Attenuation of N2 amplitude of laser-evoked potentials by theta burst stimulation of primary somatosensory cortex

Theta burst stimulation (TBS) is a special repetitive transcranial magnetic stimulation (rTMS) paradigm, where bursts of low-intensity stimuli are applied in the theta frequency. The aim of this study was to investigate the effect of neuronavigated TBS over primary somatosensory cortex (SI) on laser-evoked potentials (LEPs) and acute pain perception induced with Tm : YAG laser stimulation. The amplitude changes of the N1, N2, and P2 components of LEPs and related subjective pain rating scores of 12 healthy subjects were analyzed prior to and following continuous TBS (cTBS), intermittent TBS (iTBS), intermediate TBS (imTBS), and sham stimulation. Our results demonstrate that all active TBS paradigms significantly diminished the amplitude of the N2 component, when the hand contralateral to the site of TBS was laser-stimulated. Sham stimulation condition had no significant effect. The subjective pain perception also decreased during the experimental sessions, but did not differ significantly from the sham stimulation condition. The main finding of our study is that TBS over SI diminished the amplitude of the N2 component evoked from the contralateral side without any significant analgesic effects. Furthermore, imTBS produced responses similar to those observed by other forms of TBS induced excitability changes in the SI.
Introduction
Functional neuroimaging studies have provided unequivocal evidence of the participation of the primary somatosensory cortex (SI), secondary somatosensory cortex (SII), and insula in pain processing (Talbot et al. 1991; Casey et al. 1994; Apkarian et al. 1999; Gelnar et al. 1999). Evidence suggests that the nociceptive input into these regions at least partially underlies the perception of sensory features of pain (Bushnell et al. 1999; Coghill et al. 1999; Peyron et al. 1999; Chen et al. 2002; for a reviews see: Peyron et al. 2000; Apkarian et al. 2005).
Electrophysiological studies have also confirmed the participation of the SI in pain processing, contralateral to the stimulated side. Tarkka and Treede (1993) first reported pain induced-activity in the SI using electroencephalogram (EEG) and applying brain electric source analysis (BESA). In their model, the peak latency measured at the SI was very similar to that of SII. In a combined magnetoencephalogramm (MEG) and laser-evoked potential (LEP) study Ploner et al. (1999) reported SI activity also contralateral to the side of stimulation and further to this proposed a parallel pain processing in SI and SII. This parallel activation of SI and SII was confirmed by other studies (Ploner et al. 2000, 2002) and other groups (Timmermann et al. 2001; Kanda et al. 2000; Inui et al. 2003; Nakata et al. 2004). However, some previous LEP dipole modelling studies showed that a dipole source in SI area was necessary to explain the scalp LEP topography, none of them reported a clear correspondence between the SI activity and a definite LEP component (Tarkka and Treede 1993; Ploner et al. 2002; Kanda et al. 2003). Kanda et al. (2000) detected SI activity following painful stimulation by recording intracranial EEG, but recently, intracerebral depth recordings in an epileptic patient have shown no reliable LEP response from the area 3b of the SI after painful laser stimulation, although a reliable N2–P2 response could be recorded at Cz (Valeriani et al. 2004). Inui et al. (2003) reported the absence of activation from area 3b of the SI after noxious electrical stimulation as well, however they found activity in the area 1 of SI. Other studies found no pain-related activation of the SI at all (for a review see: Garcia-Larrea et al. 2003; Kakigi et al. 2005).
Recent studies showed, that several kinds of external stimulation methods such as single-pulse transcranial magnetic stimulation (TMS) (Kujirai et al. 1993), 1 Hz repetitive TMS (rTMS) (Enomoto et al. 2001), paired associative stimulation (PAS) (Tsuji and Rothwell 2002; Wolters et al. 2005), transcranial direct current stimulation (tDCS) (Matsunaga et al. 2004; Dieckhofer et al. 2006) or theta burst stimulation (TBS) (Ishikawa et al. 2007) modulate the amplitude of cortical components of median nerve somatosensory evoked potentials (SEPs). It was recently reported that, cathodal tDCS over the SI (Dieckhofer et al. 2006) significantly reduced the N20 amplitude of median nerve SEPs. Furthermore, cathodal polarization over the SI induced a prolonged decrease of tactile discrimination (Rogalewski et al. 2004) and diminished acute pain perception and the amplitude of the N2 component of LEPs (Antal et al. 2007).
Recently Huang et al. (2005) developed a special “theta burst” paradigm to modulate human motor cortex (M1) excitability using low intensity, repetitive bursts of magnetic stimuli. The authors distinguished three stimulation patterns, which were proved to have different effects over M1 activity, when it was monitored by the amplitude of transcranial motor evoked potentials (MEPs). Continuous TBS (cTBS) caused a significant reduction in MEP amplitudes, which was probably due to the inhibition of specific excitatory circuits (I1-wave inputs to corticospinal neurons), as later confirmed by another study (Di Lazzaro et al. 2005). In contrast, intermittent TBS (iTBS) facilitated M1 activity and produced increase in MEP amplitudes. Interestingly, intermediate TBS (imTBS) had no effect at all. In addition, TBS has also been shown to have an effect on the human premotor (Mochizuki et al. 2005) and visual cortex (Franca et al. 2006).
Non-invasive cortical stimulation of M1 for the treatment of certain kinds of chronic and experimentally induced pain has recently attracted much interest. Both low and high frequency rTMS was reported to reduce subjective pain perception and has been used experimentally to reduce chronic pain (for reviews see: Leo and Latif 2007; Fregni et al. 2007). However, although the involvement of SI in pain perception and processing is reported by several imaging studies (for reviews see: Peyron et al. 2000; Apkarian et al. 2005), the magnetic stimulation of this cortical area in order to modify pain perception is neglected. Therefore, the aim of our study was to investigate the effects of cTBS, iTBS, and imTBS, on the early (N1) and late (N2, P2) components of LEPs and related subjective pain perception when applied over the left SI. We hypothesized that the three TBS types, which have short durations (maximum 190 s), would affect LEP components in a specific, paradigm-dependent manner similarly to the effect over M1 as revealed by Huang and et al. (2005).
Methods
Subjects
Nineteen healthy volunteers between 18 and 35 years were informed about all aspects of the experiments and signed an informed consent. Three subjects chose not to continue the experiment after the first or second session and during off-line EEG analysis four further subjects were dropped out because of their hardly detectable LEP components. Hence, twelve of the subjects (six male, six female; mean age = 26.33 ± 3.17 years) were included in the final analysis. We conformed with the Declaration of Helsinki and the experimental protocol was approved by the Ethics Committee of the University of Göttingen. None of the subjects suffered from chronic pain syndromes, nor took any medication regularly. None had a history of neurological or psychiatric illness. All of them participated in all four sessions, the three TBS and sham stimulations.
TBS stimulation
Theta burst stimulation was applied over the hand area of the left SI using a standard, figure-of-eight-coil (MCF-B65 Butterfly Coil) and MagPro stimulator (Medtronic, Denmark) with an outer half-coil radius of 75 mm, with a posterior–anterior–posterior current flow in the coil. Stimulus intensity was 80% of active motor threshold (AMT) (Huang et al. 2005).
For AMT determination, the coil was placed tangentially to the scalp, with the handle pointing backwards and laterally 45° from mid-line. MEPs of the right abductor digiti minimi muscle (ADM) were recorded by Ag-AgCl-electrodes in a belly tendon-montage before each stimulation. The signals were amplified and filtered (1.59 Hz-1 kHz, sampling rate of 5 kHz), digitalized with a micro 1401 AD converter (Cambridge Electronic Design, Cambridge, UK), recorded by a computer using SIGNAL software (Cambridge Electronic Design, Version 2.13). Complete muscle relaxation was controlled though auditory and visual feedback of EMG activity. AMT was defined as the minimum intensity eliciting a MEP of a superior size when compared to spontaneous moderate muscular activity in at least three of six pulses.
The pattern of TBS consisted of bursts containing three pulses at 50 Hz which were repeated at 200 ms intervals (i.e., 5 Hz) for up to 600 pulses for 40 s continuously (cTBS), or triads repeated at 200 ms intervals for 2 s intermittently with 8 s breaks for up to 600 pulses (iTBS). In the case of imTBS the triads (three pulses at 50 Hz) were repeated at 200 ms intervals for 5 s, in every 15 s up to 600 pulses (Huang et al. 2005). In separate experimental sessions, sham stimulation was applied with the cTBS protocol using the same coil held over the same position but tilted to a 90° angle (one-wing 90°) (Lisanby et al. 2001) with only the margin of the coil in contact with the scalp.
Determination of the primary somatosensory cortex (SI)
Anatomical magnetic resonance imaging (MRI) (Siemens 3 T, T1 weighted) dataset was used to determinate the coil localization for the stimulation of SI for all subjects using the Brainsight neuronavigation system (Rogue Research Inc., Montreal Quebec, Canada). The hand area was determined by previous fMRI studies (Bushnell et al. 1999; Blankenburg et al. 2003) and was located and marked in the MRI dataset as the target for TMS application (Fig. 1).
