Cell bioreactor culture of Orostachys cartilaginous A. Bor. and involvement of nitric oxide in methyl jasmonate-induced flavonoid synthesis

Orostachys cartilaginous A. Bor. is a high-value medicinal plant, whereas flavonoids are important secondary metabolites. Bioreactor cell culture is an alternative method for the mass production of flavonoids in O. cartilaginous . This study investigated the adaption of culture conditions using bioreactors with different sizes to provide a reference for the pilot-scale culture of O. cartilaginous cells in the future. Results showed that cell fresh and dry weights per culture medium among the balloon-type airlift bioreactors of 3, 5, and 10 L did not change. Moreover, approximately equal amounts of total flavonoids were synthesized in bioreactors with different sizes, indicating that the culture conditions optimized in a bioreactor of certain size can be used in bioreactors of other sizes. This study used methyl jasmonate (MeJA) as an abiotic elicitor to treat 25-day-old O. cartilaginous cells, and an event whether nitric oxide (NO) was involved in flavonoid synthesis in MeJA-induced flavonoid synthesis was investigated to improve flavonoid accumulation. The contents of total flavonoids and flavonoid monomers including quercetin, kaempferide, epicatechin gallate, quercetin-3-O-glucose, and kaempferol-3-rutinoside, were significantly improved by MeJA treatment, reaching the maximum value at 48 h after elicitation. During the MeJA elicitation, NO burst in the early stage and NO content peaked at 6 h. In addition, nitrate reductase (NR) inhibitors of tungstate and glutamine blocked NO generation and inhibited flavonoid synthesis in MeJA-stimulated cells. However, such inhibition of flavonoid synthesis was relieved by a NO donor (sodium nitroprusside), thereby suggesting that NO was involved in MeJA-induced flavonoid synthesis through NR pathway. The present finding has a critical significance for understanding the mechanism of the defense response stimulated by the MeJA elicitor and can provide a new strategy that regulates NO burst and flavonoid synthesis by controlling the NR activity.