Chemical Changes in Lolium temulentum L. after Treatment with (2-Chloroethyl) Trimethylammonium Chloride (CCC)

Two experiments with an inbred line of the summer annual Loliumtemulentum L. are described. The plants were grown under 18-hourdays in 8.5 cm pots containing pure vermiculite. The first experimentwas concerned with the effect of (z-chloroethyl) trimethylammoniumchloride (CCC) upon soluble carbohydrate content, and the secondwith the combined effects of CCC and nitrogen upon soluble carbohydrate,free amino-N, and chlorophyll content. Concentrations of CCCfrom 0.01–0.5 M inhibited growth and induced large increasesin the free sugar content of the plant. With 0.05 M CCC andabove, large amounts of fructosan were formed. Adequate nitrogensupply lowered the free sugar level and large amounts of free amino-N appeared. Crude protein content (per cent dry weight)was increased by CCC at low nitrogen levels but was less affectedwhen N was adequate. Chlorophyll production was stimulated inthe presence of CCC. The metabolic implications of the CCC-inducedchemical changes are discussed and a possible scheme for CCCaction is suggested.     

Effects of 2-Chloroethyl Trimethylammonium Chloride (CCC) and 2-Chloroethylphosphonic Acid (CEPA) on Dry Matter Distribution in the Cowpea (Vigna Unguiculata L.)

Effects of foliar applications of CCC and CEPA on dry matterdistribution in two cowpea cultivars which exhibit differentdegrees of premature abscission of fruits were investigated.At the concentration of 0-1 mg1^–1, CCC increased stemweight in the cultivar Adzuki, but had no effect on the cv.Mala (which exhibits a relatively high degree of premature abscission).While CCC did not significantly influence root weight in Adzuki,the concentrations of 1 and 10 mg 1^–1 increased root weightin Mala. In both cultivars, the 1000 mg1^–1 treatment decreasedseed weight, with a greater decrease in Adzuki. All concentrations of CEPA, up to 1000 mg 1^–1, decreasedstem and root weights in Mala, and leaf weight in Adzuki. The10 mg1^–1 concentration increased seed weight in Mala by100 percent, and leaf weight by 28 percent, without any significanteffect on pod weight, or on the weight of the whole plant. Therewas also no increase in pod number. Overall, Mala was more responsive than Adzuki to the growthregulators, particularly CEPA. In the low concentration responses,CEPA increased seed weight not by increasing gross plant weight,but mainly by causing a change in distribution of dry matter.     

Morphogenetic Effects on Vegetative Plants of Poa pratensis L. of 6-Azauracil, (2-Chloroethyl)- phosphonic acid, and (2-Chloroethyl) trimethyl-ammonium chloride and their Interaction with Gibberellic Acid

Fresh and dry weights and leaf size of Poa pratensis were reducedwhen treated with 6-azauracil (AzU), (2-chloroethyl)phosphonicacid (CEPA), or (2-chloroethyl)trimethylammonium chloride (CCC).AzU and CEPA inhibited epidermal cell division without inhibitingcell elongation, while CCC inhibited mainly cell elongationand cell division to a small extent. The ratio of blade lengthto sheath length and the blade length/width ratio were reduced,but leaf emergence and tillering were increased by AzU and CEPA.CCC affected only the latter three features. Like GA₃, CEPAinduced stem formation, but internodes were shorter. GA₃ was ineffective in preventing leaf-growth inhibition byAzU, which inhibited Ga₃-induced cell elongation. The inhibitoryeffect of CEPA on leaf growth was apparently reversed by GA₃,but this was due solely to increased cell elongation, the reductionin cell number being unaffected. Ga₃ reversed the effect ofCCC on leaf length, as well as on cell size and number. Simultaneousapplication of the inhibitors produced a complex interactionin reducing leaf length and number and size of epidermis cells.It is postulated that AzU, CEPA, and CCC have different modesof action because they have specific effects on plant growthand different effects on GA₃-induced cell elongation.     

