Light Effects on the Synthesis of Ribulose-1,5-Bisphosphate Carboxylase in Lemna gibba L. G-3

Placing light-grown Lemna gibba L. G-3 into the dark results in a changed pattern of protein synthesis. Although the amount of protein in the tissue and the over-all rate of incorporation of [³⁵S]methionine into protein does not significantly decline during four days of darkness, the rate of synthesis of three polypeptides declines dramatically. One of these polypeptides is the chlorophyll a/b-binding protein and the two others are the large and small subunits of ribulose-1,5-bisphosphate carboxylase. The changed rates of synthesis of the two subunits were examined after transitions of plants from light to dark and dark to light. The in vivo synthesis of both subunits, while declining to a low level during four days of darkness, increases rapidly upon returning the plants to white light. In addition, the level of poly(A) mRNA coding for the precursor polypeptide of the small subunit of the enzyme falls to a low level in the dark and increases rapidly in response to white light. The increase in translatable mRNA for the small subunit is rapid enough to account for a major part of the increased synthesis of this subunit.     

White Light Effects on the mRNA for the Light-Harvesting Chlorophyll a/b-Protein in Lemna gibba L. G-3

Translation products of poly(A) mRNA isolated from Lemna gibba L. G-3 include a major polypeptide of 32,000 daltons which is immunoprecipitated by antiserum to chlorophyll a/b-protein from Chlamydomonas. This 32,000 dalton polypeptide represents a precursor to the light-harvesting chlorophyll a/b-protein of molecular weight 28,000 found in the thylakoid membranes of Lemna gibba. The amount of this translatable mRNA decreases relative to other translatable mRNAs when green plants grown in continuous white light are placed in darkness. This decrease occurs rapidly. The most rapid decline occurs during the first day; after 4 days of darkness, only a low level of this mRNA can be detected by in vitro translation. When the plants are returned to white light there is an increase in the relative level of this mRNA which can be easily detected within two hours. The in vivo synthesis of this protein has been assayed under the different light conditions. The light effects on the in vivo synthesis of the chlorophyll a/b-protein reflect the light effects on the translatable mRNA for the polypeptide. The results indicate that light induced changes in the synthesis, processing, or degradation of chlorophyll a/b-protein mRNA could account for the light-induced changes observed in the effective synthesis rates for the chlorophyll a/b-protein in vivo.     

Effect of Cycloheximide on the Inverse Control by Phytochrome of the Expression of Two Nuclear Genes in Barley (Hordeum vulgare L.)

The accumulation of mRNAs encoded by two phytochrome-regulatedgenes in barley (Hordeum vulgare L.) was examined after a singlered light pulse in presence of cycloheximide. The initial increasein mRNA encoding the major light harvesting chlorophyll a/bbinding protein (LHCP) could still be observed indicating thatno protein synthesis-requiring step is essential for the transductionchain. The phytochrome-induced decrease in mRNA encoding NADPH:protochlorophyllideoxidoreductase was considerably slowed. (Received February 22, 1988; Accepted June 8, 1988)     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with ¹³C₆-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR₁) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR₁ has the same amount of DNA per nucleus as the parent line (PL). All jsR₁ cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR₁ at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR₁ had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR₁ and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR₁ had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity.     

Spectral dependences of flowering in Lemna perpusilla and L. gibba

Floral induction in Lemna perpusilla and L. gibba was determinedunder continuous irradiation with monochromatic light in spectralranges from 396 to 765 nm. In the former it was induced underwavelengths from about 400 to 550 nm and longer than 700 nm,while in the latter with wavelengths near 400 nm and from about550 to 650 nm. The patterns of these spectral dependences werenearly mirror images and corresponded to the P_fr level in thephotostationary states of phytochrome. (Received December 3, 1974; )     

Antioxidant capacity of Lemna gibba L. exposed to wastewater treatment

In this study, the amounts of antioxidant vitamins (A, E and C), selenium (Se), reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and malondialdehyde (MDA) that is the indicator of lipid peroxidation were determined in Lemna gibba L. plants placed in the secondary clarifier and grown in natural water. The amounts of antioxidant vitamins (A, E and C), GSH, GSSG and MDA were determined with HPLC (high performance liquid chromatography) and the amounts of Se were determined fluorimetrically. While significant decrease in amounts of antioxidant vitamins (A, E and C) and Se takes place between the first and the second or third day depending on species and insignificant decrease takes place after that day, the amount of GSH/GSSG ratio decreases until the second day (p < 0.05) and insignificantly increases after that day. Amounts of vitamins (A, E and C), Se and GSH/GSSG ratio for the plants placed in the secondary clarifier are much less than that for control group while an opposite trend was observed in MDA level. The MDA for the plants placed in secondary clarifier has maximum value at the second day, which can be considered as the maximum stress occurring at that day. Consequently, the first two days of treatment time can be taken as acclimation time and after the acclimation time the plant lives in the hostile environmental condition with the certain amount of oxidative stress. As a result, it is determined that wastewater decreases the lifetime of plant by causing metabolic stress on it and affects antioxidant capacity. The lifetime of the plants in the secondary clarifier was determined to be five days since fading toward yellow color (necrosis) in the plants was observed at the fifth day.     

