Mechanism of calcium in melatonin enhancement of functional substance-phenolic acid in germinated hulless barley

Abstract

Phenolic acid is a physiologically active substance that has a variety of effects on humans. Barley sprouts are often used as food ingredients to enrich phenolic acids and to further produce functional foods rich in phenolic acids. In this study, the mechanism of Ca²⁺ involvement in regulating phenolic acid biosynthesis and plant growth in barley by melatonin (MT) under NaCl stress was investigated. According to the studies, MT (25 μM) increased total calcium content, induced Ca²⁺ burst, and up-regulated the gene expression of calcium-regulated protein-dependent protein kinase and calcium-binding protein transcription-activating protease in NaCl-stressed (60 mM) barley. Exogenous MT and its combined CaCl₂ (0.4 mM) significantly promoted phenolic acid biosynthesis by increasing the activity of C4H and PAL, and induced gene expression of PAL and F5H. The addition of exogenous CaCl₂ and MT caused systemic tolerance in NaCl-stressed barley, as determined by a decrease in the fluorescence intensity of hydrogen peroxide and oxygen radical anions as well as an enhancement in the antioxidant enzyme, thus significantly increasing sprout length and fresh weight. In addition, combined use of MT with Ca²⁺ antagonists (lanthanum chloride or ethylene glycol tetraacetic acid), impaired all impacts as mentioned above. These findings imply that Ca²⁺ participated in MT-induced phenolic acid biosynthesis and growth improvement in NaCl-stressed barley.

Fig. 1. Effect of MT–Ca²⁺ on the total phenolic acid content of barley sprout under NaCl stress. Different letters indicate the significant differences in indicators among treatments at the same germination time using Tukey's test (p < 0.05). N: NaCl; NM: NaCl + MT; NC: NaCl + CaCl₂; NMC: NaCl + MT + CaCl₂; NL: NaCl + LaCl₃; NML: NaCl + MT + LaCl₃; NE: NaCl + EGTA; (8) NME: NaCl + MT + EGTA.

Fig. 2. Effects of MT–Ca²⁺ on the (I) growth performance, (II) sprout length, (III) fresh weight, and root tip staining of (IV) O₂˙⁻ and (V) H₂O₂ of barley sprout under NaCl stress. The scale length is 100 μm. Different letters indicate the significant differences in indicators among treatments at the same germination time using Tukey's test (p < 0.05). N: NaCl; NM: NaCl + MT; NC: NaCl + CaCl₂; NMC: NaCl + MT + CaCl₂; NL: NaCl + LaCl₃; NML: NaCl + MT + LaCl₃; NE: NaCl + EGTA; (8) NME: NaCl + MT + EGTA.

Fig. 3. Effects of MT–Ca²⁺ on the activities of (I) POD, (II) SOD and (III) CAT of barley sprout under NaCl stress. Different letters indicate the significant differences in indicators among treatments at the same germination time using Tukey's test (p < 0.05). N: NaCl; NM: NaCl + MT; NC: NaCl + CaCl₂; NMC: NaCl + MT + CaCl₂; NL: NaCl + LaCl₃; NML: NaCl + MT + LaCl₃; NE: NaCl + EGTA; (8) NME: NaCl + MT + EGTA.

Fig. 4. Effect of MT on Ca²⁺ metabolism of barley sprouts under NaCl stress. (I) Total calcium content and root tip staining of (II) intracellular free calcium. The scale length is 100 μm. Different letters indicate the significant differences in indicators among treatments at the same germination time using Tukey's test (p < 0.05). N: NaCl; NM: NaCl + MT; NC: NaCl + CaCl2; NMC: NaCl + MT + CaCl₂; NL: NaCl + LaCl₃; NML: NaCl + MT + LaCl₃; NE: NaCl + EGTA; (8) NME: NaCl + MT + EGTA.

Fig. 5. Effect of MT–Ca²⁺ on the gene expression of Ca²⁺-ATP (I), CaMK1 (II), CaMK2 (III), CDPK (IV), and CaMT (V) of barley sprouts. The gene expression in germinating barley treated with NaCl was used as the control. Different letters indicate the significant differences in indicators among treatments at the same germination time using Tukey's test (p < 0.05). N: NaCl; NM: NaCl + MT; NC: NaCl + CaCl₂; NMC: NaCl + MT + CaCl₂; NL: NaCl + LaCl₃; NML: NaCl + MT + LaCl₃; NE: NaCl + EGTA; (8) NME: NaCl + MT + EGTA.

Fig. 6. Effects of MT–Ca²⁺ on activity of PAL (I) and C4H (II) and gene expression of PAL (III), 4CL (IV), C4H (V), C3H (VI), F5H (VII), and COMT (VIII) in barley sprouts. The gene expression in germinating barley treated with NaCl was used as the control. Different letters indicate the significant differences in indicators among treatments at the same germination time using Tukey's test (p < 0.05). N: NaCl; NM: NaCl + MT; NC: NaCl + CaCl₂; NMC: NaCl + MT + CaCl₂; NL: NaCl + LaCl₃; NML: NaCl + MT + LaCl₃; NE: NaCl + EGTA; (8) NME: NaCl + MT + EGTA.

Fig. 7. A simulation of how combined MT and Ca²⁺ affected the phenolic acid biosynthesis and enhanced tolerance in barley under NaCl stress. CAT: catalase; SOD: superoxide dismutase; POD: peroxidase; PAL: phenylalanine ammonia lyase; 4CL: 4-coumarate-CoA ligase; ROS: reactive oxygen species.