Chloroplast-dependent and light-independent expression of the tobacco rbcS promoter–GUS chimeric gene in black spruce

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

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(285) Characterization of a Composite Promoter from Genomic Sequences of Grapevine

HortScience ◽

10.21273/hortsci.41.4.1053c ◽

2006 ◽

Vol 41 (4) ◽

pp. 1053C-1053

Author(s):

Manjul Dutt ◽

Zhijian T. Li ◽

Sadanand Dhekney ◽

Dennis J. Gray

Keyword(s):

Mosaic Virus ◽

Genomic Sequence ◽

Fusion Gene ◽

Core Promoter ◽

Gus Activity ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

2S Albumin ◽

Wide Range ◽

Constitutive Gene Expression

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

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Enhancer Trapping Identifies TRI, an Arabidopsis Gene Up-Regulated by Pathogen Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.10.1086 ◽

2004 ◽

Vol 17 (10) ◽

pp. 1086-1094 ◽

Cited By ~ 7

Author(s):

Ingela Fridborg ◽

Alan Williams ◽

Aidong Yang ◽

Stuart MacFarlane ◽

Katherine Coutts ◽

...

Keyword(s):

Fluorescent Protein ◽

Tobacco Rattle Virus ◽

Defense Responses ◽

Gus Activity ◽

Enhancer Trap ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Resistance Response ◽

Enhancer Element ◽

Enhancer Trap Line

Enhancer trap Arabidopsis thaliana plants were screened for genes up-regulated by virus infection. The plants carried T-DNA insertions comprising a minimal -60-bp Cauliflower mosaic virus 35S promoter fused to the β-glucuronidase (GUS) reporter gene. Approximately 12,000 plants were assayed for GUS activity before and after rub-inoculation with Tobacco rattle virus (TRV) tagged with the green fluorescent protein (GFP). One plant and its progeny consistently showed upregulation of GUS activity in response to TRV-GFP infection, indicating that a virus-responsive enhancer element was “tagged” by the T-DNA in this line. Other viruses, bacteria, and oomycetes, but not wounding, up-regulated GUS activity in the enhancer trap line, indicating that the response was not specific to TRV-GFP infection. A pathogen-inducible, alternatively spliced gene was identified, which we have termed TRI for TRV-induced gene. A pathogen-responsive element was localized to a 1.1-kb region upstream of the T-DNA insertion, and two different cis-acting elements, both implicated in defense responses, were found in the sequence upstream of TRI. Sequence analyses revealed that TRI is similar to ACRE169, a gene that is up-regulated in Cf-9-expressing tobacco when treated with Avr-9, the Cladosporium fulvum elicitor of the Cf-9 resistance response.

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Nematode-Responsive Activity of the Cauliflower Mosaic Virus 35S Promoter and Its Subdomains

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.3.189 ◽

1999 ◽

Vol 12 (3) ◽

pp. 189-196 ◽

Cited By ~ 20

Author(s):

David J. Bertioli ◽

Matthew Smoker ◽

Paul R. Burrows

Keyword(s):

Mosaic Virus ◽

Giant Cells ◽

Gus Activity ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Host Gene Expression ◽

Root Knot Nematode ◽

Cyst Nematodes ◽

Feeding Sites ◽

Cauliflower Mosaic Virus 35S

Root-knot and cyst nematodes are obligate plant parasites that induce complex biotrophic feeding structures in host roots. The mechanisms by which nematodes regulate host gene expression to produce feeding sites are unknown. The cauliflower mosaic virus (CaMV) 35S promoter has been reported to be repressed strongly in the feeding sites of both root-knot and cyst nematodes. In contrast, other work has indicated that this promoter is partially active in some feeding sites. Considering the importance of the 35S promoter in biotechnology, we have defined the nematoderesponsive nature of this promoter in more detail. Transgenic tobacco harboring various 35S-uidA constructs was assayed for β-glucuronidase (GUS) activity after infection by root-knot nematodes (Meloidogyne incognita) and cyst nematodes (Globodera tabacum subsp. tabacum). The entire 35S promoter (-343 to +8) was active in giant cells induced by M. incognita and, to a lesser extent, the syncytia of G. tabacum subsp. tabacum. In the latter case, activity decreased as the feeding sites matured. Subdomains of the 35S promoter were also active in feeding sites, particularly B4 and B5 in giant cells. However, subdomain B3 was strongly down-regulated in gall tissue and syncytia. In total, 14 constructs were studied and nematode-responsive expression was always stronger and more consistent with the root-knot nematode than the cyst nematode.

