The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

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.     

Distribution of legume arabinogalactan protein-extensin (AGPE) glycoproteins in symbiotically defective pea mutants with abnormal infection threads

The interface between the host cell and the microsymbiont is an important zone for development and differentiation during consecutive stages of Rhizobium-legume symbiosis. Legume root nodule extensins, otherwise known as arabinogalactan protein-extensins (AGPEs) are abundant components of infection thread matrix. We have characterized the origin and distribution of these glycoproteins at the symbiotic interface of root nodules of symbiotically defective mutants of pea (Pisum sativum L.) by using immunogold localization with MAC265 an anti-AGPE monoclonal antibody. For mutants with defective growth of infection threads, the AGPE epitope was abundant in the extracellular matrix surrounding infected host cells in the central infected tissue of the nodule, as well as in the lumen of Rhizobiuminduced infection threads. This seems to indicate a mistargeting of AGPE as a consequence of abnormal growth of the infection threads. Furthermore, mutants in the gene sym33 showed reduced labeling with MAC265 and, in some infection threads and droplets, the label was completely absent, a phenomenon that is not observed in wild-type nodules. This suggests an alteration in the composition of the infection thread matrix for sym33 mutants, which may be correlated to the absence of endocytosis of rhizobia into the host cytoplasm.     

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.     

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of ∼1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction. Received: 12 May 1998 / Accepted: 24 June 1998     

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

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). Received: 28 October 1996 / Accepted: 22 January 1997     

<Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> bacteroid appendages expressed in senescing and argon-treated soybean nodules

Bradyrhizobium japonicum bacteroids in soybean nodules expressed fibrillar appendages during senescence. In both scanning and transmission electron microscopy (SEM and TEM), these structures were observed to connect adjacent bacteroids, and bacteroids to symbiotic membranes. They were 20–25 nm in diameter, 100–2,500 nm in length and were linear, branched, or part of a web-like matrix. Bacteroids expressing appendages were not uniformly distributed, but were abundant within localized regions in the senescing nodule. The root systems of nodulated greenhouse-grown plants flushed with argon induced the appendages at earlier plant ages, and they were more prolific and wide spread than those in untreated nodules. Bradyrhizobium japonicum symbiotic appendages appear to be a response to an environmental niche within senescing nodules.     

Rhizobium leguminosarum strains forming nitrogen-rich nodules inPisum sativum

The nodules which developed on the roots ofPisum sativum after inoculation withRhizobium leguminosarum bv.viciae strains showing a Hup⁺ symbiotic phenotype and/or a high relative efficiency of electron transfer to dinitrogen (RE) had a high nitrogen content of their tissue. In comparison with the nodules initiated by a strain possessing the Hup⁻ symbiotic phenotype or by an indigenous soilRhizobium population, the nitrogen-rich root nodules contained up to four times more nitrogen (9.2% of dry mass). In the nitrogen-rich nodules, total amino acid, and especially Ala, Lys and Phe contents were significantly increased. The nitrogen-rich nodule symbiotic phenotype (Nrn) was well expressed, irrespective of pea cultivars and host plant age. If a strain showing the Nrn phenotype was applied in double-strain inocula, a significant correlation was found between increasing rates of the strain and increasing percentage nitrogen content of nodule tissue in the nodulated plants.     

Immunocytological evidence for abnormal symbiosome development in nodules of the pea mutant line Sprint2Fix− (sym31)

Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix⁻ (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane, or were found within a vacuole-like compartment. In wild-type nodules, the monoclonal antibody JIM18, which recognizes a plasmalemma glycolipid antigen, bound to the juvenile peribacteroid membrane, and did not recognize the mature peribacteroid membrane. However, in the mutant, the antibody bound to all peribacteroid membranes within the nodule, suggesting that differentiation of the peribacteroid membrane was arrested. Another antibody, MAC266, recognized plant glycoproteins which normally accumulate in symbiosomes at a late stage of nodule development. Binding of this antibody was much reduced within mutant nodules, labelling only a few mature cells. Similarly, MAC301, which normally recognizes a lipopolysaccharide epitope expressed on differentiated bacteroids prior to the induction of nitrogenase, failed to react with rhizobial cell extracts isolated from nodules of thesym31 mutant. On the basis of these developmental markers, the symbiosomes ofsym31 nodules appeared to be blocked at an early stage of development. The distribution of infection structures was also found to be abnormal in the mutant nodules. Models of symbiosome development are presented and discussed in relation to the morphological and developmental lesions observed in thesym31 mutant.     

The Sym31Gene Responsible for Bacteroid Differentiation Is Involved in Nitrate-Dependent Nodule Formation in Pea Plants

The effects of exogenous nitrate on the number of developing nodules and their leghemoglobin content in the original pea (Pisum sativumL.) line and its symbiotic mutants were studied. Mutation in the Sym31gene conferred the tolerance to nitrate in the corresponding pea line and manifested itself as the number of nodules independent of the nitrate concentration. Thus, the Sym31gene was identified as the only known symbiotic gene involved in both the differentiation of symbiotic compartments and the nitrate-dependent process of nodule formation. The presence of leghemoglobin in double mutants (sym13, sym31) indicates the possibility of the complementary contribution of these genes in the control of leghemoglobin synthesis.     

