An Enzymatic method to Isolate Salt Gland from the leaves of Acanthus ilicifolius L. for Structural Characterization with Auto-fluorescence Imaging and Inorganic Ion Concentration Studies

Abstract

Excess salt regulation, an intricate mechanism in halophytic plants, has long fascinated researchers. In this paper, we focused on a typical mangrove-associated plant Acanthus ilicifolius L. (family-Acanthaceae), to investigate the structural organization of its salt glands. The investigation encompassed a multi-faceted approach involving enzymatic isolation, ultrastructure studies under electron microscopy (SEM) and light microscopy, autofluorescence activity assessment, and analysis of inorganic ion concentrations. We applied an enzymatic method to isolate individual glands from embedded epidermal salt glands. Isolated glands and epidermal peels were stained with safranin and toluidine blue and ultrastructural detailing was done using light microscopy and scanning electron microscopy. Analysis of the leaf ion concentration was done by flame photometry and cellular inorganic ion content by X-ray microanalysis (Edax). The multicellular asterid-type gland consists of eight secretory cells, three stalk cells, and basal cells. The glands are located on both of the leaf epidermis and emit yellowish-green fluorescence light from secretory cells. The cells showed an accumulation of high concentrations of inorganic ions such as sodium, chloride, calcium, and potassium under high salinity (17.5–19.5 ppt) stress conditions. Therefore, this study concentrated on the structural composition, ultrastructure, autofluorescence activity, and ion concentration of the salt glands (SGs) in the transverse section. In this study, the first on A. ilicifolius salt gland, a holistic understanding of the glandular architecture, and its complicated cellular structure have been examined more comprehensively.