The PDE4 inhibitor rolipram reverses object memory impairment induced by acute tryptophan depletion in the rat

Rationale The selective type IV phosphodiesterase inhibitor, rolipram, has been shown to improve long-term memory and can reverse the cholinergic deficit caused by scopolamine. However, the underlying mechanisms of action of rolipram remain obscure.
Introduction
Inhibition of phosphodiesterase type 4 (PDE4) leads to an increase in intracellular cyclic adenosine monophosphate (cAMP) availability (Silvestre et al. 1999). cAMP is an important second messenger molecule in the process of intracellular signal transduction mechanisms (Bailey et al. 1996). Several studies have shown that rolipram, a selective PDE4 inhibitor, improves cognitive performance in young rats (Blokland et al. 2006; Imanishi et al. 1997; Rose et al. 2005; Zhang et al. 2000, 2004) and ameliorates scopolamine-induced memory deficits (Egawa et al. 1997; Imanishi et al. 1997; Rutten et al. 2006; Zhang and O’Donnell 2000).
In a previous study, we have shown that rolipram reversed a scopolamine-induced deficit in an object recognition task (ORT) in male Wistar rats (Rutten et al. 2006). It was suggested that these effects may be related to the effects of elevated cAMP levels on neurotransmitter release. There is also evidence that there is a link between PDE4 inhibition and serotonergic neurotransmission (Schoffelmeer et al. 1985; West and Galloway 1996). The adenylate cyclase activator forskolin, as well as 8-bromo-cAMP, can enhance the electrically evoked release of 3H-5-hydroxytryptamine (Schoffelmeer et al. 1985). Thus, elevating cAMP levels by a selective PDE4 inhibitor could exert a general facilitatory effect on 5-HT release. Based on these findings, we argued that rolipram may also reverse memory deficits induced by a lowered 5-HT neurotransmission.
Acute tryptophan depletion (ATD) is a well-established model to assess the role of serotonin in cognitive and affective functioning (Booij et al. 2003). The neurotransmitter serotonin is synthesized from its amino acid precursor tryptophan (TRP), which is obtained from our food. Free TRP is transported into the brain across the blood–brain barrier, but has to compete for entrance with five other large neutral amino acids (LNAAs: valine, leucine, isoleucine, phenylalanine, and tyrosine). The ratio of TRP and these other LNAAs (TRP/ΣLNAA ratio) is thought to be a more sensitive index of brain TRP availability (Fernstrom 1981; Wurtman et al. 1980). As 5-HT is synthesized from TRP, through the rate-limiting enzyme TRP-hydroxylase, the availability of TRP in the brain determines the amount of central 5-HT (Wurtman et al. 1980). Due to its reversible and non-intrusive effects, the method of ATD can be repeatedly used in animals and humans.
In humans, acute tryptophan depletion has a negative effect on memory consolidation (Park et al. 1994; Riedel et al. 1999; Schmitt et al. 2000). Furthermore, memory impairments in the ORT have been observed in rats after ATD (Lieben et al. 2004b, 2005). Therefore, we hypothesized that rolipram could reverse an ATD-induced memory deficit in rats by elevating 5-HT release in the brain.
In addition to this aim of the study, we further examined the relation between peripheral TRP and memory performance. We investigated the effects of different pretreatment times of the TRP-depleted mixture on object recognition memory. These data should provide more experimental support for a relation between plasma TRP and, consequently, central 5-HT and memory performance.
Materials and methods
Animals
All experimental procedures were approved by the local ethical committee of the Maastricht University for animal experiments according to governmental guidelines. A total of 42 4-month-old male Wistar rats (Charles River, The Netherlands) were used (410–450 g). Rats were randomly assigned to either the biochemistry (n = 18) or the behavior group (n = 24). In the biochemistry groups, the animals were randomly subdivided over three treatment groups, i.e., saline, TRP+, or TRP− (n = 6/group). The animals were housed individually in standard type 3 Makrolon cages on sawdust bedding in an air-conditioned room (about 20°C). They were kept under a reversed 12/12-h light/dark cycle (lights on from 18:00 to 6:00 hours) and had free access to water. Rats were housed in the same room as where they were tested. A radio, which played softly, provided background noise in all rooms. All testing were done between 9:00 and 17:00 hours.
Drugs and chemicals
The gelatin hydrolysate (Solugel C) was obtained from PB Gelatins (Tessenderlo, Belgium). Glucodry 210 was obtained from the Amylumgroup (Koog aan de Zaan, The Netherlands). l-Tryptophan and rolipram were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Kaliumchloride (KCl), calciumchloride-dihydrate (CaCl2·2H2O), and 5-sulfosalicylic acid dihydrate were purchased from Merck (Darmstadt, Germany).
Treatment
During a period of 2 weeks preceding the experiment, the rats were handled and habituated to oral injections with normal tap water (10 ml/kg). On experimental days, the rats were not fed 14 h before treatment until the testing period was completed. This was done to minimize the availability of TRP from food, which would counteract the effects of the ATD treatment. The rats were orally treated with a protein–carbohydrate mixture containing TRP (TRP+ group; in which 0.28% TRP of the total protein was added to the mixture) or one lacking TRP (TRP− group; no TRP added to the mixture), or with saline. The composition of the nutritional mixture is shown in Table 1.
Table 1Composition of mixture and determination of the amino acids in gelatin-based protein (mol) MixtureComposition Protein (Solugel) in 100 ml water100 g Aspartic acid4.8 Glutamic acid10.3 Hydroxyproline11.4 Serine3.4 Glycine23.2 Histidine0.8 Arginine10.0 Threonine2.0 Alanine10.4 Proline12.0 Tyrosine0.4 Valine2.2 Methionine0.7 Isoleucine1.0 Leucine2.5 Hydroxylysine0.9 Phenylalanine1.1 Lysine2.9Carbohydrate (Glucodry 210) in 80 ml water50KCl0.094CaCl2·2H2O2.32l-Tryptophan (TRP−group)0l-Tryptophan (TRP+ group)0.28The composition of the mixture (g) used in this experiment is described in italic. The amino acid spectrum (%) of the Solugel protein was obtained from PB Gelatins, Tessenderlo, Belgium.
In every experiment, each dose contained Solugel C at 4.0 g/kg and Glucodry at 2.0 g/kg of the body weight and was given in a dose of 10 ml/kg between 8:30 and 12:30 hours. Blood samples were taken at baseline (20 min before treatment) and 60, 180, and 360 min after the first treatment. For the behavioral studies in the object recognition task, the mixture was administered at different time points (see below).
Rolipram was freshly suspended in 5% ethanol, 1% tylose (methyl-cellulose), and 94% distilled water on every experimental day. The following doses of rolipram were tested: 0, 0.01, 0.03, or 0.1 mg/kg always in combination with the TRP− mixture (3 h before the first trial, T1). Rolipram was always administered 30 min before T1 (i.p. injection, dose 2 ml/kg).
Biochemistry
For the determination of plasma amino acid levels, blood samples were taken at resting values and repeated at several points in time. Blood sampling was done via a tail-incision method (Fluttert et al. 2000). Promptly after collection of blood in sodium heparin tubes (Microvette® CB 300, Sarstedt, Germany), the samples were kept on ice. After centrifugation of the blood samples (at 4°C for 15 min at 3,000×g in a Hettich EBA 12 centrifuge), plasma was deproteinized with cups containing dry 5-sulfosalicylic acid (6 mg/100 μl plasma), and the protein was spun down. Samples were frozen in liquid nitrogen and stored at −80°C. Before analysis, samples were thawed at 4°C, vortex-mixed vigorously, and centrifuged at 50,000×g in a Hereaus Model Biogufe Stratos for 10 min at 4°C. From the clear supernatant, 20 μl was mixed with 1,960 μl water and 20 μl norvaline and stored in the cooled (7°C) sample compartment until analysis.
In addition to total plasma TRP, the concentrations of several other amino acids were determined with a fully automated high-performance liquid chromatography system after precolumn derivatization with ophthaldialdehyde (OPA; Van Eijk et al. 1993). OPA amino acids derivates were quantified with fluorescence detection. The concentrations of the total plasma amino acids were expressed as micromole per liter.
Object recognition memory
The object recognition test was performed as described elsewhere (Prickaerts et al. 2002).The apparatus consisted of a circular arena, 83 cm in diameter. Half of the 40-cm high wall was made of gray polyvinyl chloride, the other half of transparent polyvinyl chloride. A light bulb was switched on during testing only and provided a light intensity (20 lx) which was equal in the different parts of the apparatus. Two objects were placed in a symmetrical position about 10 cm away from the gray wall. We used four different sets of objects. The different objects were: (1) a cone consisting of a gray polyvinyl chloride base (maximal diameter 18 cm) with a collar on top made of brass (total height 16 cm), (2) a standard 1-l transparent glass bottle (diameter 10 cm, height 22 cm) filled with sand, (3) a massive metal cube (10.0 × 5.0 × 7.5 cm) with two holes (diameter 1.9 cm), and (4) a massive aluminum cube with a tapering top (13.0 × 8.0 × 8.0 cm). The objects could not be displaced by a rat.
In the first week, the animals were handled daily and were adapted to the procedure in 2 days; that is, they were allowed to explore the apparatus (without any objects) twice for 3 min each day. In the two following weeks, the rats were adapted to the testing and i.p. administration procedure by a saline injection (0.4 ml) 30 min before the first trial until they showed a stable discrimination performance, i.e., good object discrimination (a d2 value of about 0.30, see below) at a 1-h interval on two successive sessions. Subsequently, testing of the drugs began.
