Cyclic adenosine 3':5'-monophosphate in moss protonema: a comparison of its levels by protein kinase and gilman assays

From the protonema of the moss Funaria hygrometrica (L.) Sibth, a factor indistinguishable from cyclic adenosine 3′:5′-monophosphate (cAMP) has been isolated. The factor stimulated the activity of protein kinase from rabbit skeletal muscle and co-chromatographed with authentic cAMP in two solvent systems. Its ability to stimulate protein kinase activity was completely abolished by 3′:5′-cyclic nucleotide phosphodiesterase, the rate of inactivation being similar to that of authentic cAMP. Based on these properties, this factor is identified as 3′,5′-cAMP. Cyclic AMP could be readily removed from the cells and washing the cells with water reduced the endogenous level of cAMP by 2- to 3-fold. A comparison of cAMP levels by protein kinase and Gilman assays was made. The intracellular levels determined by protein kinase assay were about 7-fold lower than the values obtained by Gilman assay. This discrepancy was due to the presence of unidentified compounds which were completely degraded by 3′:5′-cyclic nucleotide phosphodiesterase. Although these displaced labeled cAMP in the Gilman assay, they did not stimulate the protein kinase activity. The protonema may contain cyclic nucleotides other than cAMP; these will not be detected in the protein kinase assay due to the specificity of this reaction. The crude extracts were found to be unsuitable for assaying cAMP by either method.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Additive and synergistic effects of kinetin and ethrel on germination, thermodormany, and polyribosome formation in lettuce seeds

The inhibition of germination of Grand Rapids lettuce (Lactuca sativa L.) seeds at 35 C was removed to a marked extent by kinetin and 2-chloroethylphosphonic acid (ethrel). When both compounds were used together, an additive effect was observed. A synergistic effect was, however, noted when ethrel promoted the kinetin reversal of abscisic acid inhibition of seed germination (light- as well as gibberellic acid-, induced). Both kinetin and ethrel increased the total ribosomal material and the percentage of polyribosomes in lettuce seeds imbibed in the light for 24 hours. A combination of the two compounds showed a synergism in polyribosome formation only at high ethrel concentration. The inability of ethrel to reverse abscisic acid inhibition indicates that kinetin action cannot always be substituted by ethrel. The possible mechanisms involved in the enhanced response by a combination of kinetin and ethrel are discussed.     

Interaction of carbon dioxide and ethylene in overcoming thermodormancy of lettuce seeds

The combination of ethylene with CO₂ will completely overcome the thermodormancy of lettuce (Lactuca sativa L.) seeds at 35 C. This combination is effective if it is added to seeds either at the start or after several days of imbibition. The action of ethylene is dependent upon the CO₂ level present in the atmosphere surrounding the seeds. When CO₂ is trapped by KOH the ethylene effect is essentially nil.     

Susceptibility of germinating cruciferous seeds to <Emphasis Type="Italic">Bagrada hilaris</Emphasis> (Hemiptera: Pentatomidae) feeding injury

Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae) is a serious pest that attacks both germinating and seedling stages of a variety of cruciferous crops grown in the Central Coast of California. B. hilaris feeding on germinating seeds can cause severe stunting and plant mortality, and little is known about the feeding preference of B. hilaris for germinating seeds of major cruciferous hosts and varieties of hosts. No-choice and choice experiments were conducted in which germinating seeds in soilless and soil settings were exposed to B. hilaris adults for 7 days. Susceptibility scores were developed using B. hilaris feeding injury sites, distorted leaves, and deformed and dead plants to determine the overall B. hilaris preference for germinating host seeds. Based on the scores, the order of preference was arugula (Eruca sativa L.)?>?turnip (B. rapa L. var. rapa)?>?mizuna (B. rapa L. nipposinica)?>?kale (B. oleracea L. acephala)?>?choi (Brassica rapa L. var. chinensis)?>?broccoli (B. oleracea var. italica Plenck)?>?cauliflower (B. oleracea L. var. botrytis)?>?lettuce (Lactuca sativa L.)?>?sweet alyssum (Lobularia maritima [L.] Desv.). The lowest feeding injury was recorded on germinating lettuce and sweet alyssum seeds. Furthermore, no-choice and choice experiments were conducted with four varieties each of arugula and mizuna, twelve varieties each of kale and choi, and nine varieties/types of leafy Asian greens. The arugula varieties ‘Wild Rocket’ and ‘Spirit’ were more damaged by B. hilaris than other varieties tested. Among mizuna varieties, ‘Beira F1’ was more attractive to B. hilaris than ‘Scarlet’ or ‘Starbor F1.’ The choi varieties ‘Tokyo Bekana,’ ‘Feng Qing Choi F1,’ ‘Joi Choi F1,’ and ‘Win-Win Choi F1’ were more attractive than ‘Rosie F1.’ The leafy Asian greens variety ‘Carlton F1’ was more attractive to B. hilaris than ‘Yukina Savon,’ ‘Tatsoi OG,’ ‘Komatsuna Summerfest F1,’ ‘Red Rain F1,’ and ‘Shungiku.’ Therefore, the results suggest that not all varieties were equally susceptible to B. hilaris feeding and possibly be utilized for further field evaluation as a trap crop or developing more resistant varieties to B. hilaris.     

Application of chemicals in organic solvents to dry seeds

Various chemicals were applied to dry seeds by means of organic solvents. The gibberellic acid-treated (1 mm) lettuce seeds (Lactuca sativa L.) germinated nearly 100% in the dark even after prolonged storage, and those treated with abscisic acid (1 mm or 0.5 mm) failed to germinate in the light. The seedlings emerging from morphactin-treated (1 mm) cucumber seeds (Cucumis sativus L.) exhibited profound changes in morphology. Different combinations of hormones applied to lettuce seeds caused a promotion or an inhibition of germination. Germination promotion or inhibition studies showed that the applied chemicals could be removed by washing with an organic solvent or water. Progressively larger amounts of chemicals were removed with increasing periods of washing. Thus the chemical appeared to penetrate the seed to some degree. The potential of the organic solvent method is discussed.     

Failure to Detect Cyclic 3', 5'-Adenosine Monophosphate in Healthy and Crown Gall Tumorous Tissues of Vicia faba

Attempts were made to provide proof for the occurrence of cyclic 3′,5′-adenosine monophosphate in healthy and crown gall tissues of Vicia faba. Although our purified extracts gave positive readings in the Gilman binding assay for cyclic AMP, they were not digested by a specific cyclic 3′,5′-adenosine monophosphate phosphodiesterase from beef heart. The extracts were digested, however, by a partially purified cyclic nucleotide phosphodiesterase from carrot tissue, which attacks both cyclic 2′,3′- and 3′,5′-nucleotides. The data indicate that the substances detected in the V. faba extracts are perhaps cyclic 2′,3′-nucleotides, a possible RNA degradation product.     

Antagonistic effects of high and low temperature pretreatments on the germination and pregermination ethylene synthesis of lettuce seeds

Red light-induced germination of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C was inhibited if the seeds were first imbibed at 30 C for 36 hours. This effect was counteracted by exogenous ethylene and associated with a reduction in the rate at which the seeds produced ethylene throughout the pregermination period. A chilling treatment reversed the effect of a prior imbibition at 30 C on both germination and ethylene production. The possibility that the pretreatments influence germination through their effects on ethylene production is discussed.     

The Effect of Arsenate and Other Inhibitors on Early Events during the Germination of Lettuce Seeds (Lactuca sativa L.)

