Increase of anthraquinone content in Rubia cordifolia cells transformed by native and constitutively active forms of the AtCPK1 gene

Abstract

Overexpression of both native and mutant forms of AtCPK1 in Rubia cordifolia cells increased anthraquinone production and transcript abundance of the RcIPPI, RcOSBL, RcOSBS , and RcICS genes to different extents. Calcium-dependent protein kinases (CDPKs) are involved in various cell processes and are regulated by a calcium signal system. CDPKs also function in plant defense against stress factors such as pathogens, temperature, and salinity. In this study, we compared the effect of heterologous expression of two forms of the Arabidopsis AtCPK1 gene, native and constitutively active (Ca(2+)-independent), on anthraquinone production in transgenic Rubia cordifolia cells. Significant qualitative and quantitative differences were found in the content of anthraquinone derivatives in control and AtCPK1-transgenic calli. Expression of the AtCPK1 gene increased anthraquinone production by 3 and 12 times for native and constitutively active forms, respectively, compared with control cells. In addition, we identified and quantified the expression of genes encoding key enzymes of the anthraquinone biosynthesis pathway, including isochorismate synthase (ICS), o-succinylbenzoate synthase (OSBS), o-succinylbenzoate ligase (OSBL), and isopentenyl diphosphate isomerase (IPPi). In all AtCPK1-transgenic cell lines, expression of ICS, OSBS, OSBL, and IPPi increased considerably at 14-15 days of subculture and decreased at the end of cultivation (30 days). The results suggest that both native and constitutively active AtCPK1 forms induced anthraquinone accumulation at the logarithmic growth stage via enhancement of expression of genes involved in the metabolism of anthraquinones or their regulatory mechanisms.

Full-Length Transcriptome Survey and Expression Analysis of Cassia obtusifolia to Discover Putative Genes Related to Aurantio-Obtusin Biosynthesis, Seed Formation and Development, and Stress Response

“…Anthraquinone is made up of three benzene rings named by A, B, and C. In the early stage of anthraquinones formation, the rings of A and B are derived from 1,4-dihydroxy-2-naphthoc acid via isochorismic acid and α-ketoglutaric acid, whereas ring C is derived from isopentenyl diphosphate (IPP)/3,3-dimethylallyl diphosphate (DMAPP) via the MVA/MEP pathway. To date, though a variety of genes involving Isochorismate, MVA, and MEP pathways have been identified in plants, a limited number of genes encoding enzymes in the biosynthesis of anthraquinone have been identified from anthraquinone-containing plants, including DXS encoding 1-deoxy- d -xylulose 5-phosphate synthase from Morinda citrifolia [ 27 ], DXR encoding, ICS encoding isochorismate synthase from Rubia tinctorum [ 28 ] and Rubia cordifolia [ 28 ], OSBS encoding o -succinylbenzoate synthase from Rubia cordifolia [ 29 ], SK encoding shikimate kinase from Cassia obtusifolia [ 30 ], OSBL encoding o -succinylbenzoate ligase from Rubia cordifolia [ 30 ], IPPI encoding isopentenyl-diphosphate isomerase in Cinchona robusta [ 31 ] and Rubia cordifolia [ 29 ], and Polyketide synthases III from Rheum emodi [ 32 ] and Aloe arborescens [ 33 ]. In the late stage of anthraquinones biosynthesis, the backbone of anthraquinones then undergoes various modifications mediated by cytochrome P450s, SAM-dependent methyltransferases, UDP-glycosyltransferases (UDPG), and other enzymes.…”

Biosynthesis and Cytotoxic Properties of Ag, Au, and Bimetallic Nanoparticles Synthesized Using Lithospermum erythrorhizon Callus Culture Extract

“…Studies were conducted with a BK-39 callus culture of Lithospermum erythrorhizon, which was obtained and described earlier [143]. Calluses were cultivated on solid "W" medium [144], supplemented with 2.0 mg/L kinetin, 0.2 mg/L indole-3-acetic acid, and 0.25 mg/L CuSO 4 , in the dark at 25 • C with 30 day subculture intervals. Freshly collected or dried (60 • C for 5 h) 30 day old BK-39 calli were used to obtain the extracts.…”

Engineered Fungus Thermothelomyces thermophilus Producing Plant Storage Proteins

“…Seeds of maize (Zea mays) and amaranth (Amaranthus hypochondriacus) were purchased from the local seed market and germinated under appropriate conditions. Total RNA was isolated from 2-week-old seedlings by using LiCl method as described previously [27]. The first-strand cDNA synthesis was conducted with MMLV RT kit (Evrogen, Moscow, Russia) according to manufacturer's instruction.…”