ABSTRACT
Two S-adenosylmethionine synthetase (SAMS) cDNAs, PcSAMS1 and PcSAMS2, have been identified in Pinus contorta. We found that the two genes are differentially expressed during root development. Thus, PcSAMS1 is preferentially expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development, whereas PcSAMS2 is expressed in roots as well as in shoots and is down-regulated during adventitious root formation. The expression of the two SAMS genes is different from the SAMS activity levels during adventitious root formation. We conclude that other SAMS genes that remain to be characterized may contribute to the observed SAMS activity, or that the activities of PcSAMS1 and PcSAMS2 are affected by post-transcriptional regulation. The deduced amino acid sequences of PcSAMS1 and PcSAMS2 are highly divergent, suggesting different functional roles. However, both carry the two perfectly conserved motifs that are common to all plant SAMS. At the protein level, PcSAMS2 shares about 90% identity to other isolated eukaryotic SAMS, while PcSAMS1 shares less than 50% identity with other plant SAMS. In a phylogenetic comparison, PcSAMS1 seems to have diverged significantly from all other SAMS genes. Nevertheless, PcSAMS1 was able to complement a Saccharomyces cerevisiae sam1 sam2 double mutant, indicating that it encodes a functional SAMS enzyme.
Subject(s)
Cycadopsida/genetics , Methionine Adenosyltransferase/genetics , Plant Roots/genetics , Amino Acid Sequence , Cycadopsida/enzymology , Cycadopsida/growth & development , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genetic Complementation Test , Hypocotyl/drug effects , Hypocotyl/enzymology , Hypocotyl/genetics , In Situ Hybridization , Indoleacetic Acids/pharmacology , Indoles/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Methionine Adenosyltransferase/metabolism , Molecular Sequence Data , Mutation , Phylogeny , Plant Roots/drug effects , Plant Roots/growth & development , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue Distribution , Transcription, GeneticABSTRACT
A gene designated swin1.1 has been isolated by screening a Salix viminalis genomic library with a heterologous probe, win3 from Populus. The region sequenced included the entire coding sequence for a protein with 199 amino acids plus the promoter and terminator. At the 5' end of the coding region is a sequence that encodes a hydrophobic region of 25-30 amino acids, that could form a signal peptide. A putative TATAA box and polyadenylator sequence were identified. Introns were absent. The gene product showed similarities with serine protease inhibitors from the Kunitz family and especially with win3 from wounded leaves of Populus. Southern blot analysis indicated that swin1.1 is a member of a clustered gene family, swin1. An oligonucleotide corresponding to the putative hypervariable region towards the carboxyl end when used as a probe in Southern hybridization showed high specificity for swin1.1. Expression of the swin1.1 gene was enhanced in wounded leaves. The swin1.1 coding region without the signal sequence was highly expressed in Escherichia coli and the protein showed inhibitory activity against trypsin but at most slight activity against the other proteases tested. A systemically induced protein, SVTI, with inhibitor activity against trypsin, was isolated from Salix leaves by affinity chromatography on a column of trypsin-Sepharose 4B and N-terminal sequenced. It corresponded with the translated swin1.1 gene at 16 of the 19 amino acid sites, suggesting that SVTI is encoded by another member of the swin1 gene family.