The Interaction of (2-Chloroethyl) trimethylammonium Chloride and Gibberellic Acid in Strawberry

Applications of (2-chloroethyl) trimethylammonium chloride (CCC), by foliar spray or to the soil, shortened petioles and decreased top and root growth of strawberry plants. Application of gibberellic acid increased petiole length and fresh weights of tops but not of roots. Applied together gibberellic acid overcame the depression of growth in weight of tops induced by CCC and countered the depression in petiole lengths. Gibberellic acid induced elongation of internodes of the vegetative stem and the elongation was increased substantially by concurrent application of CCC. This synergism in stem growth indicates a lack of antagonism between CCC and exogenous gibberellic acid in strawberry. The implications are discussed.     

Growth and nitrogen distribution in callus and crown-gall tobacco tissue cultures treated with kinetin and (2-Chloroethyl)trimethylammonium chloride (CCC)

1. 1.
   Kinetin at a supra-optimal dose of 1.0 mg/l reduces the growth inhibitory effect of CCC at 5 × 10^?2 M on tobacco callus and increases it on tobacco crown-gall.     
   
   

The Effect of 2-Chloroethyltrimethyl Ammonium Chloride (CCC) on Stomatal Diffusive Resistance in Tomato

CCC (2-chloroethyltrimethyl ammonium chloride) at a concentration of 6.3 mM was applied to tomato plants (cv. Grosse Lisse) grown in a controlled environment. There was an increase in adaxial leaf diffusive resistance but not in abaxial resistance, the effect being apparent before any growth retardation was measurable. The partial closure of adaxial stomata in response to CCC reduced transpiration from that leaf surface. In plants deprived of water, leaf water potential was higher when CCC was applied and both adaxial and abaxial stomatal closure was delayed. The data do not suggest that CCC influenced the relationship between leaf water potential and conductance for either abaxial or adaxial stomata.     

Partial Reversal by Cytokinin and (2-Chloroethyl)-Trimethylammonium Chloride of Near-Ultraviolet Inhibited Growth and Morphogenesis in Callus Cultures

Callus cultures of Haplopappus gracilis, Nicotiana tabacum and Allium cepa var. proliferum were in varying degrees inhibited by blue to near-UV light obtained from fluorescent tubes. The inhibition was considerably reduced for Haplopappus cultures by 6-(3-methyl-2-buten-1-ylamino)-purine (2 iP) and (2-chloro-ethyl)-trimethylammonium chloride (CCC) in combination. Even separately these compounds stimulated growth in blue but not in white light. A high concentration of 2 iP reduced the inhibiting effects of near-UV on tobacco tissue cultures, and a synergism was observed between 2 iP and CCC in respect to shoot formation in blue light. Allium callus was not significantly affected by CCC. It was also observed that the concentration of indol-3-acetic acid (IAA) was more important for the growth of tobacco cultures in blue than in white light. It is believed that the light-inhibition of growth is partly due to a photoinactivation of IAA and that 2 iP and CCC might be active through processes controlling the levels of IAA and other growth hormones.     

The Inhibitory Effect of (2-Chloroethyl)-trimethylammonium Chloride on Chlorophyll and Protein Synthesis in Lettuce Cotyledons, and its Reversal by Potassium

Germinated seeds of Lactuca sativa (L.) were placed in Petri-dishesin (2-chloroethyl)-trimethyl-ammonium chloride (CCC; 0.005–0.05M), KN0₃ (0.01 M), and KC1 (0.01 M) solutions, and incubatedfor 2 or 5 days under continuous light. CCC strikingly arrestedchlorophyll accumulation, and retarded cotyledon growth relativelylittle. The retardant inhibited ¹⁴C-leucine incorporation intobulk proteins of the cotyledons. KN0₃ and KC1 promoted cotyledongrowth and chlorophyll synthesis per cotyledon by about 150per cent, and about doubled protein synthesis. Potassium saltscompletely reversed the inhibitory effects of CCC on chlorophylland protein synthesis. It is suggested that the inhibition ofgreening by CCC is dependent on a prior inhibition of proteinsynthesis.     

