Increase in the Amount of celA1 Protein in Tobacco BY-2 Cells by a Cellulose Biosynthesis Inhibitor, 2,6-dichlorobenzonitrile

The biochemical analysis of cellulose biosynthesis by plantshas been a difficult problem due to the lack of a reliable assayprocedure for cellulose synthase activity. Recently, the celAlgene was cloned from cotton fiber, and this gene was identifiedfrom the rsw1 mutant of Arabidopsis as a catalytic subunit ofcellulose synthase (Arioli et al. 1998). The cloning of thesegenes enables us to obtain specific antibodies against cellulosesynthase. A highly specific antibody against celAl protein wasprepared and used to detect the protein from microsomal fractionof tobacco BY-2 cells. The quantity of celAl protein in microsomalfraction of normal BY-2 cells was under the detection limit,although they contained a large quantity of cellulose. In contrast,cells habituated to 1 µM DCB (a specific inhibitor ofcellulose biosynthesis) produced 1/10 of cellulose content ofthe normal cells, but had much more celAl protein than the normalcells. The amount of polysaccharides in the EDTA-soluble fractionwas relatively increased in habituated cells. The results suggestthat celAl protein is stabilized upon DCB binding and that thecrystallization of cellulose microfibrils is inhibited simultaneously. (Received January 28, 1998; Accepted May 7, 1998)     

Infection and Nodule Development in Aotus ericoides (Vent.) G. Don, A Woody Native Australian Legume

Infection and nodule development were studied by light and electronmicroscopy in Aotus ericoides, a woody native Australian legume,inoculated with a slow-growing field isolate of Rhizobium. Rhizobiabound to straight, but not deformed, root hairs, as detectedby immunofluorescence. Neither markedly curled root hairs norroot hairs with infection threads were seen. Nodules were indeterminate(astragaloid), with a peripheral meristematic layer, few vasculartraces and both infected and uninfected cells in the centralinfected zone. Infection threads containing contorted bacteriawere present throughout the nodule. Swollen, rod-shaped bacteriain infected cells were in groups in vesicles bounded by plasmalemma-derivedperibacteroid membranes. Senescence in infected cells was associatedwith accumulation of a fibrillar matrix inside peribacteroidmembranes, distortion of bacteria and destruction of most cytoplasmiccontents of the bacteria and host cells; however, most bacterialand plant membranes and plant cell walls remained intact. Ineffectivenesswas associated with relatively little, short-lived infectedtissue. Events in infection and nodule development were similarto those in most herbaceous legumes but showed characters ofboth determinate and indeterminate nodules. Key words: Bacteroids, Legume, Nitrogen-fixing, Nodule, Rhizobium     

Immunological Studies on Glutamine Synthetase using Antisera Raised to the Two Plant Forms of the Enzyme from PhaseolusRoot Nodules

Antisera specific for glutamine synthetase (GS) have been raisedto the two forms of the enzyme from the plant fraction of rootnodules of Phaseolus vulgaris. The two antisera recognized bothforms of plant nodule GS and also the enzyme from some otherhigher plant tissues. However, the antiserum did not cross-reactwith GS from free-living or bacteroid Rhizobium Phaseolinorwith the enzyme from representatives of green algae, fungi,mammals and bacteria. Results are presented which suggest thatone of the forms of nodule GS is closely related to the rootenzyme whereas the other, the 'nodule specific' form, has someantigenic differences Key words: Phaseolus Vulgaris, Legume/Rhizobium symbosis, Glutamine synthetase, Immunology     

Crystallization of Photosystem I Complexes from the Thermophilic Cyanobacterium, Synechococcus vulcanus

PSI complexes were isolated from the thermophilic cyanobacterium,Synechococcus vulcanus, by mild detergent treatment of the thylakoidmembranes, purified by sucrose-density gradient centrifugation,and then crystallized. High resolution SDS-PAGE revealed thepresence of the product of the psaI gene in S. vulcanus PSIcomplexes and crystals. Crystals of the PSI complexes retainedall of the components of electron carriers and subunit polypeptides(including PsaX) known in cyanobacteria. (Received July 22, 1998; Accepted October 19, 1998)     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

