K. Novák

Early action of pea symbiotic gene NOD3 is confirmed by adventitious root phenotype.

A supernodulating and Nts (nitrate-tolerant symbiosis) symbiotic mutation of pea (Pisum sativum L.) line RisfixC was found to retain its expression in the distant genetic background of pea lines Afghanistan L1268, Zhodino E900, and cv. Arvika. This finding allowed for reliable scoring for the trait in mapping crosses. The RisfixC mutation was localized 8.2cM apart from SYM2 and cosegregated with molecular markers for SYM2-NOD3 region Psc923 and OA-1. Grafting experiments showed that supernodulation is root-determined, consistently with mutants in the NOD3 locus. Therefore, the mutation of RisfixC can be localized in gene NOD3. Like in other published nod3 alleles, the RisfixC mutation determines supernodulation when it is expressed in the root but not in the shoot. Supernodulated adventitious roots that are spontaneously formed in the wild-type scions on mutant rootstocks indicate that the descending systemic signal, which is inhibitory to nodule formation, is absent in this type of chimeric plants. Since the descending signal production in the wild-type shoot reflects the presence of the ascending root signal, the nod3-associated lesion must be located in the beginning of the systemic circuit regulating nodule number.

Optimization of rhizobialnod gene-inducing activity assay in pea root exudate

ThelacZ transcriptional fusion assay of the activation of rhizobialnod genes has been adapted for the microtitration plates. The assay can be performed reproducibly on a microscale, including induction, lysis, and spectrophotometry. The technique facilitates processing of a large number of samples in physiological studies. Using the microplate method, the time course of the release ofnod-gene inducers from the root of noninoculated, hydroponically cultivated, pea (Pisum sativum L.) plants was monitored till the 16th day after germination. The released activity increased with plant age but a high variability among individual plants and days was observed.

Visualization of nodulation gene activity on the early stages of Rhizobium leguminosarum bv. viciae symbiosis

A technique was optimized for thein situ detection of nodulation (nod) gene activity inRhizobium leguminosarum bv.viciae symbiosis with compatible plant hostsVicia tetrasperma (L.)Schreb. andPisum sativum L. The transcription ofnodABC-lacZ fusion was visualized as β-galactosidase (β-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the back-ground of the indigenous β-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment.V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model ofV. hirsuta (L.) S.F.Gray: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent ofnod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the peasym8 locus. Judging from thenod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.

PEA MUTANT RISNOD27 AS REFERENCE LINE FOR FIELD ASSESSMENT OF IMPACT OF SYMBIOTIC NITROGEN FIXATION

ABSTRACT In the case of ideal behavior of nodulation mutants under field conditions, the impact of nitrogen fixation on plant growth can be easily determined by subtracting the yield of a mutant from the yield of the original cultivar forming wild-type symbiotic nodules. Therefore, four symbiotic mutants of pea (Pisum sativum L.) cv. Finale termed Risnod2, Risnod27, RisfixO, and RisfixM were tested as reference lines for the field assessment of the contribution of biological nitrogen fixation to seed yield. In 1996 and 1997 field trials, the stable non-nodulation of Risnod2 and Risnod27 was confirmed, as well as the ineffectiveness of RisfixO nodules. Phenotype of RisfixM was found to be unstable since this mutant, previously classified as ineffectively nodulating under controlled conditions, possessed residual nitrogenase activity under field conditions. Growth and yield of Risnod27 and RisfixM on non-limiting (saturating) level of mineral nitrogen in soil were indistinguishable from the wild-type plants. This equivalency indicates absence of deleterious pleiotropic effects of the main mutation or the effect of multiple mutations that might adversely influence nitrogen metabolism or plant development. This result was independent of the level of organic matter in the soil. On the other hand, Risnod2 and RisfixO do not seem to be physiologically equivalent to the wild-type plants, as judged by their fairly weak response to mineral nitrogen. For this reason, both the latter mutants are not considered as suitable for reference lines. An additional reason for the exclusion of Risnod2 and RisfixM was lower field emergence rate. Neither differential susceptibility to Fusarium infection under natural infectious background nor obvious susceptibility to other pathogens was observed in all the tested lines. It is concluded that the non-nodulating mutant Risnod27, paired with its parent cultivar Finale, may serve as a reference line in routine field estimates of the contribution of symbiotic nitrogen fixation to pea yield. Under the experimental plot conditions (České Budějovice in the Czech Republic, Orthic Luvisol with 13.9 mg NO3 −, and 4.4 mg NH4 + per kg dry soil), the benefit from N2 fixation varied from 34 to 66% of the value of individual wild-type yield characteristics.

