Green tissue-specific analysis of a cloned rbcS promoter from Lemna gibba

Many plant genetic engineering taskss require the spatial expression of genes which in turn depends upon the availability of specific promoters. The present paper analyses the green-tissue characteristics of a new L. gibba rbcS promoter driving the expression of the gus gene in transgenic tobacco. A 1491 bp rbcS (small subunit of ribulose bisphosphate carboxylase) promoter was isolated from Lemna gibba. The sequence analysis revealed that this promoter is different from the previously reported rbcS promoter and is named SSU5C. A 1438 bp fragment of the SSU5C promoter was fused with the gus gene and transgenic tobacco plants were generated. The analysis of T₁ tobacco p1438-gus revealed that GUS expression driven by the SSU5C promoter was detected in the green part of vegetative organs. The promoter deletion analysis confirmed a region from position –152 to –49 relative to the start of transcription containing boxes X, Y and Z, while a positive regulatory region conferred green tissue-specific expression. Further functional analysis of constructs of box-X, Y, Z, which was fused with the basal SSU5C promoter, confirmed that the boxes X, Y and Z represent the new minimized functional promoter, respectively, and are able to direct green tissue-specific expression. This promoter may be used for gene expression in a tissue-specific manner in plant molecular breeding.

Differential Expression of rolC Results in Unique Plant Phenotypes

As residential lot sizes decrease, there is an increased demand for new, small-statured landscape plants to fit into the smaller lots. One promising method to create smaller plants is by introducing a dwarfing gene into a plant of interest. A dwarfing gene that has been identified is the rolC gene from Agrobacterium rhizogenes. Expression of rolC in plants has been shown to cause decreased height and internode length, increased branching, and modified leaf size in a several species. Although the effects of the rolC gene have been well characterized for many plant species, most research has concerned the native promoter or the CaMV 35S promoter. Less research has been done with additional promoters or comparing the results from different promoters. In this study we examined the effects of three separate gene constructs, all containing rolC driven under either the 35S promoter, the light inducible rbcs promoter, or the native rolC promoter in tobacco. Plants transformed with these constructs ranged widely for height and other phenotypic traits. Representative plants were crossed back to wild-type tobacco. Plants from this next generation, six with the 35S promoter, six with the rbcs promoter and four with the native rolC promoter, were measured for traits such as height, days to flower, number of branches and internode length. RolC RNA expression levels were also measured in roots, stems, and leaves to determine correlations between rolC expression level in specific tissues and the observed phenotype. Information about these relationships can be used to provide insight into the use of rolC in ornamental plants and the potential to modify its phenotypic effects by controlling expression level.

Chloroplast-dependent and light-independent expression of the tobacco rbcS promoter–GUS chimeric gene in black spruce

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

Tissue-specific and light regulation of a larch ribulose-1,5-bisphosphate carboxylase promoter in transgenic tobacco

A chimeric gene composed of an eastern larch (Larix laricina (Du Roi) K. Koch) ribulose-1,5-bisphosphate carboxylase (RbcS) promoter linked to the β-glucuronidase (GUS) coding sequence was transferred to tobacco (Nicotianatabacum (L.)) via Agrobacteriumtumefaciens transformation. Based on GUS activity the larch RbcS promoter functioned in an organ-specific and light-regulated manner. Histochemical analysis revealed high levels of GUS activity in photosynthetically active tissues and low or undetectable activity in xylem and root tissues. Fluorometric analysis of GUS activity demonstrated that the larch RbcS promoter was expressed at a 10-fold higher level in leaf blades than in root tissue. Light-grown transgenic plants expressed GUS at a two-fold higher level than dark-grown individuals. These results suggest evolutionary conservation of tissue-specific RbcS promoter activity between gymnosperms and angiosperms but only weak conservation of the transduction mechanism for light regulation.