ABSTRACT
The frequency of lateral root initiation in tomato (Lycopersicon esculentum Mill cv. VFN8) seedling roots is increased over eightfold in response to 1.6 microM alpha-naphthalene-acetic acid (NAA). To identify genes that are activated during lateral root initiation, a cDNA library was made with RNA from roots treated with auxin and differentially screened with radioactive probes made from RNA isolated from treated and untreated roots. A cDNA clone, TR132, was identified that hybridized to a transcript that was induced within 4 h of auxin treatment and increased tenfold by 72 h. A gene (RSI-1) corresponding to the TR132 cDNA was cloned and characterized with regard to its nucleotide sequence, transcription start site and chromosomal map position. Approximately 1 kb of the 5' flanking DNA was linked to the beta-glucuronidase (GUS) protein coding region and tested for expression in transgenic tomato seedlings. GUS activity was observed in both lateral and adventitious root initials, including very early initials, and persisted until shortly after the lateral emerged from the parent tissue. In roots from seedlings with high activity, GUS expression was also observed in the root cap and vascular tissue. The predicted RSI-1 protein is rich in cysteine, lysine and proline, and includes an N-terminal region with characteristics of a signal peptide. The putative mature protein exhibits 79% amino acid identity to a protein encoded by a gene (GAST1) that is induced by gibberellic acid in tomato shoots.
Subject(s)
Genes, Plant , Plant Growth Regulators/genetics , Plant Proteins/genetics , Vegetables/genetics , Amino Acid Sequence , Base Sequence , Conserved Sequence , Gene Library , Glucuronidase/metabolism , Indoleacetic Acids/pharmacology , Molecular Sequence Data , Plant Proteins/chemistry , Plants, Genetically Modified , Protein Sorting Signals/genetics , Recombinant Fusion Proteins , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic/drug effectsABSTRACT
A cDNA clone corresponding to a transcript that was induced by auxin in tomato seedling roots encodes a 223 amino acid protein belonging to the proteinase inhibitor II family of tomato and potato. The TR8 open reading frame has an N-terminal region with characteristics of a signal peptide and three copies of a 64 amino acid segment that is also present in multiple copies in other PI-II family members. Comparison to other serine proteinase inhibitors indicates that all three domains are trypsin-specific. Transcripts homologous to TR8 increased in abundance within 24 h of auxin treatment and continued to increase through 72 h, at which point the induction was approximately ten-fold. Auxin induction was observed in roots and hypocotyls, but not in cotyledons or epicotyls. Induction may therefore be correlated with lateral and adventitious root initiation, which occurs over a similar time frame in the same tissues. RFLPs detected with the TR8 probe were mapped to the long arm of chromosome 11.
