Endogenous lectin from cultured soybean cells. Chemical characterization of the lectin of SB-1 cells

A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max. This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA. This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall. Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide. It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit. The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found. Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000. These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.     

Influence of growth conditions on the composition of cell wall polysaccharides from cultured tobacco cells

The sugar composition of cell wall polysaccharides of two tobacco varieties obtained from mesophyll, regenerating protoplasts and cells grown under various conditions were compared. Regenerating protoplasts developed an unusual cell wall with a low cellulose and a high non-cellulosic glucan content. In the presence of different phytohormones compact and friable calli were obtained with cell walls containing low and high arabinose/xylose ratios. The cell walls of compact calli were comparable to those of genuine mesophyll cells. The sugar constituents of cell walls obtained from cells grown in liquid media were different from those of solid calli. The cell wall composition of suspension cultured cells was hardly affected by various combinations of phytohormones, but was altered by high osmolarity of the medium.     

Enzymatic degradation of cell wall polysaccharides from soybean meal

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.     

Cytokinins from a variant strain of cultured soybean cells

A strain of soybean cells capable of growing on a tissue culture medium lacking a cytokinin produced at least 3 compounds active in the soybean cytokinin assay. The characteristics of these compounds were consistent with their being zeatin in the free form, zeatin ribonucleoside and zeatin ribonucleotide. Although the conversion from a cytokinin dependent to independent condition in this strain parallels the change of normal cells to crown gall tumor state in terms of the capacity to synthesize cell division substances, the soybean factors are distinct from the nicotinamide derivatives reported for tumor cells of Vinca.     

Endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of Rhizobium japonicum

Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells. This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr approximately 30,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin. Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells. These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin. This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.     

Chloroplast and mitochondrial DNA polymerases from cultured soybean cells

DNA polymerases were purified from chloroplasts and mitochondria of cultured Glycine max cells. The chloroplast enzyme exists in two forms which are indistinguishable from each other biochemically. All three organellar enzymes have an estimated molecular weight of 85,000 to 90,000 and prefer poly(rA)dT_12-18 over activated DNA as a template in vitro. Maximum activity of the chloroplast and mitochondrial DNA polymerases requires KCl and a reducing agent, and the enzymes are completely resistant to inhibitors of DNA polymerase α. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases γ. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Comparative studies of the neutral sugar composition of cell wall polysaccharides between cultured cells and mother plant tissues of soybean and carrot

The cell walls obtained from cultured cells grown under variousconditions and their mother plant tissues or organs were comparedwith regard to the neutral sugar composition of polysaccharides.In the pectic fraction of cell walls from soybean hypocotyl,greater amounts of galactose than other neutral sugars weredetected, and the relative sugar ratio did not change in theelongating and non-elongating portions of the tissue. On theother hand, more arabinose than other sugars was detected inthe pectic fraction of cell walls from cultured cells from soybeanhypocotyl tissue, and no significant change in the sugar ratioof polysaccharides was found in this fraction during the cultureperiod. A higher arabinose content in cell walls from culturedcells from root tissues as compared to the mother tissues ororgans was also detected in carrot. The neutral sugar ratioof cell walls from cultured cells was similar in cells grownon agar or liquid medium, with indoleacetic acid, or with orwithout 2,4-dichlorophenoxyacetic acid (2,4-D). These factssuggest that the high arabinose content of cell walls is notcommon to the cell walls of young, growing, undifferentiatedcells, and that 2,4-D is not required for the maintenance ofthe higher content of arabinose in cell walls of cultured cells. ¹A preliminary report of this work was presented for the annualmeeting of the American Society of Plant Physiologist at OhioState University, August, 1979. [Plant Physiol., 63(5) 118 (1979)]. ⁴Department of Agricultural Chemistry, Washington State University,Pullman, Washington 99164, U.S.A. (Received October 6, 1979; )     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

Properties of acid phosphatase in the cell wall of tobacco cells cultured in vitro

A cell-wall fraction containing 10.0% dry weight of proteinwas isolated from tobacco (Nicotiana tabacum L.) XD-6 cellscultured in suspension. The fraction possessed high acid phosphataseactivity toward p-nitrophenyl phosphate, phenyl phosphate, inorganicpyrophosphate, adenosine diphosphate, nucleoside triphosphates,and bis- (p-nitrophenyl) phosphate; much less activity towardnaphthyl phosphate, ß-glycerophosphate, glucose-6-phosphate,fructose-6-phosphate, fructose-l,6-diphosphate, thiamine pyrophosphate;and no activity toward glucose-1-phosphate and inositol hexaphosphate.Metallic ions were not required for activation. The enzyme wasextracted from the wall with NaCl solution. The solubilizedenzyme resulted in a single peak on Sephadex G-150 chromatographywith activity toward p-nitrophenyl phosphate, pyrophosphate,and bis(p-nitrophenyl) phosphate. The activity of the solubilizedenzyme toward p-nitrophenyl phosphate was identical with thebound enzyme in its pH optimum, substrate specificity, ion inhibitionand Km value, but it was more sensitive to pH, substrate difference,inhibition, and heat treatment. ¹Present address: Department of Biology, Japan Women's University,Mejiro-dai, Bunkyo-ku, Tokyo, Japan (Received September 13, 1972; )     