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Evaluation of Constitutive Viral Promoters in Transgenic Soybean Roots and Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-8-1027 ◽

2008 ◽

Vol 21 (8) ◽

pp. 1027-1035 ◽

Cited By ~ 26

Author(s):

Manjula Govindarajulu ◽

James M. Elmore ◽

Thomas Fester ◽

Christopher G. Taylor

Keyword(s):

Mosaic Virus ◽

Hairy Roots ◽

Target Genes ◽

Root Nodules ◽

Gus Activity ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Symbiotic Interaction ◽

Strawberry Vein Banding Virus ◽

Viral Promoters

The efficiency of β-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.

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Tissue-specific and light regulation of a larch ribulose-1,5-bisphosphate carboxylase promoter in transgenic tobacco

Canadian Journal of Forest Research ◽

10.1139/x94-218 ◽

1994 ◽

Vol 24 (8) ◽

pp. 1689-1693 ◽

Cited By ~ 1

Author(s):

Michael A. Campbell ◽

David B. Neale ◽

Peter Harvie ◽

Keith W. Hutchison

Keyword(s):

Light Regulation ◽

Evolutionary Conservation ◽

Gus Activity ◽

Larix Laricina ◽

Histochemical Analysis ◽

Bisphosphate Carboxylase ◽

Tissue Specific ◽

Rbcs Promoter ◽

Organ Specific ◽

Root Tissues

A chimeric gene composed of an eastern larch (Larix laricina (Du Roi) K. Koch) ribulose-1,5-bisphosphate carboxylase (RbcS) promoter linked to the β-glucuronidase (GUS) coding sequence was transferred to tobacco (Nicotianatabacum (L.)) via Agrobacteriumtumefaciens transformation. Based on GUS activity the larch RbcS promoter functioned in an organ-specific and light-regulated manner. Histochemical analysis revealed high levels of GUS activity in photosynthetically active tissues and low or undetectable activity in xylem and root tissues. Fluorometric analysis of GUS activity demonstrated that the larch RbcS promoter was expressed at a 10-fold higher level in leaf blades than in root tissue. Light-grown transgenic plants expressed GUS at a two-fold higher level than dark-grown individuals. These results suggest evolutionary conservation of tissue-specific RbcS promoter activity between gymnosperms and angiosperms but only weak conservation of the transduction mechanism for light regulation.

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Four Mutant Alleles Elucidate the Role of the G2 Protein in the Development of C4 and C3 Photosynthesizing Maize Tissues

Genetics ◽

10.1093/genetics/159.2.787 ◽

2001 ◽

Vol 159 (2) ◽

pp. 787-797

Author(s):

Lizzie Cribb ◽

Lisa N Hall ◽

Jane A Langdale

Keyword(s):

Cell Types ◽

Bundle Sheath ◽

Primary Role ◽

Ribulose Bisphosphate Carboxylase ◽

Sheath Cell ◽

Mesophyll Cells ◽

Phenotypic Data ◽

Bisphosphate Carboxylase ◽

Bundle Sheath Cell

Abstract Maize leaf blades differentiate dimorphic photosynthetic cell types, the bundle sheath and mesophyll, between which the reactions of C4 photosynthesis are partitioned. Leaf-like organs of maize such as husk leaves, however, develop a C3 pattern of differentiation whereby ribulose bisphosphate carboxylase (RuBPCase) accumulates in all photosynthetic cell types. The Golden2 (G2) gene has previously been shown to play a role in bundle sheath cell differentiation in C4 leaf blades and to play a less well-defined role in C3 maize tissues. To further analyze G2 gene function in maize, four g2 mutations have been characterized. Three of these mutations were induced by the transposable element Spm. In g2-bsd1-m1 and g2-bsd1-s1, the element is inserted in the second intron and in g2-pg14 the element is inserted in the promoter. In the fourth case, g2-R, four amino acid changes and premature polyadenylation of the G2 transcript are observed. The phenotypes conditioned by these four mutations demonstrate that the primary role of G2 in C4 leaf blades is to promote bundle sheath cell chloroplast development. C4 photosynthetic enzymes can accumulate in both bundle sheath and mesophyll cells in the absence of G2. In C3 tissue, however, G2 influences both chloroplast differentiation and photosynthetic enzyme accumulation patterns. On the basis of the phenotypic data obtained, a model that postulates how G2 acts to facilitate C4 and C3 patterns of tissue development is proposed.

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