Abstract: * A comparative analysis of nodule morphogenesis was carried out for three symbiotically defective pea (Pisum sativum) mutants that show abnormalities in nodule development. * In the wild-type lines, resumption of cell proliferation in the pericycle and inner cortex results in the development of a nodule primordium, within which are found proliferating cells that harbour infection threads. However, this class of cell is not observed in the mutants RisFixA (sym41) and SGEFix(-)-2 (sym33) where nodule development is arrested at the point of formation of the apical nodule meristem. It is proposed that the presence of proliferating cells harbouring infection threads is a prerequisite for normal formation of the nodule meristem. * In mutant SGEFix(-)-1 (sym40), nodule development does not differ from that of wild-type plants in the early stages but is blocked at the stage after nodule meristem persistence. * A scheme is proposed for the sequential functioning of pea symbiotic genes Sym33, Sym40 and Sym41 in the programme of nodule development.
Abstract: BACKGROUND AND AIMS: To date, there are no crop mutants described in the literature that display both Cd accumulation and tolerance. In the present study a unique pea (Pisum sativum) mutant SGECd(t) with increased Cd tolerance and accumulation was isolated and characterized. METHODS: Ethylmethane sulfonate mutagenesis of the pea line SGE was used to obtain the mutant. Screening for Cd-tolerant seedlings in the M2 generation was performed using hydroponics in the presence of 6 microm CdCl2. Hybridological analysis was used to identify the inheritance of the mutant phenotype. Several physiological and biochemical characteristics of SGECd(t) were studied in hydroponic experiments in the presence of 3 microm CdCl2, and elemental analysis was conducted. KEY RESULTS: The mutant SGECd(t) was characterized as having a monogenic inheritance and a recessive phenotype. It showed increased Cd concentrations in roots and shoots but no obvious morphological defects, demonstrating its capability to cope well with increased Cd levels in its tissues. The enhanced Cd accumulation in the mutant was accompanied by maintenance of homeostasis of shoot Ca, Mg, Zn and Mn contents, and root Ca and Mg contents. Through the application of La(+3) and the exclusion of Ca from the nutrient solution, maintenance of nutrient homeostasis in Cd-stressed SGECd(t) was shown to contribute to the increased Cd tolerance. Control plants of the mutant (i.e. no Cd treatment) had elevated concentrations of glutathione (GSH) in the roots. Through measurements of chitinase and guaiacol-dependent peroxidase activities, as well as proline and non-protein thiol (NPT) levels, it was shown that there were lower levels of Cd stress both in roots and shoots of SGECd(t). Accumulation of phytochelatins [(PCcalculated) = (NPT)-(GSH)] could be excluded as a cause of the increased Cd tolerance in the mutant. CONCLUSIONS: The SGECd(t) mutant represents a novel and unique model to study adaptation of plants to toxic heavy metal concentrations.
Abstract: Establishment of symbiosis between legumes and rhizobia requires bacterial Nod factors (NFs). The concentration of these lipochitooligosaccharides in the rhizosphere is influenced by plant enzymes. NFs induce on pea (Pisum sativum) a particular extracellular NF hydrolase that releases lipodisaccharides from NFs from Sinorhizobium meliloti. Here, we investigated the ability of non-nodulating pea mutants to respond to NodRlv factors (NFs from Rhizobium leguminosarum bv viciae) with enhanced NF hydrolase activity. Mutants defective in the symbiotic genes sym10, sym8, sym19, and sym9/sym30 did not exhibit any stimulation of the NF hydrolase, indicating that the enzyme is induced via an NF signal transduction pathway that includes calcium spiking (transient increases in intracellular Ca(2+) levels). Interestingly, the NF hydrolase activity in these sym mutants was even lower than in wild-type peas, which were not pretreated with NodRlv factors. Activation of the NF hydrolase in wild-type plants was a specific response to NodRlv factors. The induction of the NF hydrolase was blocked by alpha-amanitin, cycloheximide, tunicamycin, EGTA, U73122, and calyculin A. Inhibitory effects, albeit weaker, were also found for brefeldin A, BHQ and ethephon. In addition to this NF hydrolase, NFs and stress-related signals (ethylene and salicylic acid) stimulated a pea chitinase that released lipotrisaccharides from pentameric NFs from S. meliloti. NodRlv factors failed to stimulate the chitinase in mutants defective in the sym10 and sym8 genes, whereas other mutants (e.g. mutated in the sym19 gene) retained their ability to increase the chitinase activity. These findings indicate that calcium spiking is not implicated in stimulation of the chitinase. We suggest that downstream of Sym8, a stress-related signal transduction pathway branches off from the NF signal transduction pathway.
Abstract: Two symbiotic pea (Pisum sativum L.) mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread formation in symbiotic root nodules were characterised with respect to dynamics of arbuscule development at 15 degrees C and 24 degrees C. Mutation of sym33 decreased mycorrhiza colonisation at both temperatures and delayed arbuscule development at 15 degrees C, whereas mutation of sym40 accelerated mycorrhiza colonisation and arbuscule senescence at 24 degrees C. The differences between the mutants and the wild-type were more pronounced at 24 degrees C, a temperature close to the optimum for pea growth. The results demonstrate that both pea genes are important in the control of arbuscular mycorrhiza development and can be considered necessary for the tripartite symbiosis in pea.
Abstract: Comparative phenotypic analysis of pea (Pisum sativum) sym35 mutants and Lotus japonicus nin mutants suggested a similar function for the PsSym35 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 PsNin to the same chromosome arm as the PsSym35 locus and direct evidence demonstrating that PsNin is the PsSym35 gene was subsequently obtained by cosegregation analysis and sequencing of three independent Pssym35 mutant 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 the PsNin 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.
