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A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies.

Tan WK, Lim TM, Loh CS - Plant Methods (2010)

Bottom Line: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures.Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands.Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore. dbslohcs@nus.edu.sg.

ABSTRACT

Background: Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.

Results: Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).

Conclusions: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

No MeSH data available.


A simple method for the isolation of salt glands from A. officinalis. (A) The lower epidermal layer (*) of the leaf was removed with a razor blade to reveal the mesophyll-palisade cell layers (**) prior to enzyme digestion. Bar = 1 cm. (B, C) The leaf with the lower epidermal layer removed were cut into strips and incubated in enzyme solution (B) to obtain the adaxial epidermal peels (C). The epidermal peels appear green (C; left image) due to the presence of mesophyll-palisade cells underneath the epidermal layer, which can be easily removed to obtain peels devoid of chlorophyll-containing cells (C; right image). Bars = 1 cm. (D-G) Salt glands (arrows) were then released through the grinding of these peels (C; right image) followed by a series of purification processes, which include filtering through a 100 μm (pore size) cell strainer (D). The isolated salt glands were finally collected on a 20 μm (pore size) cup filcon (E). All isolated salt glands (D-G) were observed under Olympus inverted light microscope (IMT-2), with close-up observations of purified salt glands viewed under different magnifications (F, G). Bars in D and E = 200 μm, bar in (F) = 80 μm, and bar in (G) = 40 μm.
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Figure 2: A simple method for the isolation of salt glands from A. officinalis. (A) The lower epidermal layer (*) of the leaf was removed with a razor blade to reveal the mesophyll-palisade cell layers (**) prior to enzyme digestion. Bar = 1 cm. (B, C) The leaf with the lower epidermal layer removed were cut into strips and incubated in enzyme solution (B) to obtain the adaxial epidermal peels (C). The epidermal peels appear green (C; left image) due to the presence of mesophyll-palisade cells underneath the epidermal layer, which can be easily removed to obtain peels devoid of chlorophyll-containing cells (C; right image). Bars = 1 cm. (D-G) Salt glands (arrows) were then released through the grinding of these peels (C; right image) followed by a series of purification processes, which include filtering through a 100 μm (pore size) cell strainer (D). The isolated salt glands were finally collected on a 20 μm (pore size) cup filcon (E). All isolated salt glands (D-G) were observed under Olympus inverted light microscope (IMT-2), with close-up observations of purified salt glands viewed under different magnifications (F, G). Bars in D and E = 200 μm, bar in (F) = 80 μm, and bar in (G) = 40 μm.

Mentions: This report presents a simple and fast isolation method that enables us to obtain large numbers of isolated salt glands that are freed from the interference of neighbouring epidermal cells and the mesophyll-palisade cell layers. (Figure 2; see Methods for full descriptions). By removing the abaxial epidermal cell layers from the leaf (Figure 2A) and incubating the resulting leaf tissues in an enzyme solution (Figure 2B), the mesophyll-palisade tissues can be easily detached from the adaxial epidermal peels after an hour of enzymatic digestion. Remnants of mesophyll-palisade cells (as observed by their green appearance due to the presence of chloroplasts) underneath the adaxial epidermal peels can be removed with ease (Figure 2C) to minimize contamination with other cell types prior to salt gland isolation. The salt glands can then be released through the grinding of these epidermal peels.


A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies.

Tan WK, Lim TM, Loh CS - Plant Methods (2010)

A simple method for the isolation of salt glands from A. officinalis. (A) The lower epidermal layer (*) of the leaf was removed with a razor blade to reveal the mesophyll-palisade cell layers (**) prior to enzyme digestion. Bar = 1 cm. (B, C) The leaf with the lower epidermal layer removed were cut into strips and incubated in enzyme solution (B) to obtain the adaxial epidermal peels (C). The epidermal peels appear green (C; left image) due to the presence of mesophyll-palisade cells underneath the epidermal layer, which can be easily removed to obtain peels devoid of chlorophyll-containing cells (C; right image). Bars = 1 cm. (D-G) Salt glands (arrows) were then released through the grinding of these peels (C; right image) followed by a series of purification processes, which include filtering through a 100 μm (pore size) cell strainer (D). The isolated salt glands were finally collected on a 20 μm (pore size) cup filcon (E). All isolated salt glands (D-G) were observed under Olympus inverted light microscope (IMT-2), with close-up observations of purified salt glands viewed under different magnifications (F, G). Bars in D and E = 200 μm, bar in (F) = 80 μm, and bar in (G) = 40 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965709&req=5

Figure 2: A simple method for the isolation of salt glands from A. officinalis. (A) The lower epidermal layer (*) of the leaf was removed with a razor blade to reveal the mesophyll-palisade cell layers (**) prior to enzyme digestion. Bar = 1 cm. (B, C) The leaf with the lower epidermal layer removed were cut into strips and incubated in enzyme solution (B) to obtain the adaxial epidermal peels (C). The epidermal peels appear green (C; left image) due to the presence of mesophyll-palisade cells underneath the epidermal layer, which can be easily removed to obtain peels devoid of chlorophyll-containing cells (C; right image). Bars = 1 cm. (D-G) Salt glands (arrows) were then released through the grinding of these peels (C; right image) followed by a series of purification processes, which include filtering through a 100 μm (pore size) cell strainer (D). The isolated salt glands were finally collected on a 20 μm (pore size) cup filcon (E). All isolated salt glands (D-G) were observed under Olympus inverted light microscope (IMT-2), with close-up observations of purified salt glands viewed under different magnifications (F, G). Bars in D and E = 200 μm, bar in (F) = 80 μm, and bar in (G) = 40 μm.
Mentions: This report presents a simple and fast isolation method that enables us to obtain large numbers of isolated salt glands that are freed from the interference of neighbouring epidermal cells and the mesophyll-palisade cell layers. (Figure 2; see Methods for full descriptions). By removing the abaxial epidermal cell layers from the leaf (Figure 2A) and incubating the resulting leaf tissues in an enzyme solution (Figure 2B), the mesophyll-palisade tissues can be easily detached from the adaxial epidermal peels after an hour of enzymatic digestion. Remnants of mesophyll-palisade cells (as observed by their green appearance due to the presence of chloroplasts) underneath the adaxial epidermal peels can be removed with ease (Figure 2C) to minimize contamination with other cell types prior to salt gland isolation. The salt glands can then be released through the grinding of these epidermal peels.

Bottom Line: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures.Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands.Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore. dbslohcs@nus.edu.sg.

ABSTRACT

Background: Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.

Results: Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).

Conclusions: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

No MeSH data available.