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Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.

The structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a tubule-guided mechanism, assemble into long tubular structures at the surface of the infected protoplast. Electron microscopy and immunogold analysis confirmed the presence of both MP and virus particles in the tubules induced by AMV and BMV. The significance of the tubule-forming properties of these viral MPs is discussed.

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produces cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.

An infected root nodule cell may contain several thousand rhizobial symbionts, each enclosed in a membrane envelope, the peribacteroid membrane (PBM). The PBM is derived from the host plasma membrane, but shares properties with the vacuolar membrane and contains several nodule-specific proteins (nodulins) that perform unique functions for symbiosis.

Metaphase chromosomes of Vicia faba were exposed to antibodies recognizing defined acetylated isoforms of histone H4. After indirect immunostaining with antibodies directed against H4 acetylated on lysines 5, 8 and 12 respectively, the entire chromosome complement was labelled. The brightest signal appeared at the nucleolus organizing region (NOR). The large genetically inert heterochromatic regions, which are composed of late replicating tandemly repetitive DNA sequences, remained unlabelled. Thus, the chromosomal distribution of histones H4 acetylated at positions of lysine 5, 8 and 12 is broadly correlated with the intensity of transcription and the sequence of replication of the field bean chromatin during interphase. Antibodies against H4 acetylated at lysine 16 also caused a strong signal at the NOR but otherwise a uniform fluorescence along the chromosome.

Primed in situ DNA labelling (PRINS) labels the telomeres of Avena, Triticum, Secale, Hordeum, Lolium, Festuca and Trifolium when primers are used that correspond to the repeat unit of Arabidopsis telomeres. There are interstitial sites labelled in a Lolium x Festuca hybrid.

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H2O2 and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post-translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed.

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.

Stomatal conductances of Vicia faba leaves were recorded over a day. Coordinately, (a) guard cells dissected from leaflets were assayed, providing total sucrose content, and (b) guard cells dissected from rinsed epidermes were also assayed, providing symplastic sucrose content. Compared with that of pre-dawn samples, apoplastic sucrose content increased 4.8-5.2 x (2 experiments), reaching 1,130-1,300 fmol.guard-cell-pair-1 at midday, when conductance was highest (ca. 0.13 mol.m-2.s-1); symplastic sucrose content increased 2.5-3.5 x, reaching 350-390 fmol.guard-cell-pair-1. Thus, there is a correlation between transpiration and guard-cell sucrose content, particularly that portion localized to the apoplast. Moreover, apoplastic sucrose is apparently a source of guard-cell nutrition and, possibly, osmoticum.

Ultrastructural localization of intranucleolar DNA in Vicia faba root-tip cells was investigated by an immunocytochemical method consisting of in vitro bromodeoxyuridine (BUdR)-labelling with the aid of terminal deoxynucleotidyltransferase and successive immunocytochemistry using an anti-BUdR antibody. The nucleolus usually had many low electron-opaque patches (LEPs). The fibrillar centers (FCs) occupied their peripheral region leaving a central electron-transparent space (central space). The immunoelectron microscopic technique showed that in the nucleolus the label was mostly confined to the FCs whilst the central space was completely free from the label. In other nucleolar regions the label appeared to gradually increase towards the FCs. In addition to nucleoli, this method also localized a small amount of DNA in mitochondria.

To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.

In the nitrogen-fixing root-nodule symbiosis of Rhizobium leguminosarum biovar viciae and its host plants pea and vetch, the bacteria enter one root cortical cell after another via a tip-growing structure, the infection thread. Rhizobial Nod (nodulation) factors induce the formation of preinfection thread structures (Van Brussel, A.A.N., R. Bakhuizen, P.C. van Spronsen, H.P. Spaink, T. Tak, B.J.J. Lugtenberg, J.W. Kijne, Science 257, 70-72 (1992)), but formation of infection threads requires the presence of bacterial cells. Passing of an infection thread from cell to cell requires local cell wall degradation. We compared at the ultrastructural level local cell wall changes in the outer root cortex of pea and vetch related to preinfection thread formation and infection thread formation, respectively. Cell wall modifications in the outer periclinal walls of root cortical cells induced by Nod factors appeared to be similar to those induced by rhizobia. These modifications take place opposite cytoplasmic bridges and are probably related to induction of tip growth. However, complete cell wall degradation was never observed in the absence of rhizobia. We propose a two-step cell wall degradation process for infection thread formation. The first step is a local cell wall modification by plant enzymes, induced by rhizobial Nod factors. The second step is complete cell wall degradation in the presence of rhizobia.