Isoflavonoid phytoalexin pisatin is not recognized by the flavonoid receptor NodD ofRhizobium leguminosarum bv.viciœ

Isoflavonoid (+)-pisatin, the main phytoalexin of pea (Pisum sativum), was tested for its inducer and suppressor activity in respect of thenodABCIJ operon ofRhizobium leguminosarum bv.viciœ, a pea symbiont, in the presence of a flavonoid receptor-coding genenodD of this biovar. Nonod gene-inducing activity of pisatin could be detected up to 100 μmol/L concentration. The suppressor activity as determined against 0.1 μmol/L naringenin was expressed only above 10 μmol/L pisatin,i.e. at a concentration higher by a one order of magnitude, than with the potent flavonoid suppressor rhamnetin and by two orders above the isoflavonoid, suppressor daidzein. Analogously, only a weak suppression was observed with NodD1 ofR. meliloti, the alfalfa symbiont. The results do not support the notion about the role of plant defence substances in the control of rhizobial symbiosis.

Plant growth, photosynthetic pigments, and nitrate assimilation in symbiotic mutants of garden pea

Abstract A set of 21 pea (Pisum sativum L.) non‐nodulating mutants and a supernodulating mutant were compared to the parental cv. ‘Finale’ by leaf nitrate reductase activity, nitrogen (N) content in the shoot, content of photosynthetic pigments, and growth. The plants were cultivated asymbiotically at the growth‐saturating nitrate (NO3) level (10 mM) to detect the nitrate assimilation faults. Both positive and negative deviations were revealed in all traits, indicating pleiotropy of symbiotic mutations and/or the deleterious effects of multiple mutations. Only four mutants were indistinguishable from the control plants by all traits. The supernodulating line RisfixC demonstrated lower shoot growth and increased N content in the shoot, in spite of the asymbiotic cultivation.

Comparison of twoPisum sativum nodulation mutants with their parental cultivar

In comparison with the parental cv. Finale the ‘RisfixC’ supernodulator exhibited higher, continuously increasing nodule number and fresh mass accumulation, but substantially lower individual nodule fresh mass, leghemoglobin concentration, and specific acetylene reduction activity of nodule tissue. There were no substantial differences between Finale and ‘RisfixC’ in total acetylene reduction, nodule leghemoglobin accumulation per nodulated root, total and specific CO2 evolution from nodulated roots and gross CO2 respiratory costs of acetylene reduction. The ‘RisfixC’ also exhibited a substantially lower plant dry mass production (by 30%), but nitrogen concentration in shoots and carotenoid concentration in leaf tissue were significantly higher by 33 and 14%, and the chlorophylla+b content insignficantly higher than in the parental cultivar. In contrast, the nodulation mutant ‘Risnod29’, exhibited a somewhat higher nodule fresh mass accumulation (by 21%) and individual nodule fresh mass (by 23%), total and specific acetylene reduction (by 49 and 19%) and a somewhat more rapid plant dry mass accumulation compared with the cv. Finale.

