The dark side of green fluorescent protein

Zhou, Xin; Carranco, Raúl; Vitha, Stanislav; Hall, Timothy C.

doi:10.1111/j.1469-8137.2005.01489.x

Cited by 57 publications

(51 citation statements)

References 33 publications

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“…Bright-field and fluorescence pictures were merged with Adobe Photoshop 7.0 (Adobe Systems). Prior to fluorescence microscopy, leaf blades were extracted with 95% ethanol according to Zhou et al (2005). Leaf blades were harvested from 30-cm-tall F. bidentis grown in the greenhouse.…”

Section: Confocal Laser Scanning Microscopy and Fluorescence Microscopymentioning

confidence: 99%

Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes

et al. 2012

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The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C 3 plants but is restricted to bundle sheath cells in C 4 species. GDC is also required for general cellular C 1 metabolism. In the Asteracean C 4 species Flaveria trinervia, a single functional GLDP gene, GLDPA, encodes the P-subunit of GDC, a decarboxylating Gly dehydrogenase. GLDPA promoter reporter gene fusion studies revealed that this promoter is active in bundle sheath cells and the vasculature of transgenic Flaveria bidentis (C 4 ) and the Brassicacean C 3 species Arabidopsis thaliana, suggesting the existence of an evolutionarily conserved gene regulatory system in the bundle sheath. Here, we demonstrate that GLDPA gene regulation is achieved by an intricate interplay of transcriptional and posttranscriptional mechanisms. The GLDPA promoter is composed of two tandem promoters, P R2 and P R7 , that together ensure a strong bundle sheath expression. While the proximal promoter (P R7 ) is active in the bundle sheath and vasculature, the distal promoter (P R2 ) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 59 untranslated leader of P R2 -derived transcripts is inefficiently spliced and apparently suppresses the output of P R2 by eliciting RNA decay.

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Section: Confocal Laser Scanning Microscopy and Fluorescence Microscopymentioning

confidence: 99%

Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes

et al. 2012

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“…In 7 DAG (days after germination) plants, the levels of GFP fluorescence were high in the vascular bundle sheath and stomatal guard cells, but relatively low in mesophyll cells of the APX:gfp, PGD1:gfp and R1G1B:gfp leaves. Levels of GFP fluorescence in mesophyll cells could have been underestimated due to quenching of GFP fluorescence in chlorophyll-containing tissues, as described by Zhou et al (2005). Levels of GFP fluorescence in root apex, root cap and elongating regions of PGD1:gfp roots were comparable with those of OsCc1, Act1 and ZmUbi1:gfp roots.…”

Section: Activity Of Promoter:gfp Constructs In Various Organs and Timentioning

confidence: 74%

Analysis of the APX, PGD1 and R1G1B constitutive gene promoters in various organs over three homozygous generations of transgenic rice plants

Park

Bang

Jeong

et al. 2012

Planta

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We have previously characterized the constitutively active promoters of the APX, PGD1 and R1G1B genes in rice (Park et al. 2010 in J Exp Bot 61:2459-2467). To have potential crop biotechnology applications, gene promoters must be stably active over many generations. In our current study, we report our further detailed analysis of the APX, PGD1 and R1G1B gene promoters in various organs and tissues of transgenic rice plants for three (T₃₋₅) homozygous generations. The copy numbers in 37 transgenic lines that harbor promoter:gfp constructs were determined and promoter activities were measured by real-time qPCR. Analysis of the 37 lines revealed that 15 contained a single copy of one of the three promoter:gfp chimeric constructs. The promoter activity levels were generally higher in multi-copy lines, whereas variations in these levels over the T₃₋₅ generations studied were observed to be smaller in single-copy than in multi-copy lines. The three promoters were further found to be highly active in the whole plant body at both the vegetative and reproductive stages of plant growth, with the exception of the APX in the ovary and R1G1B in the pistil and filaments where zero or very low levels of activity were detected. Of note, the spatial activities of the PGD1 promoter were found to be strikingly similar to those of the ZmUbi1, a widely used constitutive promoter. Our comparison of promoter activities between T₃, T₄ and T₅ plants revealed that the APX, PGD1 and R1G1B promoters maintained their activities at comparable levels in leaves and roots over three homozygous generations and are therefore potentially viable alternative promoters for crop biotechnology applications.

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“…GFP expression was better visualized in roots when compared to other plant tissues due to chlorophyll interference in GFP detection. Even with the appropriate filters, interference of chlorophyll with GFP detection could occur through an overabundance of red fluorescence or competition between GFP and chlorophyll for the same excitation wavelengths of light (Zhou et al 2005). Therefore, GFP expression in a low-expressing clone can be of difficult observation.…”

Section: Resultsmentioning

confidence: 98%

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and Agrobacterium system

et al. 2010

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An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2D wrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes' integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T 1 progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.

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The dark side of green fluorescent protein

Cited by 57 publications

References 33 publications

Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes

Regulation of the Photorespiratory GLDPA Gene in C4 Flaveria: An Intricate Interplay of Transcriptional and Posttranscriptional Processes

Analysis of the APX, PGD1 and R1G1B constitutive gene promoters in various organs over three homozygous generations of transgenic rice plants

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and Agrobacterium system

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