Fluorometric quantification of green fluorescent protein in tobacco leaf extracts

Robić, Goran; Lacorte, Cristiano; Miranda, Everson Alves

doi:10.1016/j.ab.2009.05.016

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…2B and 2C ). The fluorescence of MOS9-YFP was considerably lower in the tt4-11 background, even though the absence of flavonoids in these tissues should, if anything, provide for an enhanced fluorescence signal from YFP ( Mercuri et al, 2001 ; Robic, Lacorte & Miranda, 2009 ). The finding that MOS9 transcript levels were not statistically different in this line ( Fig.…”

Section: Resultsmentioning

confidence: 93%

Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus

Watkinson

Bowerman

Crosby

et al. 2018

PeerJ

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Plant flavonoid metabolism has served as a platform for understanding a range of fundamental biological phenomena, including providing some of the early insights into the subcellular organization of metabolism. Evidence assembled over the past three decades points to the organization of the component enzymes as a membrane-associated complex centered on the entry-point enzyme, chalcone synthase (CHS), with flux into branch pathways controlled by competitive protein interactions. Flavonoid enzymes have also been found in the nucleus in a variety of plant species, raising the possibility of alternative, or moonlighting functions for these proteins in this compartment. Here, we present evidence that CHS interacts with MOS9, a nuclear-localized protein that has been linked to epigenetic control of R genes that mediate effector-triggered immunity. Overexpression of MOS9 results in a reduction of CHS transcript levels and a metabolite profile that substantially intersects with the effects of a null mutation in CHS. These results suggest that the MOS9–CHS interaction may point to a previously-unknown mechanism for controlling the expression of the highly dynamic flavonoid pathway.

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Section: Resultsmentioning

confidence: 93%

Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus

Watkinson

Bowerman

Crosby

et al. 2018

PeerJ

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“…Firstly, the presence of some agent that can mask the GFP fluorescence, and/or is opaque to the excitation signal, represents an obstacle that could complicate the monitoring of the fluorescence emitted from particular tissue (Robic et al 2009). Consequently, untransformed plant tissues were used as negative controls for adjusting the parameters to ensure the absence of GFP fluorescence in nontransformed leaves.…”

Section: Discussionmentioning

confidence: 99%

Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy

Cai

Tan

et al. 2010

Plant Cell Tiss Organ Cult

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The green fluorescent protein (GFP) has become an ideal visual marker to monitor and quantify the expression of the transgene. It can be targeted to specific subcellular locations, including the endoplasmic reticulum, mitochondria, actin cytoskeleton and nuclei through the addition of signal peptides. Our previous work has resulted in transgenic citrus plants expressing cytoplasmic targeted GFP (Cy-GFP) or endoplasmic reticulum targeted GFP (Er-GFP) gene. To evaluate the localization of three different subcellular targeted GFP, i.e., Cy-GFP, Er-GFP and mitochondria targeted GFP (Mt-GFP) in citrus tissues and to utilize cell lines containing Mt-GFP for basic research in cell fusion, the plasmid pBI-mgfp4-coxIV encoding the Mt-GFP gene was successfully transferred into embryogenic callus of Valencia sweet orange (Citrus sinensis (L.) Osbeck) via Agrobacterium tumefaciens-mediated transformation. Furthermore, we compared the specific expression of these three different subcellular localized GFP constructs in cells of different mature leaf tissues (upper epidermis, palisade parenchyma, spongy parenchyma and lower epidermis) by a confocal laser scanning microscope (CLSM). Cytoplasmic-localized GFP expression was observed throughout the cytoplasm but appeared to accumulate within the nucleoplasm. The Er-GFP occurred within a layer very close to the cell wall. In addition, a stable fluorescence on the ER network throughout the guard cells was detected. Interestingly, the Mt-GFP specifically expressed in the guard cells to particles of about 1-2 lm within the cytoplasm in this case. To verify that the fluorescent particles observable in the guard cells are indeed mitochondria, we co-localize the Mt-GFP fusion protein with a mitochondrial-specific dye in citrus protoplasts. These results demonstrate that the subcellular distribution of the three subcellular targeted GFP is very distinct in citrus leaf cells and the cell lines containing Mt-GFP gene can be further used in citrus basic cell fusion research.

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“…Fluorometric measurements were done according to Robic et al29 by using an F‐4500 fluorescence spectrophotometer (Hitachi, Japan) set to a 480‐nm excitation wavelength with an excitation slit opening of 5 nm and a 513‐nm emission wavelength with a 20‐nm emission slit opening. As a reference sGFP(S65T) was used, which was prepared as described in our previous publication 29. One unit of sGFP fluorescence was defined as the fluorescence of 1 ng/mL of sGFP in 50 mmol/L sodium phosphate buffer pH 7.00.…”

Section: Methodsmentioning

confidence: 99%

Application of electrochemically produced aluminum hydroxide gel for prepurification of recombinant synthetic green fluorescent protein produced in tobacco leaves

Robić

Lacorte

Rech

et al. 2011

Biotechnology Progress

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The use of recombinant proteins has increased greatly in recent years, as also have increased the number of techniques and materials used for their production and purification. Among the different types of bioreactors being studied, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. In the present work, the adsorption to electrochemically produced aluminum hydroxide gel was applied as a prepurification step for recombinant synthetic green fluorescent protein (sGFP), also referred to as enhanced green fluorescent protein, produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. As electrochemical preparation of aluminum hydroxide gel is a cost-effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest.

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Fluorometric quantification of green fluorescent protein in tobacco leaf extracts

Cited by 7 publications

References 27 publications

Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus

Identification of MOS9 as an interaction partner for chalcone synthase in the nucleus

Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy

Application of electrochemically produced aluminum hydroxide gel for prepurification of recombinant synthetic green fluorescent protein produced in tobacco leaves

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