Author for correspondence: Stephen C. Fry Tel: +44 131 650 5320 Fax: +44 131 650 5392 Email: S.Fry@ed.ac.uk
• Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility.
• Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of 3H2O and [14C]mannitol from a ‘bed’ of cultured cells. Osmotic potential was measured by depression of freezing point.
• OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID50 of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells’ ability to acidify the apoplast (medium), which may be necessary for ‘acid growth’.
• As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.
Oxaziclomefone [OAC, IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one, previously called MY-100; Fig. 1] is a potent herbicide recently released onto the Japanese market by Bayer CropScience. OAC is effective in controlling cockspur (Echinochloa crus-galli) and other grasses and annual sedges that can substantially reduce the yield of rice in paddy fields. OAC inhibits the growth of gramineous monocots but is relatively ineffective on most dicots (Jikihara et al., 1997; Suzuki et al., 2003). It is uncharged and relatively nonpolar, with a maximum solubility in water of ≈ 480 nm at 25°C (Jikihara et al., 1997); it is likely to penetrate membranes readily, so that it could act either at the cell surface or in any subcellular compartment.
OAC is particularly effective at inhibiting cell expansion in gramineous roots. In its presence, lateral roots continued to be initiated but failed to grow out; treated roots thus developed numerous short lateral protuberances (Miller et al., 2001). Staining of the root-tip cell walls with toluidine blue and ruthenium red was diminished after the roots had been in OAC for 24 h. OAC was deduced to have a novel mode of action clearly distinct from that of any established class of herbicide (Miller et al., 2001). There is considerable commercial and theoretical interest in novel herbicide targets.
We investigated possible mechanisms by which cell expansion might be inhibited by OAC. Cell expansion is driven by turgor pressure, and in healthy tissues is usually limited by the extensibility of the cell wall or sometimes by the wall yield threshold (Cosgrove, 1993; Kutschera, 2001). Theoretically, therefore, a herbicide could inhibit cell expansion by decreasing the turgor pressure (Mostowska, 1998). In the present paper we have tested for possible effects of OAC on processes that govern turgor and wall acidification.
Turgor pressure is generated when the osmotic pressure of the cell sap exceeds that of the apoplast. The establishment of such an osmotic gradient depends on membrane integrity and on ATP to fuel the synthesis or accumulation of protoplasmic solutes. The development of turgor pressure depends additionally on water influx through the membrane, a process which is partially mediated by aquaporins (Maurel, 1997; Chaumont et al., 1998). OAC is not lethal over a period of days, and thus seems unlikely to act by damaging membranes. However, OAC could, in principle, block the synthesis or accumulation of osmotically active protoplasmic solutes, which we have therefore assayed.
Materials and Methods
Maintenance of cell-suspension cultures
Cell-suspension cultures of maize (Zea mays Black Mexican) were maintained at 220 ml per 500-ml flask in a standard medium [Murashige and Skoog inorganic salts (Sigma Chemical Co.) 4.7 g l−1; sucrose 20 g l−1; 2,4-D 4.4 µm; pH before autoclaving 5.6–5.8 with NaOH] and subcultured fortnightly by 11-fold dilution into fresh medium.
Application of OAC
OAC was dissolved, for example at 10 mm, in dimethylsulphoxide (DMSO) and then added to aqueous media to give the desired final concentration – usually 480 nm unless otherwise stated. All experimental controls received the same concentration of DMSO (< 1%) as was used to dispense the OAC.
Effect of culture age on susceptibility (ID50) to OAC
The ID50 (concentration of OAC that inhibits 50% of cell growth) was determined on maize cell cultures of various ages. Aliquots (4 ml) of the cultures were transferred into sterile, loosely capped 60-ml Sterilin beakers (cylindrical containers, Bibby Sterilin Ltd, Stone, Staffordshire, UK), supplemented with OAC to various concentrations (each in triplicate), and incubated at 25°C for 6 d.
Estimation of cell expansion and cell number
The settled cell volume (SCV) of each culture was estimated by sedimentation at 1g for 15 min in a graduated centrifuge tube. This method provides a measurement related to the total cellular volume within the culture (Lorences & Fry, 1990).
Maize cells were difficult to macerate by conventional agents (CrO3, H2O2 ± glacial acetic acid, hot aqueous trifluoroacetic acid, chelating agents or commercial preparations of cellulase, pectinase or ‘hemicellulase’) without also damaging the cells beyond recognition. However, acceptable results were obtained by gentle shaking of the cells in 0.1 m NaOH at room temperature for 1 wk. The NaOH was then neutralized with acetic acid and the cells were counted in a haemocytometer under phase contrast.