A testing session comprised two trials. The duration of each trial was 3 min. During the first trial (T1) the apparatus contained two identical objects (samples). A rat was always placed in the apparatus facing the wall in the center of the transparent front segment. After the first exploration period, the rat was put back in its home cage. Subsequently, after a delay interval, the rat was put back in the apparatus for the second trial (T2), but now with two dissimilar objects, a familiar one (the sample) and a new one. The times spent exploring each object during T1 and T2 were recorded manually with a personal computer.
Exploration was defined as follows: directing the nose to the object at a distance of no more than 2 cm and/or touching the object with the nose. Sitting on the object was not considered exploratory behavior. To avoid the presence of olfactory trails, the objects were always thoroughly cleaned with alcohol (70%). Moreover, each object was available in triplicate so neither of the two objects from the first trial had to be used as the familiar object in the second trial. In addition, all combinations and locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations or objects.
As we expected ATD to impair memory performance, we needed a delay interval at which normal rats discriminate between the novel and familiar object. Therefore, we selected a delay interval of 1 h, as Wistar rats show good discrimination between the two objects after this interval (Rutten et al. 2006). Furthermore, we hypothesized that rolipram may reverse deficits induced by ATD in a 1-h delay ORT. In 1 week, three testing sessions were given, with a 48-h washout period in between. Tests were always conducted on Mondays, Wednesdays, and Fridays.
In the first behavioral experiment, we investigated the effects of administration with TRP+ or TRP− mixture at different time points (i.e., 30 min, 1, 3, or 6 h) before testing in the ORT. Each rat always participated in each condition, and the order of test conditions was decided at random.
The second behavioral experiment examined the effect of rolipram (30 min before T1) on object recognition performance in combination with the TRP− mixture at the most effective time point (i.e., 3 h before T1). Thus, five treatment conditions were tested, i.e., saline, vehicle and TRP−, rolipram 0.01 mg/kg and TRP−, rolipram 0.03 mg/kg and TRP−, and rolipram 0.1 mg/kg and TRP−. Each rat was tested in each condition, and the order of treatment conditions was decided at random. The same animals (n = 24) were used for both behavioral experiments; thus, control conditions (TRP+) from experiment 1 and saline from experiment 2 can be compared to each other.
Statistical analysis
Biochemistry
The mean concentrations of plasma amino acids were determined for each treatment and time condition separately. The extent of reduction (expressed in absolute values and in percentage decline from resting values) was calculated for total plasma TRP concentrations and for the TRP/ΣLNAA ratio. Extreme values, as determined by an extremity test (see Dixon 1959), were excluded from statistical analysis. Differences in plasma amino acid concentrations were analyzed with a general linear model, with main factor ‘treatment’ and repeated measure factor ‘time’. Separate one-way analyses of variance (ANOVA) were performed to analyze differences between treatment conditions per time point. A post-hoc Bonferroni test was used to further characterize the differences between treatment conditions. Differences were regarded as statistically significant if P < 0.05.
Behavior
The basic measures were the times spent by rats exploring an object during T1 and T2. Table 2 shows the measures involved in the object recognition task (Prickaerts et al. 1997). e1 and e2 are measures of the total exploration time of both objects during T1 and T2, respectively. d2 was considered as index measures of discrimination between the new and the familiar objects. d2 is a relative measure of discrimination which corrects the difference between exploring the old and the novel object for exploration activity (e2) and appears to be independent of the total exploration times (see Şık et al. 2003). In the object recognition task, results are not reliable if an animal explores both objects in the second trial for less than 5 s (Şık et al. 2003). Therefore, in every session, animals that explored less than 5 s in the second trial (i.e., e2 < 5) were excluded from analysis. For all parameters, the effects of the different treatment, time, and interactions were analyzed with univariate ANOVA for the first experiment and one-way ANOVA for the second experiment. Significant effects were analyzed in more detail using the Bonferroni correction. To compare control conditions, i.e., the TRP+ (3 h before the first trial) condition of the first experiment with the saline condition of the second experiment, a t test was performed. Differences were regarded as statistically significant if P < 0.05.
Table 2Measures involved in the object recognition testExplorationDiscriminatione1 is the measure of the time spent in exploring both identical objects (a1 and a2) in T1; e2 is the measure of the time spent in exploring both the familiar (a) and new object (b) in T2; d2 is the measure of discrimination between the new and familiar objects.
Results
Plasma TRP values
The plasma TRP/ΣLNAA ratio is shown in Fig. 1. TRP/ΣLNAA levels changed over the 6 h [F(3,60) = 4.92; P < 0.01]. There was a treatment effect for TRP/ΣLNAA ratio [F(2,60) = 36.72, P < 0.01]. Post hoc analysis showed that the TRP− condition was significantly different than the TRP+ and the saline condition (Bonferroni, P < 0.05). In addition, the time × treatment interaction was also significant for the TRP/ΣLNAA ratio [F(6,60) = 7.80; P < 0.01]. Separate one-way ANOVA analyses for each treatment time point showed differences between treatment conditions at the 1-h time point [F(2,17) = 31.44; P < 0.01] and the 3-h time point [F(2,16) = 6.79; P < 0.01]. However, no differences between treatment conditions were observed at the baseline time point (−20 min) [F(2,15) = 2.03; n.s.] or the 6-h time point [F(2.17) = 3.33; n.s.] (see Fig. 1). Furthermore, post hoc analysis showed that the TRP/ΣLNAA ratio of the TRP-treated animals was lower than the TRP+- or saline-treated animals at the 1-h time point (Bonferroni; P < 0.01) and the 3-h time point (Bonferroni; P < 0.05).
Fig. 1The effects of treatment time on the ratio plasma TRP/ΣLNAA (mean values and SEM). Percentages indicate the difference from baseline levels in the TRP− conditions. Asterisks indicate significant differences from baseline (*P < 0.05; **P < 0.01)
Behavioral experiment 1
The effects of ATD on ORT performance are shown in Fig. 2 and Table 3. There was a general treatment effect on ORT performance [F(1,165) = 36.44; P < 0.01]. In addition, a time effect was observed on ORT performance [F(3,165)  =  2.86; P < 0.05]. The time × treatment interaction was also significant for the memory performance in the ORT [F(3,165) = 4.47; P < 0.01]. Separate analysis per treatment condition showed that there was a significant time effect for the TRP− condition [F(3,87) = 5.41; P < 0.01] in memory performance, but not for the TRP+ condition [F(3,84) = 1.77; P = n.s.]. Post hoc analysis showed that object recognition performance was significantly impaired when the TRP− mixture was administered 3 h before the first trial.
Fig. 2The effects of treatment time of TRP+ or TRP− on the performance in object recognition task. Asterisks indicate a significant impairment in object recognition performance (*P < 0.05)Table 3Exploration times in the object recognition task at baseline and after ATD T1—30 minT1—1 hT1—3 hT1—6 hTRP+e126.62 (2.09)32.30 (1.37)27.82 (2.06)29.00 (2.50)e233.64 (2.40)49.78 (3.11)*28.61 (2.39)28.37 (2.15)TRP−e126.97 (2.11)30.39 (1.78)29.29 (1.66)24.98 (1.53)e239.26 (4.17)37.32 (2.40)30.06 (1.77)28.91 (2.69)*P < 0.05 (indicates significant differences to the baseline group)
Behavioral experiment 2
The effects of ATD in combination with rolipram on object recognition performance are depicted in Fig. 3 and Table 4. A dose-dependent increase in discrimination performance was observed after treatment with rolipram [F(4,104) = 6.85; P < 0.01]. Post hoc analysis showed that a dose of 0.1 mg/kg rolipram reversed the effects of TRP− treatment (P < 0.05). Rolipram treatment decreased exploration times in the first trial [F(4,106) = 16.24; P < 0.01], but had no effect on exploration times in the second trial [F(4,106) = 1.53; n.s.; see Table 4). Post hoc analysis showed that for all of the rolipram doses the exploration in the first trial was lower than the saline control condition.
Fig. 3The effects of rolipram treatment on ATD-induced deficits in the object recognition task. Asterisks indicate a significant difference in object recognition performance compared with the TRP− and vehicle condition (**P < 0.01). The 0.1-mg/kg dose of rolipram reverses the ATD effectTable 4Exploration times in the object recognition task after treatment with rolipram and TRP− mixture SalineVehicle0.01 mg/kg0.03 mg/kg0.1 mg/kgSalineTRP−TRP−TRP−TRP−e128.72 (2.52)26.18 (1.72)19.15 (1.66)*18.68 (1.35)*12.02 (1.53)*e228.73 (1.42)27.43 (1.80)21.68 (1.96)27.89 (2.63)27.29 (2.70)*P < 0.05 (indicates significant differences to the baseline group)
A comparison between the d2 values of the control groups of the first and second experiment (TRP+ 3 h vs saline) showed that there was no difference between these two control groups [t(38) = 0.906; n.s.].
Discussion
The present study shows that acute tryptophan depletion results in reliable lowering of plasma TRP and the TRP/ΣLNAA ratio. When rats were administered one dose (10 ml/kg) of the TRP− mixture, depletion was maximal (48%) 1 h after the treatment. The present data provide strong evidence that blood TRP levels predict object recognition performance and that ATD has only temporary effects on TRP levels and memory performance. When injected 3 h before T1, object recognition was maximally impaired. This is in support of the results from the blood TRP values (see above). Thus, TRP levels in the blood seem to be related with behavioral performance in rats. As previous studies have shown (Lieben et al. 2004a) a clear relation between plasma TRP and central 5-HT, these data provide further support that ATD can be used as a serotonergic-deficit model in the object recognition task.