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.

The genome of pea enation mosaic virus (PEMV) is composed of two taxonomically unrelated RNAs, interacting to create what has traditionally been considered a bipartite virus. The cohesiveness of this interaction was assessed by examining the autonomy of each RNA in viral replication, coat protein expression and systemic invasion. Using a pea protoplast system, in vitro transcripts of RNA1 were found to be capable of initiating RNA2-independent replication, including the formation of the distinctive nuclear membrane-based replication complex associated with wild-type PEMV infection. Western blotting and electron microscopic analysis demonstrated that the synthesis of the RNA1-encoded coat protein, as well as virion assembly, was also independent of RNA2-directed functions. Mechanical inoculations with transcripts of RNA1 failed to establish a systemic RNA1 infection, whereas inoculations with RNA2 were able to establish a largely asymptomatic systemic infection. Combined inoculum containing RNA1 and RNA2 transcripts were able to recreate wild-type PEMV symptomatology, demonstrating the dependence of RNA1 on RNA2 for mechanical passage. With the notable exception of the adaptation of PEMV to establish a true systemic invasion, these data further strengthen the analogy between PEMV and the helper-dependent complexes associated with members of the luteovirus group.

Endo-1,4-beta-glucanase induced by treatment of pea seedlings with 2,4-D was extracted from a preparation of the walls of epicotyl cells. The beta-glucanase was purified by chromatography on DEAE-cellulose, affinity chromatography on Con A-Sepharose and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The activity of beta-glucanase was retained after removal of SDS and extraction from polyacrylamide gels. The band of a protein (46 kDa), that corresponded to the activity of endo-1,4-beta-glucanase, was injected directly into mice for preparation of antiserum and the protein was also subjected to amino acid sequencing after blotting onto a membrane. Western blot analysis showed that the antiserum obtained bound to a 46-kDa polypeptide and recognized endo-1,4-beta-glucanase. The N-terminal sequence of the 46-kDa polypeptide revealed some homology to abscission endo-1,4-beta-glucanases of bean and avocado fruit.

The tropical legume Sesbania rostrata can be nodulated by Azorhizobium caulinodans on both its stem and its root system. Here we investigate in detail the process of root nodulation and show that nodules develop exclusively at the base of secondary roots. Intercellular infection leads to the formation of infection pockets, which then give rise to infection threads. Concomitantly with infection, cortical cells of the secondary roots dedifferentiate, forming a meristem which has an "open-basket" configuration and which surrounds the initial infection site. Bacteria are released from the tips of infection threads into plant cells via "infection droplets," each containing several bacteria. Initially, nodule differentiation is comparable to that of indeterminate nodules, with the youngest meristematic cells being located at the periphery and the nitrogen-fixing cells being located at the nodule center. Because of the peculiar form of the meristem, Sesbania root nodules develop uniformly around a central axis. Nitrogen fixation is detected as early as 3 days following inoculation, while the nodule meristem is still active. Two weeks after inoculation, meristematic activity ceases, and nodules then show the typical histology of determinate nodules. Thus, root nodule organogenesis in S. rostrata appears to be intermediate between indeterminate and determinate types.