Elution rate of 3H2O from cultured maize cells
A 90-ml sample of 6-d-old maize cell culture was allowed to sediment, and 60 ml of medium was removed. A mixture of 3H2O (370 kBq) and [14C]mannitol (74 kBq; 2.0 MBq µmol−1) was added to the remaining cells, which were incubated with gentle shaking at room temperature for 1 h. Aliquots (5 ml) of culture were then passed through empty PolyPrep columns (10-ml disposable polypropylene chromatography columns; Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) to form 1.8-ml beds of cells. Fresh medium was passed through the cell bed by means of a peristaltic pump at ≈ 1 ml min−1. Fractions of effluent medium were collected every 10 s for up to 15 min and assayed for radioactivity. Eluted solutions were assayed for 3H and 14C in a Beckman LS6500 scintillation counter after the addition of 10 vol OptiPhase scintillation fluid (Wallac, Milton Keynes, Buckinghamshire, UK). Data were collected simultaneously in preset 3H and 14C windows, then the contribution of the measured 14C to counts in the 3H window (‘false 3H’) was subtracted from the observed 3H count.
In some experiments OAC was added (to 480 nm) to the cultures 18 h before the radioisotopes. In others, Triton X-100 was added (to 1%, w/v) immediately before the isotopes.
Estimation of osmotic pressure
Duplicate 200-ml samples of 6-d-old maize cell-suspension cultures were treated with or without 480 nm OAC. After 2, 4, 8, 24 and 48 h, 2-ml samples of each culture were filtered through a small piece of glass fibre paper which had been inserted into the outlet of a 2-ml syringe barrel. The plunger was then inserted into the barrel, and depressed to remove excess medium and to force a small volume of air through the cell bed, care being taken not to squash the cells. The culture filtrate (≈ 1 ml) was collected and frozen. The cells in the syringe barrel were frozen at −80°C overnight, then thawed and quickly squashed (by means of the syringe plunger). Sap (≈ 1 ml) from the squashed cells was collected, filtered through glass wool and frozen. The osmotic pressure of each sample was measured with a depression-of-freezing-point osmometer (Roebling, Camlab, Cambridge, UK). We thank Dr Wieland Fricke, The University of Paisley, for use of the osmometer and for helpful advice. To estimate the turgor pressure of the cells, we subtracted the osmotic pressure of the medium from that of the cell sap.
Effects of OAC on growth in maize, spinach and rose cell-suspension cultures
Low concentrations of OAC inhibited cell expansion (measured as SCV) in cell cultures of maize (ID50≈ 4.8 nm; Fig. 2) and spinach (Spinacia oleracea; ID50≈ 30 nm; data not shown). It had little effect on the growth of rose (Rosa sp.) cultures except at high concentrations (ID50≈ 250 nm). After 1 wk, OAC had caused some loss of chlorophyll from the spinach cells, which became paler green than normal (at > 30 nm OAC) or yellow-green (at > 300 nm). After 1 wk in OAC (> 30 nm in maize or > 300 nm in rose), the achlorophyllous cultures had become white, whereas healthy cells of these species are cream-coloured.
When maize cell cultures of various ages were supplemented with a range of OAC concentrations and then incubated for a further 6 d, the ID50 was found to be lower in young cultures than in older ones (Table 1). The greater susceptibility of young cultures appeared to be caused mainly by their lower cell number at the time of treatment, rather than directly by their age. Thus 0-d-old cultures that had been adjusted to a higher cell density had a lower susceptibility to OAC (ID50≈ 8 nm), similar to that of a normal 3- or 5-d-old culture. It therefore appeared that OAC susceptibility was related to the capacity of the medium to support further growth – cultures that had not much further to grow required higher OAC concentrations to prevent that growth; while longer-term growth was effectively blocked by a low OAC concentration.
Table 1. Effect of culture age on susceptibility of maize (Zea mays Black Mexican) cell-suspension cultures to oxaziclomefone (OAC)
Culture age at time of OAC treatment (d after subculture)
Fold-growtha (of OAC-untreated control) during following 6 d
Effect of OAC on mean cell volume in maize cultures
Growth, defined as cell expansion (Thimann, 1969) and estimated from SCV, is quite distinct from cell division. The latter could a priori be inhibited, unaffected or even promoted by growth-inhibiting doses of OAC. Thus, during the early phases of growth inhibition, the mean volume per cell could be increased, unaffected or decreased. In the long term, blocking growth will inevitably prevent further cell division as the volume per cell cannot continue to decrease indefinitely. To investigate which of these possibilities applies during the early phases of OAC action, we performed cell counts on cultures treated with 0 or 100 nm OAC, and hence calculated the mean volume per cell. During the first 40 h of culture, a small (20%) but highly significant increase in cell volume occurred in the absence of OAC (Fig. 3a). OAC almost completely blocked this early cell expansion (Fig. 3a). During the same time interval there was a 40% increase in cell number (regardless of the presence of OAC), and therefore a decrease in mean volume per cell (Fig. 3b). These observations indicate that the major initial effect of OAC is an inhibition of cell expansion rather than of cell division.