Fig. 1Three dimensional anatomical MRI of a single subject. Anatomical magnetic resonance imaging (MRI) (Siemens 3 T, T1 weighted) dataset was used to determinate the coil localization for the stimulation of SI by all subjects using the Brainsight neuronavigation system. The hand area was located and marked in the MRI dataset as target for TMS application. The black point indicates the hot-spot of the stimulation coil
Laser stimulation
A Tm : YAG laser system (WaveLight Laser Technologie AG, Erlangen, Germany) was used to induce painful stimulation. The thulium laser emits near-infrared radiation (wavelength 2,000 nm, pulse duration 1 ms, laser beam diameter 7 mm) with a penetration depth of 360 μm into the human skin and allows a precise restriction of the emitted heat energy to the termination area of primary nociceptive afferents without affecting the subcutaneous tissue (Treede et al. 2003). The distal handpiece of the laser was positioned 30 cm from the radial part of the dorsal surface of the hand. The pain threshold was determined on both hands at the beginning each session before baseline EEG recording by applying laser stimuli from 200 mJ in 50 mJ steps. During EEG recordings, each laser stimulus was delivered with an intensity of 1.4–1.6 times the threshold intensity to a slightly different spot in a 5 × 5 cm square on the dorsum of the hand in order to reduce receptor fatigue or sensitization by skin overheating (Treede et al. 2003). Skin temperature of the stimulated area was checked prior to every switch between hands, and corrected with a heating lamp if it fell below 35°C.
Psychophysical evaluation
We used the verbal analogue score (VAS) to assess the subjective intensity of pain. The subjects were instructed to pay attention to the laser stimuli and to rate the perceived pain verbally (1-warm, 1.1 smallest pain, and 1.9 most intense pain) about 2–3 s after each laser-impulse. The values were individually averaged separately for both hands in each session and conditions. The ears of the subjects were plugged during the measurements to avoid auditory artifacts accompanying laser stimulation.
Electrophysiological recordings
The EEG was recorded using a 64-channel montage applying 64 ring electrodes (inner diameter: 6 mm, outer diameter: 12 mm) (EasyCap; Falk and Minow GmbH, Münich, Germany). The electrodes were placed in accordance with the extended international 10–20 system. The impedance was kept <5 kΩ. The Fz was used as reference, the ground was placed 2 cm anterior to the tragus of the right ear. Data were collected at a sampling rate of 1,000 Hz with the BrainAmp system (Brain Products GmbH, Munich, Germany) and were analyzed offline. The obtained data were re-referenced to the connected mastoids (TP9-TP10). A 0.5 Hz low-cutoff as well as a 30 Hz high-cutoff filter was used. In addition to automatic artifact detection (200 μV amplitude criterion) all epochs were visually inspected, and those containing eye blinks or muscle movement artifacts were excluded. Baseline correction was performed on the basis of the 100 ms prestimulus interval. The amplitudes of N1 (referring to Fz) and N2–P2 (referring to TP9-TP10) components were measured.
Although we recorded data on 64 channels, we assessed LEPs according to the scalp distribution on the analyzed peaks. The N2 is a negative component (referring to TP9-TP10) was peaking around 160–240 ms. The amplitude of the early N1 negative peak which came before N2 on T7 and T8 channels (referring to Fz) was analyzed. The P2 positive component after N2 was peaking around 300–360 ms. The N2 component is largest over the lateral temporal and fronto-central areas on electrodes Fz, Cz, CPz, F1–F4, FC1–FC6, C1–C6, T7, T8, and CP1–CP6. In contrast, the P2 peak has its maximum amplitude over the vertex on electrodes FCz, Cz, CPz, Pz, F1–F4, FC1–FC4, C1–C4, CP1–CP4, and P1–P4. For the analysis of LEPs according to regional distribution, we defined three distinct areas with pooling the data: central (with all the mid-line electrodes such as Fz, FCz, Cz, CPz, and Pz), left (F3, FC3, C3, CP3, P3, FC5, C5, CP5, and T7) and right (electrodes according to the left side) instead of separate electrodes.
Experimental design
The subjects were sitting in a reclining chair. In case of all sessions first the EEG cap was placed on the head. After pain threshold determination the baseline LEP measurements were performed. Every run for the LEP recording consisted of 40 epochs of laser stimulation on each hand. The interstimulus interval of the stimulation ranged from 8 to 15 s (Raij et al. 2003). Thus the LEP recording lasted for 8–10 min for the first hand and also for 8–10 min for the second hand laser-stimulation. In all three TBS and sham TBS conditions, the right hand was stimulated first in half of the cases and the left hand was stimulated first in the other half. This order was kept for the subjects for all conditions. After baseline LEP recording the AMT was measured (∼15–20 min) and the TBS were applied for 40–190 s through the cap. After TBS the impedance of the EEG electrodes were retested and corrected below 5 kΩ if it was necessary. The TBS was followed by a post-stimulation LEP recording in ∼5 min after TBS. Thus, the interval between the two LEP recordings with regard to the same hand was about 30–35 min including all subjects and conditions.
The experimental sessions were separated from each other by at least 5 days. The subjects were blinded as to the type of magnetic stimulation. The order of the sessions was randomized across subjects.
Data analysis
The individually averaged VAS values and LEP amplitudes were entered into a repeated-measures-ANOVA for both hands and LEP components separately [four TBS CONDITION (cTBS, iTBS, imTBS, and sham) × 2 TIME (before, after TBS)]. We considered a psychophysical or an electrophysiological change only if the CONDITION × TIME interaction was significant. In case of the LEP components we investigated if this effect was dependent on the defined areas by calculating the CONDITION × TIME × REGION interaction. Post hoc analysis was done using Student’s t tests (paired samples, two-tailed, level of significance P < 0.05).
Results
Psychophysics
The intensity of the laser stimulation (1.4–1.6 × of the pain threshold) was 19.88 mJ/mm2 for cTBS, 20.53 mJ/mm2 for iTBS, 20.52 mJ/mm2 for imTBS, and 20.33 mJ/mm2 for sham stimulation. None of the subjects reported any side-effect after the stimulation.
In case of the contralateral hand (right) stimulation, repeated-measures-ANOVA revealed no main effect of CONDITION [F(3,33) = 0.828, P = 0.488] but the TIME was significant [F(1,11) = 27.270, P < 0.001]. The CONDITION × TIME interaction was also not significant [F(3,33) = 0.080, P = 0.97]. In case of the ipsilateral hand (left) stimulation, there was no significant main effect of CONDITION [F(3,33) = 1.329, P = 0.282] but the TIME was significant [F(1,11) = 15.395, P < 0.005]. The CONDITION × TIME interaction was also not significant [F(3,33) = 0.716, P = 0.55] (Fig. 2).
Fig. 2The effects of the TBS on subjective pain perception. The VAS-values were standardized for each subject, for each condition by calculating the after/before ratio. The independent variables were the CONDITION and HAND in order to determine if there is any difference between stimulation of the two hands. There was no main effect of the stimulation CONDITION and the HAND (left or right). The interaction CONDITION × TIME was also not significant
Electrophysiology
The N1, N2, and P2 components could clearly be identified in all subjects. The LEPs are presented in Figs. 3 and 4.
Fig. 3The effects of the TBS on the N1 component. The figure shows N1 amplitude changes during the experimental sessions on the grand averages of LEPs. The N1 was analyzed over the electrodes T7 and T8 referred to Fz (international 10–20 electrode system) in case of both hands. The solid line shows LEPs before and the intermittent line after TBS interventions. There was no significant difference between stimulation conditionsFig. 4Grand averages of LEPs obtained by contralateral (right) hand laser stimulation for three scalp regions (central, left and right) re-referenced to the connected mastoids (TP9-TP10), before and after the three TBS conditions and sham stimulation. The solid line shows LEPs before and the intermittent line after TBS interventions
The N1 component
We analyzed the amplitudes of the early N1 components on channels T7 and T8 (referring to Fz). There was no significant main effect of CONDITION [contralateral hand: F(3,33) = 2.216, P = 0.105; ipsilateral hand: F(3,33) = 2.865, P = 0.052]. The TIME was significant if the contralateral hand was stimulated [F(1,11) = 6.186, P = 0.030] but not for the ipsilateral hand [F(1,11) = 3.305, P = 0.096]. The interaction of the CONDITION × TIME resulted in no significant interaction neither after the contraleteral [F(3,33) = 0.727, P = 0.543] or ipsilateral [F(3,33) = 1.694, P = 0.187] hand stimulation (Fig. 3).
The N2 component
The repeated-measures-ANOVA (ipsilateral hand) showed no significant main effect of CONDITION [F(3,33) = 0.555, P = 0.65], but the effect of TIME was significant [F(1,11) = 11.769, P = 0.006]. The CONDITION × TIME interaction resulted in no significance [F(3,33) = 0.149, P = 0.93] (Fig. 5a). In case of the (contralateral hand) the main effect of CONDITION [F(3,33) = 0.250, P = 0.86] was not significant, but the TIME was significant [F(1,11) = 32.034, P < 0.001]. The CONDITION × TIME interaction was also significant [F(3,33) = 4.058, P = 0.015] (Fig. 5b). The interaction with electrode position was not significant [F(6,66) = 1.068, P = 0.39]. The post hoc t test showed that all active TBS stimulation significantly decreased the amplitudes of the N2 component at all defined regions for the contralateral hand stimulation. Table 1 summarizes the results of t tests.
