Acute and sub-lethal effects in juvenile Atlantic salmon exposed to low µg/L concentrations of Ag nanoparticles.

Silver nanoparticles (Ag-NP) are components in numerous commercial products and are discharged into the environment in quantities that are largely unknown. In the present study, juvenile Atlantic salmon were exposed to 1, 20, and 100 µg/L (48 h, static renewal) of a commercially available Ag-NP colloidal suspension in natural (soft) lake water. A solution of AgNO(3) containing 20 µg/L Ag(I) ions was also included to discriminate the effect of NPs from that of ionic silver. Furthermore, the commercial Ag-NP suspension was compared to an in-house synthesised colloidal NP suspension prepared from AgNO(3) and NaBH(4) in citrate buffer. The size distribution of Ag in all exposure solutions was characterised by 0.22 µm filtration and 10 kDa hollow fibre cross-flow ultrafiltration in combination with ICP-MS. All exposures were characterised by a relatively high proportion of Ag-NP in the colloidal size fraction 3-220 nm. For assessment of biological effects, acute toxicity, gill histopathology, blood plasma parameters (Na, Cl, glucose, haemoglobin), and gene expression of a selection of gill biomarkers were measured. Results showed that the gills accumulated Ag in all exposure groups apart from the fish exposed to 1 µg/L Ag-NP. Accumulated Ag caused concentration-dependent response increases in general stress markers such as plasma glucose and gill gene expression of heat shock protein 70. Furthermore, induction of the metallothionein A gene indicated that Ag had been internalized in the gills, whereas a concentration-dependant inhibition of Na/K ATPase expression indicated impaired osmoregulation at as low as 20 µg/L concentrations of Ag-NP. The commercial Ag-NP suspension caused acute gill lamellae necrosis at high concentrations (100 µg/L), potentially giving rise to the substantial (73%) fish mortality at this concentration. The two different Ag-NP preparations gave comparable results for several endpoints measured, but differed in MT-A induction and mortality, thus emphasising the variation in effects that may arise from different Ag-NP preparations.

Hydroxylation of N-(Delta-Isopentenyl)adenine to Zeatin: Relative Activities of Organ Systems from Actinidia arguta.

By incubating explants from Actinidia arguta seedlings on a nutrient medium supplemented with 20 to 30 micromolar N(6)-(Delta(2)-isopentenyl)adenine (i(6)Ade) and then measuring zeatin (io(6)Ade) accumulation in tissues, the distribution of i(6)Ade hydroxylase activities in whole plants could be determined. Based on analyses with three entire plants, it is estimated that, as an organ system, roots contain approximately 68% of the plant's hydroxylase, while stems and leaves account for about 26% and 6%, respectively, of the total activity. Depending on the part of the root examined, hydroxylase activities ranged from 20 to 148 nanomoles io(6)Ade accumulated per gram fresh weight per 24 hours of incubation. Stem activities ranged from 17 to 165 nanomoles per gram fresh weight per 24 hours with the lowest activities being found at the tip. Leaf activities were substantially lower (1-10 nanomoles per leaf depending on position) than either root or stem.

Biosynthesis of zeatin from N-(Delta-isopentenyl)adenine in Actinidia: Sites and seasonal changes in activity.

In Actinidia arguta (hardy kiwifruit) plants, the potential to accumulate the cytokinin zeatin (io(6)Ade) during feeding with a precursor, N(6)-(Delta(2)-isopentenyl)adenine (i(6)Ade), varies depending on the tissue. This can be demonstrated by incubating explants for 24 hr on a basal nutrient medium supplemented with 30 muM i(6)Ade and then extracting the tissues and analyzing cytokinin contents using HPLC methodology. Under these conditions, the potential for io(6)Ade accumulation in tissue slices from growing roots was 93 nmol/g at the root tip, >200 nmol/g immediately behind the tip (1.0-mm diameter), approximately 100 nmol/g in 2-mm diameter root, and progressively lower in older tissues. A similar gradient of io(6)Ade accumulation was detected in growing stems, with relatively low activity (40 nmol/g) in the terminal 0.5 cm, approximately 170 nmol/g in the 5- to 15-cm interval, and about 25 nmol/g in stem tissues taken 15-100 cm from the tip. Growing leaves accumulated little io(6)Ade (7-26 nmol/g) during feeding, as did fruits (0-8 nmol/g) at various stages of development and maturation. Seasonally, root activity was detected as early as March 1, whereas stem activity did not appear until March 15. Thus, the activation of i(6)Ade metabolism in both of these organs preceded sap flow (March 29) and bud break (April 16) by several weeks. The results suggest that root and stem tissues may be sites of cytokinin biosynthesis in growing Actinidia plants and that cytokinin production may not be the critical factor controlling the beginning of shoot growth in the spring.

