Mechthild Rieping

The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco

SummaryA series of deletion mutants of a soybean heat shock (hs) gene promoter was generated and linked to the chloramphenicol acetyl transferase (CAT) coding sequence. These chimaeric promoter/reporter gene constructs were introduced into tobacco and thermoregulated expression of CAT activity was examined in leaf extracts. Three different types of gene fusions were tested using two differentBIN19 vector constructions: (1) translational fusion between the N-terminus of the protein coding sequence of the heat shock geneGmhsp17.3-B and CAT; (2) transcriptional fusions between the 5′ nontranslated RNA regions ofGmhsp17.3-B and CAT; and (3) promoter fusions joining the hs promoter upstream sequences to the TATA box sequence of the Δ CaMV 35S-CATter vector. Alternatively, multiple copies of a synthetic deoxyoligonucleotide with the soybean hs consensus element (HSE2) were used. Heat inducible CAT activities were detected except in plants containing a transcriptional fusion devoid of all but 18 nucleotides at the 5′ terminus of the hs gene transcript. CAT activity was detectable in these plants only during the recovery at 25° C after a hs (40° C). Overlapping HSE-like promoter sequences seem to be necessary for the induction of heat inducible transcription of linked genes; synthetic HSE2 sequences have the capacity to reconstitute a hs promoter in combination with a TATA box sequence. Effective translation during hs seems to require sequences in the 5′ nontranslated leader of the hs protein mRNA; these sequences can be functionally replaced by the 5′ leader sequence of the Δ CaMV 35S promoter.

A dominant negative mutant of PG13 suppresses transcription from a cauliflower mosaic virus 35S truncated promoter in transgenic tobacco plants.

TGA1a and PG13 constitute a family of tobacco basic leucine zipper (bZIP) proteins that bind to activating sequence-1 (as-1), which is one of the multiple regulatory cis elements of the cauliflower mosaic virus (CaMV) 35S promoter. After truncation of the CaMV 35S promoter down to position -90 (CaMV 35S [-90] promoter), transcription stringently depends on the presence of as-1, which is recognized by nuclear DNA binding proteins called ASF-1. The role of the TGA1a/PG13 bZIP family in the formation of ASF-1 and in transcriptional activation of the CaMV 35S (-90) promoter has not yet been demonstrated in vivo. We constructed transgenic tobacco plants expressing a mutant of potato PG13, which lacks its wild-type DNA binding domain. This mutant acts as a trans-dominant inhibitor of ASF-1 formation and of expression from the CaMV 35S (-90) promoter, showing that PG13 can specifically interact with proteins necessary for these processes. Although we did not observe any other obvious phenotypic changes, these transgenic plants are a potentially valuable tool in identifying whether TGA1a and PG13 are involved in controlling promoters encoded in the plant genome.

The nodule-specific VfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I of Vicia faba L. and is predominantly expressed in the interzone II-III of root nodules

A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common nodulin sequences ‘AAAGAT’ and ‘CTCTT’ were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.

The Induction of the Heat Shock Response: Activation and Expression of Chimaeric Heat Shock Genes in Transgenic Plants

The heat shock (hs) response is a highly conserved, almost universal genetic system in living organisms. This response appears to have a protective function for many types of cells and tissues which have been exposed to heat stress, but also certain other chemical and physico-chemical stressors have the capacity to elicit the hs response (for reviews see Neumann et al., 1989; Lindquist and Craig, 1988; Schoffl et al., 1988). Hence, stressed cells seem to seek protection from the detrimental effects of environmental stress, at least to some extent, by the hs response. In physical terms, the stressor is an external force causing an internal strain. The adoption of these terms to biological systems has changed the meaning in a way that the term stress is used for “any environmental factor potentially unfavourable to living organism” and “strain is any physical or chemical change produced by a stress” (Levitt, 1980). The term hs response is commonly used for the reprogramming of cellular activities which is rapidly induced by heat stress. One important feature of this response is the cte novo synthesis of a number of hs proteins (hsps). Following severe but sublethal heat stress, cells are able to recover and they may even tolerate a subsequent, higher dosage of the stressor.