The chromosomes of a field bean line with a reconstructed karyotype (ACB) were hybridized in situ with biotinylated probes of a repetitive Fok I sequence, of DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) amplified DNA from a chromosome that does not contain this sequence, and with probes containing dispersed repetitive sequences. The results were compared with Giemsa banding, DNA late replication and Fok I in situ digestion patterns. This allowed further differentiation between the chromatin types of this species. Centromeric and NOR-associated heterochromatin as well as euchromatin were shown to be free of Fok I sequence repeats. Among the interstitial late replicating Giemsa bands, subdivided into 'marker' and 'additional' bands, most of the marker bands located at mid-arm positions were composed mainly or exclusively of tandemly arranged Fok I repeats. Some of the marker bands and nearly all of the additional bands located in the vicinity of centromeres were free of FokI sequence repeats, of Fok I recognition sites, and possibly also of dispersed repetitive sequences. They are probably composed of specific, not yet defined, repetitive sequences.

Starting with a protocol originally developed for the purification of intact plastids from cauliflower buds [Journet and Douce (1985) Plant Physiol. 79, 458-467] we have modified this method to obtain intact heterotrophic plastids from etiolated barley leaves (Hordeum vulgare) and pea (Pisum sativum) and maize (Zea mays) endosperm. Two subsequent centrifugation steps on Percoll gradients were performed, the first as an isopycnic, the second as zonal, centrifugation step in a swing-out rotor. Percoll density and centrifugation time were adjusted for the various tissues. The obtained plastid preparations are characterized by a low degree of contamination with other cellular components and an intactness of at least 90%. In isolated maize endosperm amyloplasts, starch synthesis is driven by exogenously applied hexose phosphates (glucose 6-phosphate and glucose 1-phosphate) rather than by dihydroxyacetone phosphate. The hexose-phosphate-dependent starch synthesis is strictly dependent upon the intactness of the plastids and is increased up to 9-fold when ATP and 3-phosphoglyceric acid are added to the incubation medium. The occurrence of fructose-1,6-bisphosphatase and malate dehydrogenases in some plastid types is discussed in relation to their possible role in starch synthesis.

The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.

In vitro assays for the import of proteins by isolated pea thylakoids have been refined and optimised with respect to (a) the method of thylakoid preparation, (b) the concentration of thylakoids in the import assay, and (c) the pH and temperature of the import assay. As a result, the 23 kDa and 16 kDa proteins of the photosynthetic oxygen-evolving complex are imported with efficiencies approaching 100%; import of the third oxygen-evolving complex protein is also observed, albeit with lower efficiencies. We have also demonstrated import of three further thylakoid proteins: plastocyanin, the CFoII subunit of the ATP synthase, and the photosystem I subunit, PSI-N, using this import assay. Import of plastocyanin, PSI-N and the 33 kDa oxygen-evolving complex protein subunit requires the presence of stromal extract whereas the other three proteins are efficiently imported in the absence of added soluble proteins. Import into isolated barley thylakoids was achieved under identical assay conditions, although with somewhat lower efficiency than into pea thylakoids.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.

To identify regions of the mitochondrial genome that potentially could specify cytoplasmic male sterility (CMS) in Phaseolus coccineus (including P. polyanthus), and to define differences amongst P. coccineus lines, mitochondrial (mt)DNA restriction patterns and Southern blots of total DNA from sterile and fertile lines were analysed. By restriction endonuclease mapping we isolated a region which was specific to CMS lines flanking an F1-ATPase alpha-subunit (atpA) gene. DNA sequence analysis of this region showed 99.9% homology to the region previously isolated from P. vulgaris CMS Sprite. A high frequency of plants carrying the CMS-fragment was observed in a wild Phaseolus population, perhaps explaining the occurrence of inter- and intra-specific gene flow observed in the autogamous species P. vulgaris.

