Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins.

Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.

Genotype-dependent transcriptional activation of novel repetitive elements during cold acclimation of alfalfa (Medicago sativa).

In a search for cold-regulated genes that are differentially expressed in alfalfa genotypes of contrasting freezing tolerance, we screened 1036 arrayed cDNA clones. The screening resulted in isolation of cDNA clones, which demonstrated dramatic differences in expression between hardy and un-hardy alfalfa varieties. Detailed analysis revealed that these cDNAs represent parts of novel non-coding repetitive elements carrying long-terminal repeats (LTR) and other retroelement-like features. Despite strong expression under low temperatures, DNA templates remained highly methylated, and a drug-induced decrease in methylation did not activate transcription under normal temperatures. We identified that these repetitive elements represent a large family and could insert into, or be adjacent to, the unrelated polyprotein sequences of putative retrotransposons. These retrotransposons also showed low temperature-induced transcriptional activation; however, this activation was not genotype-dependent. The retroelements described in this study are the first retroelement characterized in the Medicago genus. Furthermore, they represent the only known example of genotype-specific cold-induced transcriptional activation of multiple copies of a repetitive element whose expression is associated with a genotype difference in cold acclimation.

Linear amplification coupled with controlled extension as a means of probe amplification in a cDNA array and gene expression analysis during cold acclimation in alfalfa (Medicago sativa L.).

This study describes a rapid and simple way to amplify limited amounts of probes used for cDNA array hybridization while maintaining the original representation of transcripts in the samples. The approach is based on linear amplification of cDNA-coupled controlled extension of amplified products and yielded a 50-75-fold increases in hybridization signal intensity. Controlled extension of products is achieved either by adjusting the amplification conditions or by using a digested template. Linear amplification with controlled extension generates a population of fragments consisting mainly of 3'-end portions of original transcripts and ranging in length from 200 to 800 nucleotides. cDNA array analysis revealed that amplified and non-amplified probes generate expression profiles with correlations ranging from r=0.857 to 0.895. Up to 90% of cDNA clones, differentially expressed during cold acclimation in alfalfa, could be detected with both types of probes. This amplification method should increase the utility of cDNA arrays for identifying novel differentially expressed genes as well as expression profiling in specialized tissues or cells when the amount of analysed material is limited. The possibility of diminishing cross-hybridization of long genes sharing high sequence homology and improving the hybridization kinetics of complex probes after amplification is also discussed.