Hydrogenase in pea root nodule bacteriods

Summary Hydrogen uptake has been shown to occur with pea root nodule breis and this uptake has been shown to be confined to the bacteriods. The uptake of hydrogen by washed bacteriods, in the absence of any added substrates, has been shown to be accompanied by oxygen uptake and the ratio of hydrogen uptake to oxygen uptake in these preparations has been found to be 2:1. Substrates, provided to washed bacteriods, inhibit the uptake of hydrogen and it has been found that the utilisation of substrates, as measured by carbon dioxide evolution, is inhibited by hydrogen. It is suggested that hydrogen and substrates compete for electron carriers and that electrons from the hydrogen reduce components of the electron transport pathway and ATP is produced.The action of hydrogen on nitrogen fixation in nodule breis and washed bacterioid preparations was examined and the evidence shows that some non-competitive inhibition of nitrogen fixation, by hydrogen, occurs.     

Differential expression of phenylalanine ammonia-lyase and chalcone synthase during soybean nodule development.

We have used conserved and nonconserved regions of cDNA clones for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) isolated from a soybean-nodule cDNA library to monitor the expression of members of the two gene families during the early stages of the soybean-Bradyrhizobium japonicum symbiosis. Our results demonstrate that subsets of the PAL and CHS gene families are specifically induced in soybean roots after infection with B. japonicum. Furthermore, by analyzing a supernodulating mutant line of soybean that differs from the wild-type parent in the number of successful infections, we show that the induction of PAL and CHS is related to postinfection events. Nodulated roots formed by a Nod+ Fix- strain of B. japonicum, resembling a pathogenic association, display induction of another distinct set of PAL and CHS genes. Our results suggest that the symbiosis-specific PAL and CHS genes in soybean are not induced by stress or pathogen interaction.     

Immunofluorescence localization of chalcone synthase in roots ofPisum sativum L. andPhaseolus vulgaris L. and comparable immunochemical analysis of chalcone synthase from pea leaves

Summary Chalcone synthase in roots ofPisum sativum andPhaseolus vulgaris was demonstrated enzymatically and immunochemically. In situ localization by indirect immunofluorescence revealed that chalcone synthase is chiefly present in the lateral regions of the calyptra, in the rhizodermis, and the cortex. In the central cylinder the enzyme protein is no longer detectable a short distance behind the meristem. Chalcone synthase was not found in root tips ofZea mays. Two isoforms of chalcone synthase were separated by chromatofocusing of protein extracts from pea leaves. The two forms differed in their subunit molecular masses. The smaller isoform was not detected in roots.Abbreviations CHS chalcone synthase - FITC fluorescein isothiocyanate - IgG immunoglobulin G - DTE dithioerythritol     

Regulated expression of ADPglucose pyrophosphorylase and chalcone synthase during root development in sweet potato

The phase transition in sweet potato root during tuber differentiationis a complex developmental process that involves changes in gene expression andmorphogenesis. Among the three kinds of root in sweet potato (white fibrousroot, thick-pigmented root and lateral root), ADP-glucose pyrophosphorylase(AGPase) and chalcone synthases (CHS) are expressed only in thick-pigmentedroots after 3 weeks, and this also depends on the developmental stage. Sinceexposing roots to the light or culturing under hydroponic conditions inhibitstuber formation in sweet potato, the expression of AGPase and CHS was studiedunder light and dark conditions. AGPase and CHS expression in sweet potatorootswas suppressed very sensitively by light or water stress, similar to rootdevelopmental patterns. Based on an analysis of AGPase and CHS expression indifferent kinds of root tissues and in different developmental stages, thesegenes were shown to be closely associated with the differentiation ofthickeningpigmented roots.     

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip Apeldoorn have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS sodium dodecyl sulfate Some of the results were presented at the meeting of German Botanical Society in Freiburg, FRG, September 1982, and at the meeting of the Groupe Polyphenols in Toulouse, France, September/October 1982     

Molecular evolution and functional relevance of the chalcone synthase genes of pea