Antisera raised against the major hydroxyproline-rich glycoprotein (HRGP) in carrot (Daucus carota L.) taproot, extensin-1, and a minor HRGP, extensin-2, were characterized by western blot analysis, enzyme-linked immunosorbent assay, and periodate oxidation and found to be directed against carbohydrate epitopes shared by both glycoproteins. The anti-extensin-1 antibodies (gE1) target periodate-sensitive epitopes and may recognize the terminal alpha-1,3-arabinoside of extensin-1. The anti-extensin-2 antibodies (gE2) recognize periodate-insensitive epitopes, possibly binding the reducing, internal beta-1,2-arabinosides on the carbohydrate side chains. Despite the cross-reactivity of these antibodies, immunolocalization studies of carrot taproot and green bean (Phaseolus vulgaris L.) leaf tissues reveal a spatial segregation of gE1- and gE2-labeling patterns. The gE1 antibodies bind only to the cellulose-rich region of the cell wall (J.P. Staehelin and L.A. Stafstrom [1988] Planta 174: 321-332), whereas gE2 labeling is restricted to the expanded middle lamella at three cell junctions. Periodate oxidation of nonosmicated, thin-sectioned tissue abolishes gE1 labeling but leads to labeling of the entire cell wall by gE2, presumably as a result of unmasking cryptic epitopes on extensin-1 in the cellulose layer. Purified extensin-2 protein is more efficient than extensin-1 protein at agglutinating avirulent Pseudomonas strains lacking extracellular polysaccharide. Our results indicate that extensin-2 does not form a heterologous HRGP network with extensin-1 and that, in contrast to extensin-1, which appears to serve a structural role, extensin-2 could participate in passive defense responses against phytopathogenic bacteria.

The presence of endo- and exoproteolytic activity in peroxisomes was detected in cell organelles purified from pea leaves. By PAGE using different exopeptidase substrates (L-aa-beta NA), one leucine aminopeptidase (AP) was found in peroxisomes. The peroxisomal AP was characterized as a serine protease and had a maximal activity at pH 7.5, a molecular mass of 56.8 kDa and a pI of 5.3. This enzyme was mainly present in the soluble fraction of peroxisomes. The occurrence of proteases in peroxisomes suggests that they might be involved in the protein turnover and processing of imported precursor polypeptides in peroxisomes.

Development of the legume root nodule can be divided conceptually into two parallel processes. On the one hand, there is the induction of a nodule meristem and the progressive differentiation of specialised cells and tissues. On the other hand, there is cell and tissue invasion by Rhizobium, which leads ultimately to the differentiation of intracellular bacteroids as specialised nitrogen-fixing endosymbionts. The early stages of plant-microbe communication seem to be mostly mediated by the exchange of soluble, diffusible signal molecules: flavonoid compounds are secreted by plant roots, and chitin-like lipooligosaccharides are secreted by rhizobia. Further development of the nodule structure may involve the interplay of fairly conventional plant growth regulators. Direct physical contact between the cell surfaces of the symbionts also plays a prominent role in the process of tissue and cell invasion.

An alpha-fucosidase that releases fucosyl residues from oligosaccharide fragments of xyloglucan, a plant cell wall hemicellulosic polysaccharide, was purified to homogeneity from pea (Pisum sativum) epicotyls using a combination of cation exchange chromatography and isoelectric focusing. The alpha-fucosidase has a molecular mass of 20 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha-fucosidase has an isoelectric point of 5.5. The substrate specificity of the alpha-fucosidase was determined by high performance anion exchange chromatographic analysis of oligosaccharide substrates and products. The enzyme hydrolyzes the terminal alpha-1,2-fucosidic linkage of oligosaccharides and does not cleave p-nitrophenyl-alpha-L-fucoside. The enzyme does not release measurable amounts of fucosyl residues from large polysaccharides. The subcellular localization of alpha-fucosidase in pea stems and leaves has been studied by immunogold cytochemistry. The alpha-fucosidase accumulates in primary cell walls and is not detectable in the middle lamella or in the cytoplasm of 8-day-old stem tissue and 14-day-old leaf tissue. alpha-Fucosidase activity was readily detected in extracts of 8-day-old stem tissue. No significant alpha-fucosidase activity or immunogold labeling of the alpha-fucosidase was detected in 2- and 4-day-old stem tissue indicating that production of alpha-fucosidase is developmentally regulated.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.