After > 40 h in the absence of OAC, cell expansion was rapid (Fig. 3a) and fairly closely matched by cell division, so that the mean volume per cell changed only a little (Fig. 3b). After > 40 h in the presence of OAC, little further increase in cell number occurred; and as there was also almost no cell expansion, the mean cell volume remained approximately constant at ≈ 170 picolitres per cell. These data suggest that a later, and thus presumably secondary, effect of OAC is an inhibition of cell division.
Effect of OAC on acidification of the medium in maize cell-suspension cultures
After subculture, the cells rapidly decreased the pH of their medium from ≈ 5.8 to ≈ 3.5 during the first 2 d of growth, presumably mainly because of the uptake of NH4+ in exchange for H+. OAC (up to 480 nm) did not appreciably affect this extracellular acidification during 6–8 h periods of observation (Fig. 4). In addition, when 0-, 1-, 2- or 7-d-old cultures were incubated aseptically with 10 nm OAC for a further 5 d, the pH of spent medium showed no differences from the DMSO-only controls (data not shown). We conclude that OAC did not prevent ‘acid growth’ by blocking H+ secretion.
Effect of OAC on water transport in maize cell-suspension cultures
Theoretically, a decrease in cell expansion could be brought about by an inhibition of water transport across the plasma membrane or tonoplast, for example by inhibition of aquaporin action.
Preliminary experiments established a method of assaying water transport in cultured maize cells. The cells were preincubated for 1 h in [14C]mannitol plus 3H2O, then quickly packed as a ‘cell bed’ and irrigated with nonradioactive, air-saturated medium. It was expected that the [14C]mannitol (which is membrane-impermeant) would be eluted from the bed of cells more rapidly than the 3H2O, which would have equilibrated between all subcellular compartments, and efflux of which from the cells might therefore be limited by membrane permeability. As predicted, the 14C did elute from the cell bed more rapidly than the 3H (Fig. 5a), so that the 3H : 14C ratio in the medium emerging at any given moment increased markedly with time (Fig. 5b). The difference between 3H and 14C elution was largely lost in cultures treated with Triton X-100 to permeabilize the membranes (data not shown). This shows that the relative retention of 3H within the cells was caused primarily by the presence of their membranes, rather than by a slow exchange of 3H between cellular 3H macromolecules and the solvent water.
The elution of 3H2O from living maize cells was at least as rapid in cells that had been OAC-treated for 3 or 24 h as in untreated cells (Fig. 5). Thus OAC did not hinder the movement of water across the tonoplast or vacuole. These results show that OAC did not block aquaporin action, or that aquaporins are not required for the effective movement of water across the major cellular membranes.
Effect of OAC on osmotic pressure of cell sap in maize cell-suspension cultures
At time points up to 2 d after treatment with or without OAC, cultured cells were collected, frozen and thawed. The osmotic pressure of the cell sap (OPsap) was estimated with a depression-of-freezing-point osmometer (Fig. 6a). In addition, the osmotic pressure of the spent medium (OPmedium) was assayed at each time point (Fig. 6a). It has been shown experimentally (Nobel, 1991) that 1 MPa corresponds to 407.5 mOsmol l−1, and this relationship was used here.
Assuming that the hydraulic conductivity of the cultured cells was high, as indicated by the rapidity with which 3H2O escaped from the cells (see above), we calculated the turgor pressure as OPsap − OPmedium. The results (Fig. 6b) indicate that the turgor pressure was unaffected by OAC even after 48 h. This confirms that OAC inhibits cell expansion by a mechanism other than one involving the denaturation of membranes or in any other way compromising the cells’ ability to synthesize or accumulate low-Mr solutes.
OAC inhibited the growth (cell expansion, estimated from settled cell volume) of cell cultures of the gramineous monocot maize more strongly than in those of the dicots spinach and (especially) rose. In maize cultures growth became discernible between 20 and 40 h, and even this early phase of cell expansion was blocked by OAC (Fig. 3a). In contrast, OAC did not block cell division immediately in the maize cells. As cell division continued for a while in the absence of cell expansion, the mean cell volume had decreased by 40 h, especially in the presence of OAC (Fig. 3b). After > 40 h treatment cell division was also inhibited by OAC, presumably as a secondary consequence of the blockage of cell expansion. A major inhibition of cell expansion in cell cultures by OAC corroborates the reported inhibition of cell expansion in the roots of intact plants.
