Rhizosecretion of recombinant proteins from plant hairy ... - Springer

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quently resulting in cell differentiation and the formation of hairy roots (Chilton et al. 1982). Induction of hairy roots has been demonstrated on various plant organs, leaf discs, storage organs, seedling hypocotyls and stem tissue (Golds et al. 1991). In vitro cultivated hairy roots are used in a range of research applications for plant improvement (Christey 1997), plant secondary metabolism studies (Christen et al. 1989; Rhodes et al. 1997) and plant interactions with the environment (Tone et al. 1997). Hairy root cultures were also shown to produce recombinant proteins (Wongsamuth and Doran 1997a). In the case of hairy root cultures expressing monoclonal antibodies, it was shown that antibodies were not only associated with the root tissue, but were released extracellularly (Wongsamuth and Doran 1997b). Hairy roots induced by rol genes are usually characterized by extensive secondary branching and an increase in the number of root hairs with a higher ratio of metabolically active meristem tissues. All these morphological features of hairy roots, resulting in significant expansion of the total root surface, seem to be beneficial for protein rhizosecretion. The objective of this study was to investigate the potential of A. rhizogenesinduced hairy roots as a more vigorous root system to boost the production of recombinant protein by rhizosecretion under non-sterile hydroponic conditions. Materials and methods Plant material and hairy root induction All experiments were performed using seeds of the Nicotiana tobacum (cv. Wisconsin) transgenic plant, line A1 (Borisjuk et al. 1999), transformed with the human placental SEAP gene with its own signal peptide under the control of the cauliflower mosaic virus 35S promoter. Wild type tobacco (cv. Wisconsin) was used as a control. Both transgenic and wild type seeds were surfacesterilized, germinated and grown in Petri dishes on solid Murashige and Skoog medium (Murashige and Skoog 1962) at 24C under a 14-h-light/10-h-dark regime (irradiance of 150 mE m -2 s -1 using a mixture of fluorescent and incandescent lamps). Growth medium for SEAP transformants was supplemented with 100 mg/ml kanamycin. Seedlings were then transferred to Magenta boxes for further growth. All transgenic plants used for further experiments were checked for the presence of the SEAP gene by polymerase chain reaction according to Klimyuk et al. (1993). In order to ensure genetic identity, double cuttings from single plants were generated and placed into Magenta boxes for rooting. Well-rooted plantlets were then grown in soil under standard greenhouse conditions for 6 weeks. One of the two plantlets generated from the same plant was used for the induction of hairy roots, while the other was used to produce an adventitious root system. The induction of hairy roots was stimulated by inoculation with bacterium A. rhizogenes, strain A4 (provided by Dr. Michael Lawton, Biotech Center, Rutgers University). Single colonies of A. rhizogenes propagated in liquid yeast extract-peptone medium for 48 h at 28C were inoculated into plant stems by multiple pricks using a syringe (4 ml inoculum per 8 cm stem). The inoculated area was immediately sealed off within a plastic tube (10 cm”2.5 cm, Fig. 1b) to create the high humidity favorable for root formation. Hairy roots appeared 11–12 days after bacterial inoculation. In order to stimulate the formation of adventitious roots, high humidity conditions were created by wrapping an entire tobacco plant in a plastic bag (Fig. 1a). Plastic bags were removed 2 days after the appearance of first adventitious roots on the plant stem and this stem area was sealed off in a plastic tube as described for the hairy roots. Protein and mRNA analysis Comparative determination of SEAP and total protein was carried out on hairy and adventitious roots grown on tobacco stems within the plastic chambers (Fig. 1b). For initial determination of SEAP secretion (Fig. 2), on the 5th day after the appearance of the first root, the tubes enclosing induced roots were filled up with 25–30 ml air-bubbled 1 mM CaCl 2 solution and incubated for 24 h to collect secreted proteins. After 24 h, the solution was removed and subjected to quantitative protein analysis according to Borisjuk et al. (1999). Following the solution sampling, roots were collected, weighed, frozen in liquid nitrogen, and kept at Ÿ80C for RNA extraction. To evaluate long-term SEAP secretion and total protein accumulation (Fig. 3), samples of the liquid medium were collected after 24 and 48 h as described above, and corresponding roots