α-Amanitin resistance is developmentally regulated in carrot

Abstract

Carrot cells are capable of inactivating aamanitin only in embryogenic conditions (regenerating cells and embryoids). Instead, the mutant line a3 is capable of inactivating the drug also in nonembryogenic conditions (vegetative growth). The mutation is dominant in somatic hybrids and is pleiotropic, allowing expression during vegetative growth of other embryonal functions. The inactivation of a-amanitin is due to the oxidative activity of tyrosinase.Research on plant development is much less advanced a field than its animal counterpart. For a long time this was due to the difficulty of access to the embryo that, being covered by the seed protective coats, was not amenable to experimental manipulation.Much of this difficulty could be circumvented after the definition of efficient and reproducible conditions for embryogenesis from somatic cell cultures (for a review on the developmental biology of somatic embryogenesis, see ref. 1). However, the lack of biochemical markers is still a major block, and finding a number of such markers has become a priority task. So far only two specific functions have been described during carrot somatic embryogenesis: cycloheximide resistance (2) and production of embryonal proteins El and E2 (3).In the present work we report that the capacity to inactivate a-amanitin (amai) is a developmentally regulated function: it is possessed by carrot cultured cells during organized growth (differentiating) and not during unorganized growth (in nonembryogenic medium). Moreover, the characterization of a mutant constitutively exhibiting amai will also be presented. MATERIALS AND METHODSOur wild-type line (Daucus carota var. S. Valery) and its a-amanitin-resistant mutants a3 and aS have been described (4). C1S is a double mutant showing alterations in RNA polymerase II and in hypoxanthine phosphoribosyl-transferase (5). Line WOO1C (from wild carrot) and its derivatives WOO1, (5-methyltryptophan resistant) and WCH105 (cycloheximide resistant) are described by Sung (6) and Sung et al.(2). Line HA, (var. Juwarot) and its growing conditions also have been described (7). Basal media, obtained as powder from Flow, were Gamborg's b5 for var. S. Valery and derivatives and Murashige and Skoog for WOO1C and derivatives. The hormonal addition for WOO1C was 0.1 mg of dichlorophenoxyacetic acid (2,4-D) per liter. The standard medium (b5, nonembryogenic) for our var. S. Valery lines was b5 supplemented with 0.5 mg of 2,4-dichlorophenoxyacetic acid and 0.25 mg of 6-benzylaminopurine per liter. HAT medium was bS+ containing hypoxanthine (100 ,uM), aminopterin (100 ,uM), and glycine (100 ,M). Cell plating was done on standard medium supplemented with 0.8% Oxoid agar for all lines.Cultivation was done at 250C in Erlenmeyer flasks on a gyratory shaker (80 rpm) under a light intensity of 500 lx. Cells were subcultured regularly every 15 days, inoculating 2 ml (packed cell volume after centrifugation for 10 min at 200 x g) in 50 ml of fresh medium.Plants were regenerated from 7-day-old cultures as describ...

Spermiogenesis inMarsilea vestita: A temporal correlation between centrin expression and blepharoplast differentiation

“…It is possible, but unlikely, that inactivation of ␣-amanitin occurs during development in the male gametophytes of M. vestita. In some carrot suspension culture cell lines, ␣-amanitin can be inactivated by tyrosinase activity (Pitto et al, 1985). Since we have not directly assayed for tyrosinase activity, we are unable to rule out the possibility of ␣-amanitin inactivation in M. vestita.…”

The Maternal-to-Zygotic Transition in Higher Plants

“…However, α-amanitin has not been successfully used in plant MZT experiments, possibly because the drug lacks useful effects on plant embryos. Early pioneering work in Daucus carota showed that carrot cells inactivate α-amanitin under embryonic conditions (Pitto, Schiavo, & Terzi, 1985). Two other inhibitors, actinomycin D and cordycepin, have been used in N. tabacum MZT experiments.…”

Purification of a Trypsin Inhibitor Secreted by Embryogenic Carrot Cells

“…The mechanism for this inactivation has not been clarified, although the enzyme tyrosinase (phenoloxidase) has been proposed as responsible for degradation of a-amanitin since high activity of this enzyme was observed exclusively during embryogenesis in these carrot strains (14).…”

“…During embryo formation some carrot strains have the ability to inactivate the fungal toxins cycloheximide (22) and a-amanitin (14) supplied to the culture medium. The mechanism for this inactivation has not been clarified, although the enzyme tyrosinase (phenoloxidase) has been proposed as responsible for degradation of a-amanitin since high activity of this enzyme was observed exclusively during embryogenesis in these carrot strains (14).…”