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.     

Far upstream activating promoter regions are responsible for expression of the BnC1 cruciferin gene from Brassica napus

Summary Cruciferin is the major seed storage protein in Brassica napus. As much as 1.9 kbp of the BnC1 cruciferin gene promoter have been sequenced and analyzed. Promoter fragments with 5 deletions from –2500 to –v202 were fused with the ß-glucuronidase reporter gene and used for Nicotiana tabacum transformation. ß-glucuronidase could be specifically expressed in transgenic tobacco seeds under the control of the BnC1 promoter and regulatory elements were found to be dispersed over 1903 bp. An almost 5-fold increase in ß-glucuronidase expression was obtained when the promoter length was increased from –379 to –498, and another 10-fold increase was observed when sequences between –1266 and –1903 were added. Histochemical analysis shows that the region between –844 and –1266 directs the expression of the chimeric gene specifically to the root apical meristem.Abbreviations GUS ß-glucuronidase - MU 4-methyl umbelliferone - MUG 4-methyl-umbelliferyl-ß-D-glucuronide - X-gluc 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

Positive and negative cis-regulatory regions in the soybean glycinin promoter identified by quantitative transient gene expression

Summary The 5 and 3 flanking regions of the soybean glycinin gene, Gy1, responsible for expression in seeds, were analyzed by quantitative transient expression assay. The construct containing the -glucuronidase (uidA) reporter gene under the control of the 1.12 kb Gy1 promoter and 0.74 kb Gy1 terminator was introduced into immature soybean seeds and leaves by particle bombardment. To normalize the variability of introduction efficiency, a second reporter gene, firefly luciferase, was cobombarded as an internal standard, and relative activities (GUS/luciferase) were measured. There was a seed-specific -glucuronidase (GUS) expression, as observed by X-Gluc staining. Compared with the nopaline synthase gene (nos) terminator, the Gy1 terminator enhanced the level of expression in immature seeds, indicating that the terminator region of the glycinin gene is involved in the activation of the gene expression in these seeds. To identify cis-regulatory elements in the glycinin gene upstream sequence, deleted derivatives of the promoter were fused to the luciferase reporter gene. The expression could be measured with a higher accuracy, and constructs were introduced with the internal reporter uidA gene into immature seeds. The results suggest the presence of a positive regulatory element in the –620 to ––380 region of the Gy1 promoter. A deletion which eliminates the legumin box with its RY element led to increased relative activity, suggesting that this box is negatively regulating expression of the seed storage protein gene. Analysis of mutant promoters also suggest that the RY element involves negative regulation in seeds. This quantitative transient expression assay using particle bombardment provides a reliable system for the study of seed-specific gene expression in soybeans.Abbreviations GUS -glucuronidase - Gy1 glycinin AlaB2 gene - CaMV cauliflower mosaic virus - nos nopaline synthase gene - uidA -glucuronidase gene - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Apical and lateral shoot apex-specific expression is conferred by promoter of the seed storage protein β-phaseolin gene

A previous analysis with deletion mutants of the native -phaseolin gene demonstrated that removal of a negative element 5 upstream of–107 permitted phaseolin expression in stem cortex and secondary root (Burowet al., 1992). Here we employed the -glucuronidase (GUS) reporter gene to visualize, by histochemical staining, the cell type-specificity of phaseolin expression in stem and root, and to understand further the spatial control of the -phaseolin gene. The 782 bp 5 upstream promoter and its deletion mutants were fused to the GUS gene, and these chimaeric genes were used to transform tobacco. Histochemical staining for GUS activity demonstrated that phaseolin promoters truncated downstream of –227 conferred cell-type specific expression in internal/external phloem and protoxylem of mature stem. Surprisingly, GUS staining was prominent in both apical and lateral shoot apices of plants that contain the full-length –782 promoter and mutant promoters deleted up to –64. GUS expression was extended to all cell types of shoot tips, including epidermis, cortex, vasculature, procambium and pith. Expression in vasculature of petioles was limited to plants with promoters truncated to –106 and –64. The current results are in agreement with our previous findings with the native phaseolin gene: that the major positive element (–295/–228) is sufficient for seed-specific late-temporal expression of the phaseolin gene. We conclude that the 5 upstream sequence of the -phaseolin gene directs spatially- and temporally-controlled gene expression in developing seeds during the reproductive phase, but also confers expression in shoot apices during the vegetative phase of plant development.     

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells

A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5-deletion mutants of the SWPA2 promoter fused to the -glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the –1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the –1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the –1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.     

Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene,in roots of transgenic tobacco and cowpea1

SbPRP1 is a member of the soybean (Glycine max L. Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue. To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding -glucuronidase (GUS). This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation. Nested 5-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between –1080 and –262 is required for maximal expression of this gene. Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between –1080 and –623, a region which is needed for maximal expression of the SbPRP1 promoter. Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition. These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.     

Developmental,hormonal, and pathogenesis-related regulation of the tobacco class I β-1,3-glucanase B promoter

The class I -1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric -glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I -1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I -1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from –1452 to –1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: –568 to –402 for ethylene induction of leaves; –402 to –211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and –211 to –60 for expression in roots.     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Upstream sequences regulating legumin gene expression in heterologous transgenic plants

Summary We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the -glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. -Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.     

Heat shock elements are involved in heat shock promoter activation during tobacco seed maturation

The soybean Gmhsp17.3-B heat shock promoter is developmentally regulated in transgenic tobacco, as indicated by the constitutive expression of a -glucuronidase reporter in seeds [16]. In this paper, we show that both the heat shock promoter-driven -glucuronidase activity and the mRNA of the endogenous Nthsp18P gene accumulate coincident with the onset of seed desiccation. Deletions of the soybean Gmhsp17.3-B promoter, encompassing the heat shock element (HSE)-containing regions, revealed a co-localization of sequences responsible for heat induction and developmental expression. Moreover, synthetic HSEs fused to a TATA box sequence had the potential to stimulate the developmental expression of a GUS reporter gene in seeds of transgenic plants.     

Evaluation of putative seed-specific promoters for Linum usitatissimum

The GUS reporter gene was used to test four different putativeseed-specific promoters in developing and mature seeds, leaves and roots fromlinseed flax (Linum usitatissimum). The promoters testedincluded the regulatory regions of the -ketoacyl-CoA synthase gene (KCS)and the napin protein gene from Brassica napus, thepromoter regions of the 'unknown seed protein' (USP), and a legumin proteingene(LeB4) from Vicia faba and the CaMV 35S promoter (positivecontrol). The promoter-GUS constructs were inserted into L.usitatissimum via Agrobacterium mediatedtransformation, and GUS activity evaluated using histochemical andfluorimetrical assays. All the promoters showed some activity, but only CaMV35S, LeB4 and USP exhibited an expression level high enough to be useful inlinseed flax. Plants with USP-GUS showed the earliest GUS activity at 5 to 6days after flowering (daf) and persisting until 40 daf. Expression of GUS underthe control of the LeB4 promoter was measurable 11 daf and was still detectableat 40 daf. The KCS-GUS construct showed a low level of GUS activity between 14daf and 40 daf. Plants transformed with USP-GUS or LeB4-GUS exhibited a lowlevel of GUS activity in leaves and roots of some of the transformants,indicating the need for generating large numbers of primary transformants,followed by careful evaluation and selection for ones with not only the desiredlevel of expression, but also the desired spatial and temporal expression.     

Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5 non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions –623 to –471 and from –471 to –341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position –1185 to –341, although at different levels, maintained the same pattern of GUS expression — maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from –341 to –306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.     

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a `hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.