Fig. 5The mean N2 amplitude changes during the four TBS conditions at the ipsilateral (left) (Fig. 5a) and contralateral (right) (Fig. 5b) hand laser-stimulation for the three calculated regions (central, left and right). The stars mark significant differences between before-after TBS conditions (post hoc t-tests, paired samples, two-tailed, P < 0.05)Table 1Post hoc analysis of the N2 componentcTBSiTBSimTBSshamBefore versus afterBefore versus afterBefore versus afterBefore versus afterCentralP-levels0.00330.00020.00210.3942t-values−3.730−5.499−4.005−0.887LeftP-levels0.00540.00040.00310.5921t-values−3.451−5.041−3.775−0.552RightP-levels0.03940.00850.00390.8118t-values−2.337−3.196−3.6450.244Shows the results of the Student’s t-tests (paired samples, two-tailed) in case of the right hand stimulation. The level of the significance was P < 0.05
The P2 component
The repeated-measures-ANOVA showed no significant main effect of CONDITION [contralateral: F(3,33) = 1.571, P = 0.22; ipsilateral: F(3,33) = 1.054, P = 0.38], but the TIME was significant [contralateral: F(1,11) = 17.038, P = 0.002; ipsilateral: F(1,11) = 15.362, P = 0.002]. The CONDITION × TIME interaction was also not significant [contralateral: F(3,33) = 2.669, P = 0.064; ipsilateral: F(3,33) = 0.418, P = 0.74].
The means of the different LEP components from all 12 subjects are presented in Table 2.
Table 2The mean amplitudes of the LEP componentsPeakcTBSiTBSimTBSshamBeforeAfterBeforeAfterBeforeAfterBeforeAfterLeft handN1T7−4.42 ± 2.75−4.60 ± 3.13−4.68 ± 2.52−3.71 ± 2.65−3.33 ± 3.22−3.50 ± 3.15−4.98 ± 2.70−5.20 ± 3.08T8−6.13 ± 2.17−4.78 ± 3.60−6.73 ± 3.44−5.13 ± 2.89 −4.99 ± 4.27−4.64 ± 3.62−6.56 ± 3.47−6.33 ± 3.11N2Central−9.77 ± 5.48−7.92 ± 4.91−10.29 ± 5.01−8.88 ± 3.98−9.04 ± 5.43−7.25 ± 5.52−10.02 ± 4.43−8.11 ± 6.16Left−7.13 ± 3.60−6.05 ± 3.44−7.44 ± 3.49−6.69 ± 2.91−6.81 ± 3.69−5.55 ± 3.88−7.13 ± 3.55−5.98 ± 4.28Right−6.70 ± 3.05−4.59 ± 3.25−6.42 ± 3.54−5.41 ± 2.56−5.88 ± 3.05−4.70 ± 3.19−6.11 ± 3.24−4.82 ± 3.85P2Central14.79 ± 8.0211.74 ± 7.0314.86 ± 7.6912.43 ± 7.0013.19 ± 7.7411.26 ± 8.2313.83 ± 6.7512.03 ± 6.31Left8.40 ± 3.897.18 ± 3.669.19 ± 4.74 7.74 ± 3.597.36 ± 4.806.63 ± 5.167.90 ± 3.646.84 ± 3.67Right8.80 ± 3.806.78 ± 3.038.68 ± 4.657.59 ± 3.687.91 ± 3.706.90 ± 3.988.28 ± 4.277.76 ± 4.17Right handN1T7−8.18 ± 4.79−5.98 ± 3.39−6.38 ± 4.67−5.12 ± 3.62−7.34 ± 4.70−5.88 ± 3.73−7.43 ± 3.30−6.38 ± 3.21T8−4.92 ± 4.43−3.96 ± 3.24−3.25 ± 3.29−2.98 ± 2.35−4.56 ± 4.61−3.03 ± 2.39−4.37 ± 3.53−4.15 ± 2.62N2Central−10.11 ± 3.83−7.44 ± 4.52−11.05 ± 4.87−6.69 ± 4.23−9.99 ± 5.06−6.40 ± 4.27−9.57 3.97−8.94 ± 5.40Left−7.74 ± 2.70−5.67 ± 2.77−8.62 ± 3.65−5.71 ± 3.43−7.77 ± 3.80−5.09 ± 2.64−6.79 ± 3.29−6.42 ± 3.78Right−6.58 ± 3.37− 5.12 ± 3.34−6.76 ± 3.00−4.80 ± 3.26−6.52 ± 3.59−4.24 ± 2.72−5.82 ± 2.74−6.00 ± 3.99 P2Central14.30 ± 6.5611.62 ± 5.9314.52 ± 7.1410.90 ± 5.4914.04 ± 7.7910.74 ± 7.1311.37 ± 6.3810.75 ± 5.73Left8.47 ± 3.876.95 ± 3.038.90 ± 4.147.00 ± 2.918.65 ± 4.796.64 ± 3.376.21 ± 3.456.62 ± 3.22Right8.52 ± 3.836.73 ± 2.758.29 ± 4.176.41 ± 3.328.75 ± 4.296.90 ± 3.766.84 ± 4.446.67 ± 4.04The mean amplitudes of the LEP components before and after stimulation in all four conditions. (mean ± standard deviation)
Discussion
The main finding of our study is that all theta burst paradigms over the SI were able to diminish the amplitude of the N2 component of LEPs significantly when compared to sham stimulation. Surprisingly, the imTBS condition, that is suggested to be used as a placebo condition, when it applied over the M1 (Huang et al. 2005), also caused a strong amplitude decrease. The N1 and P2 components and the subjective pain rating scores were not significantly influenced by any type of TBS.
Recent studies using the theta burst paradigm have concentrated on the effects of continuous (cTBS) and intermittent theta burst stimulation (iTBS) (Franca et al. 2006; Andoh et al. 2007; Ishikawa et al. 2007; Koch et al. 2007; Mochizuki et al. 2007). None of them investigated the intermediate (imTBS) pattern since Huang and et al. published that it has no effect over the M1 as revealed by MEPs and could thus be used as a sham condition (Huang et al. 2005).
According to our knowledge, only five studies applied TBS over non-motor cortical areas. First Franca et al. (2006) used the theta burst pattern of rTMS over the visual cortex. They found that cTBS increased phosphene thresholds whilst iTBS was found to be ineffective. In another study, both cTBS and iTBS over the left dorsal premotor cortex decreased the transcallosal inhibition revealed by pairs of transcranial magnetic stimuli (Koch et al. 2007). In a recent study, Wernicke’s area was stimulated with iTBS while the reaction time of auditory word detection was measured (Andoh et al. 2007). In this work, iTBS facilitated the detection of foreign words when compared with native words.
Concerning the SI, a shorter form of the iTBS (300 pulses) over the left SI resulted in a significant oxy-hemoglobin decrease at the contralateral SI and M1, detected by near infrared spectroscopy (Mochizuki et al. 2007). In another study cTBS of the SI resulted in a temporary decrease (13 min), whereas stimulation of the M1 caused a long-lasting increase (up to 53 min) of the amplitudes of cortical components of the median nerve SEPs (Ishikawa et al. 2007). In summary, these results suggest that cTBS has an inhibitory effect on non-motor areas; whereas the effect of iTBS is more facilitatory, but clearer results have still to emerge. Our results are the first demonstrating that all three TBS paradigms, but not by sham stimulation over the SI resulted in similar after-effects regarding the amplitude of the N2 component of LEPs evoked by the laser stimulation of the contralateral hand.
The N2 component (peaking around 160–220 ms), is generated bilaterally in the operculoinsular region and in the anterior cingulate cortex (ACC) (Garcia-Larrea et al. 2003) and reflects sensory, discriminatory processes (Garcia-Larrea et al. 1997; Iannetti et al. 2005); whilst the P2 component (peaking around 300–360 ms) arose mostly from the ACC and represents attentional, cognitive and affective factors of pain perception and processing (Treede 2003). However, other studies did not find significantly different brain sources for N2 and P2, revealing both parasylvian and ACC contributions for the N2–P2 components (Raij et al. 2003; Ohara et al. 2004). Thus, LEP changes in N2 or P2 component might result from changes in either sensory-discriminative or cognitive aspects of pain. Studies using subdural recordings (Kanda et al. 2000; Ohara et al. 2004) or MEG (Kanda et al. 2000) demonstrated that the LEP components can be recorded over the SI and SII simultaneously, and the N2 peak may indicate the arrival of input originating from nociceptors.
With regard to the N1 component which is an early LEP potential (peaking around 140–170 ms) and reflecting the early sensory-discriminative processing of pain perception (Iannetti et al. 2005), we did not find any significant change in amplitude. According to scalp topography (maximum near T3 and T4), the N1 is generated near to the SII in the fronto-parietal operculum (Treede et al. 2003). The participation of operculoinsular cortex in coding the pain intensity was recently suggested by using LEP measurements (Ianetti et al. 2005). However, another study (Gracia-Larrea et al. 1997) did not find any significant correlation between the amplitude of the N1 component and subjective pain rating.
The fact that we found a decrement of both the N2 and P2 amplitudes in the sham condition as well, should be discussed. This phenomenon is known as habituation and has been described in previous LEP studies (Spiegel et al. 2000; Tamura et al. 2004a). Still, the effects of real TBS conditions on N2 amplitudes were greater than that of the sham condition and it was significant above the stimulated left, the neighboring central and the contralateral area as well when the right hand was laser stimulated. Attention can also directly influence the N2–P2 components of LEPs as well as subjective pain rating as it was suggested by previous experimental results (Gracia-Larrea et al. 1997; Ohara et al. 2006). In our experiment the subjects were asked to pay attention to each laser stimuli and since the TBS and sham condition were applied in a randomized order and the subjects were blinded as to the type of magnetic stimulation, the significant difference between sham and the other three TBS on the N2 amplitude is more than simply habituation or the effect of the different attentional states.
