Ca²⁺ second messenger-mediated stimulation of plant marker immune gene using clinoptilolite-based Ca-alginate

Calcium, as an important structural, metabolic, and signalling element in plants, can stabilize cell membranes, and its physiological function can be achieved through its coordinated transport on the cell membrane. In present work, the potential of natural clinoptilolite (NCP) containing calcium alginate (abbreviated as Alg-Ca-NCP) with a Ca content of 9.46 wt% was explored, which can be used as a calcium secondary signal to stimulate plant marker immune genes WRKY33 (Tryptophan (W)-Arginine (R)-Lysine (K)-Tyrosine (Y)), NHL10 (Nicotiana tabacum L), and OXI1 (the oxidative stress-induced) under calcium resistance control conditions for approximately 3 times, 5 times and 3 times, respectively. The results verified that Alg-Ca-NCP had a significant effect on the Ca²⁺ transport through calcium channels. Specially, the Catalase content in leaf mustard treated with Alg-Ca-NCP was around 1970 U·g^-1·min^-1 on the 21^st day, much higher than that treated with CaCl₂ (1365 U·g^-1·min^-1) and water (1210 U·g^-1·min^-1), obviously indicating that the Alg-Ca-NCP treatment was conducive to continuously and stably improving plant immunity. Mean-while, Alg-Ca-NCP treatment significantly affected the levels of Catalase and Chlorophyll (Chla and Chlb) in leaf, which was useful to enhance the electron transfer capacity in leaf mustard, but reduced the hypoxia-induced damage. The preliminarily investigates of the fluorescein isothiocyanate (FITC)-(Alg-Ca-NCP) expression in Arabidopsis leaves and tomato roots suggested that Alg-Ca-NCP may penetrate horizontally into the xylem ducts through the endothelial layer, and transport upwards through leaves and root cells. These results suggested that Alg-Ca-NCP may directly enter the cell membrane through transmembrane transport, but the specific mechanism still requires investigation in depth.