Riboflavin Nullification of Inhibitory Actions of 3-Amino-1,2,4-Triazole on Seedling Growth.
The physiological mechanislmis involved in the toxic actions of 3-amino-1,2,4-triazole (amitrole) have been actively investigated since the chemical was first introduced as a potential herbicide in 1954 (2). Much of the research has been guided by hypotheses involving one of three major metabolic processes: A, porphyrin synthesis, B, cation functions, and more recently C, the metabolism of purines and their derivatives. The extensive literature associated with the first two categories will not be considered herein. Amitrole interferences in some of the biochemiiical processes of the third category now appear to be the actions of nmajor physiological importance in the response of plants to the herbicide. Much of the pertinent research deals with attempts to find physiological compounds capable of reducing the toxicity of amitrole to various organisms. The most effective conmpounds are purines (23, 24) or histidine (13, 23) for unicellular plants anid riboflavin or riboflavin derivatives (22, 24) for higher plants. The investigations reported herein Nwere initiated as a result of my observations that the riboflavin effect could not be denmonstrated with barley seedlings by tecliiques that had been used successfully with other herbicidemetabolite combinations (9, 14, 18). The objectives of this study were: A, to develop techniques by whiclh the riboflavin nullification of amitrole toxicity could be observed with higher plants, and to compare adenine and histidine with riboflavin by these techniques; BR, to consider the possibilitv that the riboflavin effect was the result of non-physiological processes: C, to compare the effectiveness of riboflavin in species of higher plants that differ in their response to amitrole, and D, to determine if certain other metabolites of theoretical interest might give additional reductionis in amitrole toxicity.
Expression of chloroplastic and cytosolic glutamine synthetase in barley leaves after cold-sensitive blocking of beta-carotene synthesis by amitrole or mutation
3-Amino-1,2,4-triazole (amitrole) provided to germinating barley at 20°C in the light led to bleached seedling leaves and photodynamic destruction of chloroplast structure, whereas normal greening and chloroplast ultrastructure was obtained when the seedlings developed in the presence of amitrole in the light at 30°C. Mass spectrometric analysis of the extractable herbicide demonstrated the same content of amitrole in leaves developed at 20 and 30°C. A very similar temperature-sensitive syndrome is characteristic for the nuclear gene mutant tigrina-o 34 in barley. Amitrole and the mutation were shown to inhibit the cyclization of lycopene, leading to severe deficiencies in β-carotene and its xanthophyll derivative lutein. Besides accumulation of lycopene, also its precursors phytoene, phytofluene and ξ-carotene accumulated. Inhibition of carotenoid biosynthesis by amitrole and the mutation at 20°C in the light led to a strong reduction of both transcript and protein levels for chloroplastic glutamine synthetase (GS 2 ) while transcript amount and protein of the cytosolic isoenzyme (GS 1 ) were unaffected. At 30°C increased levels of mRNA for the chloroplastic isoform GS 2 were observed in wild type, mutant and amitrole-treated seedlings, but protein levels remained unchanged. Turnover rates of the GS 2 protein were the same at 20 and 30°C. This extensive translational control of chloroplastic GS 2 synthesis was also observed in a heat shock experiment, which revealed transiently increased mRNA levels for chloroplastic GS 2 but unchanged protein levels. Permissive synthesis of β-carotene and chloroplastic glutamine synthetase (GS 2 ) at 30°C in the presence of amitrole or the tigrina-o 34 mutation might be due to two alternative pathways of ionone ring formation using either lycopene or neurosporene as substrates for cyclization.
On the fractionation of β-lactoglobulin and α-lactalbumin
Abstract Existing methods for the preparation of α-lactalbumin and β-lactoglobulin are examined critically. It is proposed that the most satisfactory method for the preparation of the whey protein fraction from bovine milk is (NH 4 ) 2 SO 4 fractionation. A number of new gentle procedures for the fractionation of the whey proteins are compared with some existing methods. The distribution of total protein at various stages is determined by total Kjeldahl nitrogen and the kind of protein by starch-gel electrophoresis. The effect of variables, such as pH, salt concentration and temperature on the separation of α-lactalbumin and serum albumin from β-lactoglobulin is studied. Differences in the behavior ( e.g. solubility) of genetic variants of β-lactoglobulin are noted. The solubility of each of the major whey proteins, under similar conditions to those used in the fractionation, is compared with its solubility in the whey protein mixture. The understanding gained of factors important in the fractionation is used in recommending new procedures for α-lactalbumin and β-lactoglobulin isolation.