Soybean nodule sucrose synthase (nodulin-100): further analysis of its phosphorylation using recombinant and authentic root-nodule enzymes.

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta.     

Seryl-phosphorylation of soybean nodule sucrose synthase (nodulin-100) by a Ca-dependent protein kinase

Sucrose synthase (SS; EC 2.4.1.13) was radiolabeled in situ by incubating detached soybean nodules with ³²Pi. Phosphoamino acid analysis indicated that SS was phosphorylated on a serine residue(s). In-vitro phosphorylation of purified nodule SS by desalted nodule extracts was Ca²⁺-dependent. This SS-kinase was partially purified (2200-fold) from nodules harvested from illuminated plants. The molecular mass of the SS-kinase was about 55 000 on a Superdex 75 size-exclusion column or in a denaturing autophosphorylation gel. With either purified nodule SS or Syntide 2 as substrate, exogenous calmodulin and phosphatidylserine showed little or no effect on the in-vitro activity of this partially purified protein kinase. However, its activity was inhibited by W-7. The purified nodule SS-kinase (or CDPK) phosphorylated nodule PEP carboxylase (PEPC; EC 4.1.1.31) in the presence of Ca²⁺. In contrast, a partially purified nodule PEPC-kinase preparation was incapable of phosphorylating nodule SS. Unlike nodule PEPC [Zhang et al. (1995) Plant Physiol. 108, 1561–1568], the phosphorylation state of SS is not likely modulated in planta by photosynthate supply from the shoots.     

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.     

Nodulin-26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment.

The peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). Antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an RNA fraction that served as a template for the synthesis of a cDNA probe for screening a nodule-specific cDNA library. Clone p1B1 was found to encode a 26.5 kDa polypeptide (nodulin-26) which is immunoprecipitable specifically with the anti-pbm serum. Nodulin-26 has features of a transmembrane protein and its structure differs from that of nodulin-24 which appears to be a surface protein of pbm. The expression of these two pbm nodulins was examined in nodules induced by Bradyrhizobium japonicum Tn5 mutants that arrest nodule development at different stages of pbm biosynthesis. Nodules that do not show release of bacteria from the infection thread express nodulin-24 at a very low level. In contrast, the expression of nodulin-26 occurs fully in nodules that form infection threads only and is not affected by the release of bacteria from the threads.     

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).     

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca²⁺-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca²⁺ (K_0.5 = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca²⁺-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with ³²P-phosphate. Overall, the results show the existence of a Ca²⁺-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca²⁺-dependent phosphorylation.     

Structural and expression analysis of uricase mRNA from Lotus japonicus

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.     

Newly featured infection events in a supernodulating soybean mutant SS2-2 by Bradyrhizobium japonicum

Supernodulating soybean (Glycine max L. Merr.) mutant SS2-2 and its wild-type counterpart, Sinpaldalkong 2, were examined for the microstructural events associated with nodule formation and development. SS2-2 produced a substantially higher percentage of curled root hairs than the wild type, especially at 14 days after inoculation with Bradyrhizobium japonicum. In addition, there was new evidence that in SS2-2, B. japonicum also entered through fissures created by the emerging adventitious root primordia. Early steps of nodule ontogeny were faster in SS2-2, and continued development of initiated nodules was more frequent and occurred at a higher frequency than in the wild type. These data suggest that the early expression of autoregulation is facilitated by decreasing the speed of cortical cell development, leading to the subsequent termination of less-developed nodules. The nodules of SS2-2 developed into spherical nodules like those formed on the wild type. In both the wild type and supernodulating mutant, vascular bundles bifurcate from root stele and branch off in the nodule cortex to surround the central infected zone. These findings indicate that SS2-2 has complete endosymbiosis and forms completely developed nodule vascular bundles like the wild type, but that the speed of nodule ontogeny differs between the wild type and SS2-2. Thus, SS2-2 has a novel symbiotic phenotype with regard to nodule organogenesis.     