We have isolated seven genomic chalcone synthase (CHS) genes and six classes of CHS cDNA from elicitor-treated pea tissues. Comparison of the nucleotide sequences of the coding regions revealed the existence of eight members of the CHS gene family in pea. These can essentially be divided into three groups (PSCHS1, 2 and 8; PSCHS3, 4 and 5; and PSCHS6 and 7) on the basis of nucleotide and/or amino acid sequence comparisons of the coding regions, introns and promoter regions. We previously reported that the accumulation of CHS mRNAs is induced by elicitor treatment. Accumulation of CHS mRNA was observed mainly in roots and very little was found in floral organs. To specifically detect expression of each CHS gene in various types of pea cells, S1 nuclease protection assays were performed. Interestingly, the classification of the eight members of the CHS gene family based on the sequence identity was found to reflect their expression patterns as determined by the S1 nuclease protection assay. The first group of CHS genes, PSCHS1, 2 and 8, was strongly induced not only by elicitor treatment and UV irradiation but is also constitutively expressed in root and flower tissues. The second group, PSCHS3, 4 and 5, was also strongly induced by elicitor treatment and UV irradiation but is constitutively expressed only in root. Expression of the third group, PSCHS6 and 7 was barely detectable in any of the organs tested and was not influenced by environmental stimuli such as elicitor or UV. Furthermore, sequence analysis of the promoter region of each member of the CHS gene family revealed that putative cis-regulatory elements, such as Box-I, Box-II and G-Box, were conserved only in PSCHS1, 2, 3, 4 and 5. From these results we propose that an ancestral CHS gene might have given rise to defense response-related (UV irradiation- and elicitor-responsive) and -unrelated (unresponsive) genes at an early stage of evolution, followed by divergence within these subclasses based upon the developmental program in pea. Received: 5 November 1996 / Accepted: 6 February 1997     

Identification and genetic regulation of the chalcone synthase multigene family in pea.

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of diverse flavonoids involved in disease resistance, nodulation, and pigmentation in pea. We describe a multigene family encoding CHS and the effects of two regulatory loci, a and a2, on the pattern of expression of three of its member genes. Two of the genes, CHS1 and CHS3, are expressed in both petal and root tissue, whereas expression of a third gene, CHS2, is detected only in roots. The products encoded by the a and a2 loci are required for the expression of the CHS1 gene and for wild-type levels of expression of the CHS3 gene in petal tissue. In root tissue, all three CHS genes are expressed and induced by CuCl2 regardless of the genotype at the a and a2 loci. These results show that the various members of the CHS multigene family interact in diverse ways with multiple genetic signals in the plant, providing a basis for the differential expression of these genes. Spatially specific genetic regulation of distinct members of a multigene family has been clearly demonstrated.     

A novel plastid localization of chalcone synthase in developing grape berry

Chalcone synthase (CHS, E.C. 2.3.1.74) is an entrance enzyme of flavonoid metabolism and a critical point to regulation of biosynthesis of different flavonoid compounds that directly contribute to color and monthfeel of grape and wine. In the present experiment, subcellular localization of CHS in developing grape berry was performed via immunogold electron microscopy technique. The result showed that CHS was localized in rough endoplasmic reticulum (ER) and cytoplasm of the skin cells, while few gold particles representing CHS were found on the cell wall. Besides, two novel localized sites of CHS were observed within a cell of developing grape berry, one being the plastid-distributed throughout developmental stages and the other being vacuole-distributed at late developmental stage. It is speculated that these novel localized patterns may relate to abundant and multi-branch flavonoid metabolism in grape berry. This work will provide new insight for the regulation of different branch pathways leading to diverse flavonoid compounds.     

Lupin leghemoglobins during root nodule development

Two yellow lupin leghemoglobins, Lb I and Lb II, were purified to homogeneity using the HPLC technique for final separation. Lb I and Lb II were identified by the N-terminal sequences and their reaction with antibodies against electrophoretically pure leghemoglobin. The third Lb species was detected by the combined method of isoelectrofocusing and PAGE of Lb I. It seems that Lb III represents a posttranslational modification of Lb I. Developmental changes in Lb multiple forms were examined using the Western blotting method. The content of leghemoglobin, first detectable approximately 3 weeks after infection, increased up to 6-7 weeks, and then it remained at the same level until 8-9 weeks after the infection. At the early stages of nodule formation Lb I prevailed over Lb II, while later Lb II became the predominant form. This suggests physiological role of particular forms and precise regulation of the expression of Lb genes.     

In situ localization of chalcone synthase inLarix needles by indirect immunofluorescence

Summary Chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis, is localized by indirect immunofluorescence in needles ofLarix decidua. In young stages of needle development CHS is present in epidermal cells and individual cells in the region of the vascular bundles which possibly contain tannins. A later developmental stage exhibits immunofluorescence predominantly in the mesophyll of the needle. The epidermis and the cells in the vicinity of the vascular bundles show a considerably weaker and only sporadically detectable fluorescence. CHS is no longer observable at the latest analyzed stages of needle development.     

