Two distinct cis-acting elements are involved in light-dependent activation of the pea elip promoter

Light activation of the pea (Pisum sativum) elip gene promoter was analysed in transgenic plants and in transiently transfected plant protoplasts. A series of promoter deletions fused to the gusA reporter was tested, and the results obtained by the two experimental approaches were in good agreement. We identified two nucleotide sequence elements involved in light-regulated expression of the elip gene. One element is similar to the GT1 binding site of the rbcS-3A gene, and the other resembles a G-box-like ACGT element. The region containing both elements was able to confer light responsiveness on a heterologous basic promoter. Electrophoretic mobility shift assays demonstrated that each element is specifically recognized by DNA-binding proteins present in nuclear extracts from pea seedlings. The G-box-like ACGT element is necessary but not sufficient for light inducibility, indicating that the two elements act together in confering light responsiveness.     

Abscisic acid promotes novel DNA-binding activity to a desiccation-related promoter of Craterostigma plantagineum

Abscisic acid-treated callus of the resurrection plant Craterostigma plantagineum tolerates extreme desiccation. Nuclear proteins from tolerant callus bind specific sequence elements in the promoter region of the ABA and desiccation-inducible CDeT27–45 gene. One specific region of the promoter, which is protected from DNAse I treatment by DNA-binding activities, is different from previously reported ABA response elements. Four complexes of nuclear proteins and this DNA region are detected by electrophoretic mobility shift assay: two of these complexes (I and II) are readily detectable in untreated samples and are increased by ABA treatment while two other complexes (III and IV) accumulate only following ABA treatment and are prevented from accumulating by protein synthesis inhibitors. When a fragment containing the novel binding site is deleted from the wild-type promoter the ABA responsiveness of the promoter is removed; however, gain of function experiments using synthetic promoters in a protoplast transient assay suggest that besides the binding site other promoter elements are required. A second region of the promoter, containing the sequence element ACGT which is found in abscisic acid response elements, is also bound by nuclear proteins. The level of this second binding activity is similar in both untreated and ABA-treated cells and promoter/reporter gene constructs which contain only the four ACGT elements of the CDeT27–45 promoter are not ABA responsive in a C. plantagineum transient assay system     

Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare L.)

Abscisic acid (ABA)-response promoter complexes (ABRCs), consisting of an ACGT core-containing element (ACGT box) and a coupling element (CE), have been shown to be necessary and sufficient for ABA induction of gene expression in cereal plants. In this work, the component elements of two ABRCs are defined in terms of base sequence, orientation, and distance from each other. The ACGT element requires the sequence 5-ACGTGGC-3 and the elements CE1 and CE3 require the sequences CCACC and GCGTGTC, respectively. The ACGT element and CE3 are next to each other in the barley ABA-inducible gene HVA1, and lengthening the distance between them gradually decreases their activity in conferring ABA response. On the other hand, the ACGT element and CE1 are separated by about 20 bp in the promoter of another ABA-inducible gene, HVA22, and need to be separated by multiples of 10 bp in order to confer high ABA induction, suggesting that these two elements have to be located in the same side of the DNA double helix. Although the coupling between an ACGT box and a CE is sufficient for ABA induction, two copies of the ACGT element are equally active. However, two copies of CE3 appear to be less active. Specific interactions between ABRC and nuclear proteins have been detected. In vitro binding activities of nuclear proteins to an ABRC and to its mutant forms appear to be proportional to the biological activities of these sequences in vivo. Our data suggest that the specific response to ABA is determined by the presence of two ACGT boxes or an ACGT box plus a CE as well as by the flanking sequences of the ACGT boxes and the CEs.     

Sequence and functional analyses of the aldehyde dehydrogenase 7B4 gene promoter in Arabidopsis thaliana and selected Brassicaceae: regulation patterns in response to wounding and osmotic stress

Key message

The core promoter of the antiquitin ALDH7B4 gene was compared between selected Brassicaceae. Conserved cis elements controlling osmotic stress and wound-induced expression were identified and analysed in Arabidopsis thaliana leaves and seeds.

