Monitoring hairy-root growth by image analysis

Hairy roots, incited byAgrobacterium rhizogenes, form a useful system for analysing the expression and phenotypic effects of foreign genes in plant root tissue. Image analysis offers a non-invasive method of describing their growth in culture. Images of pea (coarse) andBrassica (fine) hairy roots were captured, processed and analysed without difficulty using a commercially available image analysis system. The value of this method in monitoring intermediate changes in growth pattern was illustrated by following the changes in five putatively chlorsulfuron-resistantBrassicc hairy-root lines cultured with and without a selective level of chlorsulfuron. Areas of hairy-root research where this technique will be particularly useful are discussed.     

A rapid non-destructive assay to quantify soybean nodule gas permeability

The low gas permeability of a diffusion barrier in the cortex of soybean nodules plays a significant role in the protection of nitrogenase from oxygen inactivation. It may also set an upper limit on nodule respiration and nitrogen fixation rates. Two methods which have been used to quantify the gas permeability of leguminous nodules are reviewed and found to be unreliable. A new assay technique for determining both the nodule activity and gas permeability is developed and tested. This ‘lag-phase’ assay is based on the time nodules require to reach steady-state ethylene production after being exposed to acetylene. The technique is rapid, insensitive to errors in biochemical parameters associated with nitrogenase, and is non-destructive. The method was tested with intact aeroponically grown soybean plants for which the mean nodule gas permeability was found to be 13.3×10⁻³ mms⁻¹. This corresponds to a layer of cells approximately 35 um thick and is consistent with previously reported values.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

A comparison of electro-ultrafiltration and quantity/intensity measurements of soil potassium with its uptake by ryegrass in Scottish soils

Summary Electro-ultrafiltration (EUF) and quantity/intensity (Q/I) parameters of soil K were compared for 14 soils from each of three soil series. The K desorbed by EUF during the first 10 min (K₁₀) was closely correlated with the equilibrium activity ratio (AR₀) for soils of the same series, but differences between series reflected the soil K-buffering capacity, indicating that K₁₀ includes loosely held exchangeable K and is not strictly an intensity measurement. EUF values were compared with conventional soil test methods for predicting K-uptake and dry-matter yield of ryegrass grown in the glasshouse. Correlation coefficients between K uptake at the first cut were 0.80 for K₁₀, 0.88 for K_a (the initially labile K derived from the Q/I curve), 0.92 for K₃₅ (desorbed by EUF in 35 min) and 0.97 for K_ex (1.0M ammonium acetate extraction).     

A new Andricus Hartig oak gallwasp species from China (Hymenoptera: Cynipidae: Cynipini)

A new species of oak gallwasp, Andricus xishuangbannaus is described from China. The species induces leaf galls on Quercus griffithii (Fagaceae). Diagnosis, distribution, and biology of the new species are included.     

The interaction of nitrite with photosynthetic electron transport under anaerobic conditions

Chlorella cells were examined in a modulated oxygen polarograph under aerobic and anaerobic conditions. At light intensities below about 600 ergs · cm^?2 · s^?1 of 650 nm light, the oxygen yield and phase lag are lower under anaerobic conditions. Addition of 25 mM sodium nitrite increases both these parameters to values close to those found in the presence of oxygen. It is proposed that nitrite is reduced by Photosystem I thus diverting electrons from the cyclic electron transport pathway. The intersystem electron transport chain becomes more oxidized and this suppresses a backflow of electrons to the oxidizing side of Photosystem II, hence increasing the oxygen yield and the phase lag. The removal of oxygen from the bathing medium also alters the response of dark adapted _Chlorella to a series of saturating light flashes. In terms of the Kok model of Photosystem II (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) there is a large increase in the parameter α. Addition of nitrite reverses this change and virtually restores the response seen in the presence of oxygen. The deactivation of the S₂ state is greatly speeded up in the absence of oxygen but the addition of nitrite again reverses this.     

High-performance tryptic mapping of recombinant bovine somatotropin

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.     

ASW: a gene with conserved avian W-linkage and female specific expression in chick embryonic gonad

Vertebrates exhibit a variety of sex determining mechanisms which fall broadly into two classes: environmental or genetic. In birds and mammals sex is determined by a genetic mechanism. In mammals males are the heterogametic sex (XY) with the Y chromosome acting as a dominant determiner of sex due to the action of the testis-determining factor, SRY. In birds females are the heterogametic sex (ZW); however, it is not known whether the W chromosome carries a dominant ovary-determining gene, or whether Z chromosome dosage determines sex. Using an experimental approach, which assumes only that the sex-determining event in birds is accompanied by sex-specific changes in gene expression, we have identified a novel gene, ASW (Avian Sex-specific W-linked). The putative protein for ASW is related to the HIT (histidine triad) family of proteins. ASW shows female-specific expression in genital ridges and maps to the chicken W chromosome. In addition, we show that, with the exception of ratites, ASW is linked to the W chromosome in each of 17 bird species from nine different families of the class Aves. Received: 18 October 1999 / Accepted: 10 January 2000