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  • Review Article
  • Published:

A greener world: The revolution in plant bioimaging

Nature Reviews Molecular Cell Biology volume 3, pages 520–530 (2002)Cite this article

Key Points

  • This review describes exciting advances in the use of green fluorescent protein (GFP) and its spectral derivatives to conventional and new imaging techniques in plants.

  • GFP can be used in the study of plant cells as an in vivo reporter. Several of its spectral derivatives are also commonly used in bioimaging of plants.

  • These technologies can be used for transgenic screening, motif and enhancer-trap technology, and flow cytometry.

  • Organelles can be labelled with single or multiple colours. Using suitable paths and filter combinations, dual labelling can be achieved with the confocal microscope.

  • Fluorescent proteins can also be used for physiological studies. Amongst these, the analysis of protein movement within cells, including examples of organelle tracking, the analysis of protein movement within an organelle based on FRAP, FLIP and FCS, the analysis of protein–protein interaction by FRET are discussed. The potential of fluorescent proteins as transgenic Ca2+ and pH indicators are reported. However, some caution is needed when interpreting the images.

Abstract

The exploitation of fluorescent proteins has heralded a new age in the in vivo analysis of subcellular events, and has overcome many of the limitations that are associated with the investigation of cellular and molecular processes in plant cells. Recently, there have been many exciting applications of green fluorescent protein and its spectral derivatives in the study of plant cells.

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Figure 1: Examples of plant cellular structures that can be highlighted in vivo by GFP.
Figure 2: Enhancer-trap technology.
Figure 3: Fluorescent protein spectrum.
Figure 4: Visualization of different combinations of fluorescent proteins.
Figure 5: Triple colour imaging in plants.
Figure 6: Selective photobleaching in tobacco epidermal cells.
Figure 7: Quantification of fluorescence in FRAP experiments.
Figure 8: Use of cameleons in Arabidopsis.

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Acknowledgements

The Biotechnology and Biological Sciences Research Council and Oxford Brookes University are kindly acknowledged for supporting this work.

Author information

Authors and Affiliations

  1. Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK

    Federica Brandizzi & Chris Hawes

  2. Department of Plant Sciences, University of Oxford, South Parks Rd, Oxford, OX1 3RB, UK

    Mark Fricker

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  1. Federica Brandizzi
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Correspondence to Chris Hawes.

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DATABASES

LocusLink

lamin B receptor

<i>Saccharomyces</i> Genome Database

GAL4

Swiss-Prot

BFP

calmodulin

GAL4

GFP

GUS

Luc

YFP

FURTHER READING

Encyclopedia of Life Sciences

Agrobacterium tumefaciens

Arabidopsis

confocal microscopes

FRET

green fluorescent protein

Motif-trap technology

Reporter systems

two-photon excitation (2PE) FCS technique

Glossary

ANTHOZOAN

An organism that belongs to the Coelenterata, which includes the corals and sea anemones. The three principal groups or orders are Acyonaria, Actinaria and Madreporaria.

CHROMOPHORE

The part of a coloured molecule that is responsible for light absorption over a range of wavelengths. In the case of fluorescent proteins, the absorbed light is re-emitted at a longer wavelength, which gives rise to the fluorescent colour.

BIP

One of the main chaperones of the endoplasmic reticulum that binds to nascent or unfolded polypeptides and ensures correct folding before the protein continues through the secretory pathway.

HDEL

Tetrapeptide composed of histidine, aspartic acid, glutamic acid and leucine that participates in the retrieval of soluble proteins from the Golgi to the endoplasmic reticulum.

SIALYLTRANSFERASE

A mammalian Golgi enzyme that catalyses the transfer of N-acetylneuraminic (sialic) acid to an acceptor molecule, which is usually the terminal sugar residue of an oligosaccharide, glycoprotein or glycolipid.

ACETOSYRINGONE

A low-molecular-weight plant compound that stimulates the activity of Agrobacterium vir genes and might act as a chemotactic agent in nature.

