Plant Cell Reports
Plant Cell Reports (1985) 4:321-324
© Springer-Verlag1985
Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy J. Hopkinson 1, C. Moustou 2, K. A. Charlwood 1, j. E. Newbery
1 and B. V. Charlwood 2
1 Chemical Laboratories, Department of Life Sciences, University of London Goldsmiths College, New Cross, London SE 14 6NW, U K 2 Department of Biology, King's College London, 68 Half Moon Lane, London SE24 9JF, U K Received August 12, 1985 / Revised version received October 10, 1985 - Communicated by W. Barz
ABSTRACT
A method has been d e v e l o p e d to d e t e r m i n e the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the p r o t o p l a s t s r e l e a s e d from such cells by enzymatic digestion. It is d e m o n s t r a t e d that there is a smooth and r e p r o d u c i b l e t r a n s i t i o n in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of p r o t o p l a s t release during enzymatic digestion of cell wall material. INTRODUCTION
Plant protoplasts are usually isolated from cell p r e p a r a t i o n s (leaf mesophyll cells or cells c u l t u r e d in vitro) by enzymatic d i g e s t i o n of the cell wall with a m i x t u r e of cellulase and pectinase (Evans and Bravo 1983). F l u o r e s c e n c e microscopy is commonly employed to monitor the release of p r o t o p l a s t s by p r e - s t a i n i n g the cells with brighteners such as Calcofluor White ST (Nagata and Takebe 1970). A l t h o u g h this dye has a strong affinity for cell wall components, such binding is n o n - s p e c i f i c and the assay itself is rather i n s e n s i t i v e (Weber et al. 1976). The scanning e l e c t r o n m i c r o s c o p e (SEM) can be used to obtain a very d e t a i l e d picture of the cell surface, and has been w i d e l y used to i n v e s t i g a t e the r e g e n e r a t i o n of cell walls on protoplasts (Burgess and Linstead 1976). However, all SEM methods require a p r o t r a c t e d fixation protocol followed by either d e h y d r a t i o n or c r y o p r e s e r v a t i o n . Dehydration techniques can lead to gross d i s t o r t i o n of the surface t o p o g r a p h y and the introduction of artefacts, whilst cryopreservation produces a great deal of variation in the surface appearance of the p r o t o p l a s t thus r e n d e r i n g the i n t e r p r e t a t i o n of the images rather difficult (Attree and Sheffield 1984), Either technique is time consuming, expensive and unsuitable for the routine
Offprint requests to: K. A. Charlwood
m o n i t o r i n g of p r o t o p l a s t release by enzymatic digestion. This paper describes the use of internal reflectance Fourier Transform Infrared spectroscopy (reflectance FTIR) as a new technique for m o n i t o r i n g the changes at the surface of cells of higher plants in aqueous solution as cell wall material is d i g e s t e d during p r o t o p l a s t production. MATERIALS
AND
METHODS
P r e p a r a t i o n of Protoplasts: Suspension cultures of the scented cultivar Pelargonium "Rober's Lemon Rose" were obtained as p r e v i o u s l y d e s c r i b e d (Brown and C h a r l w o o d 1985). Early log phase cells (16 g fresh weight) were h a r v e s t e d 4-6 days after sub-culture, and t r a n s f e r r e d to CPW m e d i u m (Power and Davey 1979, 15 ml) c o n t a i n i n g Rhozyme HPI50 (2%), Meicelase (4%), Macerozyme (0.3%) and mannitol (13%) at pH 5.8. Incubation was carried out at 27°C for a period of up to 6 h. After an appropriate period the protoplasts which had been released were p a r t i a l l y p u r i f i e d by "sucrose flotation" (I00 x g for 5 min on 21% sucrose solution) and were m o n i t o r e d by reflectance FTIR, fluorescence m i c r o s c o p y and SEM. R e f l e c t a n c e FTIR of Plant Cells: Partially purified protoplasts were r e s u s p e n d e d in 0.5 M KCl. The very high concentration of sucrose n o r m a l l y present in protoplast media would dominate the reflectance FTIR s p e c t r u m and hence make the process of solvent subtraction much more difficult. The KCl solution provided an osmotically stable medium sufficient for protoplasts to remain intact during the period of data collection, and also reduced the risk of plasmolysis in the intact cells prior to screening. R e f l e c t a n c e FTIR spectra were recorded a liquid cell having a 45 ° ZnSe
using prism
322