Nodules elicited by Rbizobium meliloti heme mutants are arrested at an early stage of development

Summary Heme-deficient mutants of Rhizobium and Bradyrhizobium have been found to exhibit diverse phenotypes with respect to symbiotic interactions with plant hosts. We observed that R. meliloti hemA mutants elicit nodules that do not contain intracellular bacteria; the nodules contain either no infection threads (empty nodule phenotype) or aberrant infection threads that failed to release bacteria (Bar^– phenotype). These mutant nodules expressed nodulin genes associated with nodules arrested at an early stage of development, including ENOD2, Nms-30, and four previously undescribed nodulin genes. These nodules also failed to express any of six late nodulin genes tested by hybridization, including leghemoglobin, and twelve tested by in vitro translation product analysis which are not yet correlated with specific cloned genes. We observed that R. meliloti leucine and adenosine auxotrophs induced invaded Fix^– nodules that expressed late nodulin genes, suggesting that it is not auxotrophy per se that causes the hemA mutants to elicit Bar^– or empty nodules. Because R. meliloti hemA mutants elicit nodules that do not contain intracellular bacteria, it is not possible to decide whether or not the Fix^– phenotype of these nodules is a direct consequence of the failure of R. meliloti to supply the heme moiety of hololeghemoglobin. Our results demonstrate the importance of establishing the stage in development at which a mutant nodule is arrested before conclusions are drawn about the role of small metabolite exchange in the symbiosis.     

Localization of H+-ATPases in soybean root nodules

The localization of H⁺-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H⁺-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H⁺-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids. Received: 25 November 1998 / Accepted: 6 January 1999     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Coinoculation of Bacillus thuringeinsis-KR1 with Rhizobium leguminosarum enhances plant growth and nodulation of pea (Pisum sativum L.) and lentil (Lens culinaris L.)

Nodulation and the subsequent nitrogen fixation are important factors that determine the productivity of legumes. The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote plant growth of field pea (Pisum sativum L.) and lentil (Lens culinaris L.) under Jensen’s tube, growth pouch and non-sterile soil, respectively, when co-inoculated with Rhizobium leguminosarum-PR1. Coinoculation with B. thuringiensis-KR1 (at a cell density of 10⁶ c.f.u. ml⁻¹) provided the highest and most consistent increase in nodule number, shoot weight, root weight, and total biomass, over rhizobial inoculation alone. The enhancement in nodulation due to coinoculation was 84.6 and 73.3% in pea and lentil respectively compared to R. leguminosarum-PR1 treatment alone. The shoot dry-weight gains on coinoculation with variable cell populations of B. thuringiensis-KR1 varied from 1.04 to 1.15 times and 1.03 to 1.06 times in pea and lentil respectively, while root dry weight ratios of coinoculated treatments varied from 0.98 to 1.14 times and 1.08 to 1.33 times in pea and lentil respectively, those of R. leguminosarum-PR1 inoculated treatment at 42 days of plant growth. While cell densities higher than 10⁶ c.f.u. ml⁻¹ had an inhibitory effect on nodulation and plant growth, lower inoculum levels resulted in decreased cell recovery and plant growth performance. The results of this study indicate the potential of harnessing endophytic bacteria of wild legumes for improving the nodulation and growth of cultivated legumes.     

Symbiotic Tissue Degradation Pattern in the Ineffective Nodules of Three Nodulation Mutants of Pea (Pisum sativum L.)

Three symbiotic mutants of pea (Pisum sativum L.) forming ineffectivenodules were investigated for aberrations in nodule structureusing light and transmission electron microscopy. The mutantswere ordered according to the timing of the nodule developmentblock. In the mutant RisfixO, symbiotic tissue development isarrested before the late symbiotic zone (LSZ) forms, while theinfected cells of the LSZ of RisfixT lose the wild-type structureafter full differentiation. In contrast to the bacteroid degradationvia an electron-dense stage in RisfixO, lysis of symbiosomecontents prevails in RisfixT nodules. Enhancement of the lyticfunction of symbiosomes in RisfixT may be interpreted in termsof the symbiosome—lysosome homology. The weakened controlover symbiotic development in RisfixO may be responsible forthe abundant spread of the infection threads and their enlargement. Cells from the LSZ of RisfixV undergo fast collapse, resemblingdefence necrosis, after differentiation. In contrast to thenodules of RisfixO and RisfixT, degraded nodules of RisfixVdo not function as a sink for photosynthates and a source ofthe nodulation regulatory factor. This is indicated by the absenceof further starch accumulation after collapse, and by hypernodulation.Copyright1995, 1999 Academic Press Garden pea, developmental mutant, Pisum sativum, Rhizobium leguminosarum, root nodule, symbiosis, ultrastructure     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Accumulation of an Aromatic Amine, β-Phenethylamine, in Root Nodules of Adzuki Bean Vigna angularis

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.