Enhanced Secretory Activity of Atropa belladonna Hairy Root Culture Over-expressing ADP-Ribosylation Factor Gene

Abstract

Monomeric GTP-binding proteins are involved in regulating numerous essential functions of eukaryotic cells, such as cell differentiation, intracellular vesicle transport, and cytoskeleton organization. [1][2][3][4][5] Based on the amino acid homology and the deduced functions, the low molecular weight GTP-binding proteins are generally classified into several families. 2,5) It has been well known 3,4) that activation of these small GTP-binding proteins is strictly controlled by the two regulatory proteins, GDP/GTP exchanging factor (GEF) and the GTPase-activating protein (GAP). GEP interacts with inactive small G-proteins to covert them to GTP-bound active form, while small G-protein-GTP complex is changed to the inactive form by the hydrolysis of GTP to GDP by the action of GTPase-activating protein (GAP). ADP-ribosylation factor (ARF) is a member of the Arf/Sar family of small G-proteins, 1,4) and several lines of evidence suggest that these proteins function in vesicular transport from endoplasmic reticulum to the plasma membrane via the Golgi apparatus.6,7) As well as other small G-proteins, the functions of this class of are regulated by ARF-specific GEF and GAP. It has been demonstrated 8) that over-expression of ARF-specific GEF restores the inhibitor-induced defect of secretory activity. On the other hand, it has been recently shown 9) that over-expression of ARF-specific GAP appreciably inhibits the proteintrafficking processes from the endoplasmic reticulum to Golgi apparatus. Therefore, the effects of unphysiologically high concentrations of the two ARF-regulator proteins GEF and GAP have been examined in detail. In contrast, however, only very limited information is available on the biochemical properties of the transformed cells over-expressing ARF protein itself.We have reported previously 10) that genes encoding ARF proteins of carrot are organized as a small or multi-gene family in the genome, and arf-001 (GeneBank accession no. AY874441) has been isolated as a carrot ARF gene. The primary amino acid sequence of arf-001 appears to show significant homology to ARF proteins from various biological sources. In order to understand the physiological functions and effects of over-accumulation of the products of ARF genes in higher plant cells, in the present experiments, we attempted to prepare the transformed cells of Atropa belladonna over-expressing carrot arf-001. The newly developed vectors, pBCR82 and pBCR90, have been employed for over-expression of arf-001 and as the empty vector control, respectively, 11) and the transformed cells were obtained as hairy root tissues by co-expression of rol cluster in these vectors. MATERIALS AND METHODS MaterialsSeeds of A. belladonna L. were surface-sterilized in 70% (v/v) ethanol and 2% (v/v) sodium hypochlorite, successively, and, after several washings with autoclaved water, they were placed on Murashige and Skoog's agar medium 12) for germination. The sterilized seeds were then incubated at 26°C under constant illumination for germination, and the leaf se...

The influence of the fungal pathogen Mycocentrospora acerina on the proteome and polyacetylenes and 6-methoxymellein in organic and conventionally cultivated carrots (Daucus carota) during post harvest storage

“…ADP-ribosylation factor 1 (spot 23) is ADPribosyltransferase also involved in protein trafficking in the Golgi apparatus. An overexpression of carrot ADP-ribosylation factor 1 gene and accumulation of ADP-ribosylation factor protein in Atropa belladonna root cultures resulted in the enhancement of the proteins secretion by the Golgi [54]. A decrease of these two proteins may indicate a reduction of the proteins synthesis.…”

Hairy Roots, their Multiple Applications and Recent Patents

“…The addition of a retention sequence that increases cytoplasmic protein and thus prevents protein degradation in the medium may enhance overall protein yields. For example, the addition of -glucuronidase (GUS) Reporter protein N. tabacum Shake flasks [114] GFP Reporter protein N. benthamiana Shake flasks [115] Carrot ADP-ribosylation factor gene Endoplasmic reticulum (ER) targeting signal sequence Atropa belladonna Shake flasks [116] Human acetylcholinesterase Bioscavenger of organophosphate toxins N. benthamiana Shake flasks [117] Single chain murine interleukin-12 Immunomodulator N. tabacum cv. Xanthi Shake flasks, airlift reactor, and scalable mist reactor [118] Human epidermal growth factor Growth factor N. tabacum Shake flasks [119] GUS Reporter protein Corchorus capsularis L.…”

“…In contrast, the presence of a leader peptide, which directs the recombinant protein to the secretory pathway, may be useful in another system. For example, overexpression of a carrot ADP-ribosylation factor gene (arf-001) in A. belladonna HRs resulted in an evident enhancement of protein secretion to the medium [116].…”