The transcription factor VP1 regulates maturation and dormancy in plant seeds by activating genes responsive to the stress hormone abscisic acid (ABA). Although activation involves ABA-responsive elements (ABREs), VP1 itself does not specifically bind ABREs. Instead, we have identified and cloned a basic region leucine zipper (bZIP) factor, TRAB1, that interacts with both VP1 and ABREs. Transcription from a chimeric promoter with GAL4-binding sites was ABA-inducible if cells expressed a GAL4 DNA-binding domain::TRAB1 fusion protein. Results indicate that TRAB1 is a true trans-acting factor involved in ABA-regulated transcription and reveal a molecular mechanism for the VP1-dependent, ABA-inducible transcription that controls maturation and dormancy in plant embryos.

SummaryACGT-containing ABA response elements (ABREs) have been functionally identi®ed in the promoters of various genes. In addition, single copies of ABRE have been found to require a cis-acting, coupling element to achieve ABA induction. A coupling element 3 (CE3) sequence, originally identi®ed as such in the barley HVA1 promoter, is found approximately 30 bp downstream of motif A (ACGT-containing ABRE) in the promoter of the Osem gene. The relationship between these two elements was further de®ned by linker-scan analyses of a 55 bp fragment of the Osem promoter, which is suf®cient for ABA-responsiveness and VP1 activation. The analyses revealed that both motif A and CE3 sequence were required not only for ABAresponsiveness but also for VP1 activation. Since the sequences of motif A and CE3 were found to be similar, motif-exchange experiments were carried out. The experiments demonstrated that motif A and CE3 were interchangeable by each other with respect to both ABA and VP1 regulation. In addition, both sequences were shown to be recognized by a VP1-interacting, ABAresponsive bZIP factor TRAB1. These results indicate that ACGT-containing ABREs and CE3 are functionally equivalent cis-acting elements. Furthermore, TRAB1 was shown to bind two other non-ACGT ABREs. Based on these results, all these ABREs including CE3 are proposed to be categorized into a single class of cisacting elements.

SummaryTo elucidate the molecular basis of loss of self-incompatibility in Lycopersicon, S-RNases and HTproteins were analysed in seven self-compatible (SC) and three self-incompatible (SI) taxa. No or low stylar RNase activity was a common feature in most SC taxa examined, in contrast to the uniformly high levels of activity found in all SI species. The S-RNase gene is most likely deleted in the four red-fruited SC taxa (L. esculentum, L. esculentum var. cerasiforme, L. pimpinellifolium and L. cheesmanii) because S-RNase genes could not be ampli®ed from genomic DNA. S-RNase genes could, however, be ampli®ed from the genomes of the three green-fruited SC taxa examined. L. chmielewskii and L. hirsutum f. glabratum show a decreased accumulation of transcripts, possibly re¯ecting changes in the 5¢¯anking regions of the S-RNase genes. The remaining green-fruited SC species, L. parvi¯orum, has a functional S-RNase gene in its genome that is expressed at high levels in the style, suggesting a genetic factor responsible for the low S-RNase activity. Together these results argue for several independent mutations in the S-RNase gene over the course of Lycopersicon diversi®cation, and that loss of S-RNase function is unlikely to the primary cause of the loss of self-incompatibility. We also examined the HT-B genes that play a role in self-incompatibility. HT-B transcripts were markedly reduced in the styles of all the SC taxa examined. A scenario is described where a mutation causing reduced transcription of HT-B in an ancestral SI species was central to the loss of self-incompatibility in Lycopersicon.

SummaryGametophytic self-incompatibility (GSI) systems involving the expression of stylar ribonucleases have been described and extensively studied in many plant families including the Solanaceae, Rosaceae and Scrophulariaceae. Pollen recognition and rejection is governed in the style by specific ribonucleases called SRNases, but in many self-incompatibility (SI) systems, modifier loci that can modulate the SI response have been described at the genetic level. Here, we present at the molecular level, the isolation and characterization of two Solanum chacoense homologues of the Nicotiana HT modifier that had been previously shown to be necessary for the SI reaction to occur in N. alata (McClure et al., 1999). HT homologues from other solanaceous species have also been isolated and a phylogenetic analysis reveals that the HT genes fall into two groups. In S. chacoense, these small proteins named ScHT-A and ScHT-B are expressed in the style and are developmentally regulated during anthesis identically to the S-RNases as well as following compatible and incompatible pollination. To elucidate the precise role of each HT isoform, antisense ScHT-A and RNAi ScHT-B lines were generated. Conversion from SI to self-compatibility (SC) was only observed in RNAi ScHT-B lines with reduced levels of ScHT-B mRNA. These results confirm the role of the HT modifier in solanaceous SI and indicate that only the HT-B isoform is directly involved in SI.

Gametophytic self-incompatibility in the Solanaceae is controlled by a single, nultiafleic locus, the S locus.We have recently described an allele of the S locus of Lycopersicon peruvianum that caused this normally self-incompatible plant to become self-compatible. We have now characterized two glycoproteins present in the styles of self-compatible and self-incompatible accessions of L. peruvianum: one is a ribonuclease that cosegregates with a functional self-incompatibility allele (S6 allele); the other cosegregates with the self-compatible allele (S, allele) but has no ribonuclease activity. The derived amino acid sequences of the cDNAs encoding the S6 and Sc glycoproteins resemble sequences of other ribonucleass encoded by the S locus. The derived sequence for the S. glycoprotein differs from the others by lacking one of the hidine residues found in all other S-locus ribonucleases. These findings demonstrate the essential role of ribonuclease activity in self-incompatibility and lend further weight to evidence that this hisidine residue is involved in the catalytic site of the enzyme.Self-incompatibility is a major factor affecting mating systems in flowering plants (1, 2). In plants with gametophytic self-incompatibility such as members of the Solanaceae, rejection or acceptance of pollen tubes by the style is controlled by a single, multiallelic locus, the S locus. Pollen expresses its haploid S genotype, and matings are incompatible if the S allele of the pollen is matched by one of the two alleles expressed in the pistil. Thus, self-incompatibility is an example of recognition between plant cells; the underlying mechanism may be similar to other recognition systems in plants such as those involved in host-pathogen interactions (3, 4). The products of the S locus are a class of extracellular glycoproteins with RNase activity called S-RNases (5, 6). The genes that encode these proteins cosegregate with alleles of the S locus (7, 8). S-RNases are abundant proteins found in high concentrations in the transmitting tract of the style, the site at which inhibition of pollen tubes occurs during incompatible matings (9). Sequences of S-RNase alleles from different solanaceous species share a characteristic structure that includes five short stretches of highly conserved sequence (10). Two of these conserved regions correspond to the sequences surrounding the catalytic domains of fungal RNases and include both of the histidine residues essential for catalytic activity (11 LA2157 is self-compatible and has S genotype SS,; LA2163 is self-incompatible and has the S genotype 56S7 (16). L. peruvianum plants homozygous for the S6 allele were produced by self-pollinating heterozygous individuals at the green bud stage as described (16).Purification and N-Terminal Sequencing of the S6 and S, Glycoproteins. Extracts from 50 styles of plants homozygous for the S6 or S, alleles were prepared and fractionated by cation-exchange chromatography as described (18). S glycoproteins recovered from the extract after ammonium...