In the study by Lieben at al. (2004a,b), two doses of the TRP− mixture (10 ml/kg) were administered, which resulted in a 70% drop of the TRP/ΣLNAA ratio and in memory impairment in the ORT. In contrast, the present study showed that one dose of the TRP− mixture resulted in a 48% drop in the TRP/ΣLNAA ratio, which was found to induce memory impairment in the ORT. These findings suggest that a 50% reduction in plasma TRP levels is sufficient to impair object memory in rats.
In the first behavioral experiment, no effects on exploration times in the ORT were observed, except from an increase in exploration time in the second trial of the TRP+ 1 h before T1 group. However, in the second experiment, a dose-dependent decrease in the exploration time of T1 was observed after treatment with rolipram. Previous work from our group has shown that as long as exploration in trial 1 is higher than 10 s, a reliable discrimination index (d2) can be calculated (Şık et al. 2003). Furthermore, as can be seen when exploration times are compared to d2 values, effects in exploration time are independent of effects in d2 values (Şık et al. 2003). Rolipram is known to have some sedative side effects (at higher dosages >0.1 mg/kg) (Griebel et al. 1991; Silvestre et al. 1999); nevertheless, the low dosages used in the present study improved memory performance.
Rolipram has shown its pro-cognitive effects in several behavioral models. We have shown that rolipram treatment increases object memory in a time-dependent forgetting paradigm (Rutten et al. 2006). In addition, rolipram attenuated cholinergic deficits caused by scopolamine in several behavioral tasks (Imanishi et al. 1997; Rutten et al. 2006; Silvestre et al. 1999; Zhang and O’Donnell 2000). When rolipram (0.1 mg/kg) was administered in combination with the TRP− mixture (i.e., TRP− 3 h before T1 and rolipram 30 min before T1), we found that it reversed the effects of ATD. These are the first data suggesting that rolipram can reverse a serotonergic-induced memory deficit in rats.
Our previous study indicated that rolipram can reverse the cholinergic deficit caused by scopolamine in the ORT (Rutten et al. 2006). Remarkably, a similar dose of 0.1 mg/kg rolipram was capable of reversing the serotonergic and cholinergic deficits in the object recognition task, which might be explained by a non-specific working mechanism of rolipram. Although the underlying mechanisms of action of the memory-enhancing effects of rolipram in these models remain to be determined, the present data could be explained via an enhanced cholinergic and serotonergic turnover by rolipram (Imanishi et al. 1997; Schoffelmeer et al. 1985).
Alternatively, the effects of rolipram could also be explained in terms of only a cholinergic mechanism of action. Thus, previous studies have shown that the acetylcholinesterase inhibitor metrifonate ameliorates the effects of ATD as well and improves performance in scopolamine-deficit models of the ORT (Lieben et al. 2005). This suggests that the cholinergic and serotonergic systems act in a synergistic manner in memory performance (see Lieben et al. 2005 for a detailed discussion). Clearly, further research is needed to scrutinize the mechanism of action rolipram in the different animal models of memory.
Another mechanism of action of rolipram that could explain the present data is related to the intracellular signaling in long-term potentiation (LTP). In the hippocampus, rolipram has shown to improve LTP through activation of the cAMP/PKA/CREB pathway (Frey et al. 1993; Impey et al. 1996). Moreover, these mechanisms have been linked to behavioral improvement in several behavioral tasks (Barad et al. 1998; Bernabeu et al. 1997; Blokland et al. 2006; Rutten et al. 2006). Consequently, it could be argued that the present data could be explained by the effects of rolipram on the cAMP/PKA/CREB pathway. However, a recent study by our group showed that the administration time of rolipram was of critical relevance. Rolipram was only effective in the 24-h interval ORT when administered 3 h after T1 (Rutten et al. 2006), and the mechanism of late LTP was proposed to explain these effects. However, in the present study, a 1-h interval in the ORT was examined, in which no gene transcription or protein synthesis is required. Therefore, we assume that the cAMP/PKA/CREB pathway cannot explain these effects.
A recent PET study used radioactive rolipram as a measure for PDE4 binding and as an indirect index of cAMP signaling (Lourenco et al. 2006). Acute elevation of synaptic neurotransmitter levels (noradrenaline, 5-HT, and histamine) resulted in elevated cAMP levels that in turn elevated (R)-[11C] rolipram binding to PDE4 (Lourenco et al. 2006). According to this rationale, elevation of cAMP levels through inhibition of PDE4 can reverse cAMP-mediated deficits in behavior that occur due to a shortage of 5-HT, ie, reversing the ATD-induced object recognition impairment. As rolipram non-selectively facilitates several neurotransmitter systems, a broad mechanism of action can be expected. This can be favorable because influencing multiple systems increases the likelihood that several aspects of behavior could be enhanced. This may explain previous reports that rolipram was effective in depression models (Bobon et al. 1988; Norman et al. 1992; Weishaar et al. 1985). On the downside, the broad mechanism of action increases the risks for negative side effects, e.g., emesis, sedation, and nausea.
In summary, the method of ATD can be used as a reliable method for inducing object memory impairment in rats. The selective PDE4 inhibitor rolipram was able to reverse this serotonergic deficit, which could be explained by different mechanisms of action. These data provide further support that PDE4 inhibition could be considered as a potential target to improve memory performance.

pde4
rolipram
memory
tryptophan depletion
phosphodiesterase
cognition
object recognition
atd
cyclic amp


Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.
Introduction
Plants produce a wide variety of terpenoids that have primary functions as hormones (gibberellins, abscisic acid, brassinolide), sterols, pigments (carotenoids, phytol), and as parts of electron carrier moieties (ubiquinone, plastoquinone) (McGarvey and Croteau 1995). Most terpenoids, however, are secondary metabolites: over 20,000 different terpenoid structures from plants have been described (Sacchettini and Poulter 1997). Some terpenoids function in the direct defence strategy of plants as phytoalexins, which accumulate upon pathogen infection. Sesquiterpene phytoalexins (e.g., capsidiol) are characteristic for the Solanaceae (Chappell and Nable 1987; Egea et al. 1996).
Volatile monoterpenes and sesquiterpenes can function in the interaction of plants with other organisms. They are present in floral scents that attract pollinators (Knudsen et al. 1993; Langenheim 1994) and are emitted by many plant species in response to herbivory by insects or spider mites. Induced terpene emission can attract predators or parasitoids of the herbivores, a mechanism commonly referred to as the indirect defense strategy of plants. This has been studied in, for instance, Arabidopsis (Arabidopsis thaliana) (Van Poecke et al. 2001), lima bean (Phaseolus lunatus) (Dicke 1994; Takabayashi and Dicke 1996), tobacco (Nicotiana tabacum; N. attenuata), maize (Zea mays), and cotton (Gossypium hirsutum) (De Moraes et al. 1998; Pare and Tumlinson 1999), and tomato (Kant et al. 2004; Takabayashi et al. 2000).
These volatile terpenes are produced by sesqui- and monoterpene synthases of which several are induced by herbivory. For instance, nerolidol synthase activity is induced in lima bean, cucumber (Cucumis sativus), and maize (Bouwmeester et al. 1999; Degenhardt and Gershenzon 2000), whereas transcript levels of various other sesquiterpene synthases increase in, amongst others, maize (Shen et al. 2000), cucumber (Mercke et al. 2004), and wormwood (Artemisia annua) (Cai et al. 2002). In Arabidopsis, caterpillars (Pieris rapae) induce the myrcene/ocimene synthase AtTPS10 and the β-ocimene synthase AtTPS03, which coincides with increased myrcene emission (Van Poecke et al. 2001). Moreover, a β-ocimene synthase (LjEβOS) has been identified from Lotus (Lotus japonicus), which is induced by spider mite-feeding, resulting in increased emission of β-ocimene (Arimura et al. 2004b). However, artificial wounding or jasmonic acid (JA) treatment are often also sufficient to induce terpene synthases. The wound- and JA-induced synthesis of terpenes by coniferous plants is well described (Martin et al. 2002; Miller et al. 2005; Steele et al. 1995), and these treatments also mimic herbivore-induced responses in several angiosperms (Arimura et al. 2004a; Gomez et al. 2005; Schnee et al. 2002; Shen et al. 2000).
Here we describe the identification and characterization of the first two monoterpene synthases from a solanaceous species (tomato), LeMTS1 and LeMTS2. We provide evidence that these genes are differentially regulated and more importantly, that expression of linalool synthase LeMTS1 is restricted to trichomes and induced by JA.
Results
Identification of tomato terpene synthases 1 and 2 (LeMTS1 and LeMTS2)
In order to identify tomato monoterpene synthases that are induced upon spider mite herbivory, we first queried tomato EST databases (www.tigr.org and www.ncbi.nlm.nih.gov/BLAST/) with known monoterpene synthase sequences from other plant species. Retrieved tomato EST sequences were used to design a primer specific for monoterpene synthases (Fig. 1). We then used cDNA derived from spider mite-infested plants, to amplify a single 850 bp fragment with this primer and an oligo dT(18) primer. This fragment was subsequently used as a probe to screen available tomato cDNA libraries. Two different full-length cDNAs were identified; LeMTS1 (AY840091) was isolated from tomato leaves (sequence identical to the probe) and LeMTS2 (AY840092) from tomato roots and stems. The LeMTS1 and LeMTS2 open reading frames encode proteins of 609 and 590 amino acids (Fig. 1), which have predicted masses of 70.8 and 68.6 kDa, respectively, and are 71% identical. LeMTS1 and LeMTS2 contain amino acid motifs conserved in angiosperm (mono)terpene synthases. These are, based on LeMTS1 amino acid positions: R(45)R(46)W(55), required for the initial diphosphate migration (Williams et al. 1998); D(362)D(363)D(366), required for binding of a Mg2+ or Mn2+ ion, which is used as cofactor (Cane et al. 1996; Starks et al. 1997); D(505)T(509)E(513), required for binding of a second Mg2+ or Mn2+ cofactor; and D(586) in the active site cleft (Schwab et al. 2001; Whittington et al. 2002). LeMTS1 and LeMTS2 contain both putative (predicted with TargetP; Emanuelsson et al. 2000) plastid targeting signal peptides, which should be processed in the region upstream of the conserved R(45)R(46) arginine pair.