Translocation of nitrogen and expression of nodule-specific uricase (nodulin-35) in Robinia pseudoacacia

Uricase (urate oxidase, EC 1.7.3.3) activity and nodule-specific uricase II (nodulin-35) were detected in the nodules from a number of legume: Rhizobium symbioses ( Vigna unguiculata (L.) Walp., Phaseolus vulgaris L., and Kennedia coccinea Vent.) in the Phaseoleae, as well as in those of Robinia pseudoacacia L. which belongs to the tribe Robineae. Neither uricase activity nor nodulin-35 was detected in nodules from Lupinus angustifolius L., an amide-forming symbiosis of the tribe Genisteae. Nodules of R. pseudoacacia also showed high levels of allantoinase (EC 3.5.2.5) activity but activity of enzymes earlier in the pathway of ureide synthesis (xanthine dehydrogenase, EC 1.2.1.37; inosine monophosphate dehydrogenase, EC 1.2.1.14; and xanthosine nucleosidase, EC 3.2.2.1) could not be detected. Analysis of transport fluids (xylem, phloem and nodule exudates) from R. pseudoacacia found that asparagine, and, to a lesser extent, glutamine were the major translocated nitrogenous solutes. Ureides accounted for, at most, 2.6% of the N in transport fluids (tracheal xylem sap) and in nodule exudate, 0.1%. In common with nodules of the ureide-forming symbioses, those of R. pseudoacacia contained a high proportion of uninfected interstitial cells (53.7 ± 2.3%) in the central N₂-fixing tissue whereas in L. angustifolius only 2.5 ± 0.4% of cells in this tissue were uninfected. These data have been interpreted to indicate that expression of nodule-specific uricase is related to the differentiation of uninfected interstitial cells in nodules and not to the synthesis of ureides.     

Root nodule development: origin, function and regulation of nodulin genes

The symbiotic root nodule, an organ formed on leguminous plants, is a product of successful interactions between the host plant and the soil bacteria, Rhizobium spp. Plant hormones play an important role in the genesis of this organ. The hormonal balance appears to be modulated by the signals produced by bacteria. Many host genes induced during nodule organogenesis and the symbiotic state have been identified and characterized from several legumes. These genes encode nodule-specific proteins (nodulins) which perform diverse functions in root nodule development and metabolism. Formation of a subcellular compartment housing the bacteria is essential to sustain the symbiotic state, and several nodulins are involved in maintaining the integrity and function of this compartment. The bacteroid enclosed in the perbacteroid membrane behaves as an 'organelle,'completely dependent on the host for all its requirements for carbon, nitrogen and other essential elements. Thus it seems likely that the nodulins in the peribacteroid membrane perform specific transport functions. While the function of a few other nodulins is known (e.g. nodulin-100, nodulin-35), a group of uncharacterized nodulins exists in soybean root nodules. These nodulins share structural similarities and seem to have been derived from a common ancestor. Induction of nodulin genes occurs prior to and independent of nitrogen fixation, and thus is a prelude to symbiosis. Although some of the early nodulin genes are induced prior to or during infection, induction of late nodulins requires endocytotic release of bacteria.     

Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules

Nodulin-35, a protein specific to soybean root nodules, was purified under non-denaturing conditions (DEAE-cellulose followed by Sephacryl S-200 chromatography) to homogeneity. The holoprotein showed uricase (EC 1.7.3.3) activity. Analytical ultracentrifugation under non-denaturing conditions revealed a molecule of 124 kd, S°_20W = 8.1; however, under denaturing conditions a value of 33 kd, S°_20W = 1.9, was obtained. This indicated that nodulin-35 is the 33-kd subunit of a specific soybean root nodule uricase (uricase II) and that the enzyme contains four similar subunits. The native molecule contains ˜1.0 mol Cu²⁺ per mol, has an isoelectric point of ˜9.0 and a pH optimum for uricase activity at 9.5. Uricase activity found in young uninfected soybean roots is due to another form of enzyme (uricase I) which is of ˜190 kd, has maximum activity at pH 8.0 and does not contain any subunit corresponding in size to nodulin-35. Uricase I, also present in young infected roots, declines at a time when nodulin-35 appears. Monospecific antibodies prepared against uricase II (nodulin-35) showed no cross-reactivity. Uricase II was localized in the uninfected cells of the nodule tissue. These results are consistent with the concept that a nodule-specific ureide metabolism takes place in peroxisomes of uninfected cells, and suggest the participation of uricase II in this pathway.     

