Polyamines in normal and auxin-induced strawberry fruit development

The possible involvement of polyamines during strawberry ( Fragaria × ananassa Duch.) fruit development was investigated. Putrescine, spermidine, and spermine were identified in strawberry receptacles and achenes at all stages of development. Total (free) polyamine levels decreased from a maximum of 485 nmol g⁻¹ fresh weight at pollination to a minimum of 55 nmol g⁻¹ fresh weight in ripe receptacles. Total polyamine concentrations during corresponding stages of development were consistently higher in achenes than in receptacles, and ranged from 891 to 203 nmol g⁻¹ fresh weight. Removal of achenes from the surface of developing receptacles 10 days after pollination reduced receptacle growth, and re-initiation of growth by application of 1 m M α-naphtaleneacetic acid (α-NAA) was accompanied by a rapid increase in polyamine concentrations 24 h after treatment. Polyamine content per receptacle increased >3-fold in normally developing receptacles and in de-achened, auxin-treated receptacles 10 days after removal of achenes, but did not increase during this period in de-achened receptacles not treated with exogenous auxin. α-NAA increased growth and polyamine levels to a greater extent than the structurally related, but less effective auxin, β-NAA. Polyamine concentrations in receptacles with intact achenes remained similar to those of auxin depleted (de-achened) receptacles, implying that the concentration of these compounds may not be limiting following achene removal.     

Accumulation of a glycine rich protein in auxin-deprived strawberry fruits

Growth of strawberry (Fragaria ananassa Duch. cv. Ozark Beauty) receptacles is regulated by auxin supplied from the achenes. The receptacle growth can be stopped at any stage by deachening the fruits, and can be resumed by exogenous application of auxin. In our earlier studies we demonstrated auxin regulated polypeptide changes at different stages of strawberry fruit development. Removal of achenes from fruits and growing the receptacles without auxin resulted in the time-dependent accumulation of 52,000 Mr polypeptide. Amino acid analysis revealed that the protein is rich in glycine. Our studies, with normal and variant strawberry receptacles, indicate that the synthesis and accumulation of this glycine-rich protein correlates with cessation of receptacle growth. These results suggest a possible role for the glycine-rich protein in cessation of growth.     

Endogenous levels of polyamines and abscisic acid in pepper fruits during growth and ripening

The levels of polyamines (PA) and abscisic acid (ABA) in the pericarp of California variety pepper fruit ( Capsicum annuum L.) were analyzed during development and ripening. Putrescine level was 2.75 μmol g⁻¹ fresh weight 7 days after fruit set and fell during the exponential stage of growth to 1.05 μmol g⁻¹ fresh weight. During the second growth stage. PA and ABA levels remained stable and fell sharply at the beginning of maturation. The levels of spermidine and spermine decreased throughout fruit development and maturation from 0.61 to 0.05 and 0.31 to 0.02 μmol g⁻¹ fresh weight, respectively, but no changes were associated with the onset of maturation. ABA levels remained high (0.70-0.80 μg g⁻¹ fresh weight) during the stages of fruit growth and fell at the beginning of maturation to 0.12 μg g⁻¹ fresh weight, before rising again during the last stages of maturation and senescence. The decrease in putrescine and ABA levels and the subsequent increase in the latter may be responsible for controlling the processes of ripening in pepper fruit.     

Physiological adaptation during cell dehydration and rewetting of a newly-isolated Chlorella species

A new terrestrial species of green alga ( Chlorella sp. strain DT) that survives under extremely dry conditions was isolated from an arid mountainous area of Taiwan. The water content of the cells dropped to less than 3% after 3 h dehydration at 42°C. The dried cells could regrow if they were rewetted. The photosynthetic activity of the cells ceased following 2 h of dehydration, but respiratory activity was still detectable after 3 h desiccation. During the rewetting process, respiration increased immediately and then decreased to a steady level after 5 days of rewetting, matching the net photosynthetic oxygen evolution. The cells had 38% neoxanthin (1520 nmol g⁻¹ dry weight), 39% lutein (1580 nmol g⁻¹ dry weight), and 14% violaxanthin (570 nmol g⁻¹ dry weight) of total carotenoids, but only 5.1%β-carotene (210 nmol g⁻¹ dry weight), 3.8%α-carotene (150 nmol⁻¹ g dry weight) and a trace of antheraxanthin and zeaxanthin. Upon dehydration, zeaxanthin and carotene contents increased and recovered to normal levels after 8 days of rewetting. The photoprotective xanthophyll cycle was found in these cells. It appears that the energy dissipation process for preventing photodamage is perhaps one of the tolerance mechanisms in this Chlorella strain.     