Fig. 1Alignment of deduced amino acid sequences of LeMTS1 and LeMTS2 with three terpene synthases. A ClustalW alignment is shown of Mentha aquatica linalool synthase, a monoterpene synthase (C10), MaLin (AAL99381); Lycopersicon esculentum monoterpene synthase 1, LeMTS1 (AY840091); L. esculentum monoterpene synthase 2, LeMTS2 (AY840092); Mentha longifolia limonene synthase, MlLim (AAD50304), and L. esculentum germacrene C synthase, a typical sesquiterpene synthase (C15), LeGerC (AAC39432). Amino acids are shaded dark if identical in at least three of the five sequences and light if identical in LeMTS1 and LeMTS2 only. Conserved amino acid motifs typical for terpene synthases are boxed: RRx8W (Williams et al. 1998), DDxxD (Cane et al. 1996; Starks et al. 1997), Dx3Tx3E, and D (Schwab et al. 2001; Whittington et al. 2002). The presence of putative N-terminal plastid targeting peptides in the four monoterpene synthases and the primer used for amplifying the initial LeMTS fragment from cDNA of spider mite-infested leaves are indicated
Remarkably, LeMTS1 contains an extra sequence stretch of 22 amino acids starting at N170 (Fig. 1). This sequence is not found in any other terpene synthase, nor does it have homology to any protein in the NCBI database (www.ncbi.nlm.nih.gov/BLAST/). Its presence can also not be explained by alternative splicing since the flanks do not contain canonical splice sites. This extra internal element is not an artefact of the isolated cDNA since it was present in four independent cDNAs isolated from different organs (data not shown).
LeMTS1 and LeMTS2 belong to the TPSb subfamily of terpene synthases
LeMTS1 and LeMTS2 are more similar to typical monoterpene synthases from, e.g., mint and citrus than to a typical tomato sesquiterpene synthase (Fig. 1). Based on protein sequence relatedness, LeMTS1 and LeMTS2 can be classified in the TPSb subfamily (Fig. 2), a distinct group of angiosperm monoterpene synthases, according to the original classification proposed by Bohlmann et al. (1997). So, unlike the snapdragon (Antirrhinum majus) monoterpene synthases from subfamily TPSg (Dudareva et al. 2003), including an Arabidopsis and a strawberry terpene synthase, the monoterpene synthases from the solanaceous family do not seem to form a new subfamily.
Fig. 2LeMTS1 and LeMTS2 belong to the TPSb subfamily of terpene synthases. The TPS family is divided into subfamilies according to the nomenclature of Bohlmann et al. (1997). TPSa: angiosperm sesquiterpene synthases, TPSb: angiosperm monoterpene synthases, TPSc: angiosperm diterpene synthases, TPSd: gymnosperm mono-sesqui- and diterpene synthases, TPSe: a second group of angiosperm diterpene synthases, TPSf: a distinct group of (putative) linalool synthases mainly from Clarkia (Dudareva et al. 1996) and TPSg: a recently identified group of monoterpene synthases mainly from Antirrhinum (Dudareva et al. 2003). A limited amount of representative terpene synthases is shown. The bootstrapped phylogenetic tree was constructed using PAUP and TreeView software. A representative tree of 100 replicates is shown. Genbank protein accessions of terpene synthases shown are, top to bottom: TPSf: AAL24105, AAC49395; TPSe: BAC56714, AAC39443; TPSc: BAA84918, NP192187; TPSd: AAK83566, AAK39129, AAK39127, AAB71084; TPSg: NP176361, AAO42614, CAD57081; TPSa: AAU05951, AAK54279, AAC39432, AAG09949, Q40577; TPSb: NP189212, NP567511, AY840091, AY840092, AAC26018, AAV63791, AAL99381, AAD50304, AAG31435, AAM53943, AAM53945, AAF13356, AAK58723, CAC41012, AAS79351
Terpene synthase activity of recombinant LeMTS1 and LeMTS2
Recombinant LeMTS1 and LeMTS2 proteins were produced in Escherichia coli in order to characterize their enzymatic activities. The plastid targeting peptide was deleted up to one amino acid upstream of the RRX8W motif (Fig. 1) to produce a His-tagged ‘pseudo-mature’ form of the proteins, since it has been reported that this can improve protein expression without affecting activity and product specificity of the enzymes (Bohlmann et al. 1999; Williams et al. 1998). The soluble protein fractions were assayed for terpene synthase activity and products were analyzed on a GC coupled to a Time Of Flight-MS. Using the monoterpene precursor geranyl diphosphate (GPP) as substrate, LeMTS1 generated only linalool whereas LeMTS2 generated several monoterpene products: β-phellandrene, β-myrcene, and sabinene (Fig. 3a). Mass spectra and relative retention indices (Adams 2001) or authentic standards confirmed the identification of terpene reaction products. Extracts of E. coli cells expressing the empty vector control generated minor amounts of linalool (Fig. 3a) and occasionally geraniol. Thermally induced autoconversion or solvolysis of GPP to linalool has been observed before (Crowell et al. 2002; Jia et al. 1999), as well as conversion to geraniol by aspecific hydrolase or phosphatase activity in protein extracts (Crowell et al. 2002). However, LeMTS1 product levels are several hundred-fold higher than the background levels of the control (Fig. 3a).
Fig. 3Enzymatic activity of recombinant LeMTS1 and LeMTS2 proteins. (A) GC/MS chromatograms of the LeMTS1 and LeMTS2 monoterpene products and the empty vector control with GPP as substrate. (B) GC/MS chromatogram of the LeMTS1 sesquiterpene product and the empty vector control with FPP as substrate. The chromatograms show detector responses for the terpene-specific ion mass 93. Products were identified as linalool (1), sabinene (2), β-myrcene (3), β-phellandrene (4), and (E)-nerolidol (5). Representative chromatograms are shown from assays of at least two independent experiments
Enantiomer separation on an enantiomer-selective column showed that LeMTS1 produced only (R)-linalool (data not shown). LeMTS1 was also able to utilize the sesquiterpene precursor farnesyl diphosphate (FPP) as substrate to produce (E)-nerolidol (Fig. 3b), whereas LeMTS2 had no detectable sesquiterpene synthase activity. Both enzymes were unable to form terpene products from the diterpene precursor geranylgeranyl diphosphate.
LeMTS1 and LeMTS2 are differentially expressed in various plant organs
We investigated the organ specificity of LeMTS1 and LeMTS2 expression in a mature (13 weeks old) tomato plant (Fig. 4a). Transcripts of LeMTS1 were detected in young fruit, flower buds, petals, sepals, stems, petioles, and in young leaves. The expression pattern of LeMTS2 was very different: no transcripts were detected in petals, sepals, and leaves. However, LeMTS2 was expressed in roots.
Fig. 4Spatial expression patterns of LeMTS1 and LeMTS2 in tomato. (A) Expression of LeMTS1 and LeMTS2 in different organs of an adult tomato plant. (B) Expression in trichomes or complementary tissue of petioles and stems. Expression was analyzed by RT-PCR. The data from one of two independent tissue sets that gave similar results is shown. RUB1 conjugating enzyme (RCE1, www.tigr.org: TC153679) was used as constitutive control. Ethidium bromide-stained agarose gels are shown
For plants that contain glandular trichomes, monoterpene production is considered to be localized exclusively in these organs (Gershenzon et al. 1992; Iijima et al. 2004; Kutchan 2005; Turner et al. 1999; Turner and Croteau 2004). Tomato plants possess several types of trichomes including glandular trichomes that are present in high density on leaves, petioles, and stems and that contain terpenes (Maluf et al. 2001; Snyder and Carter 1985). Therefore, we investigated the location of LeMTS1 and LeMTS2 expression in trichomes of petioles and stems. This showed that LeMTS1 is expressed in trichomes, whereas LeMTS2 is expressed in the complementary tissue (Fig. 4b).
LeMTS1 but not LeMTS2 is induced by spider mite feeding, artificial wounding, and jasmonic acid
To study whether LeMTS1 and LeMTS2 were induced upon spider mite feeding, changes in transcript levels of intact plants were analyzed by RT-PCR since LeMTS gene-expression was too low to determine by RNA gel-blot analysis. Expression of LeMTS1 was indeed induced in leaves by spider mite-feeding, but LeMTS2, which expression was very low in leaves, was not (Fig. 5a). Spider mites damage plant tissue and induce JA- and SA-responsive genes such as a wound-induced proteinase inhibitor (WIPI-II) and a pathogenesis related protein (PRP6), respectively (Kant et al. 2004). Therefore, we investigated the effects of artificial wounding and exogenous application of JA and SA (Fig. 5a). Both artificial wounding and JA induced LeMTS1. In contrast, LeMTS2 was not induced by these treatments (Fig. 5a). SA-treatment induced the SA-marker PRP6 but neither LeMTS1 nor LeMTS2 was increased. Induction of LeMTS1 and WIPI-II was validated by real-time Q-RT-PCR. This showed consistent and clear induction of both genes by JA, artificial wounding and spider mites, although there was large variation in the level of induction between independent experiments (Fig. 5b). We routinely used the tomato cultivar Moneymaker for our experiments. However, in our earlier work (Ament et al. 2004; Kant et al. 2004) the cultivar Castlemart (CM) was used because it has the same genetic background as the defenseless1 (def-1) mutant (Howe et al. 1996). Here, we show that spider mite- and JA-induced LeMTS1 expression occurred both in CM and Moneymaker (Fig. 5b).
