Relative quantification of membrane-associated calcium in red spruce mesophyll cells

Abstract

AbstractmWe describe a method for localizing and comparing relative amounts of plasma membrane-associated calcium ions (mCa) in complex tissues and verify the procedure for mesophyll cells of red spruce (Picea rubens Sarg.) needles. This technique incorporates epifluorescence microscopy using the fluorescent probe chlorotetracycline (CTC) with computer image processing and analysis. Using an appropriate standardization for image brightness, the procedure allows relative quantitative comparison of CTCfluorescence in the plasma membrane-cell wall region that corresponds to relative amounts of mCa. The technique effectively discerned mCa differences among red spruce needle sections exposed to treatments designed to alter mCa levels in vitro. Estimates of mCa for nine red spruce seedlings, were highly repeatable over a 6 week period in late summer. This repeatability verifies that the described methods produce reliable and reproducible estimates of foliar mCa in woody plant foliage. By incorporating image analysis, this technique allows for relative quantitative estimates of mCa specific to the physiologicallyimportant and labile pool of Ca associated with the plasma membrane-wall complex. Such measurements have not previously been reported for woody plant tissues and thus may provide new insights into the relative roles and responsiveness of mCa vs total foliar Ca pools.

Long-term calcium addition increases growth release, wound closure, and health of sugar maple (Acer saccharum)trees at the Hubbard Brook Experimental Forest

“…Approximately 12 sun-exposed leaves were collected from each branch, sealed into plastic bags, and kept cool for transport to the laboratory. Four leaves per tree were selected for measurement of foliar mCa levels using wellestablished fluorescence microscopy and computer image analysis methods (Borer et al 1997;DeHayes et al 1999;Schaberg et al 2000) as modified for sugar maple leaves (Schaberg et al 2006). …”

Associations of calcium and aluminum with the growth and health of sugar maple trees in Vermont

“…Sectioning, staining, microscopy, and image analysis procedures used were adapted from Borer et al (1997) and DeHayes et al (1997). These procedures incorporate epifluorescence microscopy using the fluorescent probe chlorotetracycline (CTC) with computer image processing to quantify the intensity of mCa-specific fluorescent emissions.…”