNN, coordinated via NIN and miR2111. We further characterized the mutants’ phenotype by studying nodule distribution and nodulation efficiency. E107 was similar to wild-type (WT) in its nodule distribution, but E132 had an extended nodulation zone with nodules forming distally on its lateral roots. Moreover, we tested whether their shoots produced a compound inhibitory to nodulation. We made ethyl-acetate extracts of roots and shoots of both mutants and WT, which we applied to rhizobia-inoculated WT seedlings and to pure rhizobial cultures. Whereas free-living bacteria were unaffected by any of the extracts, WT treated with shoot extracts from either inoculated mutant had fewer nodules than that of control. E107 and E132 shoot extracts led to a 50% and a 35% reduction in nodule number, respectively. We propose that E107 and E132 belong to a new sub-class of AON mutants, i.e., hypo-nodulators, and that their respective gene products are acting in the AON descending branch, upstream of TML signaling.

Mutational analysis indicates that abnormalities in rhizobial infection and subsequent plant cell and bacteroid differentiation in pea (Pisum sativum) nodules coincide with abnormal cytokinin responses and localization

Background and Aims Recent findings indicate that Nod factor signalling is tightly interconnected with phytohormonal regulation that affects the development of nodules. Since the mechanisms of this interaction are still far from understood, here the distribution of cytokinin and auxin in pea (Pisum sativum) nodules was investigated. In addition, the effect of certain mutations blocking rhizobial infection and subsequent plant cell and bacteroid differentiation on cytokinin distribution in nodules was analysed. Methods Patterns of cytokinin and auxin in pea nodules were profiled using both responsive genetic constructs and antibodies. Key Results In wild-type nodules, cytokinins were found in the meristem, infection zone and apical part of the nitrogen fixation zone, whereas auxin localization was restricted to the meristem and peripheral tissues. We found significantly altered cytokinin distribution in sym33 and sym40 pea mutants defective in IPD3/CYCLOPS and EFD transcription factors, respectively. In the sym33 mutants impaired in bacterial accommodation and subsequent nodule differentiation, cytokinin localization was mostly limited to the meristem. In addition, we found significantly decreased expression of LOG1 and A-type RR11 as well as KNOX3 and NIN genes in the sym33 mutants, which correlated with low cellular cytokinin levels. In the sym40 mutant, cytokinins were detected in the nodule infection zone but, in contrast to the wild type, they were absent in infection droplets. Conclusions In conclusion, our findings suggest that enhanced cytokinin accumulation during the late stages of symbiosis development may be associated with bacterial penetration into the plant cells and subsequent plant cell and bacteroid differentiation.

Hairy roots of pea mutants with a modified morph leaf type

In the present study, some pea mutants rich in protein with a modified morph leaf type with af and tl genes were genetically transformed using different strains of Agrobacterium rhizogenes A-4, A-48, 15834. Even though a higher total protein content was obtained with the A-48 strain, maximum of the probable transformants were obtained with the A-4 strain but of transient character. The content of the total protein in hairy roots cultures, depending on the composition of the nutrient media and the growth phase of the culture, has been refined.

Analysis of epitope distribution of arabinogalactan protein-extensins in pea (Pisum Sativum) nodules of wild-type and mutants impaired in infection thread growth

Background. During the colonization of root and nodule tissues of legumes by rhizobia, bacterial cells are immersed in a plant extracellular matrix which includes arabinogalactan protein-extensins (AGPE). Materials and methods. Immunogold electron microscopy with monoclonal antibodies MAC204 and MAC236 was used to analyse the distribution and abundance of epitopes of AGPE in wild-type and symbiotically defective pea mutants. Results. In the nodules of the wild-type line SGE, both AGPE epitopes were detected to the same extent in the matrix of infection threads and infection droplets. In the nodules of the mutant line SGEFix-1 (sym40), the level of labelling by MAC204 was significantly higher than with SGE in both infection threads and infection droplets, but the level of labelling by MAC236 was only increased in the infection droplets. In the mutant line SGEFix-2 (sym33-3), a relatively high level of both epitopes was observed among all analysed genotypes. The double mutant line RBT3 (sym33-3, sym40) showed an intermediate level of labelling for both epitopes in infection threads compared with the parental mutants. In SGEFix-1, an abnormal distribution of both epitopes was observed in the intercellular space matrix. The MAC204 epitope was found in the cell walls of SGEFix-1 and in the infection thread walls of SGEFix-2, whereas in RBT3 this epitope was detected in both types of walls. Conclusions. The sym33-3 and sym40 mutations have different effects on the accumulation of AGPE epitopes recognised by MAC204 and MAC236. This indicates that both the Sym33 and the Sym40 genes affect the composition of AGPE in the matrix of infection threads and infection droplets.