"
z < ~E
Plant Cell Reports (1985) 4:321-324
© Springer-Verlag1985
Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy J. Hopkinson 1, C. Moustou 2, K. A. Charlwood 1, j. E. Newbery
1 and B. V. Charlwood 2
1 Chemical Laboratories, Department of Life Sciences, University of London Goldsmiths College, New Cross, London SE 14 6NW, U K 2 Department of Biology, King's College London, 68 Half Moon Lane, London SE24 9JF, U K Received August 12, 1985 / Revised version received October 10, 1985 - Communicated by W. Barz
ABSTRACT
A method has been d e v e l o p e d to d e t e r m i n e the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the p r o t o p l a s t s r e l e a s e d from such cells by enzymatic digestion. It is d e m o n s t r a t e d that there is a smooth and r e p r o d u c i b l e t r a n s i t i o n in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of p r o t o p l a s t release during enzymatic digestion of cell wall material. INTRODUCTION
Plant protoplasts are usually isolated from cell p r e p a r a t i o n s (leaf mesophyll cells or cells c u l t u r e d in vitro) by enzymatic d i g e s t i o n of the cell wall with a m i x t u r e of cellulase and pectinase (Evans and Bravo 1983). F l u o r e s c e n c e microscopy is commonly employed to monitor the release of p r o t o p l a s t s by p r e - s t a i n i n g the cells with brighteners such as Calcofluor White ST (Nagata and Takebe 1970). A l t h o u g h this dye has a strong affinity for cell wall components, such binding is n o n - s p e c i f i c and the assay itself is rather i n s e n s i t i v e (Weber et al. 1976). The scanning e l e c t r o n m i c r o s c o p e (SEM) can be used to obtain a very d e t a i l e d picture of the cell surface, and has been w i d e l y used to i n v e s t i g a t e the r e g e n e r a t i o n of cell walls on protoplasts (Burgess and Linstead 1976). However, all SEM methods require a p r o t r a c t e d fixation protocol followed by either d e h y d r a t i o n or c r y o p r e s e r v a t i o n . Dehydration techniques can lead to gross d i s t o r t i o n of the surface t o p o g r a p h y and the introduction of artefacts, whilst cryopreservation produces a great deal of variation in the surface appearance of the p r o t o p l a s t thus r e n d e r i n g the i n t e r p r e t a t i o n of the images rather difficult (Attree and Sheffield 1984), Either technique is time consuming, expensive and unsuitable for the routine
Offprint requests to: K. A. Charlwood
m o n i t o r i n g of p r o t o p l a s t release by enzymatic digestion. This paper describes the use of internal reflectance Fourier Transform Infrared spectroscopy (reflectance FTIR) as a new technique for m o n i t o r i n g the changes at the surface of cells of higher plants in aqueous solution as cell wall material is d i g e s t e d during p r o t o p l a s t production. MATERIALS
AND
METHODS
P r e p a r a t i o n of Protoplasts: Suspension cultures of the scented cultivar Pelargonium "Rober's Lemon Rose" were obtained as p r e v i o u s l y d e s c r i b e d (Brown and C h a r l w o o d 1985). Early log phase cells (16 g fresh weight) were h a r v e s t e d 4-6 days after sub-culture, and t r a n s f e r r e d to CPW m e d i u m (Power and Davey 1979, 15 ml) c o n t a i n i n g Rhozyme HPI50 (2%), Meicelase (4%), Macerozyme (0.3%) and mannitol (13%) at pH 5.8. Incubation was carried out at 27°C for a period of up to 6 h. After an appropriate period the protoplasts which had been released were p a r t i a l l y p u r i f i e d by "sucrose flotation" (I00 x g for 5 min on 21% sucrose solution) and were m o n i t o r e d by reflectance FTIR, fluorescence m i c r o s c o p y and SEM. R e f l e c t a n c e FTIR of Plant Cells: Partially purified protoplasts were r e s u s p e n d e d in 0.5 M KCl. The very high concentration of sucrose n o r m a l l y present in protoplast media would dominate the reflectance FTIR s p e c t r u m and hence make the process of solvent subtraction much more difficult. The KCl solution provided an osmotically stable medium sufficient for protoplasts to remain intact during the period of data collection, and also reduced the risk of plasmolysis in the intact cells prior to screening. R e f l e c t a n c e FTIR spectra were recorded a liquid cell having a 45 ° ZnSe
using prism
322
"
z < ~E