The Spirulina platensis Adenylate Cyclase Gene, cyaC, Encodes a Novel Signal Transduction Protein

A cyaC gene encoding an adenylate cyclase of the filamentouscyanobacterium Spirulina platensis was se-quenced. The predictedamino acid sequence of the C-ter-minal region of cyaC is similarto the catalytic domains of adenylate cyclases in other cyanobacteriaand eukaryotes. The sequences of other regions are similar tothose of proteins consisting of the bacterial two-componentsignal transduction system: the sensory kinase and the responseregulator. The predicted gene product of cyaC contains, fromthe N-terminal end, a receiver domain of the response regulatorprotein (Rl), a domain similar to the ETR1 of Arabi-dopsis thaliana,a transmitter domain of the sensory kinase protein, a receiverdomain of the response regulator protein (R2), and a catalyticdomain of adenylate cyclase. The cyaC gene was expressed asan affinity-tagged protein in Escherichia coli, and the recombinantprotein was purified. The purified protein had adenylate cyclaseactivity which was activated by Mn²+. The results of Westernblotting using an anti-CyaC antiserum and the S. platensis cellextract confirmed that cyaC gene is expressed in S. platensis (Received February 27, 1997; Accepted April 26, 1997)     

Wavelength options for monitoring leghaemoglobin oxygenation gradients in intact legume root nodules

Nodule oximetry, based on spectrophotometric measurements ofleghaemoglobin (Lb) oxygenation in intact nodules, has providednumerous insights into legume nodule physiology. Fractionaloxygenation of Lb (FOL) has been monitored at various wavelengths,but comparisons among wavelengths have not been published previously.Changes in transmittance were monitored simultaneously at 660nm and either 560 or 580 nm as FOL was manipulated by changingthe O₂ concentration around nodules of Medicago sativa L. orLotus comiculatus L. Video microscopy at 580 nm was used togenerate two-dimensional maps of FOL gradients in intact nodules.In general, all three wavelengths gave similar results. Smalldiscrepancies between 660 and 580 nm, sometimes seen in noduleswith high O₂ permeability, may indicate interference by theferric Lb peak at 625 nm. A slightly longer wavelength, forexample 670 nm, might be preferable. No significant discrepanciesamong wavelengths were seen in nodules whose O₂ permeabilityhad been reduced by a 48 h exposure to 10 mM nitrate. Minorgradients in FOL were seen in nodules of M. sativa and Trifoliumrepens L. under air and steeper gradients could be induced byvarious treatments. The existence of these gradients indicatesat least some restriction of longrange O₂ diffusion within theinfected zone. The FOL maps do not have enough spatial resolutionto measure gradients within infected cells. Key words: Leghaernoglobin oxygenation, nodules, spectrophotometry, nodule oximetry     

Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.

The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.     

Comparisons of the Respiratory Effluxes of Nodules and Roots in Six Temperate Legumes

The specific respiration rates of nodulated root systems, ofnodules and of roots were determined during active nitrogenfixation in soya bean, navy bean, pea, lucerne, red clover andwhite clover, by measurements on whole plants before and afterthe removal of nodule populations. Similar measurements weremade on comparable populations of the six legumes, lacking nodulesbut receiving abundant nitrate-nitrogen, to determine the specificrespiration of their roots. All plants were grown in a controlled-environmentclimate which fostered rapid growth. The specific respiration rates of nodulated root systems ofthe three grain and three forage legumes during a 7–14-dayperiod of vegetative growth varied between 10 and 17 mg CO₂g^–1 (dry weight) h^–1. This mean value consistedof two components: a specific root respiration rate of 6–9mg CO₂ g^–1 h^–1 and a specific nodule respirationrate of 22–46 mg CO₂ g^–1 h^–1. Nodule respirationaccounted for 42–70 per cent of nodulated root respiration;nodule weight accounted for 12–40 per cent of nodulatedroot weight. The specific respiration rates of roots lackingnodules and utilizing nitrate nitrogen were generally 20–30per cent greater than the equivalent rates of roots from nodulatedplants. The measured respiratory effluxes are discussed in thecontext of nitrogen nitrogen fixation, nitrate assimilation. Glycine max, Phaseolus vulgaris, Pisum sativum, Medicago sativa, Trifolium pratense, Trifolium repens, soya bean, navy bean, pea, lucerne, red clover, white clover, nodule respiration, root respiration, fixation, nitrate assimilation     