Space experiments permit to understand better some phases of the gravitropic reaction which occurs when the orientation of the root changes in the gravitational field. In gravisensing cells (statocytes in the root cap), the nucleus is attached to the cell periphery, close to the plasma membrane, by actin filaments. The location of the amyloplasts (statoliths) depends also greatly on these elements of the cytoskeleton. A short period in microgravity (5 min.) modifies the location of the nucleus and of the amyloplasts in the statocytes. The tensions exerted by these very dense organelles on the actin network disappear and this network undergoes a relaxation. The kinetics of gravitropic curvature is also better understood. In fact, gravitropic reaction is regulated by a mechanism depending on gravity. In roots grown in space, then stimulated for 1 h on a 1 g centrifuge, and replaced in microgravity, the regulation limiting the curvature does not occur. It is hypothesized that the sedimentation of the amyloplasts on the endoplasmic reticulum placed at the basal pole of the statocytes could be responsible for this regulation. The contacts between these two organelles should have also a role in root growth. This hypothesis will be tested in our next space experiment (July 94). The experiments in near weightlessness also permit to determine the presentation time which is the duration of stimulation necessary to evoke a slight but significant curvature. Presentation time is 27 s. This short period allows a slight movement of the amyloplasts only (around 0.45 micrometer). The sequence of events leading to the curvature of the root is now well established: the first signal is the separation of the endoplasmic reticulum and the amyloplasts, when the root is subjected to a change in orientation. It is followed by the pressure of these organelles on the actin network which transmits this mechanical effect to the plasma membrane. The transduction of the effect occurs then by the activation of the ions channels (Ca++) and the carrier of a growth inhibitor (auxin), both located in the plasma membrane. This growth inhibitor provokes an asymmetrical growth in the distal part of the meristem and the proximal part of the cell elongation zone. At last, when the root tip reaches the direction of gravity, the amyoloplasts sediment on the endoplasmic reticulum and induce a signal of termination of the curavature.

We isolated two Tn5-generated mutants of Rhizobium meliloti whose growth was inhibited by rich medium or by exogenous tryptophan or phenylalanine. These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group. The mutant locus could not be found on either indigenous megaplasmid but was localized on the chromosome. The mutants formed ineffective nodules on alfalfa plants. They invaded nodules within infection threads and were released into plant cells enclosed within peribacteroid membranes, but once released into the plant cells they failed to differentiate into mature bacteroids. The mutants demonstrated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-phosphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis. Wild-type genes were isolated that complemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP synthase activity. Each mutant strain gave rise to linked second-site suppressor mutations that restored growth on rich medium. The suppressor mutants showed restoration of near wild-type DAHP synthase levels. One of the suppressor strains restored effective symbiosis while the other did not. Genetic complementation experiments showed that growth on rich medium, DAHP synthase activity, and effective symbiosis were all affected by the same genetic lesion. These results suggest that normal flux of metabolites through the aromatic biosynthesis pathway is essential for bacteroid development.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.

Higher-order packaging of DNA in chromatin structures could be an essential step in the complex chain of events leading to activation/repression of eukaryotic gene expression. With the goal to investigate this aspect of transcriptional regulation of plant genes involved in symbiotic interactions between legumes and rhizobia we analyze here the molecular parameters of chromatin structure in functioning root nodules, callus and radicles of pea. Morphological intactness and the typical nucleosomal organization are preserved in purified nuclei isolated from all three sources. The calculated values of nucleosomal repeat changed from 185 +/- 5 bp in the nuclei of radicles to 168 +/- 5 bp and 195 +/- 6 bp in nodules and callus respectively. The observed changes are due to alterations in linker DNA lengths. The core histones are identical in all cases, but the subfractional composition of H1 linker histone is subjected to quantitative alterations. The most pronounced is the several-fold increase in content of the lowest-molecular-weight subfraction H1-6 which takes place during nodule development.

A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.

A plasmid containing fragments of rp12 and rps19 genes from the chloroplast genome of Arabidopsis thaliana was developed. The presence of inverted repeats in the chloroplast DNA of A. thaliana and Vicia sativa, and their absence from two species of Fabaceae family (Lathyrus sativus, Lens esculenta) were shown with the help of this plasmid.

Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.

When conventional, high ionic strength buffers were used for the isolation of polysomes from pea roots, only about 10% were retained in the detergent-insoluble pellet, and they were not degraded by endogenous RNase. A low ionic strength, cytoskeleton-stabilizing buffer increased retention to 60%, but polysomes were severely degraded. The RNase inhibitors, ribonucleoside-vanadyl complexes, heparin, KCl and ammonium sulphate lessened degradation but caused release, while Tris-HCl at 15-25 mM was able to prevent degradation without causing release. Cosedimentation of polysomes with the cytoskeleton is not an artefact of adsorption or trapping since isolated polysomes labelled through their nascent polypeptides and added to unfractionated tissue were not retained in the cytoskeletal pellet.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl-, K+ and Ca2+ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.

Karyological polymorphism is generally the rule in related species of genus Vicia, as has been frequently shown in many entities of its comprehensive species; therefore a correspondence of phenotypic and karyotypic characters is rather rare. Two cases are described here, in which both external morphological and internal karyological features, are correlated. These data have been obtained through biometric and karyotypic studies, carried out both on the perennials V. onobrychioides and V. altissima, and on the annuals V. villosa ssp. varia and V. benghalensis. In both perennials the chromosomes were rather long, the nucleolar constriction was on the longest couple of chromosomes, and even the satellite was of the same type; in both annuals the chromosomes were short, the nucleolar constriction was on the shortest couple of chromosomes, and the satellite was large in comparison to the arm to which it was connected. These similarities of the karyotypes are important because they may make easier a possible experimental cross-breed of the abovementioned entities.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.

The existence of a relationship between clofibrate-induced peroxisome proliferation and oxidative stress mediated by activated oxygen species was studied in intact peroxisomes purified from Pisum sativum L. plants. Incubation of leaves with 1 mM clofibrate produced a remarkable increase in the peroxisomal activity of acyl-CoA oxidase and, to a lesser extent, of xanthine oxidase, whereas there was a nearly complete loss of catalase activity and a decrease in Mn-superoxide dismutase. Ultrastructural studies of intact leaves showed that clofibrate induced a five- and twofold proliferation of the peroxisomal and mitochondrial populations, respectively, in comparison with those in control leaves. Prolonged incubation with clofibrate produced considerable alterations in the ultrastructure of cells. In peroxisomal membranes, the NADH-induced generation of O2- radicals, as well as the lipid peroxidation of membranes, increased as a result of treatment of plants with clofibrate. In intact peroxisomes treated with this hypolipidemic drug, the H2O2 concentration was higher than in peroxisomes from control plants. These results demonstrate that clofibrate stimulates the production of activated oxygen species (O2- and H2O2) inside peroxisomes, as well as the lipid peroxidation of peroxisomal membranes. This effect is concomitant with a decrease of catalase and Mn-SOD activities, the main peroxisomal enzymatic defenses against H2O2 and O2-, and indicates that in the toxicity of clofibrate, at the level of peroxisomes, an oxidative stress mechanism mediated by activated oxygen species is involved.

An Exo- mutant of Rhizobium leguminosarum biovar trifolii was isolated which did not produce acidic exopolysaccharide and induced defective, non-fixing nodules on clover plants. The nodules were defective at a late stage of development, they contained infection threads and bacteria were released into the host cells. Cosmid pARF136 capable of complementing the Exo- mutation was isolated from a cosmid bank made from total R. trifolii DNA. Hybridization between DNA of pARF136 and plasmids of R. trifolii strains separated by Eckhardt's technique suggested that the exo locus is located on a 300 kb megaplasmid, and nodDABC and nifKDH genes are located on another 180 kb pSym plasmid. A 5.4 kb BamH1 fragment of the recombinant cosmid pARF136 was able to restore exopolysaccharide synthesis in Exo- mutant of R. trifolii 93 but it did not complement the symbiotic defect.