Allelopathic activities of three carotenoids, neoxanthin, crocin and ß-carotene, assayed using protoplast co-culture method with digital image analysis.

Allelopathic activities of three carotenoids of a natural pigment group, neoxanthin, crocin and ß-carotene, were assayed by the protoplast co-culture method with digital image analysis (DIA-PP method). Effects on three different growth stages of lettuce protoplasts, i.e., cell wall formation, cell division, and yellow pigment accumulation, were investigated using 96-well culture plates. Cell division was inhibited 65-95% by all three carotenoids at 33-100 µM. Inhibition of cell division stage was stronger than at the cell wall formation stage in neoxanthin, and the water-soluble carotenoid, crocin, whose yellow pigment was incorporated into the vacuole of lettuce protoplasts. Neoxanthin at 33 µM and crocin at higher than 100 µM inhibited more than 100% of the yellow pigment accumulation. By contrast, at low concentrations (0.01-1 µM) ß-carotene stimulated growth at the cell division stage. At high concentrations of ß-carotene (100-500 µM), inhibition was prominent at all three stages, and also in neighboring wells of zero control, which suggested emission of a volatile compound by ß-carotene. They were compared with the report of the volatile compound, tulipalin A. Differences in patterns of inhibition of carotenoids on lettuce protoplast growth were compared with reports of another natural pigment, anthocyanin, and anthocyanin-containing red callus cultured in the light, and with that of neoxanthin-containing yellow callus cultured in the dark.

Establishment of Pluripotent Cell Cultures to Explore Allelopathic Activity of Coffee Cells by Protoplast Co-Culture Bioassay Method.

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

In vitro bioassay of allelopathy of Arabidopsis thaliana by sandwich method and protoplast co-culture method with digital image analysis.

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were ß-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

Allelopathy in a leguminous mangrove plant, Derris indica: protoplast co-culture bioassay and rotenone effect.

To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 µM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 µM 2,4-D and 0.1 µM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

Effect of purine alkaloids on the proliferation of lettuce cells derived from protoplasts.

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 µM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 µM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

A protocol for axenic liquid cell cultures of a woody leguminous mangrove, Caesalpinia crista, and their amino acids profiling.

Callus induction, maintenance and protoplast cultures were achieved from immature seeds of a woody leguminous mangrove, Caesalpinia crista. Axenic cultures were possible during 1.5 months of pod storage in 0.1% benzalkonium chloride solution. Callus induction was achieved using 1 mL liquid medium in a 10 mL flat-bottomed culture tube. Protoplasts were isolated using Cellulase R10, Hemicellulase, and Driselase 20 in 0.6 M mannitol solution and sub-culturable calluses were obtained in 50 µL liquid medium using a 96-microplate method. The optimal hormonal concentration was 10 µM each of 2,4-dichlorophenoxyacetic acid and benzyladenine in liquid Murashige and Skoog's basal medium for both callus induction and maintenance, and protoplast cultures. Similarities and differences in amino acid profiles and culture conditions are discussed among woody mangrove species and non-mangrove leguminous species. Caesalpinia crista cultures were unique as they secreted a large amount of amino acids, including proline, into the liquid culture medium.

Effect of NaCl on ionic content and distribution in suspension-cultured cells of the halophyte Sonneratia alba versus the glycophyte Oryza sativa.

The effect of a high concentration of NaCl on the intra- (cytoplasmic matrix and vacuole) and extracellular (cell wall) distribution of Na, Cl, K, Mg, Ca, S, and P was investigated in suspension-cultured cells of the mangrove halophyte Sonneratia alba and compared to cultured cells of glycophytic rice (Oryza sativa). No significant differences were observed in ultrastructural features of cluster cells of both species cultured with and without 50mM NaCl. Quantitative X-ray microanalysis of cryosections of the cells cultured in the presence of 50mM NaCl showed that the Na concentration ([Na]) and Cl concentration ([Cl]) significantly increased in all three cell components measured. In S. alba, the [Na] was highest in the vacuole and lowest in the cytoplasmic matrix, while the [Cl] was highest in the cell wall and lowest in the cytoplasmic matrix. In O. sativa, however, the [Na] and [Cl] were highest in the cell wall, and the [Na] was lowest in the cytoplasmic matrix. Thus, the possible activities for Na and Cl transport from the cytoplasmic matrix into the vacuole were greater in S. alba than in O. sativa, suggesting that halophilic mangrove cells gain salt tolerance by transporting Na and Cl into their vacuoles. In O. sativa, the addition of NaCl to the culture medium caused no significant changes to the intracellular concentrations of various elements, such as K, P, S, Ca, and Mg, which suggests the absence of a direct relationship with the transport Na and Cl. In contrast, a marked decrease in the Ca concentration ([Ca]) in the cytoplasmic matrix and vacuole and an approximately two-fold increase in the P concentration ([P]) in the cytoplasmic matrix were found in S. alba, suggesting that the decrease in the [Ca] is related to the halophilic nature of S. alba (as indicated by the inward movement of Na(+) and Cl(-)). The possible roles of a Na(+)/Ca(2+) exchange mechanism in halophilism and the effect of the [P] on the metabolic activity under saline conditions are discussed.

An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 µM 2,4-dichlorophenoxyacetic acid, 0.1 µM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate.

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1-0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 µg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.