Pleiotropy of pea RisfixC supernodulation mutation is symbiosis-independent

Mutations affecting the development of root symbiosis between legume plants (Fabaceae) and nodule bacteria (rhizobia) are often associated with pleiotropy. This might either primarily be caused by the mutation or develops as a physiological consequence of a changed nodule structure, number and activity. Three pleiotropic traits were revealed in the pea (Pisum sativum L.) mutant RisfixC which is of supernodulation/nitrate-tolerant symbiosis (Nts) type. They comprise shortened internodes, reduced shoot dry mass accumulation and increased nitrogen content in the root tissues when compared to the wild type. The changes were expressed in the same degree in asymbiotic and nodulated plants when the effect of symbiotic nitrogen on plant growth was abolished with a saturating nitrate level. Consequently, the pleiotropic traits are inherently associated with the mutation. In RisfixC, the pleiotropy coincided with the presumed absence of the systemic feedback factor regulating nodule number. However, no differences were detected in the comparison of nonnodulating mutant Risnod27 (sym8) with the wild type and of inoculated with noninoculated wild-type plants although these pairs also differ in the presence of the systemic factor. Therefore, the pathway leading from the RisfixC mutant product to pleiotropic changes appears to be independent of systemic nodule number regulation. Implications for the genetic improvement of growth and yield parameters of supernodulating breeding lines are discussed.

Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner

BACKGROUND Fluorescent tagging of nodule bacteria forming symbioses with legume host plants represents a tool for vital tracking of bacteria inside the symbiotic root nodules and monitoring changes in gene activity. The constitutive expression of heterologous fluorescent proteins, such as green fluorescent protein (GFP), also allows screening for nodule occupancy by a particular strain. Imaging of the fluorescence signal on a macro-scale is associated with technical problems due to the robustness of nodule tissues and a high level of autofluorescence. SCOPE These limitations can be reduced by the use of a model species with a fine root system, such as Vicia tetrasperma. Further increases in the sensitivity and specificity of the detection and in image resolution can be attained by the use of a fluorescence scanner. Compared with the standard CCD-type cameras, the availability of a laser source of a specified excitation wavelength decreases non-specific autofluorescence while the photomultiplier tubes in emission detection significantly increase sensitivity. The large scanning area combined with a high resolution allow us to visualize individual nodules during the scan of whole root systems. Using a fluorescence scanner with excitation wavelength of 488 nm, a band-pass specific emission channel of 532 nm and a long-pass background channel of 555 nm, it was possible to distinguish nodules occupied by a rhizobial strain marked with one copy of cycle3 GFP from nodules colonized by the wild-type strain. CONCLUSIONS The main limitation of the current plant model and GFP with the wild-type emission peak at 409 nm is a sharp increase in root autofluorescence below 550 nm. The selectivity of the technique can be enhanced by the use of red-shifted fluorophores and the contrasting labelling of the variants, provided that the excitation (482 nm) and emission (737 nm) maxima corresponding to root chlorophyll are respected.

A mutation affecting symbiosis in the pea line Risnod27 changes the ion selectivity filter of the DMI1 homolog

After identifying regions of cDNA conserved between the symbiotic gene DMI1 of the model species Medicago truncatula and the homologous genomic region of Arabidopsis thaliana, universal primers were designed from 8 of 12 exons to allow the routine amplification of plant homologs. As an example, the complete homologous sequence from the pea (Pisum sativum L.) was amplified and sequenced, although the poorly conserved 5′-end and 5′-flanking region of the gene had to be amplified using a modified TAIL-PCR strategy. The identity of this amplified homolog with the SYM8 gene was independently confirmed by the presence of a single nucleotide change in the coding sequence of the mutant line Risnod27 (sym8) that cosegregated with the asymbiotic phenotype. Five insertions in pea introns responsible for increasing the total length of SYM8 by 1443 bp, compared to the M. truncatula homolog DMI1, belong to known transposon and retrotransposon families of pea and legumes in general. In view of the predicted function of SYM8 as an ion channel, the Risnod27 mutation (His309Tyr) appears to be localized in the selectivity filter domain. This finding confirms the essential role of histidine 309 in the symbiotic function of SYM8 and provides a guide to its ionic specificity. In view of the Risnod27 symbiotic phenotype, we hypothesize that SYM8 does not have identical functions in the transduction of rhizobial and mycorrhizal signals. The variability of the N-proximal region of the known legume homologs of DMI1 suggests an interaction with a variable ligand.