Zeatin biosynthesis from N-(Delta-isopentenyl)adenine in Actinidia and other woody plants.

Tissue cultures of Actinidia kolomikta can be maintained as callus through repeated passages on a nutrient medium devoid of cytokinin but containing inorganic nutrients, sucrose, and other basal organics plus auxin. Under these conditions, actively growing callus contained 2 and 0.5 nmol of the cytokinins zeatin [io(6)Ade; 6-(4-hydroxy-3-methylbut-2-enylamino)purine] and N(6)-(Delta(2)-isopentenyl)adenine (i(6)Ade), respectively, per gram (fresh weight). When tissues were transferred from cytokininless medium to 30 muM i(6)Ade, endogenous io(6)Ade increased linearly to 160 nmol/g (fresh weight) during 8 hr, and i(6)Ade increased to 5 nmol/g (fresh weight) in 2 hr and then declined. The apparent K(m) for i(6)Ade in A. kolomikta and Actinidia chinensis x Actinidia arguta callus and in tissue slices of A. arguta stems was 12 muM. In addition, the reaction(s) converting i(6)Ade to io(6)Ade was O(2)-requiring and specific for i(6)Ade versus N(6)-(Delta(2)-isopentenyl)adenosine (i(6)A). When A. kolomikta callus was fed 30 muM i(6)A, io(6)Ade increased and reached a concentration corresponding to 6 nmol/g (fresh weight) in 8 hr. Ribosylzeatin (io(6)A) did not increase. Under N(2) during i(6)A feeds, i(6)A accumulated rather than being metabolized to i(6)Ade, suggesting that i(6)A normally may be metabolized via i(6)AMP and i(6)Ade to io(6)Ade. A survey of 30 species of woody plants in 20 families of dicotyledonous angiosperms indicated that the ability to accumulate io(6)Ade (>/=10 nmol/g) in 24-hr feeds with 30 muM i(6)Ade was restricted to certain systematic groups-e.g., order Ericales, families Oleaceae and Rubiaceae. This suggests that plants may differ in their pathways for io(6)Ade biosynthesis and that cytokinin biochemistry has potential as a taxonomic character above the species and genus levels.

Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues.

Tissue cultures grown from stem explants of three Actinidia species and a hybrid species rapidly converted N6-isopentenyladenine (i6Ade) to zeatin (io6Ade), a potent hydroxylated cytokinin. Within 24 h on 50 uM i6Ade, callus tissues of A. chinensis x arguta accumulated 83 +/- 6 nmol/g io6Ade which was purified using HPLC and identified by its characteristic UV and mass spectra. Activity converting i6Ade to io6Ade was also demonstrated in stem segments from intact plants where it was low in the tip (3 cm), highest in the region corresponding to rapid leaf growth and very low in the mature stem. Root segments converted i6Ade to io6Ade almost as rapidly as the most active region of the stem while leaf petioles produced little io6Ade. Fruits of A. arguta and A. chinensis produced little or no io6Ade, respectively.

Citrus Tissue Culture : AUXINS IN RELATION TO ABSCISSION IN EXCISED PISTILS.