The Effects of Foliar Applications of (2-Chloroethyl)-trimethylammonium Chloride on Leaf Area and Dry Matter Production by the Brussels Sprout Plant

When CCC was applied as a spray to the leaves of Brassica oleraceaL. (Brussels sprout) grown in pots, plant height and mean internodelength were reduced. The effects appeared more slowly and wereless pronounced than those previously observed when CCC hadbeen applied to the soil; other differences were that root growthwas not inhibited, stem weight was only significantly reducedat the highest rate of application (2 per cent), and stomatalnumber per unit area of lower leaf epidermis was not affected.In common with soil applications, leaf thickness, stem diameter,and the percentage moisture contents of the leaves were allincreased by foliar applications.In a further experiment theprogress of wilting was observed in untreated plants and inplants treated with CCC applied either to the leaves or to thesoil. The rates of water loss and the moisture contents of theleaf laminae of the treated plants, after a period of wilting,were not significantly different from the controls. The treatedplants, however, looked less ‘wilted‘ for the changein angle of the leaf lamina to the stem was less and their leaveswere therefore held more upright.     

Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity

Rhizobium sp. strain WR1001, isolated from the Sonoran Desert by Eskew and Ting, was found to be able to grow in defined medium containing NaCl up to 500 mM, a concentration approaching that of sea water. Therefore, it is a valuable strain for studying the biochemical basis of salt tolerance. Intracellular free glutamate was found to increase rapidly in response to osmotic stress by NaCl. It accounted for 88% of the amino acid pool when the bacterium was grown in 500 mM NaCl. The role of glutamate dehydrogenase in glutamate biosynthesis was examined in several Rhizobium strains. Both NADH- and NADPH-dependent glutamate dehydrogenase activities in various Rhizobium strains were observed. The range of activity differed considerably depending on the particular strain. KCl (500 mM) did not stimulate glutamate dehydrogenase activity, as reported in a number of bacterial strains by Measures. The low activity of glutamate dehydrogenase in Rhizobium sp. strain WR1001 apparently cannot fulfill a biosynthetic function of glutamate formation in response to medium NaCl concentrations.     

Differences in Salinity Tolerance and Gene Expression Between Two Populations of Atlantic Cod (Gadus morhua) in Response to Salinity Stress

Populations of marine fish, even from contrasting habitats, generally show low genetic differentiation at neutral genetic markers. Nevertheless, there is increasing evidence for differences in gene expression among populations that may be ascribed to adaptive divergence. Studying variation in salinity tolerance and gene expression among Atlantic cod (Gadus morhua) from two populations distributed across a steep salinity gradient, we observed high mortality (45% North Sea cod and 80% Baltic Sea cod) in a reciprocal common garden setup. Quantitative RT-PCR assays for expression of hsp70 and Na/K-ATPase α genes demonstrated significant differences in gene regulation within and between populations and treatment groups despite low sample sizes. Most interesting are the significant differences observed in expression of the Na/K-ATPase α gene in gill tissue between North Sea and Baltic cod. The findings strongly suggest that Atlantic cod are adapted to local saline conditions, despite relatively low levels of neutral genetic divergence between populations.     

Enzymes of Choline Synthesis in Spinach (Response of Phospho-Base N-Methyltransferase Activities to Light and Salinity)

In spinach (Spinacia oleracea L.), choline is synthesized by the sequential N-methylation of phosphoethanolamine -> phosphomono- -> phosphodi- -> phosphotrimethylethanolamine (i.e. phosphocholine) followed by hydrolysis to release choline. Differential centrifugation of spinach leaf extracts shows that enzymes catalyzing the three N-methylations are cytosolic. These enzymes were assayed in leaf extracts prepared from plants growing under various light/dark periods. Under a diurnal, 8-h light/16-h dark photoperiod, the activity of the enzyme catalyzing the N-methylation of phosphoethanolamine is highest at the end of the light period and lowest following the dark period. Prolonged dark periods (exceeding 16 h) lead to a further reduction in the activity of this enzyme, although activity is restored when plants are reexposed to light. In contrast, the activity of the enzyme(s) catalyzing the N-methylations of phosphomono- and phosphodimethylethanolamine does not undergo comparable changes in response to light/dark treatments. Salt shock of plants with 200 mM NaCl results in a 2-fold increase in all three N-methylation activities relative to nonsalinized controls but only in plants exposed to light. Thus, light is required for the salt-responsive up-regulation of choline synthesis in spinach.     

