Experiments investigated changes in leaf development in young soybean plants progressing into P stress. The apical meristem and leaf structure were examined anatomically to evaluate the involvement of cell division and cell expansion in the restriction of leaf number and individual leaf size. Seedlings were deprived of P for 32 d following germination. Leaf initiation rates declined noticeably after about 2 weeks, even though the apical dome was of similar size and had a similar number of cells as controls. Primordia appeared morphologically similar also. Expansion of primary and the first three trifoliolate leaves of -P plants was severely reduced, and expansion of each leaf ceased, uniformly, when an area of about 40 cm(2) was obtained. Leaf epidermal cell size in the lateral plane was unaffected. The results indicate that expansion of leaves under P stress was limited by the number of cell divisions, which would imply control of cell division by a common regulatory factor within the leaf canopy.
A novel automated image collection and analysis system was used to compare two new soybean (Glycine max (L.) Merr.) promoters with the cauliflower mosaic virus 35S (CaMV35S) promoter, which was used as an expression standard. For expression comparisons, various permutations of a soybean polyubiquitin (Gmubi) promoter, a soybean heat shock protein 90-like (GmHSP90L) promoter and the CaMV35S promoter were placed upstream of a green fluorescent protein (gfp) gene. DNA constructs were introduced via particle bombardment into excised cotyledons of germinating lima bean (Phaseolus lunatus L.) seeds, which were arranged in Petri dishes for automated image capture and image analysis. The automated system allowed monitoring and quantification of gfp gene expression in the same piece of tissue over time. The Gmubi promoter, with its intronic region intact, showed the highest expression that was over five times stronger than the CaMV35S promoter. When an intronic region was removed from the Gmubi promoter, GFP expression was reduced, but was still over two times greater than with the CaMV35S promoter. The full-length soybean GmHSP90L promoter was four times stronger than the CaMV35S promoter. Truncation of the GmHSP90L promoter resulted in stepwise decreases in promoter strength, which appear to correspond to removal of regulatory elements. Automated image capture and analysis allowed the rapid and efficient evaluation of these new promoters.
A transgenic approach was used to alter soybean seed phytate content by expressing a soybean phytase gene (GmPhy) during seed development to degrade accumulating phytic acid (IP6). An expression vector containing the soybean phytase cDNA controlled by the seed-specific beta-conglycinin promoter (alpha'-subunit) was used to transform embryogenic soybean cultures. Plants from four independent transgenic lines were analyzed for transgene integration and seed IP6 levels. The reduction in IP6 levels in transgenic seeds compared to control 'Jack' soybeans ranged from 12.6 to 24.8 as determined by HPLC. A low copy transformant was propagated to the T4 generation and examined in more detail for phytase expression and enzyme activity during seed development. Expression of phytase mRNA and phytase activity increased during seed development, consistent with the use of an embryo-specific promoter. Ectopic phytase expression during seed development offers potential as an effective strategy for reducing phytate content in soybean seed.
The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3'-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), gammab (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and gammab to the 3'-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.
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