Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate

Abstract

The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphate (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) and Pi uptake. Addition of 2 mM Pi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Pi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having M(rs) of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results re consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape ( Brassica napus ) suspension cell cultures

“…With 2 mM Phi, no growth or APase induction occurred following subculture of the B. napus cells into the )Pi medium. This corroborates recent studies documenting the detrimental influence of Phi on )Pi, but not +Pi, vascular plants (including B. napus suspension cells; Carswell et al 1996Carswell et al , 1997Foerster et al 1998;Ticconi et al 2001;Varadarajan et al 2002) andyeast (McDonald et al 2001b). 31 P-NMR spectroscopy has demonstrated substantial intracellular Phi accumulation by )Pi plant and yeast cells cultured in the presence of Phi (Carswell et al 1996;McDonald et al 2001b).…”

“…6b). Similarly, Pi starvation of B. napus suspension cells (±2 mM Phi) did not significantly influence the extractable activity of pyruvate kinase or ATP-dependent phosphofructokinase, relative to +Pi controls (Carswell et al 1997).…”

“…Although Phi is non-metabolic, it greatly exacerbates the harmful effects of Pi deficiency by specifically preventing the derepression of plant and yeast genes encoding PSI proteins (i.e., APase, high-affinity plasmalemma Pi transporters, etc. ; Carswell et al 1997;Plaxton 1999;McDonald et al 2001aMcDonald et al , 2001bTicconi et al 2001;Abel et al 2002;Varadarajan et al 2002). Phi's prevention of the otherwise massive enhancement of Pi influx capacity that occurs in )Pi B. napus suspension cells provided a rationale for the approx.…”

“…; Plaxton 1999;Abel et al 2002). Although Phi is not a substrate in enzyme-catalyzed phosphoryl transfer reactions, other plant and yeast Pi-binding proteins, such as Pi transporters participating in Pi uptake or signal transduction components involved in sensing Pi status, do not appear to effectively discriminate between the Pi and Phi anions (Carswell et al 1997;McDonald et al 2001b;Ticconi et al 2001;Varadarajan et al 2002). Examination of the influence of Phi on two yeast pho regulon mutants indicated that Phi targets PHO84, a high-affinity Pi transporter and putative component of a Pi-sensor complex (McDonald et al 2001b).…”

“…In fact, low Phi concentrations are quite deleterious to the development of Pi-starved ()Pi), but not Pi-sufficient (+Pi), vascular plants (Carswell et al 1996(Carswell et al , 1997Foerster et al 1998;Ticconi et al 2001;Varadarajan et al 2002) and yeast (Saccharomyces cerevisiae) (McDonald et al 2001b). Phi treatment negates the acclimation of plants and yeast to Pi deficiency by specifically preventing the derepression of genes encoding proteins typical of their Pi starvation response (i.e., repressible acid phosphatase (APase), high-affinity plasmalemma Pi transporters, etc.…”