In theory, cell expansion could be inhibited via a decrease in water uptake, and one possible mechanism for this would be the inhibition of aquaporin action. Aquaporins are membrane-localized proteins that act as water channels, facilitating the movement of water across cell membranes (Maurel, 1997; Chaumont et al., 1998). The rate of water transport across a plant cell membrane is normally very rapid, for example 300 µm3 water (µm−2 membrane) s−1, such that a protoplast typically adjusts to its new volume within < 10 s after being shifted from 0.45 to 0.3 m sorbitol (Suga et al., 2003). This makes it improbable that the rate of water uptake could be reduced sufficiently to limit maize cell expansion (which typically involves a doubling of cell volume in ≈ 1 d) in cultures that are adequately supplied with external water. Furthermore, the t1/2 for diffusion of water into or out of plant tissues has been estimated by use of 2H- or 3H-labelled water. In 5-mm segments of oat coleoptiles the t1/2 was 8–9 min (Ordin & Bonner, 1956); in 1-mm-thick discs of potato tuber it was ≈ 1.5 min (Thimann & Samuel, 1955); and in Vicia root segments it was ≈ 0.6 min (Ordin & Kramer, 1956). These t1/2 values are very short compared with the time required for a cell to show any appreciable growth, and again suggest that it is implausible for a blockage of aquaporin action to mediate the growth-inhibiting effect of OAC.
Nevertheless, there have been repeated suggestions that aquaporins can control plant cell expansion. For example, Siefritz et al. (2004) observed that diurnal changes of growth rate in tobacco, associated with epinastic leaf movements, correlated with levels of an aquaporin, supporting the authors’ opinion that the transport of water across membranes by aquaporins is an important component of rapid plant cell elongation. The water permeability of petiole protoplasts was highest during the leaf-unfolding process. Furthermore, diurnal growth movements were diminished in transgenic tobacco lines defective in aquaporin expression (Siefritz et al., 2004). In related studies the overexpression of an aquaporin in tobacco cultures increased the cells’ volume, although it did not affect the rate of growth (Reisen et al., 2003).
To place this argument on an experimentally verifiable footing in the present context, we tested the effect of OAC on the 3H2O permeability of maize cells. The escape of 3H2O was expected to be rapid (see t1/2 values quoted above), but theoretically could have been slowed by OAC. The measured rate of 3H2O efflux was strongly enhanced by detergent, suggesting that we were observing water movement though membranes rather than simply boundary effects. However, OAC had no inhibitory effect on measured water exchange. Some water permeability is an intrinsic property of all biological membranes, and this is probably adequate for enabling the gradual influx of water required for cell expansion.
Regardless of the water permeability of the membranes, it was possible that OAC might decrease turgor pressure by preventing the synthesis or accumulation of low-Mr, osmotically active solutes within the protoplasts. At its simplest, this could involve membrane denaturation or even cell death. Alternatively it could involve a blockage of cell metabolism such that the ATP supply was diminished (Gruenhag & Moreland, 1971; Decleire & Decat, 1981; Peixoto et al., 2003), precluding the active transport or synthesis of solutes. However, the osmotic pressure gradient between the cell sap and the culture medium was maintained even after 48 h in the presence of 480 nm OAC.
As cell expansion is blocked by OAC despite the maintenance of turgor pressure, we conclude that OAC treatment of cells results in cell walls that are less susceptible to turgor-driven expansion. This could be achieved by a promotion of wall tightening and/or inhibition of wall loosening. One possible mechanism by which OAC could interfere with wall loosening involves a blockage of H+ secretion into the medium, a low apoplastic pH being regarded as necessary for some types of wall expansion (Cleland, 1992). However, OAC had no short- or long-term effects on the pH of the medium in maize cell cultures. We therefore conclude that OAC does not act by blocking wall acidification.
OAC is a novel herbicide that is unusual in that it appears to block plant growth, reversibly, primarily by inhibiting cell expansion. Effects on cell division are manifested later and appear to be secondary. However, the precise mode of action of OAC is unknown. OAC does not prevent water passing through the cell membrane, nor prevent the cells building up an osmotic gradient. Our observations therefore focus attention on wall loosening and tightening as the most likely target of OAC action.
We thank Dr David Cole (Aventis CropScience UK Ltd, Ongar, Essex, UK) for supplying OAC and for valuable discussions, and Dr Wieland Fricke (Paisley University, UK) for help with osmometry. We are grateful to Graham Wright and Graham Clark for advice on microscopy. N.O’L. thanks the BBSRC and Aventis CropScience UK Ltd for funding a studentship.