Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot.

To study the regulation of gene expression during embryo development, we isolated a gene, DC 59, expressed in embryos but not in mature carrot plants. Sequence and S1 analysis showed that the gene was composed of one exon encoding a polypeptide of 19 kilodaltons and was highly homologous to the lipid body membrane protein gene L3 from maize. The plant hormone abscisic acid regulated the accumulation of DC 59 mRNA. To understand the mechanism of embryo-specific and hormonal regulation of DC 59, 5' DNA fragments were incubated with nuclear proteins. Two adjacent regions (from -706 to -235) interacted with nuclear extracts from embryos, resulting in the formation of four complexes (C1, C2, C3, and C4). Factors involved in the formation of the C3 and C4 complexes could be competed with sequences upstream of DC 8, a gene that is coordinately expressed with DC 59 during embryo development. DNase I footprinting analysis revealed that nuclear extracts from embryos bound to four AT-rich sequences, and the protected motifs within fragment V were located in the highly homologous upstream regions of DC 59 and DC 8 genes.     

麻疯树MIPS基因启动子的分离及在烟草原生质体中瞬时表达活性分析

根据麻疯树MIPS基因序列,设计特异性的巢式引物,运用TAIL-PCR法两次步移得到MIPS基因5＇端侧翼序列,序列分析显示含有多个胁迫应答相关元件,如ABRE、HSE等。以该序列为基础,PCR扩增得到5个5＇端不同长度的缺失片段,分别插入pBI221载体置换CaMV35S启动子,构建的表达载体在PEG介导下转入烟草叶片原生质体进行瞬时表达,检测GUS报告基因的活性。经GUS活性荧光定量检测发现,分离到的MIPS基因侧翼序列5＇端不同缺失片段都能启动GUS报告基因表达,启动活性最高的是WQ1区（-565bp）,核心区位于-565～-449bp。在100μmol·L-1ABA诱导下启动活性增强,但不同区段的增长幅度不同。WQ1区增长幅度最大,比未处理时提高41.4%。     

Interaction between two cis-acting elements,ABRE and DRE,in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.     

Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A–GUS fusion genes using tobacco protoplasts revealed novel types of positive cis/-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis/-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner.     

The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.     

Gibberellin-responsive elements in the promoter of a barley high-pI alpha-amylase gene.

Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal alpha-amylase promoter (-41) conferred both GA- and ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pI alpha-amylase genes. The TAACAAA box also appears to be the site of action of ABA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

A wheat histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to ?1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (?1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division-dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division-independent detected in flower tissues including the anther wall and the pistil. In this study, replication-dependent expression occurring in non-dividing cells which undergo endoreduplication could not be discriminated from strict replication-independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5′ sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, ?908H3/GUS and ?185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from ?909 to ?1711 contains the positive cis-acting element(s) and that the proximal promoter region (up to ?185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division-dependent and -independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division-dependent expression of plant genes is discussed.     

Induction of a wheat Em promoter by ABA and optically pure ABA analogs in white spruce (Picea glauca) protoplasts

In white spruce ( Picea glauca ) protoplasts, abscisic acid (ABA) and optically pure ABA analogs induced expression of a reporter gene under regulation of a wheat ABA-responsive promoter. A fusion of a 650 bp promoter fragment from the wheat Em gene promoter and the Escherichia coli uidA sequence encoding β -glucuronidase (GUS) was linked in the plasmid pBM 113Kp. Expression of the Em-uidA fusion varied among 6 white spruce genotypes. Protoplasts from 4-day-old embryogenic suspension cultures gave the highest GUS activity relative 10 other stages in the 7-day growth cycle of suspension cultures. Racemic ABA [R.S-(±)-ABA] induced a significant increase of protoplast GUS activity over background at a concentration of 1 × 10⁻⁵ M , but maximum GUS activity was found at 1 × 10⁻³ M , ABA stereochemistry had a significant effect on gene expression. The natural isomer of ABA [S-(+)-ABA] was an effective inducer at a concentration as low as 1 × 10⁻⁷ M , but a concentration of greater than 1 × 10⁻⁴ M was required for induction by [R-(—)-ABA]. Moreover, analogs with the same configuration at C-l¹ as that of natural ABA were more effective for induction of expression from the Em-uidA . insert at 1 × 10⁻⁴ M than were their enamiomers. Plasnud pBI511. carrying the chloramphenicol acety] transferase (CAT) gene driven by the constitutively expressed, tandemly duplicated cauliflower mosaic virus 35S promoter, was co-electroporated with pBM113Kp for monitoring Ihe influence of addition of exogenous ABA or ABA analogs on heterologous gene expression in protoplasts. CAT activity was not significantly affected by the presence or absence of ABA or the analogs used.     

Abscisic acid-responsive sequences from the em gene of wheat.

We demonstrate that a chimeric gene containing the beta-glucuronidase (GUS) reporter gene linked to a 646-base pair 5' fragment (-554 to +92) from the abscisic acid (ABA)-regulated Em gene from wheat is correctly expressed in transgenic tobacco. We observe high activity only in embryos of mature seeds, and immature seeds cultured on ABA show enhanced expression. Using a rice transient assay, we identify a 260-base pair fragment (-168 to +92) that accounts for the ABA-specific 15-fold to 20-fold increase in GUS expression. A 50-base pair sequence (-152 to -103) fused 5' in either orientation to a truncated cauliflower mosaic virus promoter (35S) increases GUS activity threefold in the presence of ABA. Insertion of the Em 5'-untranslated region (+6 to +86) between the 35S promoter and the ATG of GUS results in a 10-fold increase in GUS activity in the absence of ABA. These results suggest the following two functional fragments of the Em 5' region: an ABA response element from -152 to -103 and an element between +6 and +86 that quantitatively increases the ABA response.     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.