Comparison of the formation of nicotinic acid conjugates in leaves of different plant species.

There are three metabolic fates of nicotinic acid in plants: (1) nicotinic acid mononucleotide formation for NAD synthesis by the so-called salvage pathway of pyridine nucleotide biosynthesis; (2) nicotinic acid N-glucoside formation; and (3) trigonelline (N-methylnicotinic acid) formation. In the present study, the metabolism of [carbonyl-(14)C]nicotinamide was investigated in leaves of 23 wild plant species. All species readily converted nicotinamide to nicotinic acid, and only a fraction of nicotinic acid was utilised for NAD and NADP synthesis. The remaining nicotinic acid is converted to the nicotinic acid conjugates. Only one plant species, Cycas revoluta, produced both nicotinic acid N-glucoside and trigonelline; the other 22 species produced one or other of the conjugates. The nicotinic acid N-glucoside-forming plants are Cyathea lepifera, Arenga trewmula var. englri, Barringtonia racemosa, Ilex paraguariensis, Angelica japonica, Scaevola taccada and Farfugium japonicum. In contrast, trigonelline is formed in C. lepifera, Ginkgo biloba, Pinus luchuensis, Casuarina equisetifolia, Alocasia odora, Pandanus odoratissimus, Hylocereus undatus, Kalanchoe pinnata, Kalanchoe tubiflora, Populus alba, Garcinia subelliptica, Oxalis corymbosa, Leucaena leucocephala, Vigna marina, Hibiscus tiliaceus and Melicope triphylla. The diversity of nicotinic acid conjugate formation in plants is discussed using these results and our previous investigation involving a few model plants, various crops and ferns. Nicotinic acid N-glucoside formation was restricted mostly to ferns and selected orders of angiosperms, whereas other plants produce trigonelline. In most cases the formation of both nicotinic acid conjugates is incompatible, but some exceptions have been found.

Pyridine metabolism and trigonelline synthesis in leaves of the mangrove legume trees Derris indica (Millettia pinnata) and Caesalpinia crista.

The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (= Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 microg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.

Spinning of a gigantic bundle of hollow fibrils by a spirally moving higher plant protoplast.

A unique fiber spinning was found in protoplasts from white birch (Betula platyphylla) leaves under an acidic medium containing high concentration of Ca(2+). After expanding from 10 to 100 microm in diameter under the culture condition, the protoplast started secreting a gigantic fiber while moving in a spiral way. Real time video analyses elucidated that the orientation, rate and pattern of the motion were directed due to the inverse force of the fiber spinning. Moreover, observation using several microscopic methods accompanied with histochemical staining and nuclear magnetic resonance (NMR) analysis indicated that the fiber was composed of 400-500 nm wide (1-->3)-beta-glucan hollow sub-fibrils. This entire phenomenon may be a response against the stress imposed. The observation presented provides an understanding of the unique relationship between fiber spinning and the bottom-up fiber fabrication from nano to micro scales.

Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer.

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 mum, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for beta-1,4-glucan and beta-1,3-glucan.

Identification of a novel factor, vanillyl benzyl ether, which inhibits somatic embryogenesis of Japanese larch (Larix leptolepis Gordon).

In contrast to angiosperms, some gymnosperms form well-developed suspensors in somatic embryogenesis. This characteristic makes it easy to study suspensor biology. In cultures with high cell densities, somatic embryogenesis of Japanese larch, especially the suspensor development, is strongly inhibited due to factor(s) that are released by the cells into the culture medium. In this study, we purified and identified one of the inhibitory factors present in high-cell-density conditioned medium (HCM) of larch cells. The factor with the strongest inhibitory activity was purified by dialysis, extraction by ethyl acetate, octadecylsilyl (ODS) column chromatography and high-performance liquid chromatography (HPLC). The inhibitory factor was identified as vanillyl benzyl ether (VBE) by physicochemical analysis. This compound was first isolated from natural resources. Authentic VBE inhibited somatic embryo formation in Japanese larch, and the inhibitory effect in the suspensor was stronger than in the embryo proper. Furthermore, quantification of VBE by HPLC demonstrated that VBE accumulates at high concentrations in HCM. These results suggest that VBE is a novel negative regulator of somatic embryogenesis.

Histological analysis in shoot organogenesis from hypocotyl explants of Kandelia candel (Rhizophoraceae).

In vitro culture of hypocotyl explants from Kandelia candel, a common mangrove species, on hormone-free Murashige and Skoog (MS) medium resulted in shoot formation. Since the hypocotyls showed good potential for in vitro shoot multiplication, the process of bud primordium formation was analyzed from a histological viewpoint. A wound periderm first appeared at the top, exposed cut surface of the explants. The wound-induced meristem continued to divide giving rise to suberized cells oriented towards the cut surface. After formation of the suberized cell layers, the meristem and its inner derivatives differentiated into multilayered, uniformly packed parenchyma cells. Bud primordia differentiated from the dense cytoplasmic cells of the wound-induced meristem just beneath the suberized layer near the severed vascular bundles. Each explant produced several visible shoot buds. Furthermore, histological sections revealed that additional bud primordia were present within the explant just underneath the suberized cells and that these bud primordia appeared to be arrested in their development. The fact that additional bud primordia were present within the explant suggests that further manipulation of the explant is helpful to maximize the potential of this system.