were sampled for the determination of total protein. At 48 h, tobacco stems were cut just below the area of induced root growth, and the top rooted transplants were placed in larger chambers filled with aerated 0.5”Hoagland nutrient solution (Hoagland and Arnon 1938) for an additional 12 days of cultivation (Fig. 1f). The Hoagland solution was changed every 3 days. After 12 days (14 days from the beginning of the experiment), the root system was again transferred into air-bubbled 1 mM CaCl 2 solution, and medium samples were collected after 24 h incubation for immediate analysis of SEAP. Roots were then collected, weighed and subjected to total soluble protein determination as described below. In an experiment aimed to determine the rates of protein accumulation in root tissues and its distribution between root tissues and cultivation medium (Fig. 4), root material was initially sampled from airborne roots at day 2 and day 4 after roots formed within the sealed tubes. At day 5, the tubes were filled with CaCl 2 solution, and samples of both roots and cultivation media were collected at day 6 and day 8. CaCl 2 solution was replaced every day. Collected samples of CaCl 2 cultivation media were concentrated 20-fold by ultrafiltration through a Centricon column with a 10-kDa cut-off membrane (Amicon, Beverly, Mass.). SEAP quantification was performed using a chemiluminescence assay (Clontech, Palo Alto, Calif.) as described earlier (Borisjuk et al. 1999). Total protein was determined by using the Bio-Rad (Melville, N.J.) dye-binding assay procedure (Bradford 1976). Roots were ground in liquid nitrogen, and total protein was extracted by the addition of buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) and subsequent centrifugation. The supernatant was used to determine the total protein and SEAP amounts in the samples. RNA was isolated from roots samples as described by Chomczynski and Sacchi (1987), and subjected to northern blot analysis. DNA probes were gel purified and radio-labelled with 32 P by random priming (Sambrook et al. 1989). Statistical analysis Statistical analyses of protein accumulation and protein secretion data were carried out by ANOVA tests. Significance was assigned at P
1190 Fig. 1a–g Induction and hydroponic propagation of hairy and adventitious tobacco roots. a Tobacco plant wrapped in plastic bag in order to create highly humid conditions that stimulate the formation of adventitious roots. b Sealed plastic chamber creates local high humidity favorable for growth of hairy and adventitious roots on tobacco stem. c Five-day-old adventitious roots formed on lower tobacco stem. d–e Fiveday-old hairy roots grown on tobacco stem. f A hydroponic set-up for growing experimental tobacco plants under greenhouse conditions in aerated Hoagland solution. g Hairy roots of the plant grown hydroponically in Hoagland solution for 10 days after the stem below the inoculation zone had been severed (Fig. 1a, b). Hairy roots appeared 11–12 days after inoculation (Fig. 1d, e) and were enclosed in the cylindrical plastic chamber (Fig. 1b). The density of the hairy root formation positively correlated with the extent of wounding and the density of the inoculum. Adventitious roots appeared on lower stems after 4–5 days of high humidity created by enclosing the plant in a plastic bag (Fig. 1a, c). Morphological differences were noticed between the adventitious and hairy roots (Fig. 1c, d). The adventitious roots were longer while the hairy roots had more extensive branching and root hair growth (Fig. 1e). No differences were noticed between hairy or adventitious root types induced on wild type and transgenic tobacco plants. Secretion and accumulation of SEAP and total protein To compare the production of recombinant protein by hairy roots and adventitious roots formed on the same genotype of SEAP-transgenic plant, corresponding pairs of plants with 5-day-old roots, induced at high humidity, were analysed for SEAP secretion. The age of roots was calculated from the time of the first root emergence. Rhizosecreted SEAP was collected for 24 h inside the plastic tubes filled with 1 mM CaCl 2 solution that enclosed a portion of the stem with either hairy or adventitious roots induced on a transgenic plant. SEAP production was calculated per gram of root dry weight. Results from 10 pairs of individual transformants, each pair originating from a single transformation event, showed that the levels of SEAP secretion were signifi-
Page 1: Plant Cell Rep (2003) 21:1188-1193
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Rhizosecretion of recombinant proteins from plant hairy ... - Springer

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