The roles of redox processes in pea nodule development and senescence

Nodule senescence is triggered by developmental and environmental cues. It is orchestrated through complex but poorly characterized genetic controls that involve changes in the endogenous levels of reactive oxygen species (ROS) and antioxidants. To elucidate the importance of such redox control mechanisms in pea root nodule senescence, redox metabolites were analysed throughout nodule development in a commercial pea variety ( Pisum sativum cv. Phoenix) inoculated with a commercial rhizobial strain ( Rhizobium leguminosarum bv. viciae ). Although a strong positive correlation between nitrogenase activity and nodule ascorbate and glutathione contents was observed, the progressive loss of these metabolites during nodule senescence was not accompanied by an increase in nodule superoxide or hydrogen peroxide. These oxidants were only detected in nodule meristem and cortex tissues, and the abundance of superoxide or hydrogen peroxide strongly declined with age. No evidence could be found of programmed cell death in nodule senescence and the protein carbonyl groups were more or less constant throughout nodule development. Pea nodules appear to have little capacity to synthesize ascorbate de novo . l -galactono-1, 4-lactone dehydrogenase (GalLDH), which catalyses the last step of ascorbate synthesis could not be detected in nodules. Moreover, when infiltrated with the substrates l -galactono-1, 4-lactone or l -gulonolactone, ascorbate did not accumulate. These data suggest that ROS, ascorbate and glutathione, which fulfil well recognized, signalling functions in plants, decline in a regulated manner during nodule development. This does not necessarily cause oxidative stress but rather indicates a development-related shift in redox-linked metabolite cross-talk that underpins the development and aging processes.     

Cloning of a full-length symbiotic hemoglobin cDNA and in situ localization of the corresponding mRNA in Casuarina glauca root nodule

We have characterized a full-length cDNA ( hb -Cg1F) that represents symbiotic mRNA hemoglobin ( hb ) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb -Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [³⁵S]-labeled probes was compared. The expression of hb -Cg1F gene is induced in young infected host cells prior to the detection of Frankia nif H mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O₂ concentration in the cytoplasm of the host cells before the nif genes are expressed.     

Gibberellin biosynthesis mutations and root development in pea

Dwarf mutants of pea (Pisum sativum), with impaired gibberellin (GA) biosynthesis in the shoot, were studied to determine whether the roots of these genotypes had altered elongation and GA levels. Mutations na, lh-2, and ls-1 reduced GA levels in root tips and taproot elongation, although in lh-2 and ls-1 roots the reduction in elongation was small (less than 15%). The na mutation reduced taproot length by about 50%. The roots of na plants elongated in response to applied GA(1) and recombining na with mutation sln (which blocks GA catabolism) increased GA(1) levels in root tips and completely restored normal root development. In shoots, Mendel's le-1 mutation impairs the 3beta-hydroxylation of GA(20) to the bioactive GA(1), resulting in dwarfism. However, GA(1) and GA(20) levels were normal in le-1 roots, as was root development. The null mutation le-2 also did not reduce root GA levels or elongation. The results support the theory that GAs are important for normal root elongation in pea, and indicate that a 3beta-hydroxylase gene other than LE operates in pea roots.     

Immunogold localization of callose and other cell wall components in pea nodule transfer cells

Summary Transfer cells are located adjacent to xylem and phloem elements in pea nodule vascular tissues. The composition of the labyrinthine wall intrusions was investigated by immunogold labeling using specific antibody probes. Callose antigen was found at the base of newly formed cell wall intrusions and also in adjacent plasmodesmata. Sections through developed labyrinthine intrusions revealed that wall ingrowths had an internal structure with small domains of callose suggesting the presence of channels or vents. Xyloglucan and pectin antigens were uniformly distributed within the wall, but the distribution of extensin antigens was variable, with different antigens being detected in different regions of the wall ingrowth. A lectinlike glycoprotein, PsNLEC-1, was localized in intercellular spaces associated with nodule transfer cells. Previously, expression of this component was observed in other types of cells showing complex involution of the plasma membrane, namely root cortical cells harboring arbuscular mycorrhizae and nodule cells harboring nitrogen-fixing rhizobia.     

NADH-Glutamate synthase in alfalfa root nodules. Immunocytochemical localization

In root nodules of alfalfa (Medicago sativa L.), N₂ is reduced to NH₄⁺ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH₄⁺ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N₂.