Fig. 5Induction of LeMTS1 in tomato leaves in response to spider mites, wounding, and jasmonic acid (JA). (A) Gene expression analysis by semi-quantitative RT-PCR of LeMTS1 and LeMTS2 after various treatments, compared to the well known JA- and SA-marker genes WIPI-II (Graham et al. 1985) and PRP6 (van Kan et al. 1992), respectively. Treatments: JA, SA, and artificial wounding (W) after 24 h and spider mite-infestation (M) after 48 h. A representative result from three independent experiments is shown. RUB1 conjugating enzyme (RCE1) was used as constitutive control. Ethidium bromide-stained agarose gels are shown. (B) Validation of LeMTS1 and WIPI-II induction by real-time Q-RT-PCR. Two different tomato cultivars were used to verify induction by mites and JA. Values represent expression levels relative to controls, calculated after correction for expression of the control gene RCE1 (TC153679, www.tigr.org). Average values and standard errors are shown from three to five independent replicates
To investigate whether spider mite-induced expression of LeMTS1 was dependent on JA or SA we made use of the def-1 mutant, which is impaired in induced JA-accumulation (Howe et al. 1996) and the SA-deficient, transgenic NahG tomato line (Brading et al. 2000). In plants with the def-1 mutation, LeMTS1 was not induced by spider mites, whereas the expression could be induced by exogenous JA (Fig. 6a). In contrast, the NahG plants showed higher and clearly induced LeMTS1 expression (Fig. 6b). This shows that induction of LeMTS1 expression is dependent on JA but not on SA.
Fig. 6Spider mite-induced LeMTS1 expression is dependent on jasmonic acid (JA). (A) Expression of LeMTS1 in wild type (wt) or JA deficient def-1 mutant plants infested with spider mites (M) or treated with JA for 2 days. (B) LeMTS1 expression in wild type and SA deficient NahG plants in response to 2 days of spider mite-feeding. Gene expression was analyzed by RT-PCR, a representative result from one out of two independent experiments is shown. RUB1 conjugating enzyme (RCE1) was used as constitutive control. Ethidium bromide-stained agarose gels are shown
JA induces expression of LeMTS1 in trichomes
Since basal LeMTS1 expression in stems and petioles seemed to be restricted to trichomes (Fig. 4b), we addressed the question whether JA would induce expression of LeMTS1 solely in trichomes or also in other tissues. Indeed, JA-induced LeMTS1 expression was strongest in trichomes (Fig. 7). Interestingly, WIPI-II expression showed the opposite pattern. WIPI-II expression is very low in trichomes and basal as well as strongly induced WIPI-II expression occurred mainly in the complementary stem tissues.
Fig. 7LeMTS1 is induced in trichomes by JA. Expression analysis of LeMTS1 and WIPI-II by real-time Q-RT-PCR. Intact plants were treated with JA, and subsequently trichomes were separated from remaining tissues of stems. The results from four independent experiments are shown as mean values and standard errors. Expression levels are indicated as expression relative to the control gene RCE1
Linalool accumulates in glandular trichomes and linalool emission is induced by wounding, jasmonic acid, and by ectopic LeMTS1 overexpression
Figure 4b showed that, in petioles and stems, LeMTS1 was expressed specifically in the trichomes. Extraction of terpenes from the same tissues of that experiment demonstrated that linalool was produced predominantly in trichomes (Fig. 8). (E)-nerolidol and β-phellandrene also accumulated in trichomes. Conversely, the volatile benzenoid methyl-salicylate was not produced in trichomes and was also clearly detected in roots, in contrast to most terpenes.
Fig. 8Linalool accumulation in trichomes. Relative levels of linalool, a selection of other terpenes and methyl salicylate in extracts of petioles and stems before and after removal of trichomes compared to total leaf and root extracts. Equal amounts of the different tissues were used (freshweight, 0.5 g), and trichome material was corrected for freshweight of the original petiole or stem. Averages of two measurements and min/max are shown
Because LeMTS1 was induced by spider mite-infestation, wounding and JA, we investigated whether the emission of its in vitro products was induced concomitantly. Analysis of volatile production during 2 days of wounding or JA-treatment, conditions that induce LeMTS1 expression (Fig. 5), revealed that plants emitted more linalool after both treatments (Fig. 9a), while nerolidol emission was not induced.
Fig. 9Increased linalool emission. (A) Linalool and (E)-nerolidol emission by intact tomato plants that were wounded (W) or treated with 1 mM jasmonic acid (JA) twice a day, for 2 days. The bar-graph shows the average (n = 3) emission over 2 days compared to the control and standard errors. Data (corrected for unequal variances) were analyzed by means of ANOVA followed by a Fishers-LSD post hoc-test. Linalool emission for both JA and wounding treatments was significantly increased compared to the control (P < 0.05), whereas (E)-nerolidol emission was not. (B) Linalool emission by a transgenic tomato line overexpressing LeMTS1. Headspaces were sampled from desiccators containing two plants each and compared to that of plants transformed with the empty vector. The graph expresses average values and standard deviations of four measurements. Emission of (E)-nerolidol, β-phellandrene, and methyl-salicylate did not significantly differ between plants. LeMTS1 overexpression in leaves was confirmed by RNA gel-blot analysis. The expression of RUB1 conjugating enzyme (RCE1) and ribosomal RNA levels (ethidium bromide-stained gel) are shown to indicate equal loading of the gel
Next, we tried to overexpress the full length LeMTS1 cDNA (including the putative targeting peptide) in tomato plants, under control of the CaMV 35S promoter. Four transformation experiments yielded only two transformants of which one overexpressed LeMTS1. Compared to empty vector-transformed plants, LeMTS1 overexpressing plants emitted several hundred-fold more linalool into the plants headspace (Fig. 9b), while emission of other terpenes, including nerolidol, did not differ significantly from control plants. This suggests that LeMTS1 functions in planta as a true linalool synthase.
Discussion
Monoterpenes are abundantly emitted by tomato plants. For instance β-pinene, β-myrcene, 2-carene, β-phellandrene, limonene, and terpinolene are constitutively emitted, while linalool and β-ocimene emission is induced after spider mite feeding (Kant et al. 2004). In this paper, we describe the first two monoterpene synthases from tomato, LeMTS1 and LeMTS2 and show that LeMTS1 is induced by JA in trichomes.
Identification of two tomato monoterpene synthases
In vitro assays with the recombinant LeMTS1 and LeMTS2 proteins show that LeMTS1 has both (R)-linalool synthase and (E)-nerolidol synthase activity, and that LeMTS2 is a β-phellandrene/β-myrcene/sabinene synthase (Fig. 2a). LeMTS1 activity in planta seems restricted to linalool synthase activity since increased LeMTS1 expression by wounding, JA-treatment and ectopic overexpression led to increased linalool emission but did not affect nerolidol levels (Figs. 5, 9).
Generally speaking, monoterpene synthases are plastid targeted and use GPP, while sesquiterpene synthases use FPP in the cytosol. There are however some exceptions to this rule. For instance, a cytosolic strawberry nerolidol synthase (FaNES1) has both (3S)(E)-nerolidol synthase and S-linalool synthase activity and is likely responsible for both linalool and nerolidol synthesis in planta (Aharoni et al. 2004). Vice versa, when FaNES1 was targeted to the plastid in Arabidopsis it also produced both linalool and nerolidol (Aharoni et al. 2003). The authors suggested that GPP and FPP substrate pools are not strictly separated between the cytosol and plastid, which has recently been supported by similar engineering experiments in tobacco (Wu et al. 2006). These observations might be explainable by for instance cytosolic as well as plastidial targeting of GPP synthase (GPS) in Arabidopsis and Litospermum erythrorizon (Bouvier et al. 2000; Sommer et al. 1995). In addition, FPP might be transported from the cytosol into plastids (Lichtenthaler 1999) and finally, the prenyltransferases GPS, FPS or GGPS might generate multiple products. However, since induced LeMTS1 expression does not correlate with increased nerolidol emission but only with increased linalool emission (Figs. 5, 9), we consider LeMTS1 to be a linalool synthase. Furthermore, we hypothesize that the plastidial FPP concentrations in tomato are negligible.
Despite their high sequence homology (Fig. 1), LeMTS1 and LeMTS2 have different biochemical activities. It is commonly found that different terpene synthases within one plant species often have higher sequence similarity to one another than to functionally related terpene synthases of other plant species. For example, β-pinene synthase from citrus (Citrus limon) is more homologous to the citrus γ-terpinene synthase than to β-pinene synthase from wormwood (Fig. 2). It is also known from work with other terpene synthases that product specificity can depend on only a few amino acids (Kollner et al. 2004; Rising et al. 2000; Yoshikuni et al. 2006) and thus, product specificity of terpene synthases is not predictable on basis of protein sequence or specific amino acid residues. By comparing terpene synthase structures, attempts have been made to ascribe substrate binding and substrate modifications to certain residues. Modeling of the active sites of a Salvia (Salvia officinalis) sabinene synthase, a mint (Mentha citrata) limonene synthase and a mint linalool synthase led to the following observation (Crowell et al. 2002): compared to the other two enzymes, the linalool synthase has a three amino acid deletion in the active site pocket (two amino acids after the conserved D591, see Fig. 1), resulting in a more open structure of the so called J/K loop and providing an easier access of water during substrate ionization. Water access results in the premature release of the intermediate monoterpenol carbo-cation, before cyclization to a monoterpene olefin (non-hydrated) can occur. The tomato linalool synthase LeMTS1 also has a deletion in the active site pocket after the conserved D586 (Fig. 1). Although the deletion is eight instead of three amino acids, it indicates that LeMTS1 might utilize a similar structural mechanism to generate a monoterpene alcohol.