Identification and Localization of Frankia Strains in Alnus Nodules by In Situ Hybridization of nif H mRNA with Strain-Specific Oligonucleotide Probes

In situ hybridization of Frankia mRNA with specific probes wasused to localize the strains Arl3 and AcoN24d in Alnus nodulesobtained after inoculation with one or both strains. The probesconsisted of 18-mer oligonucleotides, complementary to strain-specificsequences located within the nif H gene. Sections of nodulesinoculated with only one strain revealed a specific hybridizationbetween the probe and the corresponding Frankia strain mRNA.In sections of dually-inoculated nodules the presence of thestrain AcoN24d in the nodule was clearly shown whereas thoseof the strain Arl3 could not be detected. This suggests thatthe strain Arl3 is less infective than the strain AcoN24d andis not present within the nodule. Key words: Nitrogen fixation, actinorhizae, autoradiography, histochemistry     

Effectiveness of Lotus Root Nodules: I. MORPHOLOGY AND FLAVOLAN CONTENT OF NODULES FORMED ON LOTUS PEDUNCULATUS BY FAST-GROWING LOTUS RHIZOBIA

The morphology of root nodules formed on Lotus pedunculatusby two fast-growing strains of Rhizobium, NZP2037 which formseffective (nitrogen-fixing) nodules and NZP2213 which formsineffective (non-nitrogen-fixing) nodules, has been studied.The nodules formed by NZP2037 contained a central zone of bacteroid-filledplant cells surrounded by a cortex. In contrast the nodulesformed by NZP2213 contained no Rhizoblum-infected plant cells,but rhizobia were found in localized areas on the nodule surfaceand between the outer two or three cell layers of the nodule.Electron-dense osmiophilic deposits identified as flavolans(condensed tannins) were present in the vacuoles of many uninfectedplant cells in the nodules formed by both Rhizobium strains.This is the first time that flavolans have been positively identifiedin legume root nodules. In the NZP2037 nodule flavolans werepresent in the outer cortical and epidermal cells. In the ineffecitveNZP2213 nodule fiavolans were present in many of the centralnodule cells. The concentration of flavolan in the NZP2213 nodulewas 12 times higher than in the NZP2037 nodule.     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. II. Distribution and Role in Photosynthesis

Antibody was raised against Porphyridium carbonic anhydrase(CA) which was electrophoretically recovered from the gel afterSDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of thepartially purified enzyme. The antiserum reacted with CA ofPorphyridium, but not with that of Chlamydomonas reinhardtii.Even though the antiserum did not react with CA from P. cruentumR-l in Ouchterlony's double immunodiffusion, it blocked theenzyme activity in the presence of 1% Nonidet P-40 and 1% TritonX-100. After Western blotting and enzyme-linked immunostaining(ELIS), only one band which reacted with the antiserum was detectedin the extract of low-CO₂ cells (grown under ordinary air) ofP cruentum, while no significant band was detected in that ofhigh-CO₂ cells (grown under air enriched with 1–5% CO₂).Immunogold electron microscopy of low-CO₂ cells of P. cruentumR-l using this antibody revealed that most of the CA was localizedin the chloroplast, with some in the cytoplasm. No specificbinding of gold particles was observed in the high-CO₂ cells. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan (Received May 18, 1987; Accepted September 7, 1987)     

The Promoter of a Pine Photosynthetic Gene Allows Expression of a {beta}-Glucuronidase Reporter Gene in Transgenic Rice Plants in a Light-Independent but Tissue-Specific Manner