The possible origin of the N2 component is mainly the bilateral operculoinsular region and the ACC (Garcia-Larrea et al. 2003). Therefore, when we inhibited the left SI, the activity of the pain related cortical network decreased due to the widespread cortical connections between SI and other cortical areas. However, the involvement of the contralateral SI and bilateral SII—parasylvian region in the N2 component generation was also reported (Kakigi et al. 2005; Kanda et al. 2000; Ohara et al. 2004). According to this, it is also possible that the inhibition of the SI itself may cause direct effects on LEP components. In this study we found no significant difference between the stimulation conditions with regard to subjective pain perception (Fig. 2), however, the subjective pain rating decreased during the experimental sessions after every type of TBS including the sham condition. It is important to mention that the placebo effect is high in almost every pain perception study, regardless of the paradigm used (Khedr et al. 2005; Lefaucheur et al. 2004). The explanation of this discrepancy between the electrophysiological and psychophysical changes is rather speculative, because the relationship between the anatomical origin of the N2–P2 components and their psychophysical correlates controversially discussed in the literature. In a PET study different cortical activations for pain threshold, intensity and unpleasantness have been found (Tölle et al. 1999). One possibility is that modulation the excitability of the left SI by TBS activated some elements of the pain related network that caused a decrease in the N2 amplitude. However, this stimulation intensity or duration was not strong or adequate enough to modify the subjective pain rating.
In our previous study (Antal et al. 2007), we found that cathodal tDCS over the SI, similarly to the present findings, significantly decreased the N2 component of LEPs. In contrast to the present findings the subjective pain perception in healthy subjects was also diminished. TDCS is a method that modifies the resting membrane potentials of cortical neurons intracortically (Nitsche and Paulus 2000, 2001). Cathodal stimulation decreases, whilst anodal stimulation increases cortical excitability (Nitsche and Paulus 2000, 2001). However, in that study we have used a large electrode size (5 × 7 cm) in order to optimize stimulation’s parameters (Nitsche and Paulus 2000) and therefore we might have covered a large part of the SI. It is possible that we have stimulated one part of the somatosensory association cortex (BA 5/7) that is posterior to the SI. Activation of human BA5/7 has also been linked to pain perception (Apkarian et al. 1999; Forss et al. 2005). BA 5/7 is anatomically connected to other nociceptive brain areas such as the ACC, insula, thalamus and primary motor cortices (Friedman et al. 1986). In order to increase the focality of the transcranial stimulation in the present study we used a neuronavigation system to determine the hand area over the SI.
Many of the previous pain-related studies stimulated the M1. The effects of low and high frequency rTMS of M1 on experimentally induced acute pain perception seem to depend on the type of noxious stimulation. C-fiber mediated acute pain as induced by intradermal capsaicin administration could be attenuated by 1 Hz rTMS over the M1 (Tamura et al. 2004b), whereas it increased Aδ-fiber mediated laser-induced pain in another study (Tamura et al. 2004a). Similarly, controversial effects were observed after 20 Hz rTMS (Johnson et al. 2006; Summers et al. 2004). In contrast 10 Hz rTMS over M1 increased electrically induced Aδ-fiber mediated pain threshold (Yoo et al. 2006), but others found that 10 Hz rTMS has only an effect on the unpleasantness of the pain without any effect on pain threshold (Mylius et al. 2007). In case of chronic pain the high frequency rTMS seems to more effective than the application of low frequencies (Leo and Latif 2007).
The neuronal mechanism of the theta burst paradigm is highly speculative. The results of the experiments with single trains of TBS suggest that in the human M1 TBS produces a mixture of facilitatory and inhibitory effects on synaptic transmission (Huang and Rothwell 2004). Huang and Rothwell proposed, that facilitation develops faster than inhibition, thus in case of the inhibitory cTBS, several seconds after an initial facilitation the inhibition overrides this effect. ITBS uses only the early excitatory effect in the initial 2 s and after this the stimulation is interrupted for 8 s. Most likely, the underlying mechanisms will involve many of the basic elementary mechanisms described previously in the LTP/LTD literature (Paulus 2005). Recently Huang et al. (2007) have demonstrated that the after-effects produced by both iTBS or cTBS are NMDA-receptor dependent and hence they are more likely to involve plasticity-like changes at the synapse in the M1. More recently, it was found that the excitatory effects of iTBS were reversed after NMDA receptor activation by D-cycloserin (Teo et al. 2007). This is in contrast with the findings of tDCS induced LTP where the excitatory effects are prolonged by D-cycloserin (Nitsche et al. 2004). However, it is important to note that these observations were done on the M1. It is possible that the different effectiveness observed between TBS protocols on motor and sensory cortices could be due to differences in the physiological and functional states of the stimulated cortex.
In summary, in our study we found a significant decrease of the N2 component of the LEPs after cTBS, iTBS, and imTBS when compared to sham stimulation over the SI. In addition we found, that the subjective pain perception did not show significant differences among the stimulation conditions suggesting that this method is probably not the most effective in decreasing subjective pain perception. Further findings show that imTBS resulted in more impressive modification of the LEPs, than were found in previous TMS-studies over the M1 using MEP measures (Huang et al. 2005), implying that it should not be used in further studies as ‘sham’ condition, at least with regard to LEPs and when it is applied over the SI. However these results are not directly comparable with the effects of TBS on M1, thus further studies are needed to clarify the effects of imTBS on different cortical areas. Future studies should also clarify the effectiveness of the different TBS paradigms applied over the M1 and non-motor cortical areas, such as the SII on acute pain perception and in chronic pain.

theta burst transcranial magnetic stimulation


The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis

Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested.
Introduction
During maturation, plants pass through several developmentally distinct growth phases in which the shoot gradually gains reproductive competence (Poethig 1990). After the transition from embryonic to postembryonic growth, plants undergo at least two further phase transitions, the vegetative as well as the reproductive phase change. During vegetative growth, rosette leaves are initiated at the flanks of the shoot apical meristem (SAM) with a certain frequency, referred to as plastochron (Erickson and Michelini 1957). After going through the reproductive phase transition, also known as the floral transition, the SAM starts to initiate floral buds instead of leaves. In Arabidopsis, as in many other plants showing day length dependent flowering, the floral transition is preceded by a transition from juvenile to adult growth. This switch, known as the vegetative phase change, is physiologically defined as achieving competence to respond to photoperiodic induction of flowering (Poethig 1990).
The transition from juvenile to adult growth is gradual and rather subtle but generally can be followed by several morphological markers. In Arabidopsis, for example, leaves produced in the juvenile phase have long petioles, are small, round and lack abaxial trichomes. In contrast, short petioles, elliptical anatomy and the development of trichomes on the abaxial side represent adult traits (Telfer et al. 1997).
Regulation of these developmental transitions is largely dependent on (changes in) environmental cues such as day length, light intensity and temperature, as well as on endogenous factors such as the plant hormone gibberellin (Telfer et al. 1997). Whereas the molecular genetic mechanisms underlying the floral transition are already worked out in increasing detail (Komeda 2004), it has only been recently that we begin to understand the molecular genetic basis of the vegetative phase change. Most genes suggested to play a role in promoting the latter phase change have been identified by the analysis of mutants showing a precocious onset of adult traits and intriguingly, link vegetative phase change to RNA silencing pathways. These genes include the Arabidopsis ortholog of exportin 5/MSN5, HASTY (HST; Telfer and Poethig 1998; Bollman et al. 2003), the zinc-finger-domain protein encoding locus SERRATE (SE; Clarke et al. 1999) and ZIPPY (ZIP), an AGO-family member (Hunter et al. 2003). More recently, screens for mutations with zip-like phenotypes resulted in alleles of SUPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT POLYMERASE6 (RDR6), both genes required for posttranscriptional gene silencing (PTGS) and acting in the same pathways as ZIP and HST (Peragine et al. 2004). Furthermore, a precocious vegetative phase change has also been found in dicer-like 4 (dcl4) mutants (Gasciolli et al. 2005; Xie et al. 2005; Yoshikawa et al. 2005).
One explanation for the observed effects could be that target genes of this silencing pathway play a positive role on the vegetative phase change and their down-regulation consequently promotes juvenility. Hence, mutations in genes involved in this silencing pathway, as the ones described above, cause an accelerated vegetative development.
In line with this idea, members of the plant specific SBP-box gene transcription factor family have been implicated in promoting vegetative and floral phase transitions. In particular, overexpression of the Arabidopsis SBP-box gene SPL3 leads to early flowering and a significant earlier appearance of abaxial trichomes on the rosette leaves (Cardon et al. 1997; Wu and Poethig 2006). Interestingly, together with 10 of 16 other family members, SPL3 expression is post-transcriptionally controlled by miR156 and probably also by the very closely related miR157 (Rhoades et al. 2002; Schwab et al. 2005; Wu and Poethig 2006; Gandikota et al. 2007). Consistent with its role of down-regulating SPL3 and related SPL target-genes, constitutive overexpression of miR156 encoding loci has been shown to cause the production of a significantly larger number of leaves with juvenile characteristics and a delay in flowering (Schwab et al. 2005; Wu and Poethig 2006).
Although the available data clearly point to a regulatory role for the miRNA regulated SPL genes in the temporal development of the Arabidopsis shoot, the contribution of the single genes to the described phenotypes remains to be determined. Therefore, we identified and isolated mutant alleles for single SPL genes. In comparison to other miR156 targeted SPL genes, available expression data (AtGenExpress; Schmid et al. 2005) show SPL9 and SPL15 to be already quite active in the vegetative shoot apex. Accordingly, their mutant phenotypes were found to affect vegetative development. Here we report the mutant analysis of SPL9 and SPL15, two likely paralogous members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor family (Cardon et al. 1999), and discuss their redundant regulatory role on the vegetative phase change and the temporal initiation of rosette leaves.
