Igor A. Tikhonovich

The Sym35 Gene Required for Root Nodule Development in Pea Is an Ortholog of Nin from Lotus japonicus

Comparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for thePsSym35 and LjNin genes in early stages of root nodule formation. Both the pea and L.japonicus mutants are non-nodulating but normal in their arbuscular mycorrhizal association. Both are characterized by excessive root hair curling in response to the bacterial microsymbiont, lack of infection thread initiation, and absence of cortical cell divisions. To investigate the molecular basis for the similarity, we cloned and sequenced the PsNin gene, taking advantage of sequence information from the previously cloned LjNin gene. An RFLP analysis on recombinant inbred lines mapped PsNinto the same chromosome arm as the PsSym35 locus and direct evidence demonstrating that PsNin is thePsSym35 gene was subsequently obtained by cosegregation analysis and sequencing of three independent Pssym35mutant alleles. L. japonicus and pea root nodules develop through different organogenic pathways, so it was of interest to compare the expression of the two orthologous genes during nodule formation. Overall, a similar developmental regulation of thePsNin and LjNin genes was shown by the transcriptional activation in root nodules of L. japonicus and pea. In the indeterminate pea nodules,PsNin is highly expressed in the meristematic cells of zone I and in the cells of infection zone II, corroborating expression of LjNin in determinate nodule primordia. At the protein level, seven domains, including the putative DNA binding/dimerization RWP-RK motif and the PB1 heterodimerization domain, are conserved between the LjNIN and PsNIN proteins.

IPD3 Controls the Formation of Nitrogen-Fixing Symbiosomes in Pea and Medicago Spp.

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.

Bacteria able to control foot and root rot and to promote growth of cucumber in salinated soils

The aim of the present work was to test known bacterial plant growth-promoting strains for their ability to promote cucumber plant growth in salinated soil and to improve cucumber fruit yield by protecting these plants against soil-borne pathogens. Fifty-two plant-beneficial bacterial strains were evaluated for their ability to protect plants against cucumber foot and root rot after bacterization of the seeds and infestation of salinated soil with the isolated Fusarium solani pathogen. Based on the results of initial screenings, five efficient strains were selected, namely Serratia plymuthica RR-2-5-10, Stenotrophomonas rhizophila e-p10, Pseudomonas fluorescens SPB2145, Pseudomonas extremorientalis TSAU20, and P. fluorescens PCL1751. All five strains are salt tolerant since they grow well in a medium to which 3% NaCl was added. Infestation of the soil with F. solani resulted in an increase of the percentage of diseased plants from 17 to 54. Priming of seedlings with the five selected bacterial strains reduced this proportion to as low as 10%. In addition, in the absence of an added pathogen, all five strains showed a significant stimulatory effect on cucumber plant growth, increasing the dry weight of whole cucumber plants up to 62% in comparison to the non-bacterized control. The strains also increased cucumber fruit yield in greenhouse varying from 9% to 32%. We conclude that seed priming with the selected microbes is a very promising approach for improving horticulture in salinated soils. Moreover, allochthonous strains isolated from non-salinated soil, from a moderate or even cold climate, and from other plants than cucumber, functioned as well as autochthonous strains as cucumber-beneficial bacteria in salinated Uzbek soils. These results show that these plant-beneficial strains are robust and they strongly suggest they can also be used successfully in case the climate gets warmer and the soils will become more salinated. Finally, the mechanisms by which they may exert their plant-beneficial action are discussed.

Genetic variability in tolerance to cadmium and accumulation of heavy metals in pea (Pisum sativum L.)

Ninety-nine wild growing and primitive varieties of garden pea (Pisum sativum L.) were screened for tolerance to cadmium (Cd) toxicity in quartz sand culture. Cadmium tolerance was determined by the time to plant death when treated with a lethal Cd concentration (13 mg kg-1), and by a tolerance index (TI) calculated as a ratio between biomasses of Cd-treated and untreated plants in the presence of toxic Cd concentrations (7 mg kg-1 and 5 mg kg-1). The Cd-tolerance index varied significantly between pea genotypes from 35% to 90% and from 54% to 100% in the presence of 7 and5 mg Cd kg-1, respectively. Shoot Cd concentration of tolerant and sensitive genotypes grown in the presence of 5 mg Cdkg-1 varied between 35 mg Cd kg-1and 135 mg Cd kg-1 (dry weight) and was negatively correlated with TI. Certain tolerant pea genotypes were characterized by a high Cd concentration in shoots. All varieties were also screened for their ability to take up heavy metals (HMs) from a slightly contaminated soil. The concentration of Cd, chromium, copper, nickel, lead, strontium and zinc in plant shoots varied between pea genotypes by a factor of 2.8, 4.9, 2.7, 3.5, 9.7, 3.9 and 4.0, respectively. The coefficients of variation between pea genotypes for HM concentration were high, varying from 23%to 39% depending on the metal. The distribution patterns for varieties based on Cd tolerance (sand culture) and HM concentrations (soil culture) were characterised by positive skewness coefficients, suggesting that the majority of pea genotypes was relatively sensitive to Cd toxicity and tended to avoid excessive accumulation of HMs in shoots. These results show that a high genetic variability exists in pea with regards to Cd tolerance and HM accumulation. Concentrations of different HMs in plants grown in soil correlated positively with each other, with the exception of chromium. There was no correlation between Cd tolerance of the varieties in sand culture, shoot concentration of HMs in soil culture, biomass production, subspecies and geographical origin of the varieties. The genetic systems controlling Cd tolerance, HM accumulation and morphological traits are therefore independent to some extent, suggesting a possibility for breeding pea cultivars characterised by high tolerance to and low concentration of HMs in shoots.