LeMTS1 expression correlates with induced linalool emission
The induction of LeMTS1 expression correlated with the increased emission of linalool by tomato plants. Wounding and JA application induced LeMTS1 (Fig. 5) as well as linalool emission (Fig. 9a). Induction of LeMTS1 upon spider mite-feeding (Fig. 5) coincided with linalool emission (Kant et al. 2004) in the tomato cultivar CM. Moreover, overexpression of LeMTS1 resulted in enhanced production of linalool (Fig. 9b) and no other volatile terpenes, providing evidence for in planta linalool synthase activity of LeMTS1. RNAi lines suppressing LeMTS1 expression in trichomes would provide additional evidence that LeMTS1 acts as a linalool synthase in planta.
It is generally known that successful overexpression of terpene synthases can be problematic (reviewed by Chappell 2004). Substrate limitation and product conversions can explain low-terpene production levels (Aharoni et al. 2003; Lucker et al. 2001; Ohara et al. 2003). In addition, ectopic terpene synthase overexpression might lead to product toxicity (Aharoni et al. 2006; Besumbes et al. 2004) and selection for plant lines with low transgene expression or low terpene accumulation (Diemer et al. 2001; Krasnyanski et al. 1999). We managed to regenerate only one LeMTS1 overexpressing plant line after four successive, independent transformations. The empty vector and unrelated constructs gave at least 20-fold higher transformation frequencies. Similarly, LeMTS2 overexpression was unsuccessful, seven transgenic plant lines showed only expression of truncated transcripts (data not shown).
LeMTS2 is expressed in roots, stems and petioles (Fig. 4a). In roots, only minute amounts of the main LeMTS2 product β-phellandrene were detected, hardly visible in Fig. 8 since levels are ∼300-fold lower than in leaves. The role of monoterpenes in roots remains unclear, although they might play a role in the defense response of plants. The release of the monoterpene 1,8-cineole from Arabidopsis roots is induced upon bacterial pathogen infection (Steeghs et al. 2004), although transcriptional regulation of the corresponding 1,8-cineole synthase (Chen et al. 2004) has not been shown yet.
Although LeMTS2 is hardly expressed in tomato leaves (Fig. 4a), β-phellandrene and β-myrcene are constitutively emitted by tomato plants (Buttery et al. 1987; Kant et al. 2004). This suggests that the constitutive low expression of LeMTS2 might be sufficient to generate these monoterpenes. However, like the majority of volatile terpenes (data not shown), β-phellandrene is mostly located in trichomes (Fig. 8) where LeMTS2 is not expressed (Fig. 4b). This indicates that there are other β-phellandrene/β-myrcene synthases in tomato that synthesize these terpenes.
JA-induced LeMTS1 expression occurs in trichomes
Although the presence of terpene synthases in trichomes has been well documented (Bertea et al. 2006; Iijima et al. 2004; Lange et al. 2000), the regulation of their expression in trichomes remains obscure. Here we show that JA treatment of intact plants leads to higher transcript levels of LeMTS1 mainly in trichomes (Fig. 7). In contrast, WIPI-II expression is induced by JA in the complementary tissues, indicating that there is a tissue-specific differential activation of JA-regulated genes. Basal expression of WIPI-II was very low in tomato trichomes, although constitutive expression of a WIPI-II in glandular trichomes was recently demonstrated in nightshade (Solanum americanum) (Liu et al. 2006). It is unknown whether trichome-expressed WIPI-II plays a role in the defense against herbivores. Induction of WIPI-II in non-glandular tissue (mesophyll/parenchyma) meets the expectation that it should be present in those cells that suffer from herbivory.
Previously, JA has been shown to increase trichome density on newly formed leaves of, for instance, Arabidopsis and tomato (Boughton et al. 2005; Traw and Bergelson 2003). Also, JA might induce trichome-based defenses directly. The production of acylsugars on the leaf surface (probably in trichomes) of Datura wrightii plants increased without affecting trichome density (Hare and Walling 2006). More clearly, nornicotin production on the Nicotiana repanda plant surface increased twofold within 6 h of JA treatment (Laue et al. 2000). Although it should be kept in mind that in the two above-mentioned studies, it was perhaps unjustly assumed that CHCl3 or CH2Cl2 extraction releases only trichome contents, it indicates that trichome-based secondary metabolite biosynthesis can be inducible. Results from our study show, for the first time, a specific transcriptional regulation of a gene involved in secondary metabolite biosynthesis in trichomes. Isolation of trichomes revealed that linalool synthase expression in tomato trichomes was induced more than twofold by JA after 24 h.
In this paper, we report the identification of the first two monoterpene synthases from a plant of the solanaceae family. As it has recently been described that increased volatile terpene production in transgenic plants can benefit the indirect defense (Kappers et al. 2005; Schnee et al. 2006), it will be interesting to investigate the biological effect of LeMTS1 overexpression in tomato. Furthermore, the role of LeMTS2-derived terpenes in roots and stems remains to be investigated. Finally, the specific induction of LeMTS1 in trichomes by JA can provide an excellent opportunity for identification of novel, JA-related promoter elements, and transcription factors.
Materials and methods
Isolation of LeMTS1 and LeMTS2 cDNAs
Tomato plants, Lycopersicon esculentum cv. Moneymaker (3–4 weeks old) were infested with 150 spider mites (Tetranychus urticae) as described by Kant et al. (2004). Leaves were collected and pooled after 2–5 days of infestation. RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA); cDNA was made using SuperscriptII RNAse H- (Invitrogen). A LeMTS fragment was amplified from this cDNA using the primer 5′-GATGACATTTATGATGTTTATGGC-3′ in combination with an oligo dT(18) primer. The primer was designed based on putative tomato monoterpene synthases (www.tigr.org: TC168035, TC160168, BG131411, and cLED9K13) as such that it should not anneal to sesquiterpene synthase cDNAs. The 850 bp PCR product was cloned into pGEM-T easy (Promega, Madison, WI, USA) and sequenced using the ABI PRISM BigDye terminator kit (Applied Biosystems, Foster city, CA, USA). cDNA was synthesized from leaves of 3-week-old Moneymaker plants and from hypocotyls and roots of 11-week-old Moneymaker plants with a ZAP-cDNA synthesis kit, cloned into the Lambda-ACT vector (Elledge et al. 1991) and packaged with a ZAP-cDNA Gigapack II gold cloning kit (Stratagene, La Jolla, CA, USA) 200,000 plaques of each library were screenend with the 850 bp RT-PCR product as probe, radioactively labeled by the ALL-IN-ONE Random prime labeling method (Sigma, Saint Louis, MI, USA). Filters were hybridized at 55°C in BLOTTO hybridization buffer (Sambrook and Russell 2001) and washed three times for 10 min in 1 ×  SSC, 0.1% SDS at 55°C. DNA from positive plaques was converted to pAct2 plasmids and their inserts were sequenced. LeMTS1 was obtained from the leaf cDNA library, LeMTS2 from the hypocotyl and root cDNA library. Sequences can be found in Genbank as AY840091 and AY840092, respectively. The presence of plastid targeting signals was predicted using PREDOTAR (http://genoplante-info.infobiogen.fr/predotar/predotar.html; Small et al. 2004) and TargetP (http://www.cbs.dtu.dk/services/TargetP/; Emanuelsson et al. 2000).
Expression of recombinant proteins in E. coli, enzyme assays and product analysis
LeMTS1 and LeMTS2 were subcloned into the pET32-a expression vector (Novagen, San Diego, CA, USA) after removal of the plastid targeting signal sequences up to one amino acid upstream of the conserved arginine pair (Fig. 1) by generating ‘truncated’ LeMTS1 and LeMTS2 PCR products with Pfu DNA polymerase (Stratagene). Forward primers contained NcoI- and reverse primers XhoI-restriction sites. LeMTS1 primers (5′–3′): GATCCATGGACACAAGGCGTTCAGGGAATTAC and GTACTCGAGCAAAGTAATAAAATGAAGCCTACG, LeMTS2 primers: GATCCATGGGTATCCGACGTTCAGGAAATTAC and GTACTCGAGAATAAAAGGTAATAATTCCTTGTC. pET32-LeMTS constructs were transformed into E. coli BL21(DE3) which were selected on Luria-Bertani (LB) plates containing 100 mg/l ampicillin. A single colony was grown overnight at 37°C on a plate, and transferred to 100 ml of LB with ampicillin. Cultures were grown at 37°C for 30–60 min to A600 of 0.6–1.0 before addition of 1 mM isopropyl-1-thio-β-d-galactopyranoside and grown at 20°C for an additional 8 h. Cells were harvested by centrifugation and resuspended in 5 ml monoterpene synthase assay buffer containing 50 mM HEPES pH 7.5, 10% glycerol, 5 mM DTT, 2 mM MnCl2, 10 mM MgCl2, proteinase inhibitor cocktail Complete (Roche, Basel, Switzerland), and 1 mM ascorbic acid. Cells were disrupted by addition of 1 mg/ml lysozym (Sigma), incubation on ice for 30 min and sonication. After centrifugation (10,000g) for 30 min at 4°C, 1 ml of the soluble enzyme fraction was assayed directly, or after purification of the His-tagged recombinant fusion protein on Ni-agarose beads (Novagen) for monoterpene, sesquiterpene or diterpene synthase activity using 10 μM of the substrates GPP, FPP or GGPP, respectively. The assay mix was incubated in a closed 20 ml vial at 30°C for 1 h under gentle shaking (150 rpm). Reaction products were sampled with a Solid Phase Micro Extraction fiber (SPME) for 10 min during which the vial was agitated and heated to 50°C. The SPME fiber was desorbed 1 min in an Optic injector port (ATAS GL Int., Zoeterwoude, The Netherlands) which was kept at 250°C. Compounds were separated on a DB-5 column (10 m × 180 μm, 0.18 μm film thickness; Hewlett Packard, Palo Alto CA, USA) in an 6,890 N gas chromatograph (Agilent, Amstelveen, The Netherlands) with a temperature program set to 40°C for 1.5 min, ramp to 250°C at 30°C/min and 250°C for an additional 2.5 min. Helium was used as carrier gas, the column flow was set to 3 ml/min for 2 min and to 1.5 ml/min thereafter. Mass spectra were generated with the ion source set to −70 V at 200°C and collected with a Time-of-Flight MS (Leco, Pegasus III, St. Joseph, MI, USA) at 1,671 V, with an acquisition rate of 20 scans/s. Terpene products were identified using authentic standards and comparison of ion specra and relative retention times (Adams 2001). For separation of terpene enantiomers, a Cyclosil-B column (30 m × 247 mm, 0.25 μm film thickness; Agilent) was used. The temperature program was isothermal at 115°C for 15 min, with a final ramp to 240°C at 120°C/min and 240°C for 5 min. Column flow was 0.7 ml/min.
