Betacyanin Identification And Biosynthetic Physiology Mechanism In Suaeda Salsa

As a halophyte, physiological and molecular responses of Suaeda salsa to salinity stress have been extensively studied. But nothing is known about the identification and the biosynthesis regulation of the red pigments in S. salsa. This paper focuses on the identification of the red pigments and the effects of abiotic stressors on their accumulation in S. salsa.The results showed that these red pigments were insoluble in organic solvents but free in water, the pigments were red-violet and stable under acidic condition while yellow and unstable under alkaline condition, and they absorbed the highest value at wavelength near 538 nm. These results suggested that these red pigments in S. salsa are not anthocyanins but betacyanins.The betacyanins extracted from S. salsa were sensitive to high temperature, high intensity of light, alkali and oxidant in vitro, and keep stable under low temperature, dark, acidic and free of oxidants conditions. The scavenging superoxide (O2-) ability of the betacyanins from S. salsa was lower than that of vitamin C, while the scavenging hydroxyl radical (OH·) ability of the betacyains was much higher than that of vitamin C in vitro.Light suppressed betacyanin accumulation and enhanced their decomposition in vivo. Darkness, low temperature and high salinity enhanced betacyanin accumulation, and darkness at the germination phase is one of the most important environmental factors for the betacyanin accumulation in S. salsa.Seeds, 5-d and 10-d old seedlings of C₃ halophyte S. salsa were watered, sprayed and infiltrated with 0, 0.10%, 0.33% and 1.00% H₂O₂ solution to examine whether H₂O₂ is involved in the betacyanin accumulation. H₂O₂ treatments led to the most significant betacyanin accumulation in the shoots of S. salsa seedlings when seeds were watered with H₂O₂ solution. The increase of betacyanin content was the highest and that of H₂O₂ content was the lowest in leaves, while the increase of betacyanin content was the lowest and that of H₂O₂ content was the highest in roots of seedlings obtained from seeds watered with H₂O₂ solution. When leaves of 5-d and 10-d old seedlings were sprayed and infiltrated with H₂O₂ solution, the H₂O₂ content was enhanced significantly, but the increase of betacyanin content was not notable in leaves, and the content of betacyanin and H₂O₂ was also at a lower level in roots. The H₂O₂ content in organs of S. salsa seedlings was not proportional to betacyanin content, and the betacyanin accumulation in leaves was correlated with the H₂O₂ production in roots rather than that in leaves and stems. These results suggested that the oxidative stress signal leading to betacyanin production in H₂O₂ treatments may be perceived by roots initially, then the signal was transferred to leaves; the signal transduction was performed and betacyanin accumulation was induced in leaves of S. salsa.Seeds of S. salsa were cultured both in the dark and light for three days. Addition of Ca²⁺ in the 1/2 MS nutrient solution promoted betacyanin accumulation in the dark, and Ca⁽2+ depletion by EGTA suppressed the darkness-induced betacyanin accumulation in shoots of S. salsa. Ca2+ channel blocker (LaCl₃) also inhibited the darkness-induced betacyanin accumulation. The highest activity of CaM and the maximum betacyanin content decreased by 51% and 45% in shoots of S. salsa seedlings treated with a potent CaM antagonist, chlorpromazine (CPZ), in the dark, respectively. Furthermore, another CaM antagonist, W7, also inhibited the activity of CaM and the dark-dependent betacyanin accumulation, whereas its less active structural analog, W5, had little effect on the responses to dark of S. salsa. These results suggest that Ca²⁺, Ca²⁺-regulated ion channels and CaM play an important role in the darkness-induced betacyanin accumulation in shoots of C₃ halophyte S. salsa.Seeds of the halophyte S. salsa were cultured in the dark for three days and betacyanin accumulation was induced significantly in cotyledons of S. salsa seedlings. Accumulated betacyanin in cotyledons of the dark-grown seedlings decomposed with time in light. As the betacyanin content declined, the hydroxylation and oxidation activity of tyrosinase extracted from cotyledons of the dark-grown seedlings decreased with time in light. The apparent molecular mass of tyrosinase in cotyledons of S. salsa seedlings was about 60 kDa estimated by SDS-PAGE, enzyme activity staining and Western blotting. Furthermore, the tyrosinase only synthesized in the cotyledons of dark-grown S. salsa seedlings, and declined with time in light, which was paralleled by the decreases of tyrosinase activity and betacyanin content in light. This result suggests that the specific tyrosinase is positively correlated with betalain biosynthesis, and betacyanin biosynthesis is induced by dark via synthesis of tyrosinase in S. salsa.Seeds of the halophyte S. salsa were cultured in 24 h dark and 14 h blue light/10 h dark to examine the role of blue light and the blue-light absorbing photoreceptor cryptochrome 2 (CRY2) in betacyanin accumulation, hypocotyl elongation and cotyledon opening in S. salsa seedlings. Darkness significantly promoted betacyanin accumulation and hypocotyl elongation, but inhibited cotyledon opening. Blue light suppressed betacyanin accumulation and hypocotyl elongation, but stimulated cotyledon opening. Betacyanin in S. salsa seedlings decomposed with time in blue light. Western blot analysis showed that CRY2 protein accumulated both in hypocotyls and cotyledons of S. salsa seedlings grown in dark, but degraded with time in blue light, which was paralleled by a decrease of tyrosine hydroxylation activity of tyrosinase, a key enzyme involved in the betalain biosynthesis pathway. These results suggested CRY2 protein mediates betacyanin decomposition via inactivation of tyrosinase in S. salsa seedlings, and the blue-light-dependent degradation of CRY2 protein is crucial to its function.