Complex nested promoters control tissue-specific expression of acetyl-CoA carboxylase genes in wheat.

Cis-acting regulatory elements of the wheat acetyl-CoA carboxylase (ACC) gene family were identified by comparing the promoter activity of 5' end gene fragments fused to a reporter gene in two transient expression systems: wheat protoplasts and epidermal cells of mature embryos. Expression of the plastid and the cytosolic ACC genes is each driven by two nested promoters responsible for the synthesis of two transcript types. The internal promoter is located in an intron removed from transcripts originating at the first promoter. These complex promoters, which are different for the cytosolic and plastid ACC genes, control tissue-specific expression of the enzymatic activity supplying cytosolic, plastid, and mitochondrial pools of malonyl-CoA. The activity of one such complex promoter, driving expression of one of the cytosolic ACC genes, was studied throughout development of transgenic wheat plants carrying a full-length promoter-reporter gene fusion. High activity of the promoter was detected in the coleoptile, in the upper sheath section of the leaf, on the top surface of the ovary, in some sections of the main veins in the lemma and glume, and in abaxial epidermis hair cells of the lemma, glume, and rachis. The findings are consistent with the developmental and environmental requirements for very-long-chain fatty acids and flavonoids, whose synthesis begins with the ACC reaction in the cytosol of these specific cell types.

Characterization of rbcS genes in the fern Pteris vittata and their photoregulation.

The fern Pteris vittata L. belongs to the evolutionarily highest group of vascular plants that still maintains a free-living gametophytic stage. The two-dimensional gametophytes developed under blue light exhibit higher CO2 fixation efficiency and different ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) composition when compared to the red-induced filamentous gametophytes (H. Eilenberg et al., 1991, Plant Physiol 95: 298-304). To unravel the correlation between SSU structural differences and light regulation, two rbcS genes and two additional partial cDNAs were characterized. Fern rbcS genes resemble those of higher plants in their promoter light-regulatory elements (LREs) and intron number and positions. However, the primary structure of the fern mature SSUs displays much higher divergency within the gene family. This structural variability was correlated with differential steady-state mRNA levels under red and blue light. Genes rbcS-1 and-4 and 4-to 6-fold higher transcript levels in red light while rbcS-2 and-3 contribute relatively more to the blue rbcS mRNA levels. Five of the 12 amino acids that differ between rbcS-2 and-4 affect hydrophobicity and might play a crucial role in determining the efficiency of CO2 fixation. Dendrograms of Rubisco SSUs and LSUs indicate early divergence of the fern types from the rest of the vascular plants. However, prominent higher-plant-like Rubisco features such as high carboxylation efficiency, promoter LREs and exon-intron structure, suggest that molecular specialization of the higher-plant Rubisco prototype occurred earlier than the emergence of ferns.

Variability in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunits and Carboxylation Activity in Fern Gametophytes Grown under Different Light Spectra.

Two distinct ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) populations were observed in Pteris vittata gametophytes grown under different illumination conditions. Exposure of the fern gametophytes to continuous red light (R) resulted in Rubisco SSUs that were not recognized by polyclonal antibodies raised against SSUs from spinach. Unlike the R-induced SSUs, blue light (B) induced SSUs were well recognized. This difference in SSU composition also reflected in Rubisco activity. In vitro, B-induced Rubisco exhibits a significantly higher carboxylation activity as compared to the R-induced Rubisco. Approximately a two- to threefold increase in the V(max) value of the B-induced carboxylase as compared to the R-induced one was measured. It thus seems very likely that certain domains in the SSU molecule affect enzyme activity.

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin.

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes.

Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.

A possible regulatory role of squalene epoxidase in Chinese hamster ovary cells.

Growth of Chinese Hamster Ovary (CHO) cells in the presence of 20% lipid depleted serum (LDS) for only 2 hr results in an increase in the synthesis of [14C]sterols from [14C]mevalonate and from [14C]squalene compared with cells grown under normal growth conditions in the presence of 10% fetal calf serum (FCS). This enhanced sterol synthesis increases with time of exposure of the cells to LDS. However, exposing these cells for time periods up to 42.5 hr to a growth medium containing 20% LDS did not result in enhanced [14C]sterol synthesis from [14C]2,3-oxidosqualene. Incubation of these cells with [14C]mevalonate resulted in the accumulation of [14C]squalene regardless of the presence of either LDS or FCS. These results suggest that squalene epoxidase is a regulatory enzyme in the cholesterol biosynthetic pathway in CHO.