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.

The nutritional control of root development

“…This conclusion is supported by a recent study in which phosphonate (HPO 3 2− ) was used as a tool to investigate the role of internal P status in regulating proteoid root development (Gilbert et al, 2000). Phosphonate is an analogue of inorganic phosphate that is reported to interfere with plant perception of internal P concentration (Carswell et al, 1997). Treatment of Psufficient white lupin plants with phosphonate led to a dramatic increase in the number cluster roots, and the induced rootlets displayed the full repertoire of physiological changes associated with proteoid root development (Gilbert et al, 2000).…”

Phosphite, an Analog of Phosphate, Suppresses the Coordinated Expression of Genes under Phosphate Starvation

“…This is likely to be due to a combination of reduced uptake of Phi or its inability to interfere with biochemical reactions in the presence of Pi. The competition between Pi and Phi for the common transport system is well documented in fungi and plant cells (Barchietto et al, 1989;Griffith et al, 1989;Grant et al, 1992;Carswell et al, 1997). In the presence of both Pi and Phi, organisms preferentially acquire Pi.…”

“…In contrast to this extensive documentation of antifungal action of Phi, its influence on plant metabolism is not well understood. In Brassica napus cell cultures, Phi greatly suppressed the induction of several Pi stress marker enzymes (Carswell et al, 1996(Carswell et al, , 1997. Phi has also been shown to disrupt protein phosphorylation during Pi stress, thus affecting key events common to multiple Pi stress-induced mechanisms.…”

“…One common observation in most of these studies is that the negative effects of Phi could be overcome by Pi supplementation. Because multiple responses induced by Pi deficiency are suppressed by Phi, it was postulated that the signal transduction pathway leading to Pi starvation responses might be compromised (Carswell et al, 1997). This important observation has led to the present study in our laboratory.…”

“…It also suppressed Pi starvation-induced root growth, root hair initiation, and elongation in Arabidopsis (Ticconi et al, 2001). In addition, Phi has been shown to interfere with Pi starvation responses of Brassica napus L. cv Jet Neul by down-regulating the induction of enzymes such as acid phosphatase, phosphoenolpyruvate phosphatase, inorganic pyrophosphate-dependent phosphofructokinase, and the high-affinity Pi translocator (Carswell et al, 1997). In Arabidopsis, the Pi starvation-induced nucleolytic enzyme activities and expression of Pi starvation-induced genes were suppressed by Phi (Ticconi et al, 2001).…”