The microcrystalline structure of cellulose in cell walls of cotton, ramie, and jute fibers as revealed by negative staining of sections.

With a new technique of negative staining of sections, it has been possible to observe directly, in ultrathin sections under the electron microscope, the original microcrystalline and microfibrillar structure of cellulose as it occurs in living cells. This method has advantages over the study of isolated fibers used so far by others, in that the original arrangement of microfibrils is better preserved, and their collapse into larger fibrillar units is prevented. With this method, the cell walls of ramie, jute, and cotton fibers have been studied. The size (diameter, 25 to 40 A) and the longitudinal periodicity observed in the single microfibrils and the orientation and spatial arrangement of the microcrystallite within the microfibrils are found to correspond with the latest models derived by others from data obtained by indirect methods such as X-ray diffraction. The microfibril size of about 35 A, found by measuring these structures in sections, agrees with the latest conclusions reached by others in recent work with isolated fibrils.

Lignofibrils on the external cell wall surface of cultured plant cells.

Small strands and bundles of strands extend from the outside surface of suspension-cultured cells of Daucus, Ipomoea, and Phaseolus into the medium. This fibrous cell coat is present in all samples from various growth stages but appears to increase in quantity in the order Ipomoea < Phaseolus < Daucus. The bundles are often many microns in length and display great variation in frequency, size, and form. Identification of the composition of the strands and bundles as lignin is consistent with the following observations: alkaline nitrobenzene oxidation of the strands to compounds which resemble monomers of wood lignin; resistance of the strands to pronase, trypsin, pectinase, and lipase; strong irreversible adsorption of heavy metals; deposition of silver granules by treatment with silver nitrate-hexamine reagent; extraction of the bundles with aqueous dioxane (Björkman procedure); presence in quantity of a structured form of Klason lignin; and existence of material giving a positive test with the Wiesner reagent. Large individual strands (lignofibrils) from Phaseolus show the form of a flat ribbon with very thin branches at irregular intervals. This form does not vary with preparatory techniques, although its electron opacity does. Intercellular spaces display considerable structure and sometimes contain sheets of fibrillar material merging with both the middle lamella between the cells and the surface bundles facing the medium. These sheets are probably another form of association of the lignofibrils. It is suggested that natural fibrous lignin may be a much commoner component of plant tissue than suspected hitherto.

Calcium accumulations within the growing tips of pollen tubes.

Pollen of L. longiflorum was grown in 45Ca-labeled medium and washed with nonradioactive medium. Whole, labeled pollen was then frozen and autoradiographed at -78 degrees C. The autoradiographs show striking accumulations of 45Ca in the growing tips of the pollen tubes. This result is obtained when the pollen is labeled for times as short as 1 min, or as long as 5 h. In most cases, the tip concentration is about two to four times greater than that in the bulk of the pollen tube, and extends for a length of about 20 mum. In autoradiographs of tubes longer than 1 mm, a small fraction of cells show a distinctly larger 45Ca accumulation, the tip containing more than 100 times that in the rest of the cell. The 1- to 5-h labeling experiments show that calcium is relatively concentrated within the cytoplasm of the growing tip. The 1- to 3-min labeling experiments suggest that calcium may enter the tip faster than it enters other regions. These patterns of calcium accumulation and flux may be related to the localized secretion of vesicles at the grow;ng tip.

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. I. Preprophase band development and concomitant appearance of nuclear envelope-associated tubulin.

The development of the preprophase band (PPB) of microtubules (MT) in meristematic plant cells was studied by using antibodies to pig brain tubulin and indirect immunofluorescence microscopy. The PPB is first visible as a wide band of MT that are arranged only slightly more densely than flanking MT of the cortical interphase array. MT progressively become more tightly packed together, and other cortical MT are no longer seen as the PPB matures. The surface of the nuclear envelope (NE) displays no tubulin fluorescence during interphase but begins to fluoresce in the early stages of PPB development, and its intensity progressively increases thereafter. The pattern at the NE is usually diffuse at first, suggesting the presence of nonpolymerized tubulin, but fibers along the NE can be resolved at later stages. MT, arranged either radially or as a meshwork, can occur between the nucleus and cell cortex, and sometimes appear to connect the PPB and NE directly. Isolated preprophase nuclei from cells ruptured during processing often retain the PPB in its normal orientation, indicating stable linkages between the nucleus and PPB. Fluorescent cross-bars perpendicular to the axis of the MT were resolved in some PPB, suggesting lateral linkages. This suggestion is reinforced by the presence of PPB that hold together as a ribbon of MT in certain preparations, allowing PPB to be isolated from the rest of the cytoplasm and the nucleus.

A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants.