The effect of arsenate, arsenite, 2,4-dinitrophenol, and anaerobiosis on early events in seed germination was investigated using both intact and punched seeds of lettuce (Lactuca sativa L.). It was found that punching the seed removes penetration barriers to the entrance of inhibitors without an undue loss of germination or light responses. The kinetics of the action of germination inhibitors were established by 2-hour pulse experiments. Arsenate and 2, 4-dinitrophenol have very different kinetics. The inhibition of germination in punched seeds by arsenate given in conjunction with phosphate compared with the lack of inhibition of arsenate plus phosphate on the growing seedling, suggest a distinct metabolic change in the germinating embryo at some time between the onset of germination and subsequent seedling growth.     

Trichoderma spp. alleviate phytotoxicity in lettuce plants (Lactuca sativa L.) irrigated with arsenic-contaminated water

The influence of two strains of Trichoderma (T. harzianum strain T22 and T. atroviride strain P1) on the growth of lettuce plants (Lactuca sativa L.) irrigated with As-contaminated water, and their effect on the uptake and accumulation of the contaminant in the plant roots and leaves, were studied. Accumulation of this non-essential element occurred mainly into the root system and reduced both biomass development and net photosynthesis rate (while altering the plant P status). Plant growth-promoting fungi (PGPF) of both Trichoderma species alleviated, at least in part, the phytotoxicity of As, essentially by decreasing its accumulation in the tissues and enhancing plant growth, P status and net photosynthesis rate. Our results indicate that inoculation of lettuce with selected Trichoderma strains may be helpful, beside the classical biocontrol application, in alleviating abiotic stresses such as that caused by irrigation with As-contaminated water, and in reducing the concentration of this metalloid in the edible part of the plant.     

A Comparison of the Quantitative Effects of Seed Moisture Content and Temperature on the Accumulation of Chromosome Damage and Loss of Seed Viability in Lettuce

The rate of accumulation of cells containing chromosome aberrationsin lettuce (Lactuca sativa L.) seeds is a positive functionof temperature and moisture content. It may be described byan equation similar to that for loss of seed viability. Therelative effect of temperature on the rates of loss of viabilityand accumulation of chromosome aberrations is the same. In contrast,the relative effect of moisture on the rate of loss of viabilityis greater than that for the rate of accumulation of aberrations.Hence considerably more chromosome damage accumulates beforedeath in drier lettuce seeds. Lactuca sativa, lettuce, seed storage, seed viability, seed longevity, chromosomal aberrations temperature, moisture content     

Cytogenotoxic effects of ethanolic extracts of Annona crassiflora (Annonaceae)

Infusions of the leaves and seeds of Annona crassiflora Mart. are commonly employed in the treatment of diarrhoea, snakebites, tumours and disorders of the hair and scalp. The aim of the present study was to investigate the cytotoxic and genotoxic properties of ethanolic extracts of A. crassiflora by evaluating their effects on germination, root elongation, chromosome structure and the cell division of Lactuca sativa (lettuce). The experiment followed a randomized design involving the treatment of L. sativa seeds with ethanolic extracts from leaves and seeds of A. crassiflora applied at ten concentrations (0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg/L) and with five repetitions per treatment. Seeds of L. sativa exposed for 48 h to A. crassiflora leaf extract at concentrations ≥ 0.1 mg/L, or to seed extracts at concentrations ≥ 0.2 mg/L, showed germination percentages that were significantly (p < 0.05) lower than those of seeds exposed to aqueous ethanol control. Exposure of L. sativa seedlings to leaf (but not seed) extracts of A. crassiflora produced significant (p < 0.05) reductions in the mitotic indices of root meristem cells of lettuce and induced chromosome and nuclear abnormalities in the root cells. The presence of chromosome stickiness, bridges, fragments, laggard chromosomes and nuclear condensation were also observed. The cytogenetic effects observed suggest that folkloric medicines prepared with extracts of the leaves or seeds of A. crassiflora should be employed with caution.     

