Ernest G. Jaworski

Nitrate reductase assay in intact plant tissues

Summary The measurement of nitrate reductase activity in intact plant tissue based on reduction of nitrate to nitrite is facilitated by the use of propanol and other organic solvents added to the incubation medium. Optimum conditions for the assay are defined for use with soybean leaf tissue.

Patterns of Nitrogen Utilization in the Soybean

SummaryThe patterns of nitrate uptake, nitrate reductase activity in the leaves, and nitrogen fixation by the nodules were investigated in field-grown soybeans (Glycine max (L.) Merr.) over the growing season.The level of nitrate-reductase activity generally paralleled the concentration of nitrate in the leaf tissue over the entire growing season. A precipitous drop in both parameters was noted within 2–3 weeks after flowering. These parameters decreased by 80–95% at mid-pod fill, a stage where ovule (seed) development was in the logarithmic growth phase, placing a heavy demand on the plant for both energy and fixed nitrogen.The activity of nitrogen fixation of soybean root nodules bore a reciprocal relationship to that of nitrate reductase. The maximum levels of nitrogen fixation were reached at early pod fill when nitrate reductase activity had dropped to 25% of maximum activity. A rapid loss of nitrogen fixation activity occurred shortly after bean fill was initiated, again at a time when the ovules were developing at maximal rates.The total protein content of soybean leaves increased over the season to a maximum level at mid-pod fill. This was followed by a 50% drop over the next 3-week period when the plants approached senescence. This drop corresponded to that found for nitrogen fixation. A similar pattern was noted for watersoluble proteins in the leaf.These studies suggest that there is a close and competitive relationship between the processes of nitrate reduction and nitrogen fixation, with the latter process dominating as the major source of fixed nitrogen after the plants have flowered and initiated pods. At this transitional stage, both soil and environmental effects could cause pertrubation in these processes that could lead to a nitrogen stress causing flower and pod abscission.The rapid decay of nitrogen fixation at the time of midpod fill also suggests a competition between roots (nodules) and pods for available photosynthate. This competition appears to lead to the breakdown of foliar proteins and senescence.

Messenger RNA encoding a glutathione-S-transferase responsible for herbicide tolerance in maize is induced in response to safener treatment.

SummaryGlutathione-S-transferases (GSTs) in maize represent a family of enzymes which conjugate glutathione to several major classes of pre-emergent, selective herbicides. Chemicals termed safeners have been demonstrated to increase the tolerance of maize toward such herbicides when the maize seed has been previously treated with safeners. It has subsequently been shown that corresponding increases in glutathione-S-transferase species occur. To determine whether these compounds act at a transcriptional level we have used synthetic oligonucleotide probes to isolate cDNA clones encoding the major GST polypeptide subunit, designated GST A. The identity of the clones has been confirmed by hybrid-selected mRNA translation and immunoprecipitation using antibodies made against this GST species as well as by production of active GST in yeast cells transformed with an expression vector containing the cloned DNA. GST A has been found to be encoded in a mRNA of 1.1 kb. Sequencing of cDNA products obtained by primer extension of maize mRNA using our oligonucleotide probes is consistent with this mRNA corresponding to the isolated cDNA clone. Using the clone as a probe for Northern analysis we have found a three to four-fold increase in the steady state level of this mRNA in maize tissue grown from safener-treated seeds. The level of safener which gives this induction is comparable to that required to obtain herbicide tolerance in the field.

Minimizing loss of indoleacetic acid during purification of plant extracts

SummaryPublished methods for isolation of 3-indoleacetic acid (IAA) were found to give low yields due to losses at specific steps. Loss during extraction was minimized by grinding tissue under a nitrogen atmosphere, using 0.02% sodium diethyldithiocarbamate in 80% ethanol as the extractant. When ethereal solutions of IAA were concentrated in vacuo, the hormone was lost, presumably by sublimation. This significant source of loss was eliminated by concentration at atmospheric pressure. Oxidative losses during application of extracts to chromatograms were reduced by prior application of an antioxidant to the origin of chromatograms. These precautions permitted development of a method where 10–50 μg of IAA could be recovered from soybean leaves with approximately 60% yield.

Biosynthesis of chitin during various stages in the metamorphosis of Prodenia eridania

The relationship between chitin content and activity of the chitin synthetase enzyme was investigated in final instar larvae and pupae of Prodenia eridania. A correlation was found between changes in chitin concentration and activity of the enzyme. The maximum level of chitin and the peak activity of the enzyme occurred in the late final larvae. The concentrations of hexosamines and chitodextrins are also discussed in relation to their possible significance in chitin biosynthesis.

Agreements covering exchanges of biological materials

Before the mid-1970s, scientists involved in biological research usually exchanged cells and vectors freely. Such exchanges served as a method of obtaining critical review and confirmation by peers. In addition, such exchanges often were quid pro quo, useful in obtaining similar material from others. These exchanges were not usually encumbered by con- tracts or legal restrictions; instead, they were governed by unwritten understandings and ethical standards. However, the huge increase in industrial and commercial interest in genetic engineering greatly increased the potential value of cells and vectors. Inevitably, several unwritten under- standings fell apart, creating some disappointments, some bitterness, and at least one lawsuit*. Both industry and academia responded by adopting various written agreements to control the exchange of biological material. The first few agreements were indivi- dually negotiated and agreed to by both sides. Before long, most corporations that released material to academic resear- chers developed form agreements to simplify and expedite releases that did not require individual attention. In early 1983, the Biological Sciences Group of the Mon- santo Corporate Research and Development Staff recog- nized that it needed to consider the issue of form agree- ments. Research in several fields was progressing rapidly and scientists throughout the Biological Sciences Group were anxious to be able to exchange cells and vectors with academic researchers. In particular, Monsanto received a large number of requests for plant cell transformation vectors, which were first announced in January 1983. Unlike cells and vectors which express specific metabolites, the plant cell vectors were capable of inserting any DNA sequence into the chromosomes of a wide variety of plant cells. A group of Monsanto attorneys and scientists therefore began to draft a set of agreements for use in connection with the release of biological materials. This task began with a review of the forms that were currently in use by several other corporations and institu- tions in the United States, as well as several publications L It was soon recognized that no two forms were alike; varia- tions in terminology and conditions were often subtle or unclear. It was doubtful that researchers would fully under- stand the varying implications of different forms, and likely

γ-Methylthiobutyric acid as a growth stimulant

Abstract γ-Methylthiobutyric acid, an analog of methionine, was found to stimulate the growth rate of Escherichia coli, Bacillus pumilis and Spirodela polyrhiza . The secretion of protease by B. pumilis was also enhanced. Two possible hypothesis for the observed growth stimulation are suggested.

Estrogenic activity of the bis (γ -methylthiobutyryl) ester of diethylstilbestrol

Abstract The bis (γ -methylthiobutyryl) ester of diethylstilbestrol possessed higher estrogenic activity than diethylstilbestrol based on mouse uterine bioassays. A similar comparison of these hormones in young female broiler chicks resulted in approximately the same stimulation of oviduct development, but the ester did not cause hypertrophy of the liver nor did it elevate plasma lipids to the degree found with diethylstilbestrol.