Phaseolin Promoter Research Articles

Changes in fatty acid composition in plant tissues expressing a mammalian delta9 desaturase.

Plant tissues expressing a mammalian stearoyl-CoA delta9 desaturase were reported to accumulate delta9 hexadecenoic acid (16:1), normally very minor in most plant tissues. The transgenic plants were thoroughly analyzed for alterations of individual lipids in different subcellular sites. Western blot analysis indicated that the animal desaturase was targeted to the microsomes. The delta9 16:1 was incorporated into both the sn-1 and sn-2 positions of all the major membrane lipids tested, indicating that the endoplasmic reticulum acyltransferases do not exclude unsaturated C16 fatty acids from the sn-2 position. In addition to increases in monounsaturated and decreases in saturated fatty acids, accumulation of 16:1 was accompanied by a reduction in 18:3 in all the lipids tested except phosphatidylglycerol, and increases in 18:2 in phospholipids. Total C16 fatty acid content in the galactolipids of the transgenics was significantly higher than that in the control, but those in the phospholipids were unchanged. In transgenics, delta11 18:1 was detected in the sn-1 position of the lipids tested except phosphatidylinositol and phosphatidylserine. Introduction of the animal desaturase, controlled by a seed-specific phaseolin promoter, into soybean somatic embryo resulted in a significant reduction in saturated fatty acids. Such effects were greater in cotyledons than hypocotyl-radicles. This study demonstrated that the animal desaturase can be used to decrease the levels of saturated fatty acids in a crop plant.

Cell Ablation Reveals That Expression from the Phaseolin Promoter Is Confined to Embryogenesis and Microsporogenesis.

Most previous studies of the [beta]-phaseolin (phas) gene, which encodes the major storage protein in bean (Phaseolus vulgaris L.), have shown its expression to be rigorously confined to the developing seed, both in bean and transgenic tobacco (Nicotiana tabacum L. cv Xanthi) plants. To confirm unequivocally the lack of phas expression in vegetative tissues, we placed the diphtheria toxin A-chain (DT-A) coding region under the control of [beta]-phaseolin promoter sequences. Tobacco plants transgenic for phas/DT-A were phenotypically normal until flowering, when they produced anthers that were externally normal but contained no viable pollen. Microscopic examination of immature anthers revealed a normal tapetum, but the pollen mother cells did not undergo meiosis and subsequently degenerated, resulting in male-sterile plants. This demonstration of phas expression during microsporogenesis was corroborated by the expression of [beta]-glucuronidase in pollen of plants transformed with comparable phas/uidA constructs. Although these findings suggested that similarities in phas expression may exist between seed and pollen maturation, no phas activity could be detected in bean pollen. After fertilization of the DT-A-transformed plants with pollen from wild-type tobacco, 50% of the resulting embryos aborted at the heart stage, defining this as the earliest time for phas expression during embryogenesis.

Utilization of luciferase fusion genes to monitor differential regulation of phytohemagglutinin and phaseolin promoters in transgenic tobacco

The firefly luciferase gene can be used as a very sensitive and easily assayed reporter to study promoter activity in transgenic plants. To study the regulation of the promoters of the genes for phytohemagglutinin-L and β-phaseolin, two seed storage proteins of the common bean, Phaseolus vulgaris, we constructed chimeric genes consisting of their 5′ flanking regions and the luciferase gene, and introduced the constructs into tobacco via Agrobacterium-mediated transformation. Our studies indicate that the two seed protein promoters are activated during seed development, and luciferase accumulates until seed maturation. Little or no luciferase activity could be found in other plant organs, and activity rapidly declined during seedling growth. The construct driven by the phaseolin promoter gave rise to about eight times more luciferase than the one driven by the phytohemagglutinin promoter. Thus, in addition to proper temporal and spatial regulation of promoter activity, we found that the differences in the levels of luciferase driven by the two promoters were similar to the calculated differences in the levels of protein in the bean resulting from the activities of these promoters. However, both promoters gave rise to similar levels of luciferase mRNA in the seeds of the transgenic plants. These results show that the levels of luciferase activity reflect not only the strength of the promoter but also the stability and/or translatability of the mRNA.

Phaseolin Gene from Bean Is Expressed After Transfer to Sunflower Via Tumor-Inducing Plasmid Vectors

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.