Post-translational regulation of cytosolic glutamine synthetase of Medicago truncatula

It was reported recently that the plastid-located glutamine synthetase (GS2) from Medicago truncatula is regulated by phosphorylation catalysed by a calcium-dependent protein kinase and 14-3-3 interaction. Here it is shown that the two cytosolic GS isoenzymes, GS1a and GS1b, are also regulated by phosphorylation but, in contrast to GS2, GS1 phosphorylation is catalysed by calcium-independent kinase(s) and the phosphorylated enzymes fail to interact with 14-3-3s. Phosphorylation of GS1a occurs at more than one residue and was found to increase the affinity of the enzyme for the substrate glutamate. In vitro phosphorylation assays were used to compare the activity of GS kinase, present in different plant organs, against the three M. truncatula GS isoenzymes. All three GS proteins were phosphorylated by kinases present in leaves, roots, and nodules, but to different extents, suggesting a differential regulation under different metabolic contexts. Cytosolic GS phosphorylation was found to be affected by light in leaves and by active nitrogen fixation in root nodules, whereas GS2 phosphorylation was unaffected by these conditions. Some putative GS-binding phosphoproteins were identified showing both isoenzyme and organ specificity. Two phosphoproteins of 70 and 72 kDa were specifically bound to the cytosolic GS isoenzymes. Interestingly, phosphorylation of these proteins was also influenced by the nitrogen-fixing status of the nodule, suggesting that their phosphorylation and/or binding to GS are related to nitrogen fixation. Taken together, the results presented indicate that GS phosphorylation is modulated by nitrogen fixation in root nodules; these findings open up new possibilities to explore the involvement of this post-translational mechanism in nodule functioning.     

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.     

Nodule-specific kinases phosphorylating nuclear factors in isolated nuclei.

In vitro phosphorylation of total nuclear proteins from soybean (Glycine max L) nodules formed by Bradyrhizobium japonicum 61A76 showed several differences in comparison with those from uninfected roots or embryonic-axes nuclei. Three types of protein phosphorylations were observed in nodule nuclei: Ca(2+)- and calmodulin-independent, Ca(2+)- and calmodulin-dependent, and Ca(2+)-dependent but calmodulin-independent. In addition, Ca(2+)-dependent dephosphorylation of some nuclear proteins was observed in nodule nuclei. The first and second types of phosphorylations were also present in root nuclei, but the trifluoperazine-insensitive and Ca(2+)-dependent phosphorylation (indicating calmodulin independence) occurs only in nodules. The latter appears to phosphorylate a nodule-specific protein of 65 kilodaltons and this protein was purified from other nuclear phosphorylated proteins. In addition, some nuclear proteins from uninfected tissue were found to be phosphorylated or dephosphorylated by kinases or phosphatases that originated from the nodule nuclei. These data suggest that some activities of nuclear factors in nodules may be regulated by specific phosphorylation or dephosphorylation during symbiotic interactions with rhizobia.     

Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5''-flanking regions of nodulin and leghemoglobin genes.

The nodulin-23 gene of soybean is one of the most abundantly transcribed genes induced during symbiosis with Rhizobium. Using a plasmid (pNod25) from a nodule cDNA library, we have isolated the nodulin-23 gene from a soybean genomic library. Nucleotide sequence analysis of the cDNA and of the genomic clone indicated that the coding region of this gene is 669 bp long and is interrupted by a single intron of about 530 bp. The deduced protein sequence suggests that nodulin-23 may have a signal sequence. The 5'-flanking sequence of two other nodulin genes, nodulin-24 encoding for a membrane polypeptide and one of the leghemoglobin genes (LbC3), were obtained. Comparison of these sequences revealed three conserved regions, one of which, an octanucleotide (GTTTCCCT), has 100% homology. The conserved sequences are arranged in a unique fashion and have a spatial organization with respect to order and position, which may suggest a potential regulatory role in controlling the expression of nodulin and leghemoglobin genes during symbiosis.