WUSCHEL-RELATED HOMEOBOX5 Gene Expression and Interaction of CLE Peptides with Components of the Systemic Control Add Two Pieces to the Puzzle of Autoregulation of Nodulation

In legumes, the symbiotic nodules are formed as a result of dedifferentiation and reactivation of cortical root cells. A shoot-acting receptor complex, similar to the Arabidopsis (Arabidopsis thaliana) CLAVATA1 (CLV1)/CLV2 receptor, regulating development of the shoot apical meristem, is involved in autoregulation of nodulation (AON), a mechanism that systemically controls nodule number. The targets of CLV1/CLV2 in the shoot apical meristem, the WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factors, have been proposed to be important regulators of apical meristem maintenance and to be expressed in apical meristem “organizers.” Here, we focus on the role of the WOX5 transcription factor upon nodulation in Medicago truncatula and pea (Pisum sativum) that form indeterminate nodules. Analysis of temporal WOX5 expression during nodulation with quantitative reverse transcription-polymerase chain reaction and promoter-reporter fusion revealed that the WOX5 gene was expressed during nodule organogenesis, suggesting that WOX genes are common regulators of cell proliferation in different systems. Furthermore, in nodules of supernodulating mutants, defective in AON, WOX5 expression was higher than that in wild-type nodules. Hence, a conserved WUS/WOX-CLV regulatory system might control cell proliferation and differentiation not only in the root and shoot apical meristems but also in nodule meristems. In addition, the link between nodule-derived CLE peptides activating AON in different legumes and components of the AON system was investigated. We demonstrate that the identified AON component, NODULATION3 of pea, might act downstream from or beside the CLE peptides during AON.

The effect of tryptophan present in plant root exudates on the phytostimulating activity of rhizobacteria

Aseptic tomato and radish roots were found to exude, respectively, 2.8–5.3 and 290–390 ng tryptophan per seedling per day. The inoculation of radish plants with rhizosphere pseudomonads increased the root biomass by 1.4 times. The inoculation of tomato plants with the same pseudomonads was ineffective. The beneficial effect of bacterial inoculation on the radish plants can be explained by the fact that the introduced rhizobacteria produce the plant growth–stimulating hormone indole-3-acetic acid. In pot experiments, the addition of this phytohormone to the soil increased the mass of radish roots by 36%. The phytohormonal action of the rhizosphere microflora was found to be efficient provided that the concentration of tryptophan in the rhizosphere is sufficiently high.

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

Medicago truncatula NOOT and Pisum sativum COCH were found to maintain nodule identity during symbiotic interactions with rhizobia and were identified as orthologs of Arabidopsis BLADE-ON-PETIOLE genes, which are involved in leaf and flower development. During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

THE PEA (PISUM SATIVUM L.) GENES SYM33 AND SYM40 CONTROL INFECTION THREAD FORMATION AND ROOT NODULE FUNCTION

Abstract Two novel non-allelic mutants that were unable to fix nitrogen (Fix−) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix−–1 and SGEFix−–2, form two types of nodules: SGEFix−–1 forms numerous white and some pink nodules, while mutant SGEFix−–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix−–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix−–1 does not differ from that of the parental line, SGE. White nodules of SGEFix−–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix−–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix−–1 and SGEFix−–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function.

Characterization of Bacillus subtilis HC8, a novel plant‐beneficial endophytic strain from giant hogweed

Thirty endophytic bacteria were isolated from various plant species growing near Saint‐Petersburg, Russia. Based on a screening for various traits, including plant‐beneficial properties and DNA fragment patterns, potential siblings were removed. The remaining isolates were taxonomically identified using 16S rDNA sequences and potential human and plant pathogens were removed. The remaining strains were tested for their ability to promote radish root growth and to protect tomato plants against tomato foot and root rot. One strain, Bacillus subtilis HC8, isolated from the giant hogweed Heracleum sosnowskyi Manden, significantly promoted plant growth and protected tomato against tomato foot and root rot. Metabolites possibly responsible for these plant‐beneficial properties were identified as the hormone gibberellin and (lipo)peptide antibiotics respectively. The antibiotic properties of strain HC8 are similar to those of the commercially available plant‐beneficial strain Bacillus amyloliquefaciens FZB42. However, thin layer chromatography profiles of the two strains differ. It is speculated that endophytes such as B. subtilis HC8 contribute to the fast growth of giant hogweed.

Sequential functioning of Sym-13 and Sym-31, two genes affecting symbiosome development in root nodules of pea (Pisum sativum L.)

Abstract Two Fix− mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix− (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix− in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix− line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line).