Materials and methods
Plant material and plant growth conditions
All of the genetic stocks described in the paper were in Columbia background. The T-DNA insertion lines SALK_006573 (spl9-2), SALK_074426 (spl15-1) and SALK_138712 (spl15-2) were obtained at the Nottingham Arabidopsis Stock Centre (NASC). The T-DNA insertion lines GABI-Kat 544F04 (spl9-3) and WiscDsLox 457 (spl15-3) were obtained from GABI-Kat and the Arabidopsis Biological Research Centre (ABRC), respectively. Insertion mutant information for NASC- or ARBC-lines was obtained from the SIGNAL website at http://signal.salk.edu. Plants homozygous for the T-DNA insertions were identified by PCR using T-DNA left border- and gene-specific primers. T-DNA specific left border primers for SALK, GABI-Kat and WiscDsLox T-DNAs were 5′-GCGTGGACCGCTTGCTGCAACT-3′, 5′-ATATTGACCATCATACTCATTGC-3′ and 5′-TGGCAGGATATATTGTGGTGTAAACA-3′, respectively. In combination with the respective left border primer we used the following gene-specific primers: 5′-GCTATGGCTTAAGCCTTAAGTTAAAAGG-3′ for SALK_006573, 5′-CGTAGCTGTCGTGGACTAGTGTCAATC-3′ for SALK_074426 and SALK_138712, 5′-AACCTCTGTTCGATACCAGCCACAG-3′ for GABI-Kat 544F04 and 5′-AGCCATTGTAACCTTATCGGAGAATGAG-3′ for WiscDsLox 457. The stable En-1 insertion mutant 5ABA33-H1 (spl9-1) was obtained from the ZIGIA-population (Unte 2001). Plants homozygous for a four base pair insertion in the first exon of SPL9 caused by the excision of the En-1 transposon were backcrossed with wild type twice to obtain plants exclusively containing the four base-pair insertion without any further transposon contamination. In order to identify plants containing the mutation we used the following primer combination: 5′-AGTAAGAGGAAACCACCATGGAGATGG-3′ (forward) and 5′-AACCTTCCACTTGGCACCTTGGTATA-3′ (reverse, recognises the insertion).
All plants were grown in plastic trays or pots filled with ready-to-use commercial, pre-fertilized soil mixture (Type ED73, Werkverband eV, Sinntal-Jossa, Germany). For stratification, seeds were kept on moist paper at 4°C in the dark for 4–5 days before transferring to soil (i.e. “sowing”) in growth chambers at 22°C, 50% relative humidity. Germination and cultivation of the plants in long-day conditions (16 h light, 8 h dark) were either under approx. 70 μE/cm2/s (LD1) or 175 μE/cm2/s (LD2) light provided by fluorescent tubes (L58W/840 and L58W/25 Osram, Munich, Germany). Plants in short-day (SD) conditions (8 h light, 16 h dark) were cultivated under approx. 450 μE/cm2/s light. To determine sensitivity to photoperiodic induction of flowering, stratified seeds were germinated in a modified SD with a 9 h light period. The developing plants were kept in these conditions for 21 days before they were transferred to similar growth chambers with continuous light provided by Osram HQIT 400 W lamps. Batches of plants were returned to the modified SD conditions after 1, 3 and 5 days.
Phenotypic analysis
Flowering time was measured as the time between sowing and anthesis (opening of first flower). Bolting time was recorded when the main inflorescence had reached a height of 0.5 cm. Inflorescence height was measured between the rosette and the first flower of the main inflorescence of plants with their first siliques fully ripened. In order to count the number of site shoots, all site shoots longer than 0.5 cm were scored. Abaxial trichomes were scored using a Leica MZFLIII stereomicroscope (Wetzlar, Germany). An estimation of the rosette leaf initiation rate (L/D day−1) was obtained by dividing the number (L) of rosette leaves having reached at least 0.5 cm in length through the number of days (D) between sowing and determination. Note that this value reflects but does not equalize (average) plastochron as it should be corrected for the true start of initiation of the first leaf as well as the time needed for the first adult leaf to reach a size of 0.5 cm.
Histological analysis
Apical regions were isolated from plants grown in SD for 41 days by trimming with a razor blade. The tissue was fixed in 4% formaldehyde/0.1 M PO4, pH 7.0 for 48 h and embedded in paraffin using a Leica ASP300 tissue processor (Wetzlar, Germany). Embedded apices were cut into 8 μm thin cross sections using a Jung Autocut 2055 and photographed using a Zeiss Axiophot microscope (Göttingen, Germany) equipped with a KY-F5U 28CCD camera (JVC, Yokohama, Japan). The first cross section in which the apex was visible plus two successive sections were used to determine the diameter of the apical region. From these three measured values the average was taken for comparison. In addition, cross sectional area and circularity factor (=4π A P−2, A is area, P is perimeter) of the leaf primordia, outlined by hand on the photographs, were determined as well. The measurements were performed with help of the program ImageJ 1.35s (Wayne Rasband, National Institutes of Health, USA).
GA3 treatments
Col-0, the spl9 spl15 double mutant and the 35S::MIR156b overexpressor were grown in LD1 conditions. Immediately after germination, half of the plants were treated by spraying 100 μM GA3, 0.02% Tween 20 and this was repeated twice per week until they started flowering. The other half of the plants was similarly treated with 0.02% Tween 20.
Phylogenetic comparison
Multiple alignments of amino acid sequences were generated by the program ClustalW of the MacVector 7.2.2 software package (Accelrys Ltd., Cambridge, UK) using the BLOSUM 30 matrix with an open gap penalty of 10 and an extend gap penalty of 0.05. Only the SBP-domain was used for the phylogenetic reconstruction. The tree was constructed using the neighbour-joining algorithm of the MacVector 7.2.2 software package.
Quantitative real-time PCR analysis
To perform quantitative RT-PCR (using the iQ5 real-time PCR detection system, Bio-Rad, Munich, Germany) apical regions were collected (roots and as much of the leaves as possible were removed using tweezers) of plants cultivated 5, 9, 13, 27 and 32 days after sowing in LD1 conditions. Total RNA was extracted using the RNeasy plant mini kit (Qiagen, Hilden, Germany), including an on-column DNase digestion. First-strand cDNA was synthesized using SuperScript III RNase H reverse transcriptase (Invitrogen) starting with 2 μg of total RNA primed with an oligo(T)12–18 primer (Gibco BRL, Karlsruhe, Germany). SPL9-specific primers, 5′-AGAACATTGGATACAACAGTGATGAGG-3′ (forward) and 5′-GTTTGAGTCGCCAATTCCCTTGTAGC-3′ (reverse) as well as SPL15-specific primers, 5′-TTGGGAGATCCTACTGCGTGGTCAACC-3′ (forward) and 5′-AGCCATTGTAACCTTATCGGAGAATGAG-3′ (reverse), were designed to generate a PCR product of 171 and 300 bp, respectively. Based on the analysis of Czechowski et al. (2005) PP2A expression was used as reference for transcript normalization with the primer pair 5′-TAACGTGGCCAAAATGATGC-3′ (forward) and 5′-GTTCTCCACAACCGCTTGGT-3′ (reverse). The PCR efficiencies for SPL9, SPL15 and PP2A primers were determined to be 96, 95.5 and 96%, respectively. Quantifications, in triplicate, were performed using the Brilliant SYBRGreen QPCR kit (Stratagene, La Jolla, CA, USA), according to the manufacturer’s protocol, in a final volume of 25 μl. PCR was carried out in 250 μl optical reaction vials (Stratagene) heated for 10 min at 95°C to hot-start the Taq polymerase, followed by 40 cycles of denaturation (30 s at 95°C), annealing (30 s at 58°C) and extension (30 s at 72°C).
Semi-quantitative RT-PCR analysis
Total RNA was extracted from seedlings of the Col-0 wild-type, mutant- and transgenic lines using the RNeasy plant mini kits (Qiagen). RT-PCR with equal amounts of RNA was performed using the one-step RT-PCR kit (Qiagen). SPL9 knockout lines were identified using the following primer pair: 5′-GGTCGGGTCAGTCGGGTCAGATACC-3′ (forward) and 5′-ACTGGCCGCCTCATCACTCTTGTATCC-3′ (reverse). SPL9 mRNA is expected to yield a 415 bp fragment whereas genomic SPL9 DNA is expected to yield a 1,138 bp fragment. SPL15 knockout lines were identified using the following primer pair: 5′-AGAAGCAAGAACCGGGTCAATACC-3′ (forward) and 5′-AGCCATTGTAACCTTATCGGAGAATGAG-3′ (reverse). SPL15 mRNA is expected to yield a 666 bp fragment whereas genomic SPL15 DNA is expected to yield a 1,004 bp fragment. RT-PCR of the loading control (RAN3; At5g55190) was performed with the primer pair 5′-ACCAGCAAACCGTGGATTACCCTAGC-3′ (forward) and 5′-ATTCCACAAAGTGAAGATTAGCGTCC-3′ (reverse) to yield a fragment of 531 bp when derived from RAN3 mRNA (genomic RAN3 is expected to yield a 1,314 bp fragment).
Statistics
Graphical representations of numerical data were generated with the Microsoft Excel program (Microsoft Germany, Munich) and statistical tests were performed using the Student’s t-Test within this program. P-values lower than 0.05 were considered to be statistically relevant and the data involved to represent significant differences.