Callus Induction by (2-Chloroethyl) Phosphonic Acid on Cultured Cotton Ovules

The response of cultured cotton (Gossypium hirsutum L.) ovules to the ethylene releasing growth regulator, (2-chloroethyl) phosphonic acid (ethephon), was measured. Control ovules grown on a complete liquid medium without hormones continued the differentiation normal for cotton, although at a slower rate than in vivo. When ethephon was added to the medium, normal organ and fiber development was inhibited but callus was induced from the micropylar end of the ovule. The callus was extremely friable and produced many free cells in the culture medium. Dry weight of the callused ovules increased over 4-fold and total cell number increased 56% over the controls. Apparently ethylene released from the ethephon stimulated both cell division and cell expansion in forming the callus.     

In Vivo Antiviral Activity of 1,3-Bis(2-Chloroethyl)-1-Nitrosourea

A prolongation in the lives of Swiss mice inoculated intracerebrally with lymphocytic choriomeningitis virus (LCM) was observed after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A variety of treatment schedules, including therapy once or twice daily up to 17 days and single treatments at various times after virus inoculation, were employed. Virus titers ranging to greater than 10⁴ were detected in the blood and brains of surviving drug-treated animals. In three comparative studies in which different treatment schedules were used, BCNU was shown to exert a protective effect approximately equal to that of methotrexate in LCM virus-infected mice. Tests were also carried out to investigate the activity of BCNU in mice experimentally infected with eastern equine encephalomyelitis (EEE) virus, western equine encephalomyelitis virus, Semliki Forest (SF) virus, herpes simplex virus, influenza virus strain PR8, vaccinia virus strain WR, Rous sarcoma virus, Friend leukemia virus (FLV), and poliovirus. Slight increases in life span were observed in the treated EEE, SF, and influenza PR8 virus-infected animals. Significant reduction in splenomegaly in FLV-infected animals treated with BCNU was demonstrated. The possible mechanisms of LCM virus inhibition by BCNU, on the basis of these and other studies, were postulated to be either specific antiviral activity or inhibition of “lethal” immune response to the LCM virus. Each of these postulates is discussed.     

Effect of (2-Chloroethyl)phosphonic Acid on CCC-Induced Delay of Fruit Ripening

(2-Chloroethyl)phosphonic acid (CEPA) which is known to releaseethylene in plant tissue, and (2-chloroethyl)trimethylammoniumchloride (CCC) were applied to ripening tomato and red pepperfruits. CEPA enhanced and CCC inhibited chlorophyll degradationand carotenoid formation. The inhibitory effects of CCC on fruitripening were counteracted by CEPA treatment. These resultsand those of other authors on gibberellin action in fruit ripeningsuggested that both CCC and gibberellin may interfere with theaction of ethylene in ripening fruit.     

Response of Melanthera biflora to Salinity and Water Stress

Melanthera biflora (Asteraceae) is a moderately salt-tolerantplant from the Indo-Pacific region. In laboratory studies itsgrowth was inhibited by salt above 50 mol m^–3, but itwas able to survive salinities approaching that of seawater,namely 400 mol m^–3. Shoot potassium concentrations weremaintained over a range of salinities up to 400 mol m^–3,while sodium and chloride accumulation followed closely theincrease in external osmotic pressure. In contrast, the increasein osmotic pressure of the leaf sap of Melanthera biflora, subjectedto water stress, was due mainly to a decrease in the ratio offresh weight/dry weight. 3-dimethylsulphoniopropionate (3-DMSP)and glycinebetaine were identified by fast atom bombardmentmass and ¹H -NMR spectroscopy, with 3-DMSP being the main oniumcompound and glycinebetaine absent in some accessions. Onium(quaternary ammonium and/or tertiary sulphonium) compounds andproline increased during salt and water stress due mainly toa decrease in the fresh weight/dry weight ratio of tissue, althoughpart of the increase in salt-stressed tissue was due to an increasein the accumulation of the onium compound. This salt-inducedincrease in 3-DMSP was inhibited in conditions of low sulphursupply and there was no compensatory increase in proline. Key words: Melanthera biflora, Asteraceae, salinity, glycinebetaine, 3-dimethylsulphonioproprionate