In angiosperms, the expression of the cab gene that encodesthe chlorophyll a/b-binding protein of PSII is light-regulated.However, the pine cab gene is expressed in a light-independentbut cell-type-specific manner. In the present study, the cab-6promoter (1.7 kbp) from pine was fused to a -glucuronidase (GUS)reporter gene and the chimeric gene was introduced into riceprotoplasts by electroporation. The GUS expression was studiedin the resultant transgenic rice plants. Expression of GUS ata substantial level was confirmed in primary leaves of dark-germinatedrice seedlings, and no obvious effect of light on the GUS activitywas observed. The expression of GUS was restricted to photosynthetictissues. The pine cab-6 promoter is, thus, sufficient for inductionof light-independent but cell-type-specific expression in cellsof a monocot, as is the case in the original pine cells. (Received December 17, 1993; Accepted April 22, 1994)     

Developmental Pattern of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Caulogenesis and rhizogenesis were studied in cultured leafexplants of Nicotiana tabacum cv. Xanthi nc. using both lightand scanning electron microscopy. The timing of organ appearancewas also recorded. The patterns of development seen were comparedto each other and to that in explants grown on growth regulator-freemedium. Shoots first appeared after 12 d in culture and rootsafter 7 d. In caulogenesis nodules appear at the explant edgeand from these the shoots arise. The nodules are mainly derivedfrom palisade mesophyll cells, along with some spongy mesophylland bundle-sheath cells. The nodules form a continuous row alongthe edge of the explant and their initiation appears to be centredon veins. Shoots are produced indirectly. Roots are produceddirectly from bundle-sheath and vein parenchyma cells. Withoutplant growth regulators bundle-sheath cells still divide, althoughonly a few divisions were seen. Key words: Nicotiana tabacum, in vitro, caulogenesis, rhizogenesis     

Isolation of a Tobacco cDNA Encoding Sar1 GTPase and Analysis of Its Dominant Mutations in Vesicular Traffic Using a Yeast Complementation System

The cDNA clone of NtSARl, a gene encoding the small GTPase Sar1pwhich is essential for vesicle formation from the endoplasmicreticulum (ER) membrane in yeast, has been isolated from Nicotianatabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated fromArabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205]could complement the lethality of the disruption of SARI inyeast cells in a temperature-sensitive fashion. They also suppressedyeast sec12 and sec16 temperature-sensitive mutations as yeastSARI does. Using this complementation system, we analyzed thephenotypes of several mutations in plant SAR1 cDNAs in yeastcells. The expression of NtSAR1 H74L and AtSAR1 N129I showeddominant negative effect in growth over the wild-type SARI,which was accompanied by the arrest of ER-to-Golgi transport.Such dominant mutations will be useful to analyze the role ofmembrane trafficking in plant cells, if their expression canbe regulated conditionally. (Received October 29, 1997; Accepted March 17, 1998)     

Bacteroids Isolated from Ineffective Nodules of Pisum sativum Mutant E135 (syml3) Lack Nitrogenase Activity but Contain the Two Protein Components of Nitrogenase

Pea mutant E135 (sym15) forms ineffective (Fix^–) nodulesthat lack nitrogen fixing activity. To determine the developmentalstep blocked in E135 nodules we studied the nitrogenase activitiesin isolated bacteroids and in cell-free extracts of bacteroids,and measured the two components of nitrogenase protein in bacteroids.Bacteroids prepared anaerobically from E135 nodules showed noacetylene reduction activity in the presence and absence ofmyoglobin. Furthermore, no acetylene reduction activity by cell-freeextracts of E135 bacteroids was detected in the presence ofATP-generating system and dithionite. However, immuno-blottinganalyses revealed the presence of nitrogenase components I andII in E135 nodule bacteroids. These results suggest that a hostplant gene is involved in the expression of nitrogenase activityin symbiotic bacteria. (Received May 11, 1998; Accepted August 7, 1998)