Proteomic mapping for legume nodule organogenesis

While genetic screens have identified mutants of the model legume Lotus japonicus that can nodulate in the absence of rhizobia, the lack of a proteome map is a major hindrance to understanding the functional protein networks associated with this nodulation process. In this issue of Proteomics, Dam et al. (Proteomics 2014, 14, 230–240) developed 2D gel‐based reference maps of nodules and roots of Lotus and a spontaneous nodule formation mutant (snf1). Comparative proteomic analysis of roots and two developmental stages of nodules provide useful insights into tissue‐specific mechanisms underlying nodule organogenesis. Additionally, a comparison of interspecies nodule proteomes displays that overlapping and individual mechanisms are associated with legume nodulation.     

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule,nectary, leaf and flower development

The NOOT‐BOP‐COCH‐LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE‐ON‐PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen‐fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference‐mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild‐type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.     

Soybean ureide transporters play a critical role in nodule development,function and nitrogen export

Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen‐fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long‐distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins – GmUPS1‐1 (XP_003516366) and GmUPS1‐2 (XP_003518768) – in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1‐1 and GmUPS1‐2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.     

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the “Class II” hemoglobins. Received: 28 June 1999 / Accepted: 25 November 1999     

INHIBITION OF NODULATION OF INOCULATED LEGUMES BY LEAF LEACHATES FROM PIONEER PLANT SPECIES FROM ABANDONED FIELDS

Leaf leachates of three forbs, Ambrosia psilostachya, Euphorbia supina, and Helianthus annuus, and three grasses, Arislida oligantha, Bromus japonicus, and Digitaria sanguinalis, all of which are important in the pioneer stages of old-field succession, were tested to see if they would affect nodulation of heavily inoculated red kidney bean (Phaseolus vulgaris), white clover (Trifolium repens), and Korean lespedeza (Lespedeza stipulacea) growing in soil. Leachates of all inhibitor species except Ambrosia psilostachya and Digitaria sanguinalis significantly reduced the nodule number of Korean lespedeza in at least one experiment. Nodule numbers of red kidney bean and white clover were not significantly reduced by any leachate. It is significant that Korean lespedeza is the most important legume in our revegetating old fields. In almost all experiments in which the nodule number was significantly reduced, the nodules were small and grey or at least not a bright pink as were the control nodules. Leachate of Helianthus annuus reduced the hemoglobin content of Korean lespedeza nodules per plant by 36 %, and Euphorbia supina reduced the hemoglobin content per clover plant by 24 % even though the nodule number was not changed significantly in the latter case. Based on previous research, this indicated a definite reduction in the nitrogen-fixing capacity of the test nodules. Root exudates of all six inhibitor species were previously found to reduce nodule numbers significantly in the three legume species as were very small amounts of decaying material (1 g per 454 g of soil) of each forb. It appears likely, therefore, that the inhibition of nodulation of legumes in old fields by the six species under test would slow the rate of addition of nitrogen and thus the rate of plant succession, because it was previously demonstrated that the order in which species enter our old fields is the same as the order based on increasing nitrogen requirements.     

Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants

In this paper we have examined whether the four glutamine synthetase (gln) genes, expressed in roots and nodules of Phaseolus vulgaris are substrate-inducible by ammonium. Manipulation of the ammonium pool in roots, through addition and removal of exogenous ammonium, did not elicit any changes in the abundances of the four mRNAs thus suggesting that the gln genes in roots of this legume are neither substrate-inducible by ammonium nor derepressed during nitrogen starvation. In nodules the effect of the ammonium supply on expression of the gln genes has been examined by growing nodules under argon/oxygen atmospheres, or with a number of Fix^- Rhizobium mutants, and following addition of exogenous ammonium. The results of these experiments suggest that the expression of the gln- gene, which is strongly induced during nodule development, is primarily under a developmental control. However nitrogen fixation appears to have a quantitative effect on expression of gln- as the abundance of this mRNA is about 2 to 4-fold higher under nitrogen-fixing conditions. This effect could not be mimicked by addition of exogenous ammonium and moreover is not specific to the gln- gene as mRNA from a leghaemoglobin gene was similarly affected. Taken together these results have failed to find an effect of ammonium on specifically inducing the expression of glutamine synthetase genes in roots and nodules of P. vulgaris.     

Nitrate Assimilation Function of Yellow Lupine Root Nodules?