We have identified a family of abundant peripheral plasma membrane glycoproteins that is unique to flowering plants. They are identified by a monoclonal antibody, MAC 207, that recognizes an epitope containing L-arabinose and D-glucuronic acid. Immunofluorescence and immunogold labeling studies locate the MAC 207 epitope to the outer surface of the plasma membrane both in protoplasts and in intact tissues. In some cells MAC 207 also binds to the vacuolar membrane, probably reflecting the movement of the plasma membrane glycoproteins in the endocytic pathway. The epitope recognized by MAC 207 is also present on a distinct soluble proteoglycan secreted into the growth medium by carrot (Daucus carota) suspension culture cells. Biochemical evidence identifies this neutral proteoglycan as a member of the large class of arabinogalactan proteins (AGPs), and suggests a structural relationship between it and the plasma membrane glycoproteins. AGPs have the property of binding to beta-glycans, and we therefore propose that one function of the AGP-related, plasma membrane-associated glycoproteins may be to act as cell surface attachment sites for cell wall matrix polysaccharides.

A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii.

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.

Localization of a phytohormone using immunocytochemistry.

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.

Analytical characterization of beetroot vacuole membrane.

Vacuoles from beetroot (Beta vulgaris L. var. esculenta Gürke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis. Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction. The vacuole membrane had a phospholipid protein ratio of 0.68 and a sterol:phospholipid ratio of 0.21. 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified. Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively). A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography. High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000. Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core. Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.

Accumulation, stability, and localization of a major chloroplast heat-shock protein.

Diverse higher plant species synthesize low molecular weight (LMW) heat shock proteins (HSPs) which localize to chloroplasts. These proteins are homologous to LMW HSPs found in the cytoplasm of all eukaryotes, a class of HSPs whose molecular mode of action is not understood. To obtain basic information concerning the role of chloroplast HSPs, we examined the accumulation, stability, tissue specificity, and intra-chloroplast localization of HSP21, the major LMW chloroplast HSP in pea. Intact pea plants were subjected to heat stress conditions which would be encountered in the natural environment and HSP21 mRNA and protein levels were measured in leaves and roots. HSP21 was not detected in leaves or roots before stress, but the mature, 21-kD protein accumulated in direct proportion to temperature and HSP21 mRNA levels in both tissues. All of the HSP21 in leaves was localized to chloroplasts; there was no evidence for its transport into other organelles. In chloroplast fractionation experiments, greater than 80% of HSP21 was recovered in the soluble chloroplast protein fraction. The half-life of HSP21 at control temperatures was 52 +/- 12 h, suggesting the protein's function is critical during recovery as well as during stress. We hypothesize that HSP21 functions in a catalytic fashion in both photosynthetic and nonphotosynthetic plastids.

A 72,000-mol-wt protein from tomato inhibits rabbit acto-S-1 ATPase activity.

Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP.

Insect hormones: alpha ecdysone and 20-hydroxyecdysone in bracken fern.

The two major molting hormones of insects, alpha ecdysone and 20-hydroxyecdysone, were isolated in crystalline form from dry pinnae of the bracken fern, Pteridium aquilinum (L.) Kuhn. Three unidentified substances with molting hormone activity were also detected. Bracken is the first plant found to contain both of the major insect ecdysones, and it is the first known plant source of alpha ecdysone.

Toxic substances in plants and the food habits of early man.

The widespread occurrence of toxic substances in plants must have greatly restricted their usefulness as food for primitive man. The development of cooking of plant products is suggested to have been a major evolutionary advance, making a major increase in the vegetable materials palatable to man; this technical advantage apparently occurred only in the most recent 2 percent of the anthropological record.

Natural plant chemicals: sources of industrial and medicinal materials.

Many higher plants produce economically important organic compounds such as oils, resins, tannins, natural rubber, gums, waxes, dyes, flavors and fragrances, pharmaceuticals, and pesticides. However, most species of higher plants have never been described, much less surveyed for chemical or biologically active constituents, and new sources of commercially valuable materials remain to be discovered. Advances in biotechnology, particularly methods for culturing plant cells and tissues, should provide new means for the commercial processing of even rare plants and the chemicals they produce. These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals. In the future, biologically active plant-derived chemicals can be expected to play an increasingly significant role in the commercial development of new products for regulating plant growth and for insect and weed control.

Origin of life: clues from relations between chemical compositions of living organisms and natural environments.

When elemental enrichment factors in living organisms are plotted against the ionic potential of the elements, a strikingly similar pattern is found for different groups of organisms; the pattern is also similar, in its general features, to that found in seawater. These relationships support the idea that life began in a water-rich environment interfacing with the primitive atmosphere of the earth.

Vernolepin: a new, reversible plant growth inhibitor.

