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.