Correlation between Lack of Receptacle Growth in Response to Auxin and Accumulation of a Specific Polypeptide in a Strawberry (Fragaria ananassa Duch.) Variant Genotype

Receptacle growth in strawberry (Fragaria ananassa Duch. cv.Ozark Beauty) occurred after either pollination or auxin treatment.In a strawberry variant genotype (Washington State UniversitySelection No. 12/13), pollination did not lead to receptaclegrowth but application of -naphthaleneacetic acid (NAA) at anthesisresulted in normal receptacle growth. The receptacles of OzarkBeauty retained their ability to respond to auxin at least upto 36 days after anthesis. However, delay of auxin applicationto the receptacles of the variant genotype resulted in decreasedauxin-responsive growth and auxin application after the 10thday of anthesis led to very little growth. The loss of auxin-responsivegrowth of the receptacle of the variant genotype was not associatedwith any loss of auxin binding activity of receptacle membranes.If auxin was not supplied to the receptacles of the variantgenotype at anthesis, the receptacles did not grow and a polypeptideof 52,000 M_r accumulated. Application of NAA to the receptaclesof the variant genotype at anthesis or on the fifth day afteranthesis resulted in the growth of the receptacle and the 52,000M_r polypeptide did not accumulate. Application of NAA to thereceptacles of the variant genotype on the 10th or the 15thday after anthesis led to very little growth of the receptacleand the 52,000 M_r polypeptide accumulated to high levels. Theseresults suggested a correlation between the lack of receptaclegrowth in response to auxin and accumulation of the 52,000 M_rpolypeptide. ¹ Current adress: The Institute of Applied Research, Ben GurionUniversity of the Negev, Beer-Sheva, Israel. (Received August 6, 1984; Accepted December 11, 1984)     

Polyamines in marine macroalgae: Levels of putrescine, spermidine and spermine in the thalli and changes in their concentration during glycerol-induced cell growth in vitro

The levels of putrescine (Put), spermidine (Spd) and spermine (Spm) were analyzed in naturally collected samples of the marine macroalgae Dyctiota dichotoma, Gelidium canariensis and Grateloupia doryphora . Polyamines (PAs) appeared in free (35–134 μg g⁻¹ fresh weight) and bound TCA-insoluble form (1 667–2 624 μg g⁻¹ fresh weight). Axenic in vitro cultures of sporelings from G. doryphora were established in the medium containing glycerol. This medium promoted growth and morphogenesis and also increased the free and bound PA levels in the sporelings. Tracer experiments using 70 kBq [U-¹⁴C]-glycerol showed significant quantities of radioactivity in Put, Spd and Spm after 20 h of incubation. The effects of glycerol on growth were inhibited by the ornithine decarboxylase (EC 4.1.1.17) inhibitor α -difluoromethylornithine (DFMO). The presence of DFMO in the incubation medium with [U-¹⁴C]-glycerol also reduced the radioactivity in PAs.     

Antioxidant status of anoxia-tolerant and -intolerant plant species under anoxia and reaeration

The redox potential of the cell, as well as the antioxidant status of the tissue, are considered to be important regulatory constituents in an adaptive response in plants. Here the involvement of active antioxidants ascorbic acid (AA), reduced glutathione (GSH) and α - and β -tocopherols in reactive oxygen species scavenging, and the effect of anoxic stress on their reduction state were studied in 4 anoxia-tolerant and -intolerant plant species: Iris germanica L., Iris pseudacorus L., wheat ( Triticum aestivum L. cv. Leningradka) and rice ( Oryza sativa L. cv. VNIIR). The initial antioxidant content (both AA and GSH) was higher in the rhizomes of the more anoxia-tolerant Iris spp., as compared with that of the roots of the cereals. The predominant form of ascorbate was dehydroascorbic acid (DHA) in the cereals and AA in the Iris spp. Imposition of anoxia with subsequent reoxygenation resulted in an overall depletion of the reduced forms of antioxidants. No concurrent increase in oxidised forms (DHA and conjugated glutathione) was observed in anoxic samples. α -tocopherol content in Iris spp. was in the range 1–2 μg g⁻¹ fresh weight, while β -tocopherol content was higher in the anoxia-intolerant I. germanica (7.2 μg g⁻¹ fresh weight) as compared with the tolerant I. pseudacorus (1.5 μg g⁻¹ fresh weight). In I. pseudacorus , a significant decrease in α - and β -tocopherol levels was observed only after long-term (45 days) anoxia. The results suggested exclusion of AA and GSH from the redox cycling under prolonged anoxia, and a concomitant decrease in the redox state, as well as an anoxia-induced depletion of α - and β -tocopherols.     

Ethylene synthesis and growth in embryogenic tissue of Norway spruce: Effects of oxygen, 1-aminocyclopropane-1-carboxylic acid, benzyladenine and 2,4-dichlorophenoxyacetic acid

Ethylene production from an embryogenic culture of Norway spruce ( Picea abies L.) was generally low. ca 2.5 nl g⁻¹ h⁻¹, whereas 1-aminocyclopropane-1 -carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g⁻¹ during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O₂) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 μ M ) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 μl I⁻¹) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2.4-dichloro-phenoxyacetic acid (2.4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g⁻¹ h⁻¹ within the 11-day incubation period. Although 2.4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g⁻¹, was observed in tissue treated with 2.4-D (22.5 μ M ) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent K_m was 50 μ M and V_max 2.7 nl g⁻¹ h⁻¹. Growth of the tissue was strongly inhibited by 2.4-D in the absence of BA, but weakly in the presence of BA (4.4 μ M ). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.     

Ascorbic acid and α-tocopherol levels in larvae of Atlantic halibut before and after exogenous feeding

Ascorbic acid (AA) and α-tocopherol (α-TOH) levels in whole Atlantic halibut larvae were constant during the yolk sac stage at 170 and 131 ng individual⁻¹, respectively. At hatching c . 80% of the AA and 97% of the α-TOH were contained within the yolk-sac compartment. With development, AA and α-TOH levels in the yolk decreased, at different rates. At first feeding (at 200 day degrees post hatch, D°PH)>95% of AA but <30% of α-TOH in the yolk at hatching had been transferred to the larval body. Transfer of α-TOH was completed at 360 D°PH, when the yolk was completely absorbed. The plankton offered to the larvae at first feeding (chiefly Temora longicornis ) contained 756 μg g⁻¹ AA and 120 μg g⁻¹α-TOH (dry weight). The AA content increased to 472 ng individual⁻¹ within one week after first feeding, while it declined slightly in unfed larvae. In fed larvae the AA content reached c . 3500 ng individual⁻¹ at 580 D)PH. The α-TOH content increased only slightly in the first week of feeding (206 to 431 D°PH), but then increased to > 800 ng individual⁻¹ at 483 D°PH.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

Species of Anguilla as indicators of mercury in the coastal rivers and lakes of Victoria, Australia

Mercury concentrations in the axial muscle tissue of most (243) of the 254 Anguilla australis and most (20) of the 27 A. reinhardtii collected from 30 sites in coastal rivers and lakes in Victoria, Australia, during 1975–78 were well below the Australian statutory health limit (0.5 μg g⁻¹ wet weight). For A. australis the mean mercury concentration was 0.17 μg g⁻¹ (±0.16 s.d. , range 0.01–1.60 μg g⁻¹); for A. reinhardtii the values were 0.37 ± 0.23 μg g⁻¹ (range 0.12–1.10 μg g⁻¹). Statistical analyses showed that variation in mercury concentration due to total length accounted for only 13% of the total variation in A. australis and 2% in A. reinhardtii whereas locality accounted for 54 and 68%, respectively. Both species are thus considered suitable as indicators of mercury pollution.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

The respiration of young coho Oncorhynchus kisutch at gradually declining oxygen levels and during recovery from oxygen deprivation

