Study Of The Mechanism Of Growth Regulator And Light-regulated Betacyanin Accumulation In Euhalophyte Suaeda Salsa Calli

The Chenopod C3 species Suaeda salsa L. represents a euhalophyte with significant succulence of leaves and has the characteristics such as salt and waterlogging tolerance. It grows well in the intertidal zone of the Yellow River Delta, where soil salt content is often higher than 3%, so S. salsa is one of the indicator plants of saline soil. Interestingly, shoots of S. salsa are violet-red throughout the whole growth period in the intertidal zone, but green on saline inland far from the seaside. In addition, seedlings are violet-red during the germination stage. As a euhalophyte, the physiological and molecular responses of S. salsa to salinity stress have been extensively studied in our laboratory. Recent studies have shown that the red pigments in S. salsa are betacyanins rather than anthocyanins, and the accumulation of betacyanin is mostly regulated by enviromental factors such as light, temperature and salinity. Betacyanins (red-violet pigments), together with betaxanthins (yellow pigments), are a group of chromoalkaloids known as betalains. Betalains are water-soluble nitrogenous pigments. The betalain biosynthetic pathway, which includes three enzymes, a tyrosinase, a dopa 4,5-dioxygenase and a betanidin glucosyltransferase or cDOPA 5-O- glucosyltransferase. However, our understanding of the betalain biosynthetic pathway and its regulatory mechanisms is limited and lagging behind the respective knowledge of the flavonoid pigments. Studies have suggested that betacyanin may be involved in stress resistance. Unfortunately, we could not induce violet-red phenotype of S. salsa under laboratory conditions as under natural conditions. The reason may be that the accumulation of betacyanin in S. salsa is regulated by comprehensive conditions difficult to simulate artifically under laboratory conditions, which limits our study of the regulatory mechanism of betalain biosynthesis. The major advantages of cell cultures includes (i) synthesis of bioactive secondary metabolites is running in controlled environment, independently from climatic and soil conditions; (ii) negative biological influences that affect secondary metabolites production in the nature are eliminated (microorganisms and insects). Therefore, cell cultures can be a useful tool for analyzing the regulatory mechanism of betalain biosynthesis and for the characterization of the enzymes involved. Among chemical and physical factors, growth regulators and light are especially important for plant growth and development as well as accumulation of metabolites. In the present study, we introduced the C3 halophyte S. salsa calli as a model system for studying the mechanism of growth regulators- and light-regulated betacyanin acummulation and the correlation between growth regulators and white light during betacyanin accumulation. This study laid the good foundation not only for studying the molecular mechanism of growth regulators- and light-regulated betacyanin acummulation but also for understanding the role of betacyanin involved in salt tolerance and for development and utilization of betacyanin. The main results are as follows:S. salsa calli cultured on Murashige and Skoog (MS) with 6-benzylaminopurine (6-BA) 0.5 mg L-1 and 2,4-dichlorophenoxyacetic acid (2,4-D) 0.2 mg L-1 (CIM) in darkness for 28 d were transferred to MS media containing various growth regulators under white light conditions for 10 d to investigate cell growth, betacyanin accumulation, and its relation to activity and expression of tyrosinase and expression of dopa-4,5-dioxygenase gene (DODA).In contrast with MS alone, callus growth and betacyanin production was substantially stimulated with the addition of growth regulators to MS. The optimum combination of exogenous growth regulators was 0.2 mg·L-1 2,4-D and 0.5 mg·L-1 6-BA for cell growth in a 10 d culture, with an RGR of 233.21 mg·g-1·d-1. Surprisingly, of the auxins tested, naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) had no effect on betacyanin content but 2,4-D strongly decreased betacyanin content. Cytokinins such as Thidiazuron (TDZ) and 6-BA markedly promoted betacyanin synthesis of S. salsa calli. TDZ had a greater effect on betacyanin content than the same concentration of 6-BA. The highest betacyanin content was found in the cells cultured on MS with 0.5 mg·L-1 TDZ and 1.0 mg·L-1 NAA; the betacyanin content was 28.77 nM·g-1 FW.Under white light conditions, DODA mRNA levels and tyrosinase activity were consistent with the response of betacyanin content to exogenous growth regulators. However, the protein levels of tyrosinase extracted from S. salsa calli response to exogenous