Variations in the structure and expression of shrunken alleles
--William K. Johnson and Prem S. Chourey
The shrunken (sh1) alleles from a total of 20 mutant lines (obtained mainly from O. Nelson and P. Peterson) of independent, spontaneous origin (sh-Woodman was provided by J. Woodman and derived from tissue culture) were analyzed at the DNA, RNA and sucrose synthase (SS) protein levels for variations in gene structure and expression. Approximately one-half of the mutant alleles appeared to be associated with transposable elements (Ac/Ds or En/I) and there was great variation among the restriction fragment patterns from all of the lines. Two of the alleles (sh-bz-m4 and sh-82-6466) are gene deletions, since no hybridization was detected on Southern and Northern blots and no Sh-encoded (SS1) protein was found on Western blots for either line. Eight of the sh mutants expressed multiple transcripts (from 1.9 to 7.2 kb in size), but these were not necessarily correlated with probable (-En/I control, while the two alleles [sh-76-3014 and the sh derivative of Sh revertant 1 from sh-m5933 (provided by B. Burr)] that yielded evidence for Ac/Ds control each expressed single, but aberrant-sized transcripts (2.5 or 4.4 kb respectively).
In terms of tissue-specific RNA accumulation, nearly all of the sh alleles exhibited very low levels of steady-state transcripts in 22 DAP kernels, although sh-79-4507 with wildtype sized (2.8 kb) transcripts and sh-80-5142 with truncated (2.2 kb) transcripts accumulated sh RNA to near wildtype levels. A few other alleles (sh-81-6302, sh-7107 and sh-7731) showed elevated kernel transcript levels compared to other sh mutants, but still low when compared to wildtype kernels. In seven-day-old seedling roots, the sh alleles expressed low levels of transcripts (usually about one-quarter of wildtype root levels) and anaerobic induction resulted in at least a five-fold increase in transcript levels in all cases. In general, anaerobically stressed roots provided the most abundant source of sh mRNA for Northern analysis.
In terms of protein accumulation, Western blots revealed a pattern that was very different from what was expected based on steady-state RNA levels. Only six sh mutants yielded detectable SS1 proteins (wildtype = 96 kD). The sh-7342 mutant had nearly normal levels of SS1 protein (96 kD) in both induced and control roots, while it had almost undetectable protein levels in 22 DAP kernels. The other five mutants (sh-80-5142, 70 kD; sh-76-3018, 96 kD; sh-7731, 96 kD; sh deriv. of Sh-rev1, 90 and 92 kD; and sh-79-4507, 94 kD) showed low levels of SS1 protein in 22 DAP kernels (about 5% of wildtype kernel levels), but no readily detectable SS1 protein in induced or control roots (except for sh-79-4507, which had slightly more than the other four). In no case (sh or Sh genotype) was there a detectable increase in the amount of SS1 protein in anaerobically induced roots when compared to control roots of the same genotype.
These results seem to indicate that there is some mechanism that leads to the accumulation of SS1 protein in 22 DAP kernels. Also, the control of sh gene expression is generally post-transcriptional in nature, since there were many mutants that had abundant transcripts (wildtype sized and/or aberrant sized), but undetectable levels of SS1 protein. In the case of sh-7107, sh transcripts are found in polysomes, but no SS1 protein was detectable. Finally, it is also interesting to note that the only difference in Sus (codes for SS2 protein) RNA and protein size and accumulation in the sh lines was found in the deletion mutant sh-82-6466. In this mutant, SDS-PAGE gels showed a normal-sized SS2 polypeptide, but native gels revealed a banding pattern suggestive of hetero-tetramerization among different SS2 subunits, indicating a duplication of the Sus locus as a possible explanation for these results.
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