Plant treatments, plant headspace sampling, trichome isolation, and measurement of extracts
For determining tissue specific gene expression, a 13 weeks old, untreated tomato plant was dissected to obtain material of each of the described tissues. For separation of trichomes from petioles and stems, 4-week-old plants were used. Trichomes were collected on the bottom of a 50 ml tube after vortexing several N2(l)-frozen petiole or stem segments, standing in upright position. The trichome fraction was used directly, whereas the remaining cleaned petiole or stem segments were thoroughly brushed to remove all remaining trichome material. Wounding-, spider mite-, and hormone-treatments (for both volatile sampling and gene expression analysis) were done using 3–4-week-old tomato plants. Wounding was inflicted by squeezing leaflets several times with a hemostat; a total of 150 spider mites were put on 3–4 leaflets per plant for 3 days; JA and salicylic acid were applied to plants by spraying 1 mM solutions made with tap water containing 0.05% SilwetL-77. Wounding- and hormone-treatments were done (cumulative) at 0 h and at 18 h and leaves were sampled at 24 h. Headspace sampling of plants was performed as described by Kant et al. (2004). Tenax sampling tubes were eluted with pentane : diethyl ether (4:1). The Eluted fractions were concentrated 20 times under a stream of N2 and 1 μl was injected into the GC injector port. For root-, leaf-, trichome-, stem- or petiole-extracts, 0.5 g tissue was used. The amount of trichome material was adjusted to be equivalent to the trichomes present on 0.5 g of the original stem or petiole. Tissues were ground in N2(l) and transferred to glass 20 ml vials containing 2 ml saturated CaCl2 (5 M) buffered in 100 mM sodium acetate (pH 4.5) which were capped immediately and kept at 5°C. Extracts were pre-incubated for 5 min at 60°C under agitation (500 rpm) and sampled for 10 min at 60°C on a 100 μM PDMS SPME fiber (Supelco, Zwijndrecht, The Netherlands). The SPME fiber was desorbed 1 min in the injector port of the GC/MS, which was kept at 250°C. GC/MS analysis was done as described earlier (Kant et al. 2004).
Gene expression analysis by RT-PCR and real-time Q-RT-PCR
For semi-quantitative RT-PCR, the tomato RUB1 conjugating enzyme (RCE1) was used as constitutively expressed control gene (www.tigr.org: TC153679). Initial PCR was performed with RCE1 primers, product levels were compared and individual cDNA samples were diluted accordingly, to ensure equal template concentrations. PCRs with primers of genes of interest were subsequently performed for non-saturating number of amplification cycles. RCE1 primers were always included in each experiment. Used primers (5′–3′) for RCE1: forward (F) GATTCTCTCTCATCAATCAATTCG; reverse (R) GCATCCAAACTTTACAGACTCTC, WIPI-II: F: GACAAGGTACTAGTAATCAATTATCC; R: CACATAACACACAACTTTGATGCC, PRP6: F: TCAGTCCGACTAGGTTGTGG; R: TAGATAAGTGCTTGATGTGCC, LeMTS1: F: GATGACATTTATGATGTTTATGGC; R: GGCCATCTCGAGACTTGAGAGCGAATGCAACATTAG, LeMTS2: F: GATGACATTTATGATGTTTATGGC; R: GGGTAATAATTCCTTGTCTTATTTC. Expression differences were validated by varying the amount of PCR cycles.
For real-time Q-RT-PCR, total RNA was isolated using Trizol (Invitrogen) and DNA was subsequently removed with DNAse (Ambion, Huntingdon, UK). cDNA was synthesized from 5 μg RNA using SuperscriptII (Invitrogen) in 20 μl reaction volume that was diluted to 50 μl prior to using it for PCR. PCRs were performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). About 20 μl PCR reactions contained 0.25 μM of each primer, 0.1 μl ROX reference dye, and 1 μl template. The cycling program was set to 2 min 50°C, 5 min 95°C, 40 cycles of 15 s at 95°C and 1 min 60°C, and a melting curve analysis. Primer pairs were tested for specificity and for linearity with a standard cDNA dilution curve. Primers used: LeRCE1 QF: 5′-GATTCTCTCTCATCAATCAATTCG-3′ QR: 5′-GAACGTAAATGTGCCACCCATA-3′, LeWIPI2 (K03291) QF: 5′-GACAAGGTACTAGTAATCAATTATCC-3′ QR: 5′-GGGCATATCCCGAACCCAAGA-3′, LeMTS1 (AY840091) QF: 5′-TTTGGGGACATCTTCGGATGAA-3′ QR: 5′-CTACTCGAGTTACTTGAGAGCGAATGCAAC-3′. Expression levels were normalized using RCE1 mRNA levels.
Generation of transgenic tomato plants
The complete LeMTS1 cDNA, including the putative signal peptide, was cloned into the EcoRI-digested and Klenow-blunted binary vector pGreen1K (Brandwagt et al. 2002), resulting in a CaMV35S:MTS1:nos cassette. The construct was transferred to Agrobacterium tumefaciens strain EHA105 carrying the pSoup helper plasmid (Hellens et al. 2000). Tomato plants cv. Moneymaker were transformed essentially as described by Vanroekel et al. (1993). Heterozygous and homozygous LeMTS1 overexpressing plants were used to measure linalool production by sampling the headspace of intact plants (four sets of two plants for both LeMTS1 overexpressors and control plants).

tomato
linalool
trichomes
jasmonic acid
terpenes
defence


Morphology and function of Bast’s valve: additional insight in its functioning using 3D-reconstruction

The utriculo-endolymphatic valve was discovered by Bast in 1928. The function of Bast’s valve is still unclear. By means of orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy 3D-reconstructions of the valve and its surrounding region are depicted. The shape of the duct at the utricular side is that of a flattened funnel. In the direction of the endolymphatic duct and sac this funnel runs into a very narrow duct. The valve itself has a rigid ‘arch-like’ configuration. The opposing thin, one cell-layer thick, utricular membrane is highly compliant. We propose that opening and closure of the valve occurs through movement of the flexible base/utricular membrane away from and toward the relatively rigid valve lip.
Hypothesis
The bottom or opposing wall of the uticulo-endolymphatic (Bast’s) valve is the moving and thus functional part of the valve.
Background
At the utricular end the utricular duct has a valve-like opening. This structure was discovered by Bast [3], who called it the utriculo-endolymphatic valve. It is since then also called Bast’s valve. The morphology of the valve has been researched in humans and several mammalians [1, 5, 10].
The function of Bast’s valve is still unclear. Bast himself suggested the closure of the utricular duct as the main function [3, 4]. Bachor and Karmody [2] follow Bast’s view, postulating that decreasing pressure in the whole endolymphatic system, secondary to collapse of the ductus reuniens, causes closure of the valve to prevent more loss of endolymph from the utricular system. Zechner [20] proposes that dysfunction of the valve causes endolymphatic hydrops.
In this paper orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy [6, 15–17] was used to obtain detailed three-dimensional reconstructions of Bast’s valve and the surrounding region in the intact guinea pig inner ear, as it is a technique capable of visualizing bone as well as soft tissue structures with high resolution, and is non-invasive.
Material and methods
Specimen preparation
Two healthy female albino guinea pigs (Harlan Laboratories, UK), weighing 450 g, were used. Animal care and use were approved by the Experimental Animal Committee of Groningen University, protocol No. 2883, in accordance with the principles of the Declaration of Helsinki.
The animals were terminated by lethal administration of sodium pentobarbital. After decapitation the bullas were dissected and fixated in a 10% formalin solution, neutrally buffered. Then the bullas were rinsed in aqua-dest. Decalcification in ethylenediaminetetraacetic acid 10% solution (EDTA; Sigma, ED5SS, pH 7.4) took place at a temperature of 50°C in a microwave oven (T/T MEGA microwave histoprocessor, Milestone) in eight sessions of 6 h. After decalcification the bullas were again rinsed with aqua-dest and dehydrated in a graded seven-step ethanol series (30%, 50%, 70%, 90%, 96%, 100%, and 100%).