Abstract

Aldehyde dehydrogenases metabolise a wide range of aliphatic and aromatic aldehydes, which become cytotoxic at high levels. Family 7 aldehyde dehydrogenase genes, often described as antiquitins or turgor-responsive genes in plants, are broadly conserved across all domains. Despite the high conservation of the plant ALDH7 proteins and their importance in stress responses, their regulation has not been investigated. Here, we compared ALDH7 genes of different Brassicaceae and found that, in contrast to the gene organisation and protein coding sequences, similarities in the promoter sequences were limited to the first few hundred nucleotides upstream of the translation start codon. The function of this region was studied by isolating the core promoter of the Arabidopsis thaliana ALDH7B4 gene, taken as model. The promoter was found to be responsive to wounding in addition to salt and dehydration stress. Cis-acting elements involved in stress responsiveness were analysed and two conserved ACGT-containing motifs proximal to the translation start codon were found to be essential for the responsiveness to osmotic stress in leaves and in seeds. The integrity of an upstream ACGT motif and a dehydration-responsive element/C-repeat—low temperature-responsive element was found to be necessary for ALDH7B4 expression in seeds and induction by salt, dehydration and ABA in leaves. The comparison of the gene expression in selected Arabidopsis mutants demonstrated that osmotic stress-induced ALDH7B4 expression in leaves and seeds involves both ABA- and lipid-signalling components.     

Isolation and characterization of Histone1 gene and its promoter from tea plant (Camellia sinensis)

One 1.2 kbp long sequence was cloned by using PCR with primers that were designed from cDNA sequence of CsH1 gene (Genbank: EU716314) from tea plant (Camellia sinensis). According to the 1.2 kbp sequence, a 0.6 kbp sequence was isolated from tea plant genomic DNA using DNA Walking Method. Sequence analysis revealed that the 1.2 kbp sequence is a CsH1 gene consisting of 1 exon and 2 introns, the border of exton and intron sequences conforming to the GT–AG rule, and the 0.6 kbp sequence was found to be the promoter of CsH1 gene which contains basic promoter elements, TATA-box and CAAT-box. Abscisic acid responsiveness cis-acting element, elictor-responsive element, GA response element, light response cis-acting element and TC-rich repeats were also represented. To further study the activity of this promoter, the sequence was used to drive a GUS fusion gene in Agrobacterium-mediated transformation of tea plant somatic embryos, leaf discs and calli of tobacco (Nicotiana tabacum L.) where a high level of GUS expression was both observed in the tobacco calli and tea plant somatic embryos. These results suggest that the CsH1 gene promoter isolated is capable of conferring nuclear gene expression.     

A double SORLIP1 element is required for high light induction of ELIP genes in Arabidopsis thaliana

Promoter elements that contribute to high light (HL) induction of the Arabidopsis ELIP1 gene were defined using a transgenic promoter-reporter system. Two adjacent SORLIP1 elements (double SORLIP1, dSL) were found to be essential for HL induction of a GUS reporter gene. The dSL element was also found to be essential for HL induction conferred by the ELIP2 promoter. SORLIP1 elements were enriched in ELIP promoters throughout the plant kingdom, and showed a clade-specific pattern of gain or loss that suggested functionality. In addition, two G-box elements were found to redundantly contribute to HL induction conferred by the ELIP1 promoter.     

Isolation of carrot basic leucine zipper transcription factor using yeast one-hybrid screening

Abscisic acid (ABA) is involved in various physiological and developmental processes, including stress responses and seed maturation. Many ABA-regulated genes associated with these processes have been identified and analyzed. Previously, we identified 2 important elements in the promoter of the carrotDcECP31 gene: motif X (CACACGTGGG), which is like an ABA-responsive element (ABRE), and motif Y (CACACGTATC). Together, these are sufficient for embryo-specific ABA-inducible promoter activity. We also showed that motif X functions is an enhancerlike element and that motif Y participates in ABA responsiveness. In this study, we isolated the nuclear protein that interacts with motif Y of theDcECP31 promoter. We performed yeast one-hybrid screening using integrated motif Y as bait and isolated clones. Sequence analysis revealed that clone 22 included the carboxyl-terminal half of bZIP, which contains the basic and leucine zipper domains and binds to G-boxes containing the sequence ACGT. This result supports the hypothesis that carrot C-ABI3, a homologue of theArabidopsis ABI3 protein, functions as a coactivator that interacts with the G-box via protein-protein contacts and suggests that the complex controls the expression of theDcECP31 gene.