Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L.

Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP arabinogalactan protein - 2,4-D 2,4-di-chlorophenoxyacetic acid - J4e JIM 4 epitope - PEM proembryogenic mass We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions.     

Elicitor-induced cell death and phytoalexin synthesis in Daucus carota L.

Suspension-cultured carrot cells and intact leaves respond to crude and purified protein elicitors from the non-host fungus Pythium aphanidermatum by activating the general phenylpropanoid pathway and incorporating de-novo-synthesized 4-hydroxybenzoic acid (4-HBA) into the cell wall. The cultured cells undergo a very rapid elicitor-induced cell death. Both reactions are directly correlated in their time course and their dose dependency. Cell death in elicitor-treated protoplasts resulted in early membrane damage and the digestion of DNA into oligonucleosomal fragments. The same pattern of DNA degradation could be induced in protoplasts by the G-protein activators Mas-7 or mastoparan. In cell cultures, both activators induced a rapid loss of viability without the activation of the general phenylpropanoid pathway. The elicitor-induced reactions, the loss of viability and the induction of 4-HBA biosynthesis were blocked by the calcium-channel blocker nifedipine. Neomycin and U73122, two inhibitors of phospholipase C, blocked the induction of 4-HBA biosynthesis but did not affect the loss in viability. The injection of the elicitor into the leaves of intact carrot plants confirmed the results obtained with cell cultures with regard to the induction of the hypersensitive response. The purification of the active compound revealed a 25-kDa protein which triggers both cell death and 4-HBA synthesis. The signalling pathways to both reactions could be independently blocked or induced. Received: 27 February 1998 / Accepted: 25 May 1998     

Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).Abbreviations DEAE diethylaminoethyl - MAb monoclonal antibody - NEPHGE nonequilibrium pH gradient electrophoresis We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis

Embryogenic and non-embryogenic callus lines derived from the same diploid Cyclamen persicum genotype (`Purple Flamed') were analyzed by flow cytometry and compared to the initial plant material. The DNA content of the diploid plant in the greenhouse was 1.12 pg DNA/2C as estimated in relation to the internal standards tomato nuclei and chicken erythrocytes. In both callus lines the majority of cells contained the same amount of DNA as the initial plant, indicating that no polyploidization has taken place after 5 years of culture on medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 mg/l 6-(γ-γ-dimethylallylamino)purine(zip). Thus, our data suggest that in Cyclamen callus lines there was no strict correlation between the ploidy level and the ability to produce somatic embryos. Furthermore, following the proportion of cells in the three phases of the cell cycle (G0/G1, S, G2/M) during one subculture period of 4 weeks revealed high division activity within the first 2 weeks for both callus lines cultured on the 2,4-D-containing medium. However, when transferred to hormone-free medium, the division activity of the embryogenic cell line decreased markedly, corresponding to the differentiation of somatic embryos. In contrast, for the non-embryogenic callus an increase in cells in the G2/M phase was observed. Received: 22 November 1996 / Revision received: 6 January 1997 / Accepted: 20 February 1997     

A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.

A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G₁/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 M amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 10⁶ chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole The authors thank Mrs. Jiina Eliáová for her excellent technical assistance and Dr. Slavomir Ondro for the supply of V. faba seeds. A gift sample of APM from the Mobay Corporation (Agricultural Chemicals Division, Kansas City, Mo., USA) is gratefully acknowledged.     

Expression and localization of polygalacturonase during the outgrowth of lateral roots in Allium porrum L.

The presence of polygalacturonase and its correlation with the formation of lateral roots in leek (Allium porrum L.) seedlings have been investigated. During root growth, a steady increase in polygalacturonase activity was associated with that of the lateral root primordia. Fractionation of root extract by fast protein liquid chromatography resolved at least two polygalacturonase isoforms. One of the isoforms, a 75-kdalton protein, strongly reacted on Western blots probed with a polyclonal antibody raised against tomato polygalacturonase. It also reacted with both polyclonal and monoclonal antisera raised against Fusarium moniliforme polygalacturonase. In situ localization with these three antibodies showed that polygalacturonase was present over the meristems of lateral root primordia. Antibodies against pectins (Knox et al. 1990, Planta 181, 512–521) detected large amounts of pectic material filling the area between the apex of the primordium and the mother root tissues. We suggest that a polygalacturonase plays an important role in leek root morphogenesis, particularly during lateral root outgrowth.Abbreviations FPLC fast protein liquid chromatography - RGU one unit of polygalacturonase activity - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The Authors are grateful to Dr. Dean Della Penna (Department of Vegetable Crops, University of California, Davis, Calif., USA) for generously providing the polyclonal antibody raised against the tomato polygalacturonase. This research was supported by National Research of Italy, Special project RAISA, Subproject N2, N360.     

Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress

The cell walls in the new white roots of jack pine (Pinus banksiana Lamb.) were observed to constrict around the shrinking protoplast of osmotically stressed roots, and pressure was maintained via an apparent adjustment of cell-wall size and elasticity. These elastic alterations of the cell wall permitted the root cells to maintain full turgor despite the loss of most of the water in the tissue. The constriction of the root cell wall around the dehydrating protoplasts to maintain turgor may reflect changes in cell wall structure. We found that these shrinking root cells synthesize and secrete into the intercellular fluid a set of proteins. These proteins become tightly associated (i.e. guanidine HCl- and sodium dodecyl sulfate-insoluble) with the cell wall but can be released from the matrix, after briefly boiling in 0.1% sodium dodecyl sulfate, by the combination of guanidine HCl, CaCl₂ and dithiothreitol. However, these cell-wall proteins became insoluble with time. The proteins could subsequently be destructively extracted from the wall with acid NaClO₂ treatments. After these proteins were incorporated into the cell walls, the roots adopted a new, smaller maximal tissue volume and elastic coefficients returned to normal levels. Received: 8 July 1998 / Accepted: 19 November 1998     

Soluble polypeptides from meristematic and mature cells of Allium cepa L. roots

By using electrophoresis on SDS polyacrylamide gels we have studied the soluble protein fraction from a section of the meristem, where most cells are in the division last cycle, and from the mature region of Allium cepa L. roots. In order to estimate the apparent rate of synthesis of these polypeptides we labeled a series of roots with [¹⁴C]leucine and another with [³H]leucine. Coelectrophoresis was carried out by using polypeptides from both regions, their mol.wt. being between 20,000 and 100,000 daltons. The results show that most of the polypeptides in the soluble fraction are constantly present in a cell throughout its development. These constant polypeptides are synthesized at a high rate in the meristematic region. In the mature cells these polypeptides show only a low labeling rate, while a small number of specific polypeptides appear to have a very high rate of metabolism.     

Induction of chalcone synthase in cell suspension cultures of carrot (Daucus carota L. spp. sativus) by ultraviolet light: evidence for two different forms of chalcone synthase

Two cell lines of carrot (Daucus carota L. spp. sativus), grown as cell-suspension cultures in the dark, were irradiated with ultraviolet light (315–420 nm) 10 d after the onset of cultivation. Chalcone synthase (CHS) enzyme activity was induced in both cell lines. Anthocyanin synthesis was only stimulated in the anthocyanin-containing cell line DCb. Parallel to the increase in CHS activity there was an increase with time in the amount of one CHS form with an isoelectric point of 6.5 and a molecular weight of 40 kilodaltons (kDa) per subunit. Whereas the anthocyanin-free cell line DCs failed to accumulate anthocyanin, it did stimulate another CHS form with an isoelectric point at pH 5.5 and a molecular weight of 43 kDa per subunit. Both enzyme activities could be separated by isoelectric focusing and stabilized using sodium hydrosulfite as an oxidation protectant. In carrot plants, CHS was restricted to the dark purple petals of the inflorescence (40 kDa) and to the leaves (43 kDa).Abbreviations BSA bovine serum albumin - CHS chalcone synthase - IEF isoelectric focusing - kDa kilodaltons - KP_i potassium phosphate buffer - PAL phenylalanine ammonialyase - pI isoelectric point - UV ultraviolet     

Variation of the Chemical Composition and Antimicrobial Activity of the Essential Oils of Natural Populations of Tunisian Daucus carota L. (Apiaceae)

The essential oils of Daucus carota L. (Apiaceae) seeds sampled from ten wild populations spread over northern Tunisia were characterized by GC‐FID and GC/MS analyses. In total, 36 compounds were identified in the D. carota seed essential oils, with a predominance of sesquiterpene hydrocarbons in most samples (22.63–89.93% of the total oil composition). The main volatile compounds identified were β‐bisabolene (mean content of 39.33%), sabinene (8.53%), geranyl acetate (7.12%), and elemicin (6.26%). The volatile composition varied significantly across the populations, even for oils of populations harvested in similar areas. The chemometric principal component analysis and the hierarchical clustering identified four groups, each corresponding to a composition‐specific chemotype. The in vitro antimicrobial activity of the isolated essential oils was preliminarily evaluated, using the disk‐diffusion method, against one Gram‐positive (Staphylococcus aureus) and two Gram‐negative bacteria (Escherichia coli and Salmonella typhimurium), as well as against a pathogenic yeast (Candida albicans). All tested essential oils exhibited interesting antibacterial and antifungal activities against the assayed microorganisms.     

