Treatment of tall and dwarf (3 beta-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C20-GA, GA19. In shoots of the slender (la crys) mutant, the content of C19-GAs was lower and GA19 content was higher than in those of the tall line. Metabolism of GA19 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feed-back regulation of the 20-oxidation and 3 beta-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea. The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this cDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C19-GA, GA9, which was the major product. The 13-hydroxylated substrate GA53 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20. Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA3, suggesting that there is feed-back regulation of GA 20-oxidase gene expression.

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Nucleotide sequences coding for proteins containing the tobacco etch virus (TEV) NIa proteinase were generated by polymerase chain reaction amplification and/or site-directed mutagenesis. These coding regions contained sequences for the proteinase alone or as part of higher Mr precursors. Following transcription and translation of these sequences in a cell-free system, the various polyproteins, all containing an active small nuclear inclusion protein (NIa) proteinase, were used to process a TEV substrate series. Most substrates were processed in a similar fashion by all proteolytic forms. However, one substrate which contained the TEV 50K/71K protein junction was differently processed by several of the polyproteins containing NIa proteinase. Substrates which previously had no identified TEV NIa proteinase cleavage sites also were tested and were not cleaved by any of the proteinase-containing polyprotein forms.