Remaining techniques and methods
Standard molecular biology techniques were performed as described by Sambrook et al. (1989). Graphical plots and digital photographic images were cropped and assembled using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
Results
Molecular characterization of the Arabidopsis SBP-box genes SPL9 and SPL15
Mutant alleles for the Arabidopsis SBP-box genes SPL9 (At2g42200) and SPL15 (At3g57920) were obtained from screening publicly available electronic databases and seed stock centres for transposon or T-DNA tagged SPL genes. For SPL9, we identified three insertion alleles designated as spl9-1 to -3 and confirmed the nature and position of their mutations (see “Materials and methods”; Fig. 1a). The first allele, spl9-1, was identified in the En-transposon mutagenised ZIGIA population (Baumann et al. 1998; Unte 2001) and most likely resulted from the excision of an inserted En-1 transposon leaving behind a 4-bp insertion footprint in the first exon. The result of this is a frame shift in the coding sequence and the generation of a stop-codon 86 base pairs after the insertion site. Both spl9-2 and spl9-3 represent T-DNA insertion mutant alleles identified within, respectively, the SALK collection (Alonso et al. 2003) and the GABI-Kat collection (Li et al. 2007).
Fig. 1Molecular characterization of SPL9 and SPL15. (a) Schematic representation of the genomic loci of SPL9 and SPL15. The positions of the mutations identified are indicated by open triangles, numbered according to the respective alleles. Boxes represent exons. The SBP-box sequences are depicted in black, the remaining coding sequences in grey and the untranslated 5′ and 3′ regions are left blank. (b) Changes in transcript levels of SPL9 and SPL15 in the shoot apical region during plant development in LD1 as determined with qRT-PCR and normalized against PP2A. For comparison, relative transcript levels were arbitrarily set to one for SPL9 5 days after sowing. Error bars indicate standard deviation. (c) Absence of SPL9 and SPL15 transcripts in seedlings of the respective mutants as validated by RT-PCR. Presence of the respective transcripts in Col-0 wild type seedlings is shown for comparison and the amplification of RAN3 transcript as quality control and reference for quantification. Fragment lengths are indicated on the left in base pairs (bp)
Also three independent T-DNA insertion lines for SPL15 could be obtained and confirmed (see “Materials and methods”; Fig. 1a). Two alleles designated as spl15-1 and spl15-2 were identified within the SALK collection and one, spl15-3, within the WiscDsLox T-DNA collection.
According to data available from the AtGenExpress micro-array database (Schmid et al. 2005), both SPL9 and SPL15 transcript levels increase during development and are preferentially found in the shoot apical region and in young flowers. We confirmed this temporal expression pattern with the help of qRT-PCR (Fig. 1b). In LD1 growing conditions (see “Materials and methods”), SPL9 and SPL15 transcript levels remain comparable during the first 2–3 weeks. Thereafter, the expression level of SPL9 starts to increase followed by that of SPL15. Around 32 days after sowing (DAS), at about the time Col-0 plants have undergone their reproductive phase transition, SPL9 transcript levels have become approximately two and a half times higher in comparison to SPL15 and six times in comparison to day 5. Arabidopsis lines carrying as a transgene a genomic fragment encompassing the locus for SPL15 and with a GUS reporter gene inserted downstream of the ATG start codon, confirmed the predominantly apical expression of SPL15 (Supplementary Fig. 1).
RT-PCR performed on mRNA isolated from whole seedling plants homozygous for any of the three SPL9 or SPL15 mutant alleles (see “Materials and methods”) did not result in the detection of RNA derived of the respective genes (Fig. 1c). This strongly suggests that all mutant alleles isolated represent functional null-alleles. Accordingly, plants homozygous for any of the three spl9 mutant alleles showed highly identical phenotypes, as did all three homozygous spl15 mutants (see phenotypic analysis below; Supplementary Fig. 2). Allelic tests confirmed that the observed phenotypes are indeed due to mutation in either SPL9 or SPL15, respectively (data not shown).
With over 75% of their amino acid residues identical, SPL9 and SPL15 show high similarity on the level of their proteins. Also a phylogenetic comparison based on the SBP-box of all 17 SPL genes in Arabidopsis revealed SPL9 and SPL15 as most closely related and most likely forming a pair of paralogous genes (Fig. 2). Based on this close relationship some degree of functional redundancy could be expected and, therefore, we created double mutant lines to uncover such redundancy. To ascertain that phenotypic changes in the mutant plants are solely due to the loss-of-function of SPL9 and SPL15 we generated two different homozygous double mutant lines with the allelic combinations spl9-1 spl15-1 and spl9-2 spl15-2, respectively. Both lines exhibit the same phenotype as described in the next section. For further detailed analysis the spl9-1 spl15-1 line was chosen and in the following referred to as spl9 spl15 for simplicity.
Fig. 2Phylogenetic relationship of the Arabidopsis SBP-box genes as based on the conserved SBP-domain. The orthologous sequence of Chlamydomonas CRR1 has been used as outgroup. The likely paralogous pair SPL9 and SPL15 is boxed in grey. MiR156/157 targeted SPL genes are marked with an asterisk. Only bootstrap values over 50% are shown
Phenotypic analysis of spl9 and spl15 mutants
For a phenotypic analysis, we compared spl9 and spl15 single mutants, spl9spl15 double mutants to Col-0 wild type as well as to an 35S::MIR156b transgenic line (kindly provided by D. Weigel and R. Schwab).
An interesting aspect of the MIR156b over-expressing plants, as already noticed by Schwab and co-workers (2005) is an increased rate of rosette leaf initiation which, in combination with a modest delayed flowering, results in the obvious denser rosettes of fully developed plants (Fig. 3a). In addition, advanced 35S::MIR156b plants became very bushy (Fig. 3c). We found these phenotypic aspects also displayed by the spl9 spl15 double mutant, albeit less pronounced (Fig. 3a, b). To quantify the contribution of SPL9 and SPL15 to these phenomena, we compared the number of rosette and cauline leaves of the respective single and double mutants and of the MIR156b overexpressor to wild type (Table 1). Whereas in LD2 growing conditions, the 35S::MIR156b line produced ca. eleven more rosette leaves in comparison to wild type, the single mutant lines produced, on average, only 1–2 rosette leaves more. Again, with ca. six extra rosette leaves, the spl9spl15 double mutant differed more from wild type than the single mutants and showed a stronger tendency towards the phenotype of the MIR156b overexpressor. The number of cauline leaves remained very comparable among all mutants and wild type, although some reduction may be observed particularly in the spl9 mutants.
Fig. 3Phenotypic analysis of spl9 and spl15 mutants. (a) Flowering spl9, spl15 and spl9spl15 double mutant plants shown next to Col-0 wild type and the MIR156b overexpressor. Plants shown next to each other are of the same age and grown in parallel under LD2 conditions. (b, c) Col-0 wild type, spl9spl15 double mutant (b) and MIR156b overexpressor (c) at a more advanced stage of development in comparison to the plants shown in aTable 1Phenotypic evaluation of spl9 and spl15 mutant alleles in comparison to Col wt and a 35S::MIR156b transgene under LD conditionsRosette leavesCauline leavesBolting (DAS)Anthesis (DAS)Juvenile leavesaInfloresc. heightb (cm)MeanSDMeanSDMeanSDMeanSDMeanSDMeanSDCol-0 wt13.11.13.90.516.31.220.91.55.51.212.11.3spl9-114.31.13.4c0.715.91.419.6c1.78.30.88.01.0spl9-215.61.23.5c0.516.91.220.81.49.20.98.20.8spl9-315.71.33.3c0.617.5c1.521.31.59.60.78.60.8spl15-115.61.13.3c0.416.60.820.91.07.10.710.31.2spl15-214.91.03.50.517.11.920.91.97.30.811.4c1.1spl15-316.11.03.70.717.2c0.821.81.17.60.711.1c1.3spl9-1 spl15-119.5d1.43.40.918.5d1.422.3c1.810.9d0.86.9d1.4spl9-2 spl15-218.9d1.33.3c0.619.0d1.522.8c1.810.8d0.46.8d0.835S::MIR156b24.4e2.43.2c0.819.31.822.4c2.114.8e1.12.3e0.716 plants per genotype were used for determinationDAS, days after sowing; SD, standard deviationValues significantly different from Col-0 wt at 0.001 confidence level are shown in italicsaNumber of rosette leaves formed before the first leaf with abaxial trichomesbMeasured from rosette to first flowercValues significantly different from Col-0 wt at 0.05 but not at 0.001 confidence leveldValues significantly different from single mutants at 0.05 confidence leveleValues significantly different from double and single mutants at 0.05 confidence level
Also with respect to the development of side shoots, the spl9spl15 double mutant differed more from wild type than the single mutants. In fact, spl9-1 and spl15-1 single mutants were found not to differ significantly from Col-0 plants that had formed, on average, 0.9 ± 0.6 side shoots of at least 0.5 cm in length by the time that the first siliques ripened. With an average of 2.1 ± 1.1 side shoots, the spl9spl15 double mutant did significantly differ from wild type as did the 35S::MIR156b transgenic line with, on average, 4.1 ± 0.8 side shoots. Taken together, the phenotypic data of the spl9spl15 double mutant clearly suggests a redundant function of SPL9 and SPL15 in shoot development and in the maintenance of apical dominance.