growth regulators were constant. These results suggest that betacyanin metabolism in S. salsa calli is regulated by growth regulators under white light conditions through the regulation of genes such as DODA and tyrosinase activity but not the protein amount of tyrosinase.S. salsa calli cultured on CIM in darkness for 28 d were transferred to MS media with 0.5 mg·L-1 TDZ and 1.0 mg·L-1 NAA under different light conditions for 10 d to investigate the effect of light on betacyanin accumulation and its relation to activity and expression of tyrosinase and expression of dopa-4,5-dioxygenase gene (DODA). Both light quality and quantity affected betacyanin synthesis of S. salsa calli. Futhermore, cell growth and the accumulation of betacyanin were positively correlated.The accumulation of betacyanin from calli cultured under 80μmol m-2 s-1 of white light had the highest level followed by red and blue light. However, the betacyanin accumulation in the dark was the lowest. Betacyanin content initially increased and reached the highest level at a certain light quantity, but then declined with an increase in white, red or blue light intensity. 60-100μmol m-2 s-1 of white light intensity, 40μmol m-2 s-1 of red light intensity, and 2μmol m-2 s-1 of blue light intensity were optimal for betacyanin accumulation in S. salsa calli, with a betacyanin content of 28.77 nMg-1FW, 15.70 nMg-1FW and 14.49 nMg-1FW, respectively. DODA mRNA levels and tyrosinase activity were consistent with the response of betacyanin content to different light quality. However, the protein levels of tyrosinase extracted from S. salsa calli response to different light quality were constant. These results suggest that betacyanin metabolism in S. salsa calli is regulated by different light quality through the regulation of genes such as DODA and tyrosinase activity but not the protein amount of tyrosinase.S. salsa calli cultured on CIM in darkness for 26 d or 28 d were transferred to MS or MS media with various combinations of TDZ and 1.0 mg·L-1 NAA under different light conditions for 10 d to investigate the correlation between growth regulators and white light during betacyanin accumulation. TDZ enhanced betacyanin accumulation under white light and had a concentration effect. In darkness, however, TDZ concentration gradient showed no effect on the betacyanin content. White fluorescent light intensity significantly enhanced growth regulators-induced betacyanin accumulation. The higher fluence rate of white light applied the stronger induction of betacyanin was observed on either MS or MS with various combinations of TDZ and 1.0 mg·L-1 NAA. It has been demonstrated that white light played a role of on-off of betacyanin accumulation and TDZ promoted betacyanin accumulation induced by white light in S. salsa calli. The results from experiments with three different treatment programs showed that the relationship between white light and TDZ in betacyanin accumulation of S. salsa calli seemed to be neither multiplicative nor additive coaction, but rather interaction. White light was necessary for betacyanin accumulation, and TDZ might be involved in the white light signaling pathway.It was observed that long-term exposure to light (10 d) or short-term periods of illumination (1 h) of any quality led to no increase in cytokinin levels, as compared with the dark-grown control calli. Therefore, cytokinin levels could not explain the marked stimulation of betacyanin accumulation under white light condition as compared with dark, blue or red light. Taken together, these data indicated that light regulate betacyanin accumulation by a distinct signalling pathway downstream of the cytokinin, and that a point of convergence exists between white light and cytokinin signalling pathways for this response. The signal transduction pathway of white light- and growth regulator-induced betacyanin and anthocyanin synthesis may be similar. Model for the two simultaneously occurring signalling processes in the presence of white light and cytokinins in betacyanin synthesis may be as follows: A cytokinin signal coming from the histidine kinase receptors (HKs), passing through response regulator 4 (RR4), which is a shared component with phytochrome B (phyB) signalling, and going further through multiple signalling intermediates, inhibits HY5 breakdown by an unknown mechanism. These processes lead to stable HY5 and a higher expression of genes such as tyrosinase, DOPA-4,5-dioxygenase (DODA), 5-O-glucosyltransferase (5-GT) that are involved in betacyanin synthesis. However, accurate genetic and molecular evidence is required for further investigation to the mechanism of betacyanin accumulation induced by light and exogenous growth regulators in S. salsa calli.