Primary Study On Regulation Mechanism Of Carotenoids Accumulation In Citrus Callus

Carotenoids are essential plant secondary metabolites in nature, which are required for the plant survival. They are involved in many biological processes, and essential for human health and animal/plant life. They give rich colors to the flowers and fruits which are conducive to the pollination and seed dispersal. Therefore, to exert their effective usage, it is essential to understand the regulation mechanism of plant carotenoid metabolism.Citrus callus exhibits extensive diversity of carotenoid patterns, its characteristics like short growth cycle and controllable culture conditions making it to be an ideal material for plant carotenoids metabolism study. In this study, upon various external treatments on citrus callus, including light/dark, herbicides, growth cycle, sugar and hormone, in-depth analysis on metabolic regulation of carotenoid metabolism and other related biological processes was possible. The main results are as follows:1. Four citrus callus including Red Marsh grapefruit (Citrus paradisi Macf.), Murcott tangor (C. reticulata×C. sinenis), Tarocco blood orange (C. sinensis Osbeck) and Bingtangcheng sweet orange (C. sinensis L. Osbeck) were under light/dark treatment. HPLC and Real-Time PCR analysis showed that, light has various regulation patterns of carotenoids in citrus callus depending on the genotype diversity. Under the light, carotenoid synthesis in Red Marsh grapefruit callus was blocked, while light induced carotenoid accumulation in Tarocco blood orange callus. Epoxy carotenoids were more photostability than non-epoxy carotenoids, such as antheraxanthin and violaxanthin. At the same time, it also showed the PSY was the rate limiting gene in2sweet orange callus, while light strongly induced CRTISO expression in Murcott tangor callus.2. CPTA is carotene cyclase inhibitor. In metabolic analysis on Red Marsh callus treated with0.2%(w/v) CPTA showed that not only carotenoids synthesis (particularly lycopene) were promoted, but also the synthesis of secondary metabolites such as flavonoids and alkaloids were stimulated. However, little impact on primary metabolite biosynthesis was detected. Gene microarray analysis was performed to investigate global transcriptional alterations, of which33genes were up-regulated and90genes were down-regulated such as those encoding peroxidase, resistance related and P450. On the other hand, the contents of H2O2, JA, SA and ABA were decreased in treated callus. Our result suggests that CPTA not only greatly promoted the accumulation of carotenoids, but also boosted the production of other antioxidants, especially flavonoids. It was speculated that these potent biological antioxidants play an important role in eliminating oxidative stress in callus caused by CPTA, so that the common warning signals system (hormones and peroxidase) had not been activated. Meanwhile, our study indicated IAA might have some impact on carotenoids accumulation.3. Gene microarray analysis was performed on citrus callus of10days after subculture (10DAC) and30DAC without any treatment. The results showed that, there were2505differentially expressed genes, including1154up-regulated,1351down-regulated. Real-Time PCR validation indicated a good consistence between the two methods. The primary metabolite analysis indicated that the contents of soluble sugars and organic acids were most significantly decreased, while there were slight changes in contents of amino acids and lipids, implying the lack of carbohydrates in callus in the later growth stage. Combined with microarray data analysis, we found that sugar shortage might cause the acceleration of TCA cycle to compensate for the energy supplying shortage, and the cyanide-resistant respiration associated with free radicals clearage, while the carbon chains were rechanneled to the secondary metabolize under stress. In the later growth stage, accompanied with a sharp decrease in carotenoid content, carotenogenesis were significantly increased, while the increase of H2O2indicated an oxidative stress in citrus callus. This study suggested that sugar shortage caused the increase of H2O2, which consequently impacted on carotenoids accumulation. The sugar and hormone induced carotenoids accumulation remains to be elucidated.4. To study the regulation of sugar on carotenoids, different concentrations of sucrose or glucose were added in the medium. The results showed that exogenous sugar could stimulate carotenoid synthesis, but the biosynthesis was inhibited at higher sugar concentrations. Real-Time PCR analysis showed that exogenous sugars could induce carotenogenic genes, however, carotenogenesis was inhibited by high glucose level (60mM), exogenous sugars could induce gene expression which encoding sugar transporter and sugar signals, e.g. SUT, TMT and HXK. Sugar might be taken as a signal affecting carotenoid synthesis to some extent.5. Red Mash citrus callus was treated with exogenous plant hormone ABA, IAA, GA and JA for12h,3d and10d, respectively, to investigate their effects on carotenoids biosynthesis. The results showed that carotenoids accumulated significantly after3d and10d’s treatment, in which IAA was the most effective hormone. As treatment time prolonged, stimulation effect exerted by hormone decreased, but the content of carotenoids kept higher than that of the control. Real-Time PCR analysis suggested that, different hormones had different regulation modes on carotenoid biosynthesis at transcription level. Interestingly, hormone treatments and related gene expression analysis indicated that gene AGL6belonging to MADS-box transcription factor family might be associated with the carotenoids synthesis.In summary, this study has important value in understanding the fundamental biology of plant carotenoid metabolism, and had further explored on carotenoid regulation, and provides novel and important clues in revealing the mechanism of the regulation of carotenoids.