MiR156 is assumed to target, besides SPL9 and SPL15, exclusively other SPL genes (Rhoades et al. 2002). These too were shown to be down regulated in MIR156b over-expressing plants (Schwab et al. 2005). As in comparison to the spl9 spl15 double mutant the MIR156b over-expressor displays an even more severe aberrant phenotype, it can also be deduced that in addition to SPL9 and SPL15, other miR156-controlled SPL genes act redundantly to control shoot development and apical dominance.
In addition to the number of leaves formed before the appearance of the first flowers, we also determined for the same plants the time they needed to bolt as well as to anthesis (Table 1). On average, the spl9 and spl15 single mutants behaved similar to wild type but, as expected based on the data of Schwab et al. (2005), the 35S::MIR156b line bolted and flowered somewhat later. The spl9 spl15 double mutants showed an intermediate behaviour. Whereas for the single mutants the few more leaves formed may be accounted for by the slight delay in the transition to flowering, this delay is unlikely to explain the increased rosette leaf number of the spl9 spl15 double mutant. In line with the observation of Schwab and co-workers (2005) who reported a leaf-initiation rate per day in SD of 2.2 vs. 1.4 for the MIR156b overexpressor and the wild type, respectively, this is probably best explained by assuming a shortened plastochron during vegetative growth.
To uncover a possible cause or consequence for this increased rate of leaf initiation, we microscopically examined cross sections of the vegetative shoot apex to determine size and phyllotaxy of the spl9spl5 double mutant and the MIR156b overexpressor and compared these to wild type. To this purpose, plants were grown for 41 days in SD conditions, whereafter the number of rosette leaves having reached at least 0.5 cm in length were recorded and their apices dissected, fixed and embedded in paraffin (see “Materials and methods”). At this age, Col-0 plants were found to have formed on average 24.5 leaves of 0.5 cm or more, the double mutant 33.8 and the MIR156b overexpressor plants already 43.4 (Fig. 4a). As the plants were of the same age, these differences most likely reflect differences in plastochron. Alternatively, one may assume large temporal differences per genotype concerning initiation of the first leaf and/or development of the last leaf recorded to have reached 0.5 cm in length. However, we obtained no indications for such discrepancies and noted increasing differences in rosette density during the entire vegetative growth phase of wild type and mutants. From these data, a relative 1.8-fold (43.4/24.5) increase in leaf initiation rate of the 35S::MIR156b transgenics over wild type can be deduced, a value that quite well matches the observation of Schwab et al. (2005). Leaf initiation rate of the double mutant seem to be increased by a factor of 1.4 (33.8/24.5) in comparison to wild type.
Fig. 4Leaf formation of spl9spl15 double mutant in comparison to wild type and MIR156b overexpressor. (a) Determination of the average number of rosette leaves of at least 5 mm in length formed by the primary shoot and (b) of the average diameter of the primary shoot apex of spl9spl15 double mutants, Col-0 wild type and MIR156b overexpressor plants after having grown for 41 days in SD. (c, d) Average circularity (c) and cross sectional area (d) of leaf primordia as determined from cross section through primary shoot as shown in e–g. Values represent averages of 10 subsequent primordia as indicted in different shades of grey according to the legend shown in d. (e–g) Cross sections through primary shoot apices of a Col-0 wild type (e), a spl9spl15 double mutant (f) and a MIR156b overexpressor plant (g) after having grown for 41 days in SD. Error bars in a–d indicate standard deviation (n = 6). Successive leaf primordia in e–g are sequentially numbered starting with the youngest (P1). Marginal meristem on one side of leaf number 17 (L17; counted from the centre outwards) is encircled in e and g. Scale bar in e–g represents 200 μm
After sectioning the paraffin embedded material, a small but not significant difference in average SAM-diameter of the spl9spl15 double mutant and Col-0 wild type could be observed (Fig. 4b, e–f). However, with an average diameter of 104 μm, the MIR156b overexpressor showed also a slight but yet significant (P < 0.05) decrease in its SAM size compared to Col-0 (Fig. 4b, e, g). Furthermore, both the spl9spl15 double mutant and the MIR156b overexpressor exhibited the same phyllotaxy as wild type with rosette leaves initiated either clock- or anticlockwise with an angle of divergence of about 137.5° between successive leaves and forming a spiral lattice with a parastichy pair (3,5) (Fig. 4e–g). From these observations, it is concluded that the observed shorter plastochron is neither the result nor the cause of an altered phyllotaxy in the spl9spl15 double mutant or the MIR156b overexpressor. The shortened plastochron, however, seems to correlate with a reduced SAM size.
As obvious from cross sections shown in Fig. 4e, g, the young leaves of the MIR156b overexpressor appear more roundish in shape in comparison to wild type leaves at similar positions. In particular, the vacuolated cells surrounding their midveins seem larger and the developing laminas reduced, i.e. represented by less small cytoplasm rich cells along their lateral margins. In addition, the stipules of the MIR156b overexpressor seem to be more prominent. In these aspects of leaf development, the spl9spl15 double mutant seems to behave intermediate (Fig. 4e).
To quantify the difference in shape and size of the leaf primordia, we determined their circularity and cross sectional area (see “Materials and methods”) starting from the first leaf cross section found to be separated from the apical meristem. To reduce effects due to imperfect cross sectioning, i.e. not absolute perpendicular to the longitudinal axes, as well as in correlating sequentially numbered primordia of different sections, we averaged the values obtained over 10 successive primordia. As shown in Fig. 4c, circularity of the youngest 10 leaf primordia was highly similar between the different genotypes. Circularity of subsequent older primordia decreased in all, however, more rapidly in wild type such that, on average, leaf 10–20 did differ significantly between the genotypes. Interestingly, it cannot be excluded that this difference is a direct consequence of a shortened plastochron in the mutant lines. In particular as the leaf initiation rate of the MIR156b overexpressor line lies roughly one and a half times above that of wild type. Accordingly, and with respect to absolute age, leaf 11–20 of the MIR156b overexpressor may be more comparable to leaves 6–15 of wild type and to which indeed no significant difference in circularity was found. However, in cross sectional area these young leaves differed significantly. On average, 6.5 ± 2.4 × 103 μm2 for wild-type leaves 6–15 and 26.7 ± 16.2 × 103 μm2 for leaf 11–20 of the MIR156b overexpressor.
It is known that the shape and other characteristics of newly formed leaves progressively change in correlation with the vegetative phase transition (Telfer et al. 1997). Furthermore, likely due to a changed plastochron, the correlation between leaf number and flowering seems to differ for the mutants and wild type. Therefore, we further investigated the possibility that the observed differences correlate with relative altered timing of the vegetative phase transition.
Functional analysis of SPL9 and SPL15 during the vegetative phase transition
In order to determine the timing of the vegetative phase change we used the absence or presence of abaxial trichomes on rosette leaves as a morphological marker for leaves formed during the juvenile or adult growth phase, respectively (Telfer et al. 1997). On average, in our LD2 growing conditions, the first abaxial trichomes developed on rosette leaf number six of Col-0 wild-type plants (Table 1). The spl9spl15 double mutants displayed its first abaxial trichomes on leaf number twelve and the 35S::MIR156b overexpressor on leaf number 16. Although less than the spl9spl15 double mutant, the respective single mutants also developed significantly more juvenile leaves than wild type (Table 1).
We distinguished juvenile and adult growth phase based on a phase dimorphism, i.e. in abaxial trichomes. However, on a plant-physiological level the juvenile phase in Arabidopsis is characterized as being incompetent to respond to photoperiodic induction of flowering (Poethig 1990). To determine if this competence was indeed affected, small populations of 20–22 plants of wild type, the spl9, spl15 single and double mutants and the MIR156b overexpressor were germinated and cultivated for 3 weeks in non-inductive SD conditions (see “Materials and methods”). The plants were then brought into continuous light and batchwise shifted back to SD after either 1, 3 or 5 days. Their flowering response was recorded within a 3-week period after this inductive treatment. Plants not flowering within this period also did not flower after 2 months like plants of a control group representing all genotypes that were kept continuously in SD. As shown in Table 2, the 5-day inductive treatment caused a flowering response in 100% of the plants of all genotypes. Three days also sufficed to induce all or almost all of the wild-type and single mutant plants, whereas the response of the double mutant and particular of the MIR156b overexpressor already declined. One day of continuous light, still enough to induce half or more of the wild-type and single mutant plants, did not induce flowering in any of the MIR156b overexpressor plants and only in one-tenth of the double mutants. These results thus demonstrate that also according to physiological criteria, SPL9 and SPL15 redundantly promote the juvenile-to-adult phase transition. In addition, other miR156-regulated SPL genes are expected to contribute as well based on the behaviour of the MIR156b overexpressor.
Table 2Photoperiodic floral induction in wild type and spl mutantsPercentage of plantsa induced after treatment with continuous light for1 D3 D5 DCol-0 wt60100100spl9-159100100spl15-15095100spl9 spl1598610035S::MIR156b027100D, daysaTwo populations of 10–11 plants per genotype were evaluated within a 3 week period following inductive treatment
The role of gibberellin in the function of SPL9 and SPL15
The plant hormone gibberellin (GA) is known to promote flowering in many plants and in Arabidopsis it is particularly required for flowering in SD (Wilson et al. 1992). Exogenous application of GA will induce abaxial trichomes on leaves where these are normally not present although they will not appear earlier than leaf three (Telfer et al. 1997). In order to test whether the spl9spl15 double mutant and the MIR156b overexpressor are defective in gibberellin sensitivity or biosynthesis, we exogenously applied GA3 and compared the onset of abaxial trichome production to mock treated and wild-type plants grown in LD1 (Fig. 5). Like in wild type, the GA3 treatment strongly reduced the number of rosette leaves without abaxial trichomes, i.e. juvenile leaves, with about a factor of three. This result shows that the spl9spl15 double mutant and the MIR156b overexpressor remained sensitive to GA3. However, in both the spl9spl15 double mutant and the MIR156b overexpressor the amount of GA3 applied could not reduce the number of juvenile leaves to that of obtained in GA3 treated wild type.