FEMTOSYRINGE

An extremely fine microcapillary-based syringe that enables injection of attolitre to femtolitre volumes into cells and organelles with minimal structural damage.

T1 GENERATION

The first progeny that is derived from a transformed plant.

ENHANCER

DNA sequences that are present in the genomes of higher eukaryotes and of various animal viruses. They can increase the transcription of genes to messenger RNA, but alone are not sufficient to cause expression.

FLOW CYTOMETRY

An optical technique for separation, classification and quantification of fluorescent cells or particles.

APOPLAST

The environment that is external to the plasma membrane. This includes cell walls and intercellular spaces, through which water and solutes pass relatively freely.

PEROXISOMES

Membrane-bound organelles that contain peroxidase and catalase, sometimes as a large crystal, where oxygen is used without ATP synthesis.

PREVACUOLE

A post-Golgi organelle that is responsible for delivering cargo to the vacuole.

PLASTIDS

A family of semi-autonomous plant-cell organelles. These are surrounded by a double membrane and contain elaborate internal membrane systems, DNA, RNA and ribosomes, and reproduce by binary fission. Includes amyloplasts, chloroplasts, chromoplasts, etioplasts, leucoplasts, proteinoplasts and elaioplasts.

PHYTOCHROME

A plant pigment protein that, on absorption of red light, initiates physiological responses that govern light-sensitive processes such as germination, growth and flowering.

PLASMODESMATA

Plasma-membrane-lined channels in the cell wall that interconnects adjacent plant cells. They consist of a break in the cell wall that is lined by cell membrane and contain a strand of membrane that is derived from rough ER called the desmotubule.

BREFELDIN A

A fungal metabolite that acts as a potent inhibitor of secretion. This has proved an invaluable tool in the study of membrane transport.

N-ETHYL MALEIMIDE

A sulphydryl reagent that is widely used in experimental biochemical studies to covalently modify cysteine residues in proteins.

STROMULES

Highly dynamic, chlorophyll-free, tubular-membrane interconnections between adjacent plastids, which are best observed in living tissues.

NOD FACTORS

Biologically active bacterial Nod gene products, such as chitin oligomers, with various modifications, including addition of N-linked fatty acids, which are involved in the establishment of legume-root symbioses.

DIPOLE

A molecule that has both negative and positive charges.

LEUCINE-ZIPPER DOMAIN

Certain DNA-binding proteins contain a motif of approximately 35 amino acids, with every seventh residue being a leucine. This facilitates dimerization of two such proteins to form a functional transcription factor.

CAMELEON

A fluorescent chimaera used as a Ca2+ indicator. Cameleons comprise BFP or CFP, calmodulin, a glycylglycine linker, the calmodulin-binding domain of myosin light chain kinase (M13) and a GFP or YFP. Binding of Ca2+ to the calmodulin causes intramolecular calmodulin binding to M13. This conformational change reduces the separation between the two fluorescent proteins and so increases the FRET efficiency between the shorter and the longer wavelength protein.

pKa

The pKa of an acid is the negative log to base 10 of its acid dissociation constant into a hydrogen ion and an anion.

RATIOMETRICAL

A property of certain fluorescent probes that improves quantitative measurements of intracellular ion levels by measuring shifts in the excitation or emission spectrum after binding to the ion as a ratio of two wavelengths. This approach compensates for changes in illumination intensity, probe concentration and optical path length that can cause errors with measurements at a single wavelength.

ABLATION MICROBEAMS

High-intensity focused laser beams that are used to selectively eliminate cellular structures.

OPTICAL-TRAP LASER TWEEZERS

Focused laser beams that are used to trap and move cellular structures.

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Brandizzi, F., Fricker, M. & Hawes, C. A greener world: The revolution in plant bioimaging. Nat Rev Mol Cell Biol 3, 520–530 (2002). https://doi.org/10.1038/nrm861

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