Experimentally determined sequence requirement of ACGT-containing abscisic acid response element

The sequence requirement of the ACGT-containing abscisic acid response element (ABRE) was analyzed by systematically substituting the bases surrounding the ACGT-core of motif A, the principal ABRE of the rice gene, OSEM: This was done within the context of a 55-bp promoter fragment that minimally confers ABA-responsiveness to a heterologous promoter. Based on this analysis, the sequence requirement of the ACGT-containing ABRE was determined as ACGTG G/T C, which matched very well with the consensus derived from sequence comparison of ABA-responsive promoters.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.     

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em 1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.     

Induction of Expression Instability of the nptII Gene in Transgenic Tobacco Plants

A comparative analysis of the neomycin phosphotransferase (nptII) gene expression was performed in two groups of transformed tobacco plants, one of which included plants with direct and inverted tandem uidA gene repeats in the T-DNA insertion. This insertion of inverted repeats was shown to reduce the level of stable nptII gene expression to 20%, as compared with 65% in the control transformants. The level of unstable expression of this gene substantially increased (up to 71.4% vs. 5.5% in the control group) when homologous sequences were brought together with direct tandem repeats in the genome of hybrid plants.     

Cloning and functional characterization of gpd and α-tubulin promoters from Annulohypoxylon stygium,a companion fungus of Tremella fuciformis

Annulohypoxylon stygium is an ascomycete that helps Tremella fuciformis produce the fruiting body in wild state or artificial cultivation. Although the interaction between these two fungi is well known, the underlying molecular mechanism is limited. In this study, the 981 bp and 886 bp promoter sequences of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and α-tubulin gene have been cloned, respectively. Sequence analysis showed that gpd promoter contained nine CAAT boxes, and single TGACG-motif, TATA box, ABRE motif, STRE motif, MYB motif, and W box. The α-tubulin promoter included eight CAAT boxes, three STRE, two TATA boxes and MYB, single Box 4, CAT-box, CCAAT-box, TGA-element, and ABRE. Subsequently, we evaluated the promoters' function by constructing four vectors pGEH, pGRH, pTEH, and pTRH to drive fused enhanced green fluorescent protein and hygromycin B phosphotransferase (egfp-hph) or red fluorescent protein and hygromycin B phosphotransferase (rfp-hph) expression under the control of gpd or α-tubulin promoters in A. stygium. The integration of the transformed DNA into A. stygium genome was verified by PCR, Southern blot, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). All the results indicated that the two promoters could drive egfp-hph and rfp-hph expression. This result could provide help in gene functional studies by using gpd and α-tubulin promoters to direct gene over-expression or build dual promoter silencing systems.     

Dehydration-Stress-Regulated Transgene Expression in Stably Transformed Rice Plants

To confer abscisic acid (ABA) and/or stress-inducible gene expression, an ABA-response complex (ABRC1) from the barley (Hordeum vulgare L.) HVA22 gene was fused to four different lengths of the 5′ region from the rice (Oryza sativa L.) Act1 gene. Transient assay of β-glucuronidase (GUS) activity in barley aleurone cells shows that, coupled with ABRC1, the shortest minimal promoter (Act1–100P) gives both the greatest induction and the highest level of absolute activity following ABA treatment. Two plasmids with one or four copies of ABRC1 combined with the same Act1–100P and HVA22(I) of barley HVA22 were constructed and used for stable expression of uidA in transgenic rice plants. Three Southern blot-positive lines with the correct hybridization pattern for each construct were obtained. Northern analysis indicated that uidA expression is induced by ABA, water-deficit, and NaCl treatments. GUS activity assays in the transgenic plants confirmed that the induction of GUS activity varies from 3- to 8-fold with different treatments or in different rice tissues, and that transgenic rice plants harboring four copies of ABRC1 show 50% to 200% higher absolute GUS activity both before and after treatments than those with one copy of ABRC1.     

Motif X (CACACGTGGG) and motif Y (CACACGTATC) of theDcECP31 promoter have different functions in the ABA-response pathway

We have identified two important elements in the DcECP31 promoter, the ABA-responsive element (ABRE)-like motif X (CACACGTGGG), and motif Y (CACACGTATC), which are sufficient for embryo-specific and ABA-inducible promoter activities. Using gain-of-function analysis, we demonstrated that motifs X and Y are necessary and sufficient to confer ABA-responsiveness upon a heterologous, truncated CaMV minimum promoter. Motif-exchange experiments were carried out to determine whether these motifs exerted similar effects on the DcECP31 promoter. The results suggest that motif X functions as an enhancer-like element and that motif Y participates in ABA-responsiveness.