Tissue distribution and subcellular localization of carbonic anhydrase in mature soybean root nodules indicates a role in CO2 diffusion

Tissue distribution of carbonic anhydrase (CA; EC 4.2.1.1) and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) was examined in developing soybean (Glycine max) nodules using an immunohistological approach. The data obtained indicate that in young nodules both CA and PEPC proteins are present in the parenchymatous cells, and at much lower levels, in the central nodular region. In mature nodules, high levels of CA were exclusively present in 2-3 cell layers of the inner cortical region, whereas high levels of PEPC were present both in infected and uninfected cells. Immunogold localization indicated that, in mature nodules, CA was localized in the cytoplasm of the inner cortical cells and the cell walls of the endodermal cells. These results considered together suggest that in mature nodules, CA may facilitate the diffusion of the excessive CO₂, derived from the respired bacteroids, in the rhizosphere. The distribution of CA was examined in mature nodules of soybean, grown hydroponically, in either limiting or non-limiting phosphate concentrations. The data indicated that in plants growing on non-limiting phosphate concentrations, an additional strong signal was found in cortical cells surrounding the nodule vascular bundles.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Alfalfa Root Nodule Carbon Dioxide Fixation : III. Immunological Studies of Nodule Phosphoenolpyruvate Carboxylase

Antiserum was prepared in rabbits against purified alfalfa (Medicago sativa L.) nodule phosphoenolpyruvate carboxylase (PEPC). Immunotitration assays revealed that the antiserum recognized the enzyme from alfalfa nodules, uninoculated alfalfa roots, and from soybean nodules. Tandem-crossed immunoelectrophoresis showed that the PEPC protein from alfalfa roots and nodules was immunologically indistinguishable. The 101 kilodalton polypeptide subunit of alfalfa nodule PEPC was identified on Western blots. The PEPC polypeptide was detected in low quantities in young alfalfa roots and nodules but was present at increased levels in mature nodules. Senescent nodules appeared to contain a reduced amount of the PEPC polypeptide. PEPC was also detected by western blot in some plant- and bacterially-conditioned ineffective alfalfa nodules but was not detected in bacteroids isolated from effective nodules. Alfalfa nodule PEPC is constitutively expressed in low levels in roots. In nodules, expression of PEPC polypeptide increases several-fold, resulting in increased PEPC activity. Antiserum prepared against the C₄ PEPC from maize leaves recognized the PEPC enzyme in all legume nodules and roots tested, while the antiserum prepared against alfalfa nodule PEPC also recognized the leaf PEPC of several C₄ plant species. Neither antiserum reacted strongly with any C₃ leaf proteins. The molecular weight of the PEPC polypeptide from C₄ leaves and legume nodules appears to be similar.     

Characterization of two novel nodule-enhanced α-type carbonic anhydrases from Lotus japonicus

Two cDNA clones coding for α-type carbonic anhydrases (CA; EC 4.2.1.1) in the nitrogen-fixing nodules of the model legume Lotus japonicus were identified. Functionality of the full-length proteins was confirmed by heterologous expression in Escherichia coli and purification of the encoded polypeptides. The developmental expression pattern of LjCAA1 and LjCAA2 revealed that both genes code for nodule enhanced carbonic anhydrase isoforms, which are induced early during nodule development. The genes were slightly to moderately down-regulated in ineffective nodules formed by mutant Mesorhizobium loti strains, indicating that these genes may also be involved in biochemical and physiological processes not directly linked to nitrogen fixation/assimilation. The spatial expression profiling revealed that both genes were expressed in nodule inner cortical cells, vascular bundles and central tissue. These results are discussed in the context of the possible roles of CA in nodule carbon dioxide (CO(2)) metabolism.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

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.