Green Fluorescent Protein In Plants Research Articles

A Cryohistological Protocol for Preparation of Large Plant Tissue Sections for Screening Intracellular Fluorescent Protein Expression

In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg 1 −1, respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.

Nonspecific intercellular protein trafficking probed by green-fluorescent protein in plants

Plasmodesmata mediate intercellular transport of proteins, nucleic acids, and small molecules in plants. We show that transiently produced green-fluorescent protein (GFP) trafficked intercellularly in the epidermis of sink leaves, but not of source leaves, in tobacco and cucumber. In contrast, the protein did not traffic in either sink or source leaves of tomato. On the other hand, the protein spread extensively from cell to cell in the epidermis of all leaves and stems ofArabidopsis thaliana as well as in young hypocotyls and cotyledons of tomato and cucumber. GFP could traffic from epidermis to ground tissues in hypocotyls but not in cotyledons of cucumber. GFP fused to a number of mutant forms of the cucumber mosaic virus 3a movement protein (CMV 3a MP) failed to traffic from cell to cell, suggesting that GFP does not have a specific motif for plasmodesmal trafficking. Our data, together with previous findings, indicate that plasmodesmata can mediate both specific and nonspecific intercellular trafficking of proteins. Furthermore, our data suggest that nonspecific protein trafficking is controlled by species-, development-, organ-, and tissue-specific factors. Since GFP can readily traffic from cell to cell, it raises the questions of how metabolites are compartmentalized intercellularly in a plant and of whether some endogenous plant proteins traffic nonspecifically from cell to cell to perform physiological functions yet to be elucidated.

Applications of green fluorescent protein in plants.

Green fluorescent protein (GFP) is increasingly being used in plant biology from the cellular level to whole plant level. At the cellular level, GFP is being used as an in vivo reporter to assess frequency of transient and stable transformation. GFP has also proven to be an invaluable tool in monitoring trafficking and subcellular localization of protein. At the organ level and up, many exciting applications are rapidly emerging. The development of brighter GFP mutants with more robust folding properties has enabled better macroscopic visualization of GFP in whole leaves and plants. One interesting example has been the use of GFP to monitor virus movement in and among whole plants. GFP is also emerging as a powerful tool to monitor transgene movement and transgenic plants in the field. In a proof-of-concept study, tobacco was transformed with a modified version of the GFP gene controlled by a constitutive (35S) promoter. GFP expression in progeny plants ranged from 0% to 0.5%, and approximately 0.1% GFP was the minimal amount needed for unambiguous macroscopic detection. GFP is the first truly in vivo reporter system useful in whole plants, and we project its usefulness will increase even further as better forms of GFP genes become available.

Imaging the green fluorescent protein in plants ? viruses carry the torch

The green fluorescent protein (GFP) was introduced into plant cells using potato virus X as a vector. The GFP was produced at high levels within virus-infected cells by utilising a duplication of the viral coat protein subgenomic RNA promoter sequence to direct transcription of mRNA encoding the GFP. We also exploited the ability of GFP to retain its fluorescence when fused to other proteins by fusing it to the PVX coat protein. The resultant fluorescent virus became systemic and its movement from cell to cell was traced using confocal laser scanning microscopy. Using PVX as the vector, additional fusions of the GFP were made to the movement protein of tobacco mosaic virus (TMV). The fluorescent fusion protein produced was targeted to specific wall sites thought to be plasmodesmatal pit fields. The utility of virus-based vectors for the delivery and targeting of GFP in living plant cells is discussed.