An in vitro bioassay for chemicals that affect Citrus abscission was used to identify three inhibitors of stylar abscission in lemon pistil explants incubated on defined nutrient media. The three inhibitors (picloram, 4-chlorophenoxyacetic acid, and 3,5,6-trichloropyridine-2-oxyacetic acid) are all auxins, and the most potent of them (i.e. picloram) was found to be at least 10 times more active in the bioassay than 2,4-dichlorophenoxyacetic acid. Picloram (2 micromolar) also was shown to be effective in inhibiting stylar abscission in pistil explants from other Citrus cultivars such as mandarin, Valencia, and Washington navel oranges and grapefruit. To study the physiology of auxins active as abscission inhibitors versus inactive auxins in lemon pistils, the transport and metabolism of [1-(14)C]-2,4-dichlorophenoxyacetic acid was compared with that of [2-(14)C]indole-3-acetic acid, which is without effect in the bioassay over the range from 0.1-100 micromolar. Insignificant quantities of labeled indole-3-acetic acid and/or labeled derivatives were found to reach the presumptive zone of stylar abscission under the test conditions. Labeled 2,4-dichlorophenoxyacetic acid and/or labeled derivatives also were transported slowly through pistils, but some radioactivity could be detected in the stylar abscission zone as early as 24 hours after the start of incubation. Extensive conversion of [2-(14)C]indole-3-acetic acid to labeled compounds tentatively considered to be glycoside and cellulosic glucan derivatives was found with the use of solvent extraction methodology. A significantly smaller percentage of the radioactivity in pistils incubated on [1-(14)C]-2,4-dichlorophenoxyacetic acid was found in fractions corresponding to these derivatives. Both transport and metabolism appear to be important factors affecting the activity of auxins as abscission inhibitors in the bioassay.

Citrus tissue culture: stimulation of fruit explant cultures with orange juice.

In vitro growth of explant (juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice (10% v/v optimum) to a basal medium containing Murashige and Skoog salts, 50 grams per liter sucrose, 100 milligrams per liter myo-inositol, 5 milligrams per liter thiamine.HCl, 2 milligrams per liter 2,4-dichlorophenoxyacetic acid and 0.5 milligrams per liter kinetin. In analyzing this effect of orange juice on citron explant cultures, we failed to obtain increased yields by addition of appropriate concentrations of citric acid to the basal medium but obtained growth stimulation when the medium was supplemented with juice from an "acidless" orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid is involved. Addition of the inorganic ash corresponding to 10% (v/v) orange juice to the basal medium had no effect on yields. Similarly, the stimulatory effect of orange juice could not be explained based on its content of sucrose or of organic growth factors already present in the basal medium.

Two effects of cytokinin on the auxin requirement of tobacco callus cultures.

Cytokinin affects the requirement for auxin of a strain of tobacco callus (Nicotiana tabacum) which is cytokinin-autotrophic when grown on Murashige and Skoog medium with 11.4 mum of indole-3-acetic acid but requires cytokinin 6-(3-methyl-2-butenylamino)purine (i(6) Ade) when grown on the same medium with <3 mum indole-3-acetic acid. As the exogenous concentration of cytokinin (i(6) Ade) is increased, the concentration of indole-3-acetic acid required for growth is decreased. A second effect of cytokinin, observed sporadically in cultures with 2.5 mum or 5 mum i(6) Ade, is the transformation of some of the callus pieces to auxin-autotrophic growth. Strains, both callus-forming and bud-forming tissues, that arise in this manner are not permanently altered in their auxin requirement because subcultures on medium without cytokinin still require exogenous auxin.

Ribosyl-cis-zeatin in a Leucyl Transfer RNA Species from Peas.

tRNA(6) (Leu) in Pisum sativum seed has been purified. This tRNA species contains a cytokinin-active nucleoside and accounts for approximately 7% of the total cytokinin activity in acid hydrolysates of pea tRNA. The cytokinin has been identified as ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino) -9-beta-d-ribofuranosylpurine.

Biosynthesis of cytokinins in cytokinin-autotrophic tobacco callus.

A cytokinin-autotrophic strain of tobacco callus contained cytokinin-active compounds with chromatographic mobilities on Sephadex LH-20 corresponding to ribosylzeatin, zeatin, and 6-Delta(2)-isopentenylaminopurine. Zeatin, the apparent major cytokinin, was estimated to be present at a concentration of 10(-4) mumol/kg of tissue. Cytokinin-autotrophic callus supplied with [(14)C]adenine produced radioactive components with the same chromatographic properties as zeatin and 6-Delta(2)-isopentenylaminopurine. These components were not obtained from cytokinin-dependent tissue supplied with [(14)C]adenine in the same manner.