Vernolepin (5 to 50 micrograms per milliliter), a novel sesquiterpenoid dilactone obtained from Vernonia hymenolepis, inhibits extension growth (from 20 to 80 percent) of wheat coleoptile sections. Inhibited tissues appear normal and their respiration is unaffected. If the inhibited sections are washed and subsequently treated with indole-3-acetic acid, the tissues respond to the auxin, but the degree of elongation is determined by the length of prior treatment with vernolepin. Administered simultaneously, increasing concentrations of auxin will significantly reduce the inhibitory effect of vernolepin, but there is no evidence for a competitive interaction between the two substances.

Reexamination of tetradecenyl acetates in oak leaf roller sex pheromone and in plants.

The chemistry of the oak leaf roller sex pheromone is shown by means of microozonolysis and computerized gas chromatography-mass spectrometry to be dominated by an approximate 70 to 30 ratio of E- and Z-11-tetradecenyl acetates. Tetradecenyl acetates are undetectable in highly purified oak leaf, apple leaf, and corn extracts analyzed by gas chromatography-mass spectrometry. These results reflect negatively on previous reports and on the hypothesis that plant components might govern insect chemical communication systems.

Evidence for origin of insect sex pheromones: presence in food plants.

Compounds identified as sex attractant pheromones in a number of phytophagous insects were found in a variety of host plants. These agents vary in chemical composition in different plant species, which suggests that dietary factors may provide an evolutionary mechanism for diversification of certain insect species. A theoretical framework to explain this phenomenon is postulated on the basis of experiments with the oak leaf roller moth.

Hernandulcin: an intensely sweet compound discovered by review of ancient literature.

Ancient Mexican botanical literature was systematically searched for new plant sources of intensely sweet substances. Lippia dulcis Trev., a sweet plant, emerged as a candidate for fractionation studies, and hernandulcin, a sesquiterpene, was isolated and judged by a human taste panel as more than 1000 times sweeter than sucrose. The structure of the sesquiterpene was determined spectroscopically and confirmed by chemical synthesis. Hernandulcin was nontoxic when administered orally to mice, and it did not induce bacterial mutation.

A 13-kilodalton maize mitochondrial protein in E. coli confers sensitivity to Bipolaris maydis toxin.

The Texas male-sterile cytoplasm (cms-T) of maize carries the cytoplasmically inherited trait of male sterility. Mitochondria isolated from cms-T maize are specifically sensitive to a toxin (BmT-toxin) produced by the fungal pathogen Bipolaris maydis, race T, and the carbamate insecticide methomyl. A mitochondrial gene unique to cms-T maize, which produces a 13-kilodalton polypeptide associated with cytoplasmic male sterility, was expressed in Escherichia coli. After addition of BmT-toxin or methomyl, inhibition of whole cell respiration and swelling of spheroplasts were observed in Escherichia coli cultures producing the novel mitochondrial protein; these effects are similar to those observed with isolated cms-T mitochondria. The amino-terminal region of the 13-kilodalton polypeptide appears to be essential for proper interaction with the BmT-toxin and methomyl. These results implicate the 13-kilodalton polypeptide in conferring toxin sensitivity to mitochondria of cms-T maize.

Identification of carcinogenic tannin isolated from Bracken fern (Pteridium aquilinum).

We attempted to isolate a carcinogenic substance from bracken fern (Pteridium aquilinum), a naturally occurring toxicant responsible for the production of chronic enzootic hematuria and urinary bladder cancer of cattle and carcinogenic for various target organs of several species. Hot methanol extracts of bracken fern were solubilized in water and extracted with chloroform followed by a mixture of n-butanol-butanone (1:1). That fraction was dried and triturated with ether-methanol (4:1), n-butanol, and finally absolute ethanol. The insoluble residue was dissolved in 10% aqueous methanol and passed through Dowex 1 OH-, Dowex 50 H+, or Dowex 1 OH- and then Dowex 50 H+ ion exchange resins. A condensed tannin, isolated from one ot the fractions, was identical to that isolated from bracken fern by the caffeine procedure used for the separation of tannins from other plant constituents. Three systems were used for bioassay; induction of bladder carcinoma by implantation of cholesterol pellets containing bracken fern fractions into the bladder lumens of mice; acute toxicity by ip injection of brachen fern fraction into mice; and growth inhibition of Escherichia coli. The following fractions induced significantly greater incidences of bladder carcinoma than did cholesterol pellets only: tannin, Dowex 50 H+, residue, n-butanol, and methanol. Tiliroside, a component of bracken fern fractions into the bladder lumens of mice; acute genic acid, and quercetin were not carcinogenic. Tannin was the most toxic (mean lethal dose: 0.16 mg/g) and carcinogenic. None of the carcinogenic fractions inhibited growth of E. coli.