Endogenous lectin as a possible regulator of the hydrolysis of physiological substrates by soybean seed acid phosphatase

The effects of two lectins concanavalin A (conA) and soybean agglutinin, on soybean seed acid phosphatase activity were investigated using p-nitrophenylphosphate (pNPP), pyrophosphate (PPi) and phosphoenolpyruvate (PEP) as substrates. Of the four acid phosphatase isoforms (AP1, AP2, AP3A and AP3B) purified from soybean seeds, only AP1 was activated 40 and 60% by conA and soybean agglutinin, respectively. Both lectins affected some of the kinetic parameters of AP1. The activation by lectins was not affected by 1 mM Ca2+ or Mn2+ but glucose and methylmannopyranoside (100 mM) prevented activation by conA. Under the same conditions, galactose had no effect. These results suggest that plant acid phosphatases may be regulated by lectins, the effects vary according to the substrate used.     

Isolation and characterization of one isoform of actin from cultured soybean cells

Cultured soybean cells (SB-1 cell line) were plasmolyzed and lyophilized. Extraction of the dried powder and fractionation yielded a polypeptide with the following key properties: (a) it has a molecular weight of approximately 45,000 and an isoelectric point of approximately 5.9; (b) it is immunologically cross-reactive with rabbit antibodies affinity purified against the Mr 45,000 polypeptide of calf thymus actin; (c) it is eluted from a DEAE-cellulose column at the same ionic strength as Acanthamoeba actin; (d) it yields peptide maps, after limited proteolysis with V8 protease, similar if not identical to those of rabbit muscle actin; and (e) it binds specifically to deoxyribonuclease I. These molecular and binding properties indicate that we have purified one isoform of actin from soybean cells.     

Processing of N-linked oligosaccharides in soybean cultured cells

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn.     

Kluyveromyces bulgaricus yeast lectins. Isolation of two galactose-specific lectin forms from the yeast cell wall

Incubation of galactose treated Kluyveromyces bulgaricus yeast cells in EDTA/phosphate-buffered saline led to an extract possessing hemagglutinating and yeast flocculating properties. Purification of this extract by affinity chromatography and gel filtration gave two lectin forms, Kb-CWL I and Kb-CWL II, with an apparent molecular mass of 38,000 and 150,000 Da, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Kb-CWL I and Kb-CWL II were dimeric and octameric of a subunit of 18,900 Da. At high concentration, purified Kb-CWL I associated to give Kb-CWL II. This association seemed to be independent on pH. The two lectin forms were glycoproteins, the peptide counterpart was very rich in Lys, Glu, and Gly, and the carbohydrate part represented 1% of the whole molecule and was composed of Glc, Man, and Ara. The two lectin forms (KB-CWL I and Kb-CWL II) agglutinated human red blood cells and flocculated EDTA-treated K. bulgaricus yeast cells. The activity of both lectin forms required Ca2+ ions, while Sr2+ showed some competitive inhibition. Optimal activity was obtained within a pH range of 4-6.5 for both forms. Temperatures of 80-90 degrees C for 20 min, or proteolytic treatment reduced irreversibly the activity of Kb-CWL I and Kb-CWL II. The role of the cell wall phosphopeptidomannan as a ligand and a potential physiological receptor of these lectin forms was demonstrated.     

Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Biosynthesis of mannose-containing lipid-linked oligosaccharides by solubilized enzyme preparation from cultured soybean cells

Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at −10°C for 1 month. The solubilized enzyme synthesized [¹⁴C]Man₃GlcNAc₂-pyrophosphoryl-polyprenol and [¹⁴C]Man₅GlcNAc₂-pyrophosphoryl-polyprenol when incubated with GDP-[¹⁴C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man₃GlcNAc₂-lipid and Man₅GlcNAc₂-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man₆GlcNAc₂-lipid, Man₇GlcNAc₂-lipid, and Man₈GlcNAc₂-lipid occurred when the above incubations were supplemented with dolichyl-phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, it appears likely that the first five mannose residues to form Man₅GlcNAc₂-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.