Spalteholz fluid, a 5:3 solution of methyl salicylate (Sigma, M-6752) and benzyl benzoate (Sigma, B-6630) [14], was thereafter used to achieve transparency of the specimen. The clearing process consisted of application of a succession of Spalteholz-ethanol solutions, 24 h each. The Spalteholz fluid fraction in the clearing session was 25%, 50%, 75%, 100%, 100%, respectively. Hereafter the specimen was dyed in a fluorescent dye bath of Rhodamine-B Isothiocyanate (RITC; Sigma, R-1755). RITC absorbs maximally at 570 nm and emits at 595 nm. The dye bath was prepared by dissolving 1.0 mg/ml RITC into ethanol, followed by dilution in Spalteholz fluid to a final dye concentration of 5 × 10−4 mg/ml [15, 16] (Voie 2003). The specimen was dyed for four days.
OPFOS imaging system
A schematic diagram of the optical setup of our custom-made OPFOS imaging device is shown in Fig. 1 [6]. Laser light, originating from a green frequency doubled Nd:YVO4 neodymium-laser (model DPGL-2050, Suwtech), with an emission wavelength of 532 nm and 52 mW maximal power, is expanded into a parallel beam of 28 mm diameter by a Galilean beam expander (BE, model 336, Spectra-Physics). The broadened and spatially filtered light falls onto a specially designed cylindrical lens (CL, custom-made by LiteTec Ltd., Essex, UK) of 30 × 30 mm and 80 mm focal length, which focuses the light along a single dimension (Z), thus creating a hyperbolic light pattern along the X-axis. Near the center of this focus, we can locally approximate the light intensity profile as a sheet of light in the X–Y plane, which will perform virtual slicing of a specimen object (O). In the plane where the object is sectioned by the sheet of light, the specimen emits fluorescence light, which is recorded by a CCD-camera (C, Firewire FO442BIC, Foculus) in the direction orthogonal to the light plane. In this way, a 2D image of a virtual section within the object is obtained. For the specimen to be compatible with the OPFOS method, the above described specimen preparation is necessary to make the object transparent, refraction index matched and fluorescent. In our setup the specimen is positioned inside a container with optical quality glass windows at the laser and CCD side, and filled with Spalteholz fluid, and can be positioned within the container by an object translation stage (OTS). Between recordings the object (and not the container) is moved along the Z-axis in small slicing steps with a high-precision DC-motor driven translation stage with position encoder (M112.1 High-Resolution Micro-Translation Stage with C862 Mercury II DC-Motor Controller, PI Polytec). A long working distance microscope objective lens (OL) with good numerical aperture (M Plan APO × 5, NA = 0.14, Mitutoyo) projects the fluorescence image on the CCD. A colour filter (Kenko R1 SR-60) placed before the objective lens blocks scattered laser light and transmits fluorescence light.
Fig. 1Orthogonal-Plane Fluorescence Optional Sectioning (OPFOS) set-up. (BE: beam expander, FS: field stop, CL: cylindrical achromat, O: object, OTS: object translation stage, FTS: focusing translation stage, OL: objective lens with colour filter, CCD = charge coupled device camera)
The virtual slicing is performed by a ‘plane’ with 3 μm 1/e²-thickness resolution in the center of the image and slightly thicker at the edges (less than 10 μm), with slicing steps of 2 μm, followed by storage in a personal computer. The two-dimensional stored images were processed with an IMOD (http://bio3d.colorado.edu/imod) software package for 3D-reconstruction. Input of relevant contours in each 2D-image was manually performed with a writing tablet (Wacom Cintiq 15X).
Results
Figure 2a shows an OPFOS image of a cross-section of part of the guinea pig inner ear. Figure 2b is an enlargement of the part of Fig. 2a inside the dashed box, in which Bast’s valve and its surrounding structures are clearly visible. Saccule, endolymphatic sinus, utricule and endolymphatic duct are filled with endolymph. Walls of the endolymphatic space are on the right side of Fig. 2b connected to bone (dense; non-compliant). The opposite walls are adjacent to perilymph (hypodense; compliant). Connective tissue (medium density; non-compliant) is present inside the lip of Bast’s valve and between the upper side of the utricule and bone. Figure 2c is another OPFOS image, showing Bast’s valve in detail.
Fig. 2(a) 2D OPFOS-image of the region around Bast’s valve and the utricular duct, obtained with another OPFOS setup [7] than for (c) and following. The dashed box is shown in a larger magnification in (b), (b) Dashed box in (a) shown in a larger magnification, (c) 2D OPFOS-image of Bast’s valve. The arrows point toward very narrow passages
Figure 3 shows a 3D-reconstruction of the entrance of Bast’s valve as seen from inside the utricle. The proximal end of the valve shows a rather rigid ‘arch-like’ configuration. Figure 4 shows a 3D-reconstruction of walls of Bast’s valve and the utricular duct. The shape of the utricular duct at the utricular side is that of a flattened funnel. This funnel quickly runs into a very narrow duct.
Fig. 33D-Reconstruction of Bast’s valve as seen from inside the utriculeFig. 43D-Reconstruction of walls of Bast’s valve and the utricular duct. The shape of the utricular duct at the utricular side is that of a flattened funnel. This funnel runs into a very narrow duct
Figure 5 is a 3D-reconstruction of part of the outer walls of the utricle and utricular duct. It again shows the small caliber of the utricular duct in relation to the size of surrounding structures.
Fig. 53D-Reconstruction of part of the outer walls of the utricle and utricular duct. Note the small caliber of the utricular duct in relation to the size of surrounding structures
Discussion
In the 3D OPFOS-reconstruction of the entrance of Bast’s valve, shown in Fig. 3, the lip of the valve appears as a relatively rigid structure. In a comparable scanning electron microscopy (SEM) image the entrance of the valve is visible as a narrow slit in the utricular wall [9]. The valve lip is filled with connective tissue (Fig. 2c). This is even better visible in Fig. 6, which is a light microscopy (LM) image. This figure also clearly shows that the bottom of the valve has a thickness of only one cell-layer, and is as result highly compliant.
Fig. 6Light microscopy image of lip and bottom of Bast’s valve
Schuknecht and Belal [13] showed that the corium of the valve consists of fibroblasts and fibrocytes.
Based on the study of 170 human temporal bones these authors propose that the valve is closed in normal ears and that its anatomical structure is ideally suited to permit the occasional egress of excessive accumulation of endolymph to be processed in the endolymphatic sac, while preventing an excessive loss of endolymph with the possible consequence of membrane distortions and interference with the motion mechanics of the vestibular sense organs. Opening of the valve, still according to Schuknecht and Belal [13], would then be accomplished by pressure displacement of the outer membraneous wall away from the more rigid inner valve lip.
The arrows in Fig. 2c indicate positions were the valve appears to be closed. Also in Fig. 6 valve lip and bottom touch.
Bast himself [5] was not sure about the valve being opened or closed in the normal situation. He writes: “As a matter of fact in many histological sections the epithelium of the valve is in contact with the opposing wall of the slit-like opening of the duct which suggests that such a closure may exist, at least at times, in the living ear.”
He also was not certain about the mechanism that closes the valve [5].
On the one hand he supposed that a high perilymphatic pressure could close the valve by moving the opposing wall against the valve lip, as shown in Fig. 7. On the other hand he proposed closing of the valve lip against the opposing duct wall in the case of a relatively greater intra-utricular pressure. In this case he supposes that it is not the tip of the valve that moves, because it is bulky, but that the valve lip rotates at its base, where it is made up of loose perilymphatic tissue.
Fig. 7Possible mechanism of valve closure (black arrows) or valve opening (grey arrows)
In our opinion the latter mechanism is less likely, considering the observation that the valve opening in 3-D reconstruction is a rigid arch-like structure (Fig. 3), and not a flap hinged at its base, that could close an underlying opening. The latter mechanism may be inspired by 2-D pictures, like Fig. 2b, c and 6, where the valve tip appears as a flap-like structure.
The rigidity of the valve tip was also noticed by Scheerer and Hildmann [12], who also thought it to be improbable that this structure can function as a valve.
The walls of the endolymph filled spaces in the inner are highly compliant. As a result the pressure difference between endolymph and perilymph are negligible in the normal situation [19]. If the valve is open it seems, at first impression, easy to understand how an increase of perilymphatic pressure will close the valve by compression of its bottom, as is shown in Fig. 7. However, an increasing perilymphatic pressure will also increase the endolymphatic pressure inside the utricule and force endolymph out through the slit-like opening and prevent closure of the valve [11, 18].
As can be seen in Figs. 4 and 5 the utricular duct has a diameter in the order of 10 μm at its narrowest passage, preventing rapid flow of endolymph and possibly protecting the sensitive vestibular receptors against large fluid shifts in a short time.
Konishi [8] found an open utriculo-endolymphatic valve in guinea pigs with an (experimental) endolymphatic hydrops. It is conceivable that an excess of endolymph will force the base of the valve opening away from the lip, as shown in Fig. 7.
In future work, high-resolution OPFOS (HROPFOS) with slicing, a resolution of 2 μm [6] is feasible and may reveal even more detail.
Conclusion
The use of OPFOS imaging techniques and graphical 3D-reconstruction of the uticulo-endolymphatic (Bast’s) valve and its surroundings has given some additional insight in its functioning. It is most likely that opening or closure of the valve occurs through movement of the flexible base away from or toward the relatively rigid valve lip.

bast’s valve
3d-reconstruction
utriculo-endolymphatic valve
opfos
endolymphatic system
imaging