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The two-electron reduction of sulfate to sulfite in plants is mediated by 5'-adenylylsulfate (APS) reductase, an enzyme theorized to be a control point for cysteine synthesis. The hypothesis was tested by expression in Arabidopsis thaliana under transcriptional control of the CaMV 35S promoter of the APS reductase from Pseudomonas aeruginosa (PaAPR) fused with the rbcS transit peptide for localization of the protein to plastids. PaAPR was chosen for the experiment because it is a highly stable enzyme compared with the endogenous APS reductase of A. thaliana, and because PaAPR is catalytically active in combination with the plant thioredoxins m and f indicating that it would likely be catalytically active in plastids. The results indicate that sulfate reduction and O-acetylserine (OAS) production together limit cysteine synthesis. Transgenic A. thaliana lines expressing PaAPR accumulated sulfite, thiosulfate, cysteine, gamma-glutamylcysteine, and glutathione. Sulfite and thiosulfate increased more than did cysteine, gamma-glutamylcysteine and glutathione. Thiosulfate accumulation was most pronounced in flowers. Feeding of OAS to the PaAPR-expressing plants caused cysteine and glutathione to increase more rapidly than in comparably treated wild type. Both wild-type and transgenic plants accumulated sulfite and thiosulfate in response to OAS feeding. The PaAPR-expressing plants were slightly chlorotic and stunted compared with wild type. An attempt to uncover the source of thiosulfate, which is not thought to be an intermediate of sulfate reduction, revealed that purified beta-mercaptopyruvate sulfurtransferase is able to form thiosulfate from sulfite and beta-mercaptopyruvate, suggesting that this class of enzymes could form thiosulfate in vivo in the presence of excess sulfite.

The committing step in Met and S-adenosyl-L-Met (SAM) synthesis is catalyzed by cystathionine gamma-synthase (CGS). Transgenic Arabidopsis plants overexpressing CGS under control of the cauliflower mosaic virus 35S promoter show increased soluble Met and its metabolite S-methyl-Met, but only at specific stages of development. The highest level of Met and S-methyl-Met was observed in seedling tissues and in flowers, siliques, and roots of mature plants where they accumulate 8- to 20-fold above wild type, whereas the level in mature leaves and other tissues is no greater than wild type. CGS-overexpressing seedlings are resistant to ethionine, a toxic Met analog. With these properties the transgenic lines resemble mto1, an Arabidopsis, CGS-mutant inactivated in the autogenous control mechanism for Met-dependent down-regulation of CGS expression. However, wild-type CGS was overexpressed in the transgenic plants, indicating that autogenous control can be overcome by increasing the level of CGS mRNA through transcriptional control. Several of the transgenic lines show silencing of CGS resulting in deformed plants with a reduced capacity for reproductive growth. Exogenous feeding of Met to the most severely affected plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250-fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM and that SAM may be a regulator of CGS expression.

A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.