Expression of allelopathy in the soil environment: soil concentration and activity of benzoxazinoid compounds released by rye cover crop residue

Rye (Secale cereale L.) residue is known to suppress weeds in agronomic environments and to produce benzoxazinoid (BX) compounds which are phytotoxic. Experiments were conducted to determine the duration of indicator plant inhibition and BX soil concentrations in response to field incorporated or surface rye residue. Surface rye residue was highly inhibitory to lettuce (Lactuca sativa L.) and smooth pigweed (Amaranthus hybridus L.) throughout an experimental period of 4 weeks, whereas soil removed from beneath residue and assayed in pots had little phytotoxicity, suggesting that physical rather than chemical mechanisms were involved. Incorporated residue inhibited lettuce and pigweed for approximately 2 weeks after incorporation, which corresponded to the period when elevated BX levels were detected in soil, suggesting potential allelopathy. The most toxic BX compounds, APO, DIBOA, and DIMBOA, were present at relatively low levels, whereas the less toxic compounds, BOA and MBOA, and the non-toxic compounds, HBOA and HMBOA, were the predominant BX species in amended soils. When the benzoxazolinones BOA and MBOA were exogenously added to soils to maintain extractable levels of up to 10 μg g^?1 soil (100–500 times higher than measured BX in field soils), no significant inhibition of pigweed plants was observed. This result indicated that the observed association between the duration of plant inhibition and BX from incorporated rye was not causal, and that other compounds released with similar dynamics were likely responsible. This approach provides a sound basis for demonstrating the presence of allelopathy in natural or managed ecosystems.     

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3′,4′,5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin-7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots.     

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863–871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.). Received: 20 December 1997 / Accepted: 18 March 1998     

A simple and sensitive DNA assay for plant extracts

A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4′,6′-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.     

Promotive effects of organic solvents and kinetin on dark germination of lettuce seeds

Significant promotion in dark germination was observed when Grand Rapids lettuce (Lactuca sativa L.) seeds were soaked in acetone or dichloromethane, vacuum-dried, and imbibed at 25 C. Permeation of kinetin via these organic solvents further enhanced the dark germination. Those seeds that were affected by acetone and acetone-kinetin treatments and germinated in the dark escaped red-far red photocontrol of germination. Although abscisic acid was not detected in the organic solvent leachates, they did contain other inhibitory substances affecting lettuce seedling growth. In the light, acetone and acetone-kinetin treatments also enhanced the rate of germination and the increased germination by acetone-kinetin treatment was correlated with increased polyribosome formation. The possible mechanisms involved in promotion of lettuce seed germination by organic solvents and kinetin are discussed.     

Water Content and Phytochrome-induced Potential Germination Responses in Lettuce Seeds

Grand Rapids lettuce seeds (Lactuca sativa L.) with 6 to 8% water content show no light-induced germination responses, whereas in seeds with 15% or more water content, germination is promoted or retarded by red and far red light respectively. By adjusting seed water content, persistent potentiated responses to light are induced in the seeds at seed water levels much below that required for germination itself. Alternate moistening and drying of seeds in conjunction with red and far red irradiations show that potentiated responses may be phototransformed only with sufficient seed water. However, the process of drying and remoistening has no effect on potentiated responses.     

Inhibition of germination and α-amylase induction by 6-methoxy-2-benzoxazolinone in twelve plant species

6-Methoxy-2-benzoxazolinone (MBOA) inhibited germination of rice (Oryza sativa L.), wheat (Triticum aethiopicum Jakubz), rye (Secale cereale L.), onion (Allium cepa L.), wild oat (Avena fatua L.), barnyard grass [Echinochloa crus-galli (L.) Beauv.], ryegrass (Lolium rigidum Gaudin), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), tomato (Lycopersicum esculentum Mill.), carrot (Daucus carota L.) and amaranth (Amaranthus retroflexus L) and the inhibition increased with increasing MBOA concentrations. MBOA also inhibited the induction of α-amylase in these plant seeds and the inhibition increased with increasing MBOA concentrations. There were variations in sensitivity of these plant species to MBOA, and species of family Poaceae (barnyard grass, wild oat, rice, rye, ryegrass, and wheat) were less sensitive to MBOA than the other plant species.