The effects of CO2, temperature and their interaction on the growth and yield of carrot (Daucus carota L.)

Stands of carrot (Daucus carota L.) were grown in the field within polyethylene-covered tunnels at a range of soil temperatures (from a mean of 7·5°C to 10·9°C) at either 348 (SE = 4·7) or 551 (SE = 7·7) μmol mol⁻¹ CO₂. The effect of increased atmospheric CO₂ concentration on root yield was greater than that on total biomass. At the last harvest (137d from sowing), total biomass was 16% (95% CI = 6%, 27%) greater at 551 than at 348 μmol mol⁻¹ CO₂, and 37% (95% CI = 30%, 44%) greater as a result of a 1°C rise in soil temperature. Enrichment with CO₂ or a 1°C rise in soil temperature increased root yield by 31% (95% CI = 19%, 45%) and 34% (95% CI = 27%, 42%), respectively, at this harvest. No effect on total biomass or root yield of an interaction between temperature and atmospheric CO₂ concentration at 137 DAS was detected. When compared at a given leaf number (seven leaves), CO₂ enrichment increased total biomass by 25% and root yields by 80%, but no effect of differences in temperature on plant weights was found. Thus, increases in total biomass and root yield observed in the warmer crops were a result of the effects of temperature on the timing of crop growth and development. Partitioning to the storage roots during early root expansion was greater at 551 than at 348 μmol mol⁻¹ CO₂. The root to total weight ratio was unaffected by differences in temperature at 551 μmol mol⁻¹CO₂, but was reduced by cooler temperatures at 348 μmol mol⁻¹ CO₂. At a given thermal time from sowing, CO₂ enrichment increased the leaf area per plant, particularly during early root growth, primarily as a result of an increase in the rate of leaf area expansion, and not an increase in leaf number.     

Graviresponse and the localization of its initiating cells in roots of Phleum pratense L.

Roots of Phleum pratense L. were photographed during both vertical growth and gravitropic bending, and positions of anticlinal rhizodermal cell walls were digitized on the physically upper and lower flanks of the root in the curvature plane. By using B-splines, arc lengths of these positions, i.e. distances along the root surface, values of curvature, and relative elemental rates of elongation were estimated. The whole graviresponse can be divided into phases according to growth-rate values: (i) an increase of rates on the upper side of the root and a decrease on the lower side during the first 1–11/2h after the root has been moved from the vertical to a horizontal position, (ii) a transient equality of the rates on both sides, (iii) 2–3 h after the beginning of graviresponse, the growth gradient is inverted, and (iv) finally, after about 4 h, the growth rates of both flanks are approximately equal again. Curvature begins 15–20 min after horizontal placement of the root. During the first 2 h of graviresponse, plots of curvature versus arc length show one maximum value. After 2–21/2 h, two maximum values can be observed, the apical one near the root tip always keeping the same distance from the tip, the other one drifting basipetally relative to the growing tip. By evaluating photographs of high magnification, a group of six rhizodermal cells on each side of the root was identified which are the first cells showing gravitropic bending. These cells are located at the beginning of the elongation zone, enclosing the region 480–680 m from the root tip. These cells might be target cells for a signal which the statenchyma, the site of graviperception, sends to the reacting zone of gravicurvature.Abbreviations curvature - RELEL relative elemental rate of elongation A preliminary report was presented at the Meeting of the Deutsche Botanische Gesellschaft, Regensburg, 30 Sept–5 Oct 1990This work was supported by Deutsche Forschungsgemeinschaft. We thank Dr. Brigitte Buchen and Professor Zygmunt Hejnowicz (Botanisches Institut, Universität Bonn, Bonn, FRG) for critical reading of the mansucript.     

The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var. Argenteum.

The connection between three light responses of green leaf cells-membrane potential (V_m), H⁺ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses: (i) a de- and repolarization of V_m and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H⁺-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of V_m, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid) responses were due to a stimulation of PM H⁺ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal cells where light stimulation of PM H⁺ pumps was rapid. Received: 3 September 1998 / Accepted: 15 October 1998     

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular “secretory” peroxidase activity (−73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed. Received: 14 October 1998 / Accepted: 31 May 1999     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in ‘gently lysed cells’ (obtained by treatment with 0.01% Brij-58) or ‘highly lysed cells’ (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [³H]dTTP efficiently, while highly lysed cells exhibited a low level of [³H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

Metabolism of [14C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots

Reverse-phase high-performance liquid chromatography was used to analyse ¹⁴C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [¹⁴C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [¹⁴C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate