Persistent Effects of Short-term, High Exposure to Chlorine Gas on Physiology and Growth of Pinus ponderosa andPseudotsuga menziesii

Following a single acute exposure to chlorine gas, persistenteffects on epicuticular waxes, cuticular transpiration, treegrowth and mortality were studied in foliage of Pinus ponderosaand Pseudotsuga menziesii for three growing seasons. Chlorinegas exposure caused foliar injury to both exposed foliage andfoliage that flushed after exposure (P < 0.05). The tendencyto form films of water rather than droplets was greater in directlyexposed foliage (P < 0.001). Rates of cuticular transpirationwere higher for directly and indirectly exposed foliage of Pinusponderosa up to 1 year after exposure and up to 6 months afterexposure for directly exposed Pseudotsuga menziesii(P < 0.001),after which P. menziesii needles defoliated. Total water content(TWC) and relative water content were significantly correlatedwith foliar injury (P < 0.05). TWC was lower for directlyexposed foliage up to 1 year after exposure (P < 0.001).There was no persistent negative effect on F_v/F_m ratios after1 year. Exposure to chlorine gas did not affect needle lengthor annual shoot increment growth, but exposure was correlatedwith increased bud production. Needle longevity of foliage thatflushed 2 months after exposure was reduced significantly (P< 0.001). Annual stem increment growth for both species decreasedover at least three growing seasons following chlorine gas exposure(P < 0.001), and depended on distance from the spill site.Cone production was lower for exposed Pinus ponderosa treescompared to controls (P < 0.05), and tree mortality was higherwithin approx. 50 m of the release site forPseudotsuga menziesii. Growth responses for both conifers agreed well with predictedpatterns of carbon allocation after defoliation caused by chlorinegas exposure. Copyright 2001 Annals of Botany Company Pinus ponderosa, Pseudotsuga menziesii, conifers, chlorine gas, leaf wettability, cuticular transpiration, water relations, growth, mortality     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Cytokinin metabolism in tomato plants. II. Metabolites of kinetin and benzyladenine in decapitated roots

Decapitated tomato plants were supplied via the roots with [8-¹⁴C]-kinetin or [8-¹⁴C]-benzyladenine in a nutrient solution for a period of 24 h. After this time the root material, the root sap produced during the 24 h period and the nutrient solution remaining at the end of the experiments were analysed for cytokinins. HPLC techniques and chemical treatments were used to tentatively identify radioactive metabolites formed. Uptake of kinetin and benzyladenine by the roots was found to be limited but once within the root tissues metabolism was both rapid and extensive.At least 14 metabolites of kinetin were recovered from root tissue and root sap. Many of these appeared to be degradation products. There was, however, some evidence of formation of zeatin-like derivatives. Side-chain cleavage of the original kinetin which occurs rapidly is suggested as a possible route for the eventual production of these endogenous cytokinin forms.The benzyladenine taken up by the roots was apparently both ribosylated and glucosylated. No unmetabolized benzyladenine was detected in the root tissues after 24 h. Only very low levels of radioactivity were associated with the retention time of adenine, suggesting that in the case of benzyladenine side-chain cleavage is of limited importance.The significance of these reactions in relation to the potential use of cytokinins in the regulation of plant growth is discussed.     

A new approach to polarized fluorescence using phase and modulation fluorometry

In the preceding paper, an alternative method is described for obtaining information about the reorientational behavior of a fluorophore in a membrane system from frequency domain measurements. To demonstrate this new analysis procedure, we present data for the probe-molecule 1,6-diphenyl-1,3,5-hexatriene (DPH) in l--dimyristoyl- and l--dipalmitoylphosphatidylcholine (DMPC and DPPC) obtained with two different phase fluorometers: the SLM 4800A Subnanosecond Spectrofluorometer which has only three fixed frequencies available (6, 18 and 30 MHz) and the recently constructed continuously variable multifrequency phasefluorometer (Gratton and Limkeman 1983).It will be shown that reasonable information about the anisotropy behavior of a fluorophore can be obtained even if only three frequencies are used. The phase modulation technique was also used to check the new expression for the anisotropy, r(t), called the general model and introduced by Van der Meer et al. (1984). The parameters P ₂, P ₄ and D, obtained from the nonlinear least squares fit (Bevington 1969) for this general model, confirm the results from the pulse technique of Ameloot and coworkers (Ameloot et al. 1984; Pottel et al. 1986).     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Biphasic fluence-response curves for phytochrome-mediated kalanchoë seed germination : sensitization by gibberellic Acid

The fluence-response curves for the effect of two red pulses separated by 24 hours on the germination of Kalanchoe blossfeldiana Poelln. cv Vesuv seeds, incubated on gibberellic acid (GA₃) are biphasic for suboptimal concentrations. The response in the low fluence range corresponds with a classical red/far-red reversible phytochrome mediated reaction. GA₃ induces an additional response in the very low fluence range, which is also phytochrome mediated. The sensitivity to phytochrome-far-red absorbing form (Pfr), however, is increased about 20,000-fold, so that even far-red fluences become saturating. Both in the very low and low fluence response range, the maximal responses induced by saturating fluences are modulated by the GA₃ concentration. GA₃ having no direct influence on the phytochrome phototransformations, alters the Pfr requirement and determines the responding seed population fraction in the very low and low fluence range. The effet of GA₃ appears to be on the transduction chain of the phytochrome signal.     

Ionic balance,proton efflux,nitrate reductase activity and growth ofHippophaë rhamnoides L. ssp.rhamnoides as influenced by combined-N nutrition or N2 fixation

Growth of 2-month-old nonnodulatedHippophaë rhamnoides seedlings supplied with combined N was compared with that of nodulated seedlings grown on zero N. Plant growth was significantly better with combined N than with N₂ fixation and, although not statistically significant for individual harvests, tended to be highest in the presence of NH ₄ ⁺ , a mixture of NH ₄ ⁺ and NO ₃ ^? producing the highest yields. Growth was severely reduced when solely dependent on N₂ fixation and, unlike the combined-N plants, shoot to root ratios had only slightly increased after an initial decrease. An apparently insufficient nodule mass (nodule weight ratio <5 per cent) during the greater part of the experimental period is suggested as the main cause of the growth reduction in N₂-fixing plants. Thein vivo nitrate reductase activity (NRA) of NO ₃ ^? dependent plants was almost entirely located in the roots. However, when grown with a combination of NO ₃ ^? and NH ₄ ⁺ , root NRA was decreased by approximately 85 per cent.H. rhamnoides demonstrated in the mixed supply a strong preference for uptake of N as NH ₄ ⁺ , NO ₃ ^? contributing only for approximately 20 per cent to the total N assimilation. Specific rates of N acquisition and ion uptake were generally highest in NO ₃ ^? +NH ₄ ⁺ plants. The generation of organic anions per unit total plant dry weight was approximately 40 per cent less in the NH ₄ ⁺ plants than in the NO ₃ ^? plants. Measured extrusions of H⁺ or OH^? (HCO ₃ ^? ) were generally in good agreement with calculated values on the basis of plant composition, and the acidity generated with N₂ fixation amounted to 0.45–0.55 meq H⁺. (mmol N_org)^?1. Without acidity control and in the presence of NH ₄ ⁺ , specific rates of ion uptake and carboxylate generation were strongly depressed and growth was reduced by 30–35 per cent. Growth of nonnodulatedH. rhamnoides plants ceased at the lower pH limit of 3.1–3.2 and deterioration set in; in the case of N₂-fixing plants the nutrient solution pH stabilized at a value of 3.8–3.9 without any apparent adverse effects upon plant performance. The chemical composition of experimental and field-growing plants is being compared and some comments are made on the nitrogen supply characteristics of their natural sites.