Degradation products of the mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean and transgenic petunia.

The degradation of a soybean ribulose-1,5-bisphosphate carboxylase small subunit RNA, SRS4, was investigated in soybean seedlings and in petunia plants transformed with an SRS4 gene construct. Polyacrylamide RNA gel blot, primer extension, and S1 nuclease analyses were used to identify and map fragments of the SRS4 mRNA generated in vivo. We showed that SRS4 mRNA is degraded to a characteristic set of fragments in soybean and transgenic petunia and that degradation is not dependent on position of insertion of the gene construct within the genome, on the expression level of the SRS4 mRNA, or on the rbcS promoter. Degradation products lacked poly(A) tails and fractionated with poly(A)-depleted RNA on oligo(dT)-sepharose columns. These products pelleted with polysomes and were released from polysomes prepared with EDTA. Sequences at the 5' end of the SRS4 mRNA were more stable than those at the 3' end of the mRNA. Three models for SRS4 mRNA degradation involving endonucleolytic and exonucleolytic degradation were presented to explain the origin of the 5' proximal fragments.     

Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants.

The petunia rbcS gene SSU301 was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. The time at which rbcS expression was maximal after transfer of the tobacco plants to the greenhouse was determined. The expression level of the SSU301 gene varied up to 9 fold between individual tobacco plants which had been standardized physiologically as much as possible. The presence of adjacent pUC plasmid sequences did not affect the expression of the SSU301 gene. In an attempt to reduce the between-transformant variability in expression, the SSU301 gene was introduced into tobacco surrounded by 10kb of 5' and 13 kb of 3' DNA sequences which normally flank SSU301 in petunia. The longer flanking regions did not reduce the between-transformant variability of SSU301 gene expression.     

Faithful degradation of soybean rbcS mRNA in vitro.

The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.     

Expression of the AtGH3a gene, an Arabidopsis homologue of the soybean GH3 gene, is regulated by phytochrome B

Light is one of the most crucial environmental cues for plants. Phytochrome, one of the major photoreceptors of plants, regulates expression of many genes. We screened for Arabidopsis promoter trap lines that exhibited light-dependent reporter gene expression. Molecular analysis of one such line revealed that a reporter gene was inserted near an Arabidopsis homologue of the soybean GH3 gene, AtGH3a. We performed RNA gel blot analysis to further characterize the response of the AtGH3a gene to light. In response to the end-of-day far-red light treatment, the expression increased substantially. Analysis of the phyB-deficient mutant indicated that this light response is under the control of phytochrome B. The expression was also induced by exogenous auxin. Furthermore, the light response was substantially reduced in the auxin-related axr2 mutant. Taken together, it is suggested that phytochrome B regulates the expression of genes by altering the levels of auxin.     

Production of transformed soybean plants by electroporation of protoplasts

Protoplasts obtained from immature seeds of Glycine max (L.) Merr. cv. Clark 63 (soybean) were electroporated with DNA carrying either the kanamycin or hygromycin resistance genes and the reporter genes, β-glucuronidase or opine synthesis. Antibiotic resistance could be selected for at the frequency of about one colony from 2 000 electroporated protoplasts (0.05%) and the reporter genes were expressed in from 75 to 90% of the selected colonies. Antibiotic resistance and reporter gene expression were not found in untreated protoplasts. Shoots formed within about 5 months after a number of transfers of selected portions of the callus on the regeneration medium. The shoots have been rooted to form plants which express the reporter genes and contain the transforming DNA in their leaves as shown by Southern hybridization. The reporter genes are expressed (opine synthesis) in all leaves and roots and NPTII activity was present in all leaves, indicating that the transformed plants are not chimeral. We expect these plants to set seed since untransformed plants regenerated from protoplasts did. We can obtain shoots from several of the soybean genotypes we have used so far. Thus, we should have a method for the efficient production of nonchimeral, transformed plants of the important crop plant soybean.