The occurrence of acetylcholine in Lemna gibba G3

The occurrence of acetylcholine in a long-day duckweed, Lemnagibba G3 has been demonstrated. After a preliminary purificationof the formic acid-acetone extract by Sephadex G-15 column chromatography,acetylcholine was identified by paper chromatography, pharmacologicalactivity on frog muscle, and sensitivity to acetylcholinesterase. Acetylcholine contents relative to that at the start of theexperimental culture were 0.99?0.06, 1.61?0.27, and 1.17?0.16after 2 cycles of the [9(15)], [16(8)], and [24(0)] schedules,respectively. (Received November 14, 1977; )     

Pb-Induced Avoidance-Like Chloroplast Movements in Fronds of Lemna trisulca L.

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb²⁺, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H₂O₂). In addition, we detected an enhanced accumulation of endogenous H₂O₂ after treatment of plants with lead. Interestingly, H₂O₂-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H₂O₂ can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic effect of lead on chloroplasts can include disturbances in their movement and distribution pattern.     

Expression of Genes Encoding Multi-Transmembrane Proteins in Specific Primate Taste Cell Populations

Background

Using fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed.

Methodology/Principal Findings

Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release.

Conclusions/Significance

Identification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality.     

Flowering Responses of the Long-day Plant Lemna gibba G3

Lemna gibba L., strain G3, exhibits a qualitative long-day flowering response with a critical daylength on a 24-hour cycle of about 10 hours. Evidence is presented that the onset of daughter frond formation in a given frond inhibits the activity of the flowering meristem. Consequently, flower induction can only occur in fronds smaller than about 0.05 to 0.07 mm long. Although a minimum of 1 long day seems to be sufficient to induce the formation of flower primordia, at least 6 long days are required to obtain mature flowers since long days are also required for the early stages of flower development. The critical night length on 24, 48 and 72-hour cycles is respectively 14, 16, and 18 to 22 hours. The close similarity between the critical night length for the different cycle lengths is explained in terms of an inhibitory effect of darkness both on flower initiation and flower development. A 10-hour dark period is more inhibitory to flowering on a 36-hour cycle than on 24, 48, 60 or 72-hour cycles. It is suggested that darkness inhibits flowering through the formation of a light-labile flower inhibitor which acts to inhibit the functioning of the flowering stimulus.     

Test system stability and natural variability of a Lemna gibba L. bioassay

Background

In ecotoxicological and environmental studies Lemna spp. are used as test organisms due to their small size, rapid predominantly vegetative reproduction, easy handling and high sensitivity to various chemicals. However, there is not much information available concerning spatial and temporal stability of experimental set-ups used for Lemna bioassays, though this is essential for interpretation and reliability of results. We therefore investigated stability and natural variability of a Lemna gibba bioassay assessing area-related and frond number-related growth rates under controlled laboratory conditions over about one year.

Methology/Principal Findings

Lemna gibba L. was grown in beakers with Steinberg medium for one week. Area-related and frond number-related growth rates (r_(area) and r_(num)) were determined with a non-destructive image processing system.To assess inter-experimental stability, 35 independent experiments were performed with 10 beakers each in the course of one year. We observed changes in growth rates by a factor of two over time. These did not correlate well with temperature or relative humidity in the growth chamber.In order to assess intra-experimental stability, we analysed six systematic negative control experiments (nontoxicant tests) with 96 replicate beakers each. Evaluation showed that the chosen experimental set-up was stable and did not produce false positive results. The coefficient of variation was lower for r_(area) (2.99%) than for r_(num) (4.27%).

Conclusions/Significance

It is hypothesised that the variations in growth rates over time under controlled conditions are partly due to endogenic periodicities in Lemna gibba. The relevance of these variations for toxicity investigations should be investigated more closely. Area-related growth rate seems to be more precise as non-destructive calculation parameter than number-related growth rate. Furthermore, we propose two new validity criteria for Lemna gibba bioassays: variability of average specific and section-by-section segmented growth rate, complementary to average specific growth rate as the only validity criterion existing in guidelines for duckweed bioassays.     

The Use of Chemical and Infra-red Spectral Analyses to Verify the Identification of Two Species of Duckweed, Lemna minor L. and Lemna gibba L. Grown Under Defined Conditions

Flat forms of the fronds of Lemna gibba L., used widely in laboratoryexperiments, can be easily confused with Lemna minor Chemicalanalyses of the two species, grown under defined laboratoryconditions, showed clear distinctions between the two speciesin relation to total protein content and the content of individualmonosaccharides. Infra-red spectral analysis confirmed the chemicaldata for protein content and also revealed differences in thetypes of proteins present in the two species. Chemical and infra-redspectral analyses, particularly the amide I: carbohydrate absorbanceratio, provide a rapid and reliable means of verifying betweenthe two species, which are often difficult to identify withcertainty from morphological differences. Key words: Gibbosity, infra-red spectroscopy, Lemna gibba, Lemna minor, monosaccharides, proteins