Use of Fura 2 Fluorescent Dyes as Indicator for Studying Calcium Distribution in Several Plant Tissues

Abstract

Calcium (Ca) is an essential plant nutrient and is important in determining quality of fruits in storage. The analytical method presently known to determine Ca concentration in vegetables measures total Ca, and there is a need to develop methods that can determine the distribution of Ca within vegetable tissues. The objective of this study was to determine the ability of fluorescent probes to visualize Ca location and distribution in vegetable tissues. We examined three protocols using fluorescent dyes developed to detect Ca. The first tested Fura 2 acetoximetil (AM) probe in fresh cuts from grapes at different combinations of temperatures and incubation times. The second protocol was developed with the aim of lowering the natural autofluorescense. In the third protocol, the Fura 2 probe was used in tissues previously fixed in formalin, acetic acid, and alcohol solution (FAA). Tissues were observed under an epifluorescence microscope and, to compare and complement the observations, under a scanning electron microscope (SEM). No sign of fluorescence was observed in fixed and fresh tissue using the Fura 2 AM. In fresh cuts, there was autofluorescent interference, provoked mainly by chlorophyll, but also from the vascular system and tannins. The Fura 2 probe was introduced to vegetable tissues by utilizing a pH of 4.5, and a more intense signal was observed in tissues with greater Ca concentration. This signal was also associated with Ca crystals, which might have had some degree of hydrolysis at low pH. Under a SEM microscope, Ca oxalate crystals were clearly observed in those tissues. Our results show that Fura 2 probe can be used to detect Ca and its distribution in vegetable tissues.

Calcium (Ca) is an essential plant nutrient and is important in determining quality of fruits in storage. The analytical method presently known to determine Ca concentration in vegetables measures total Ca, and there is a need to develop methods that can determine the distribution of Ca within vegetable tissues. The objective of this study was to determine the ability of fluorescent probes to visualize Ca location and distribution in vegetable tissues. We examined three protocols using fluorescent dyes developed to detect Ca. The first tested Fura 2 acetoximetil (AM) probe in fresh cuts from grapes at different combinations of temperatures and incubation times. The second protocol was developed with the aim of lowering the natural autofluorescense. In the third protocol, the Fura 2 probe was used in tissues previously fixed in formalin, acetic acid, and alcohol solution (FAA). Tissues were observed under an epifluorescence microscope and, to compare and complement the observations, under a scanning electron microscope (SEM). No sign of fluorescence was observed in fixed and fresh tissue using the Fura 2 AM. In fresh cuts, there was autofluorescent interference, provoked mainly by chlorophyll, but also from the vascular system and tannins. The Fura 2 probe was introduced to vegetable tissues by utilizing a pH of 4.5, and a more intense signal was observed in tissues with greater Ca concentration. This signal was also associated with Ca crystals, which might have had some degree of hydrolysis at low pH. Under a SEM microscope, Ca oxalate crystals were clearly observed in those tissues. Our results show that Fura 2 probe can be used to detect Ca and its distribution in vegetable tissues.