Differential expression of anthocyanin biosynthesis genes in Daucus carota callus culture in response to ammonium and potassium nitrate ratio in the culture medium

Abstract

Anthocyanins are major water-soluble and dynamic colouring plant pigment present in plant tissues with the high antioxidant properties. The role of ammonium and potassium nitrate in the culture medium on anthocyanin augmentation is probed thoroughly, but the mechanism of its biosynthesis continues to be unclear. Hence, the present study was undertaken to optimise nitrate ratio in the culture medium for anthocyanin augmentation and examination of its biosynthesis pathway in callus culture of Daucus carota. MS basal medium fortified with various ratio of NH 4 NO 3 :KNO 3 was employed to find their impact on biomass, anthocyanin augmentation and the expression profile of anthocyanin biosynthesis genes in the callus culture. The data indicated that the highest anthocyanin content (9.30 ± 0.25 mg/100 g FW) was seen in callus grown on the medium supplemented with 20.0 mM NH 4 NO 3 :37.6 mM KNO 3 and the least was seen in the medium which contained 40.0 mM NH 4 NO 3 :18.8 mM KNO 3 (2.74 ± 0.27 mg/100 g FW). This indicates an optimal concentration of NH 4 NO 3 :KNO 3 ratio is essential to produce a higher amount of anthocyanin in in vitro culture. Meanwhile, anthocyanin biosynthesis genes were differentially expressed as confirmed by qRT-PCR in the time interval of 5, 10, 15, 20 and 25 days. The transcript levels of nine anthocyanin biosynthesis genes were increased in the response of varying NH 4 NO 3 :KNO 3 ratio in the medium. The transcript level of early genes PAL, 4CL, CHS and CHI increased by 19.5, 21.0, 16.2 and 9.98-fold, respectively, compared with control. In addition, late biosynthesis genes LDOX and UFGT resulted in the transcript level of 11.3 and 13.6-fold, respectively.

Repression of Carotenoid Accumulation by Nitrogen and NH4+ Supply in Carrot Callus Cells In Vitro

“…Additionally, N starvation promoted anthocyanin accumulation in grape callus [ 42 ]. In contrary, other reports showed that the increase of total N content by doubling KNO 3 in the MS medium increased anthocyanin content by 135% [ 12 ], which was congruent with results showing the highest accumulation of anthocyanins using an elevated N amount (70 mM) in the medium [ 43 ].…”

“…Hairy roots of carrot, red beet, and madder in the presence of NH 4 + available in the amounts in the MS medium had a reduced growth [ 45 ]. A similar effect of restricted growth was observed for anthocyanin accumulating carrot callus cultured on MS [ 12 ]. No growth changes were reported only when carrot cell suspension was exposed to a doubled amount of ammonium N form than present in MS [ 46 ].…”

“…In callus induced from rose leaves, the reduction of NH 4 + and increase of NO 3 − concentrations in the MS-based medium enhanced anthocyanin accumulation [ 47 ]. Conversely, doubling the concentration of NH 4 + in the MS medium restricted the anthocyanin content by one third in carrot callus [ 12 ].…”

“…Recently, it was demonstrated that ammonium ions negatively affect carotenoid accumulation in Calendula officinalis callus cultured in vitro [ 11 ]. Another genetic research based on callus response to changes in the composition of mineral medium indicated that N supply affected pigment accumulation [ 12 ].…”

Molecular Evaluation of Kyoho Grape Leaf and Berry Characteristics Influenced by Different NPK Fertilizers

“…In the present study, of grape berries, it is clear that the development of the anthocyanin contents is firmly associated with the biosynthetic genes expression pattern. There are two classes of enzymes elaborating the biosynthesis pathways-early genes that comprise of- phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumaroyl:CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′5′-hydroxylase (F3′H), and late genes-dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), also called leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid-3- O -glucosyltransferase (UFGT) [ 76 ]. A previous study has described the ammonium nitrate and potassium nitrate effects on pathway genes of anthocyanin [ 77 ].…”