Abstract: Two pea (Pisum sativum L.) symbiotic mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread development and function in symbiotic root nodules have been characterised in terms of mycorrhizal colonisation of roots, shoot and root biomass accumulation and shoot and root phosphorus (P) content. The mutation in gene sym33 decreased mycorrhizal colonisation of roots (except arbuscule abundance in mycorrhizal root fragments, which increased) but did not change the effectiveness of mycorrhiza function. The mutation in sym40 did not affect either of these processes. Both mutants showed differences in plant development compared with the wild-type line SGE. The mutants had delayed flowering and pod ripening, and shoot/root biomass ratios and P accumulation also differed from those of SGE. These observations suggest that the gene mutations cause systemic changes in plant development.
Abstract: Twelve non-nodulating pea (Pisum sativum L.) mutants were studied to identify the blocks in nodule tissue development. In nine, the reason for the lack of infection thread (IT) development was studied; this had been characterized previously in the other three mutants. With respect to IT development, mutants in gene sym7 are interrupted at the stage of colonization of the pocket in the curled root hair (Crh- phenotype), mutants in genes sym37 and sym38 are blocked at the stage of IT growth in the root hair cell (Ith- phenotype) and mutants in gene sym34 at the stage of IT growth inside root cortex cells (Itr- phenotype). With respect to nodule tissue development, mutants in genes sym7, sym14 and sym35 were shown to be blocked at the stage of cortical cell divisions (Ccd- phenotype), mutants in gene sym34 are halted at the stage of nodule primordium (NP) development (Npd- phenotype) and mutants in genes sym37 and sym38 are arrested at the stage of nodule meristem development (Nmd- phenotype). Thus, the sequential functioning of the genes Sym37, Sym38 and the gene Sym34 apparently differs in the infection process and during nodule tissue development. Based on these data, a scheme is suggested for the sequential functioning of early pea symbiotic genes in the two developmental processes: infection and nodule tissue formation.
Abstract: Genetic and molecular mechanisms of development are compared for two major plant-microbe endosymbioses: N(2)-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi). Development from the primordia formed de novo in root tissues is common for all known types of N(2)-fixing nodules. However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripherical in legumes or central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones). In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia and located intercellularly (infection threads) and intracellularly (symbiosomes). Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps comprising four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation). The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution. The origin of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g., inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules). An analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N(2)-fixing symbioses.
Abstract: Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals. The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. by determination of 16S rRNA gene sequences. The root elongation of Indian mustard and rape (Brassica napus var. oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively. The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 microM CdCl2 in the nutrient solution. The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis. A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil. The biomass of pea cv. Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture. The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions. The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions.
Abstract: In this report, the expression of late symbiotic genes (fnrN, fixN, and nifA) of Rhizobium leguminosarum bv. viciae was studied in nodules of mutant pea lines blocked at four successive stages of nodule development. Bacterial gene expression was analyzed in situ with transcriptional gusA reporter gene fusions. As a control, a constitutively expressed gusA gene was included. In the nodules of Nop(nodule persistence) mutants (mutant in gene sym13), which had not yet exhibited signs of premature senescence, the expression patterns observed were identical to those in wild-type nodules. Normal expression of fusions also occurred in nodules defective at the infection droplet differentiation stage (mutant in gene sym40) in which bacteria are endocytosed, but infection threads and infection droplets are hypertrophied. In contrast, in Itn- (infection thread formation inside the nodule tissue) mutants (mutant gene sym33), in which there is no endocytosis of bacteria, expression of the constitutive fusion was only in infection threads and no activity was shown for the other fusions. From this it can be concluded that functionality of the plant gene Sym33, i.e., bacterial endocytosis, is a prerequisite for the expression of late symbiotic genes in the microsymbiont. No morphologically distinct interzone II-III could be detected in nodules blocked at the bacteroid differentiation stage (mutants in gene sym31). The constitutive fusion was expressed equally throughout the nodule tissue (except for the meristem), and the activity of fusions to late symbiotic genes increased gradually with a maximal expression level at the base of the nodule. This is consistent with an altered oxygen barrier previously reported for these nodules. By including double mutants, earlier results on sequential functioning of gene pairs sym33-sym40 and sym31-sym13 could be confirmed and it could be demonstrated that the developmental epistasis found at the morphological level also is reflected in the expression pattern of late symbiotic genes in the microsymbiont.
Abstract: Root nodule structural organisation of nine pea Fix(-) mutants representing seven symbiotic loci has been studied. This set of mutants has revealed lesions at four developmental stages in the pea-Rhizobium symbiosis. (i) Mutant RisFixA is affected in infection thread differentiation in nodule tissue, infection droplet formation, bacteroid differentiation and nodule persistence. (ii) Allelic mutants RisFixL and RisFixO (sym32) are blocked in bacteroid differentiation. (iii) Mutants RisFixM (sym26), RisFixN, RisFixQ (sym27), RisFixT (sym26) show premature degradation of symbiotic structures. (iv) Mutant RisFixV shows similar defects in nodule persistence but, in addition, this mutant is characterised by an abnormal and severe increase in the thickness of infection thread walls during the process of infection thread 'maturation' and senescence. Combining our new data with previously published studies of pea mutant phenotypes has allowed us to create an integrated scheme for the sequential functioning of the late pea symbiotic genes identified and characterised to date.
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, synm40 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.
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).
