Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 5;8(13):e2912.
doi: 10.21769/BioProtoc.2912.

Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds

Oded Skaliter  1 , Jasmin Ravid  1 , Alon Cna'ani  2 , Gony Dvir  1 , Rafael Knafo  1 , Alexander Vainstein  1
Affiliations

Isolation of Intact Vacuoles from Petunia Petals and Extraction of Sequestered Glycosylated Phenylpropanoid Compounds

Oded Skaliter et al. Bio Protoc. .

Abstract

Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles ( Paris et al., 1996 ; Olbrich et al., 2007 ). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids ( Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).

Keywords: Glycoside; Petal; Petunia; Phenylpropanoid volatile; Protoplast; Vacuole.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. Cutting below the corolla to remove all flower organs
Figure 2.
Figure 2.. Flower corolla placed on kenzan needle point holder with adaxial side facing up
Figure 3.
Figure 3.. Flower halves placed in a Petri dish containing enzyme solution
Figure 4.
Figure 4.. Collecting the protoplasts
Figure 5.
Figure 5.. Purple layer with living protoplasts formed after centrifugations
Figure 6.
Figure 6.. Filter flask connected to vacuum pump
Video 1.
Video 1.. Removal of debris in the suspension solution
Figure 7.
Figure 7.. Light microscopy of protoplasts.
Scale bar = 45 μm.
Figure 8.
Figure 8.. Ficoll fraction containing vacuoles after centrifugation.
Light microscopy of intact vacuoles is shown in the inset. Scale bar = 10 μm.
Figure 9.
Figure 9.. Protoplast concentration calculation.
A. The hemocytometer used in the experiment. B. Enlargement of the counting grid. Square used for cell counting is shown in red.
See this image and copyright information in PMC

References

    1. Bowles D., Isayenkova J., Lim E. K. and Poppenberger B.(2005). Glycosyltransferases: managers of small molecules. Curr Opin Plant Biol 8(3): 254-263. - PubMed
    1. A. Cna'ani, Shavit R., Ravid J., Aravena-Calvo J., Skaliter O., Masci T. and Vainstein A.(2017). Phenylpropanoid scent compounds in Petunia x hybrida are glycosylated and accumulate in vacuoles . Front Plant Sci 8: 1898. - PMC - PubMed
    1. A. Cna'ani, Spitzer-Rimon B., Ravid J., Farhi M., Masci T., Aravena-Calvo J., Ovadis M. and Vainstein A.(2015). Two showy traits, scent emission and pigmentation, are finely coregulated by the MYB transcription factor PH4 in petunia flowers. New Phytol 208(3): 708-714. - PubMed
    1. Dean J. V., Mohammed L. A. and Fitzpatrick T.(2005). The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures. Planta 221(2): 287-296. - PubMed
    1. de Michele R., McFarlane H. E., Parsons H. T., Meents M. J., Lao J., Gonzalez Fernandez-Nino S. M., Petzold C. J., Frommer W. B., Samuels A. L. and Heazlewood J. L.(2016). Free-flow electrophoresis of plasma membrane vesicles enriched by two-phase partitioning enhances the quality of the proteome from Arabidopsis seedlings . J Proteome Res 15(3): 900-913. - PubMed

LinkOut - more resources