Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription

The promoter region (?309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5′ as well as internal deletions fused to the reporter gene GUS (β-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between ?309 to ?152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position ?152 to position ?144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region ?133 to ?120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)_n element, found in other storage protein promoters, was identified. This suggest that the (CA)_n element is important for conferring seed specificity by serving both as an activator and a repressor element.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5 to 3) in the 5 region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter--glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position-173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the-326 to-173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position-173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.     

Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco

Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study, we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158 was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter.     

Analysis of the Tissue-SpecificS RNase gene promoter associated with self-incompatibility in tomato (Lycopersicon peruvianum L.)

To understand the expression pattern of theS RNase gene in the floral tissues associated with self-incompatibility (SI), promoter region of S₁₁ RNase gene was serially deleted and fused GUS. Five chimeric constructs containing a deleted promoter region of the S₁₁ RNase gene were constructed, and introduced intoNicotiana tabacum using Agrobacterium-mediated transformation. Northern blot analysis revealed that the GUS gene was expressed in the style, anther, and developing pollen of all stages in each transgenic tobacco plant The developing pollen expressed the same amount of GUS mRNA in all stages in transgenic tobacco plants. In addition, histochemical analysis showed GUS gene expression in vascular bundle, endothecium, stomium, and tapetum cells during pollen development in transgenic plants. From these results, it is speculated that SI ofLycopersicon peruvianum may occur through the interaction ofS RNase expressed in both style and pollen tissues.     

Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.