Low nitrate assimilation activity of the root nodules was demonstrated by assaying the activities of nitrate reductase, glutamate synthetase, glutamate dehydrogenase, and asparagine synthetase as well as the kinetics of ¹⁴C-labeled sucrose incorporation in the amino acids and amides of the cortex and the bacteroid-containing root nodule zones. Irrespective of the exogenous nitrogen concentration (0, 11.2, or 25 mM NO^- ₃), nitrate concentration in the nodules was low as compared to the plant roots, leaves, and stems. This allowed us to propose the presence of structural and/or metabolic barriers in the nodules limiting nitrate transport and assimilation in the nodule.     

MtZR1, a PRAF protein,is involved in the development of roots and symbiotic root nodules in Medicago truncatula

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen‐fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down‐regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down‐regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.     

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.     

Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula

Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen‐fixing soil bacteria to form a new specific organ: the nitrogen‐fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE‐domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock‐down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume‐specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation.     

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     

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant,SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.     

Impact of low pH and aluminium on nitrogen uptake and metabolism in roots of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

The effect of low pH and aluminum on nitrogen uptake and metabolism was studied in roots of Lotus japonicus grown in hydroponic cultures. The low pH slightly suppressed root elongation, and this effect was accompanied by the suppression of nitrate and ammonia uptake, as well as the nitrate reductase activity. In spite of high resistance of young Lotus plants to short-term Al application, the one-day treatment of Al strongly reduced nitrate uptake and also the activity of nitrate reductase (NRA) in the apical parts of roots. The glutamine synthetase activity was also suppressed by Al treatment, but in lower extent. On the other hand, the ammonium uptake and nitrite reductase activity stayed unchanged by Al treatment and the values were practically the same as in control plants. These results support the view that nitrate uptake and nitrate reduction might be the main processes responsible for Al induced growth retardation in Lotus plants grown in mineral acid soils.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

TIME-LAPSE PHOTOGRAPHIC OBSERVATIONS OF MORPHOGENESIS IN ROOT NODULES OF COMPTONIA PEREGRINA (MYRICACEAE)

Seedlings of the sweet fern Comptonia peregrina (L.) Coult. were grown aeroponically with their roots bathed in a nutrient mist lacking nitrogen except for 10 ppm N at the outset. The initiation and early development of root nodules capable of fixing atmospheric nitrogen were recorded with time-lapse photography through early development to the establishment of highly branched, roughly spherical nodules. In Comptonia multiple primary nodule lobes are formed at or near the site of infection with as many as 10 primary lobes occurring together. On the shoulders of the swollen primary lobes new primordia develop, forming secondary nodule lobes, which may persist without nodule root elongation, giving a coralloid appearance. The tips of the lobes may elongate, forming nodule roots which grow vertically upward, or, if disturbed, in random orientation. Nodule roots occasionally form lateral roots. The root axis upon which the nodule forms undergoes secondary thickening on the proximal side of the nodule attachment; the distal portion of the root shows no secondary thickening and later atrophies. Thus, nodules are perennial structures on a woody root system. The endophyte infects and occupies the basal cortical tissues of the primary nodule lobes and successive nodule lobes as they are formed, being restricted to the swollen bases and not infecting the elongate nodule roots. Development of the nodule is interpreted in terms of complex host-endophyte interactions involving the initiation of multiple primordia forming nodule lobes, the active inhibition of nodule lobes and finally nodule root elongation. Anatomical evidence for the endogenous origin of nodule primordium formation substantiates the view obtained from time-lapse photomacrography.     

Differential proteomic analysis of developmental stages of <Emphasis Type="Italic">Acca sellowiana</Emphasis> somatic embryos

Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins that are expressed during the different developmental stages of somatic embryos of A. sellowiana. Using high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), a high degree of similarity between protein profiles of the assayed somatic embryos was observed. Of the 74 different protein spots extracted for analysis, 60 were identified by means of 2-DE/MALDI-TOF/MS. Twelve proteins were expressed in all the assayed stages. Ten proteins were expressed in the initial stages and 22 proteins were expressed in the mature developmental stages of somatic embryos. Only one protein was expressed exclusively in the torpedo stage, whereas four were expressed in the pre-cotyledonary, and none in the cotyledonary stage. The proteins identified were involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures. The presence of essential proteins of nitrogen metabolism, such as the cytosolic glutamine synthetase protein, was also observed. The physiological implications of these findings are discussed.