The respiration of coho salmon, Oncorhynchus kisutch , weighing between 15 and 50 g was measured at gradually declining oxygen levels and at temperatures ranging between 14 and 17°C. The maximum and minimum oxygen concentrations tested were 250 and 40 μmol L⁻¹, respectively. Respiration rates were measured for 1 h periods before oxygen concentration was lowered by 12.5 or 25.0 μmol oxygen L⁻¹. At the end of these endurance tests the oxygen level was returned to normoxic conditions and respiration rates were determined for the recovery period. Under normoxic conditions (> 200 μmol L⁻¹) the respiration of coho levelled around 5.1 μmol g⁻¹ wet weight h⁻¹. At intermediate levels between 150 and 200 μmol oxygen L⁻¹, the average rate increased to 5.8 μmol g⁻¹ h⁻¹, which could be attributed to higher spontaneous activity of the test animals. At low oxygen levels (< 150 μmol⁻¹) average respiration rates dropped to values between 5.5 and 5.7 μmol g⁻¹ h⁻¹, reaching a minimum of 3.8 μmol g⁻¹ h⁻¹ at oxygen levels below 50 μmol L^μ. First mortality was observed in this range. After exposure to reduced oxygen levels the fish maintained a higher respiration rate when again exposed to normoxic oxygen levels above 200 μmol L⁻¹. Increased respiration rates were observed for a recovery period of 6 h.     

Strawberry fruit protein with a novel indole-acyl modification

Achenes and receptacle tissue of Fragaria vesca, L. cultivar Yellow Wonder were shown to contain conjugated indole-3-acetic acid (IAA) that was not soluble in organic solvents and yielded IAA after strong alkaline hydrolysis, suggestive of IAA attached to plant proteins. This solvent insoluble conjugated IAA accounted for between 0.4 and 4 ng of IAA per gram fresh weight of tissue in both achenes and receptacles. To investigate this strawberry conjugate class further, a polyclonal antibody was produced to IAA–glycine attached to BSA that detected neutral indole acid esters, monocarboxylic-amino acid IAA conjugates and IAA proteins. Using immunoblotting, both achenes and receptacles of strawberry were shown to have primarily an immuno-detectable band at 76 kDa. Two-dimensional polyacrylamide gel electrophoresis yielded a wide band that was analyzed by LC–MS/MS analysis following in-gel trypsin digestion. Peptides derived from the immuno-detectable band were tentatively identified by peptide fragment analysis as being from either a chaperonin related to the hsp60 class of proteins or, alternatively, an ATP synthase. This is one of the first reports of an IAA modified protein in fruit tissue.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Endogenous cytokinin levels and metabolism of zeatin riboside in genetic tumour tissues and non-tumourous tissues of tobacco

The cytokinin content of stem tissues, primary genetic tumours (excised from 2-month-old plants) and 3-week-old in vitro cultured genetic tumour tissues derived from Nicotiana glauca (Grah.) × langsdorffii (Weinm.) and N. suaveolens (Lehm.) × langsdorffii (Weinm.) hybrids and stem tissues derived from 2-month-old N. suaveolens and N. langsdorffii plants has been analysed by radioimmunoassay. Stem tissues of tumour-prone hybrids contain high cytokinin levels (3–3.7 nmol g⁻¹). This increase is caused mainly by increased levels of cytokinin nucleotides, particularly those of zeatin nucleotide (0.5 nmol g⁻¹) in stem tissues of parent plants and 2.4 nmol g⁻¹ in stem tissues of hybrids). All other tissues contain lower cytokinin levels (0.7–1.7 nmol g⁻¹). Cytokinin bases and ribosides are major compounds in cultured tumour tissues while the nucleotides are dominant cytokinins in all freshly excised tissues from parent plants and their hybrids. In a separate study, the metabolic fate of supplied [³Hj-zeatin riboside. which is inactivated mainly by sidechain cleavage, has been studied. The results collectively suggest that cytokinins may be involved in tumourigenesis.     

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

The cytokinin content in fruit tissue of the kiwifruit ( Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high-performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O - and 9-glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography-mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O - and 9-glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g⁻¹ fresh weight at anthesis to 41 pmol g⁻¹ fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g⁻¹ fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g⁻¹ fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.