Fig. 5Effect of GA on spl9spl15 double mutant and MIR156b overexpressor plants in comparison to wild type. The number of juvenile rosette leaves formed is shown for plants that were either regularly sprayed with GA3 (100 μM GA3) or mock treated. Error bars indicate standard deviation (n = 8)
Discussion
Transgenic plants constitutively over-expressing the plant specific miRNA156 have been described for Arabidopsis (Schwab et al. 2005; Wu and Poethig 2006) and rice (Xie et al. 2006). Recently, overexpression of a miR156 encoding locus has also been shown to be the cause of the natural maize mutant Corngrass1 phenotype (Chuck et al. 2007). Interestingly, in all three different species, overexpression of this well conserved miRNA (Axtell and Bartel 2005; Arazi et al. 2005) causes a similar phenotype suggesting an evolutionary conserved role for the function of miR156 and its SPL target genes. Generally, in comparison to the respective wild type, miR156 over-expressing plants are smaller, flower later, tend to lose apical dominance and initiate more leaves with a shorter plastochron. MiR156 targets eleven SBP-box genes in Arabidopsis but the results presented here clearly show that already simultaneous silencing of the two likely paralogous target genes, SPL9 and SPL15, well approximate the miR156 over-expressing phenotype regarding the traits mentioned above. SPL9 and SPL15 thus act as important and functionally redundant transcription factors regulating diverse processes in shoot maturation and most likely in combination with other miR156 regulated SPL genes. In agreement with this latter statement is our observation that in addition to spl9-1 and spl15-1, mutation of a third miR156 controlled gene, SPL2 (At5g43270; T-DNA insertion line SALK_022235), results in triple mutant plants showing an even better approximation to the MIR156b overexpressor phenotype (Supplementary Fig. 2; despite the absence of detectable SPL2 transcript, single homozygous spl2-1 mutant plants lack an obvious mutant phenotype, data not shown). In greenhouse LD conditions we found the triple mutant to have produced on average 17.3 ± 2.1 (n = 16) rosette leaves in comparison to 15.6 ± 2.6 for the spl9 spl15 double mutant. Col-0 wild-type and the MIR156b overexpressor plants grown in parallel produced 12.9 ± 1.7 and 22.5 ± 3.5 rosette leaves before flowering, respectively.
SPL9 and SPL15 positively regulate the juvenile-to-adult growth phase transition
It became clear with the detailed analysis of Arabidopsis MIR156b overexpressors by Wu and Poethig (2006) as well as with the description of the Corngrass1 mutant in maize by Chuck et al. (2007), that one of the major phenotypic alterations in miR156 over-expressing plants is an extended juvenile growth phase. This suggests that one of the important functions of miR156 targeted SBP-box genes is to promote the vegetative phase change. In agreement with this observation, Wu and Poethig (2006) showed that overexpression of the miRNA156 regulated gene SPL3 and its likely paralogs leads to a greatly shortened juvenile phase in Arabidopsis. Based on morphological markers (abaxial trichomes) and physiological parameters (response to photo-inductive stimulus) we found that the spl9spl15 double mutant exhibit a delayed vegetative phase transition and, therefore, conclude that both genes are very likely involved in the positive regulation of this developmental process in a redundant fashion. Most likely, because of this redundancy, photoperiodic induction of the single spl9 and spl15 mutants is not much affected. However, the effect on the appearance of abaxial trichomes as a marker for the juvenile-to-adult phase transition appears to be stronger in the spl9 mutant in comparison to spl15. This may be due to the fact that in shoot apical development expression of SPL9 starts to increase before that of SPL15 (Fig. 1b).
As SPL9 and SPL15 promote the juvenile-to-adult growth phase transition and thus competence to respond to photoperiodic induction of flowering, it is interesting to note that both SPL9 and SPL15 themselves are strongly upregulated in the shoot apex upon such induction (Schmid et al. 2003). An additional role for these genes in establishing inflorescence or floral meristem identity may thus be suggested.
SPL9 and SPL15 negatively regulate leaf initiation rate
Our data on the leaf initiation rate suggest that SPL9 and SPL15 act negatively on leaf initiation. Silencing of both genes leads to a shorter plastochron. Other miR156 regulated SPL genes may act similarly as the plastochron is even further shortened in the MIR156b overexpressor plants. A few mutants are known to cause a shortened plastochron and most of them simultaneously affect phyllotaxy. However, we found that shortening of the plastochron due to loss of SPL gene function is neither the cause nor the result of a changed spatial distribution of leaf primordia at the shoot apex. A shorter plastochron without an altered phyllotaxy has also been reported for two rice mutants, plastochron1 and -2 (pla1, -2; Itoh et al. 1998; Miyoshi et al. 2004; Kawakatsu et al. 2006). PLA1 encodes a cytochrome P450 protein, whereas PLA2 encodes a MEI2-like RNA binding protein. In both mutants the reduction in plastochron is accompanied by an increase in the size of the SAM and a higher rate of cell division. However, although the SAM of pla2 is actually smaller than that of pla1, it has a shorter plastochron. Furthermore, higher cell division activity associated with constitutive overexpression of CyclinD shortened the plastochron in tobacco without altering SAM size (Cockcroft et al. 2000). These observations suggest, as already noticed by Kawakatsu et al. (2006), that not SAM size but rather cell division rate is decisive in plastochron duration. Also our results may lend support to this hypothesis as both the spl9spl15 double mutant and the MIR156b overexpressor exhibit a clearly shorter plastochron than wild-type plants but their SAM sizes differ only marginally. Therefore, it will be of interest to determine if SPL9, SPL15 and other miR156 regulated SPL genes control cell division rate in the SAM and, if so, in particular if their role is mediated through the phytohormone cytokinin. Not only is cytokinin a major positive regulator of cell proliferation and division in plants (Werner et al. 2001) it is also, in mutual dependence of auxin, a major determinant in the outgrowth of lateral shoots (Sachs and Thimann 1967; Chatfield et al. 2000). This latter aspect may explain the reduced apical dominance observed for the spl9spl15 double mutant and the MIR156b overexpressor. Finally, mutants disrupting cytokinin signalling are known to result in reduced leaf initiation rates in addition to a smaller SAM and other effects (Nishimura et al. 2004; Higuchi et al. 2004).
Do SPL9 and SPL15 negatively regulate leaf maturation rate?
Based on their observations of the pla mutants in correlation to the expression of the respective genes in leaf primordia but not in the SAM, Kawakatsu et al. (2006) proposed that the rate of leaf maturation plays a significant role in regulating the rate of leaf initiation. In addition, these authors postulated a model in which the inhibitory effect of pre-existing leaf primordia on the initiation of the next leaf is lost as they mature. Similarly, a shortened plastochron in the spl9, spl15 mutants and the MIR156b overexpressor may also be due to precocious maturation of their leaves as suggested by our comparison of cross sections through successive leaf primordia of wild type and the MIR156b overexpressor. Even if the shape, i.e. circularity, of the primordia may not significantly differ after correction for an altered plastochron by comparing primordia based on age and not on serial sequence number, their cross sectional area seems to increase more rapidly in the MIR156b overexpressor. In particular, the cells surrounding the midvein in MIR156b overexpressor leaves appear to enlarge more rapidly.
SPL9 and SPL15 do not modulate the role of GA in the vegetative phase change
Exogenous application of GA3 has been found to accelerate abaxial trichome production in Arabidopsis suggesting that gibberellins function to regulate vegetative phase change (Telfer et al. 1997). These findings are also supported by mutant analysis. For example in spindly (spy) mutants, which undergo constitutive GA response, abaxial trichomes occur on leaves initiated significantly earlier than in wild type (Jakobsen and Olszewski 1993; Telfer et al. 1997). On the other hand, Telfer et al. (1997) found that mutants blocked in GA biosynthesis as well as GA insensitive mutants are significantly delayed in the appearance of abaxial trichomes. Loss-of-function mutants for the here examined SPL9 and SPL15 genes clearly delay the appearance of abaxial trichomes.
Our treatment of spl9spl15 double mutant and 35S::MIR156b transgenic plants with high doses of GA3 showed that, like in wild type, their number of juvenile leaves can be reduced but not to numbers equal to those found for similarly treated wild type. In fact the ratios of juvenile to adult leaves of wild type and mutant phenotypes remain highly comparable to untreated plants. From this we conclude that the role SPL9, SPL15 and other miR156 controlled SPL genes play in the vegetative phase change, is unlikely to be GA mediated although a minor contribution to GA sensitivity can not be excluded.
Outlook
MiR156 targeted members of the SBP-box family of transcription factors in both mono- and dicots appear to play an important role as positive regulators of shoot maturation and of the vegetative to reproductive phase transition in particular. Both genetic factors, i.e. miR156 and SBP-box genes, have also been suggested to be major determinants in the transition from undifferentiated to differentiated embryogenic calli of rice (Luo et al. 2006). As the interaction between SBP-box genes and miR156 is of ancient origin in land plants (Arazi et al. 2005; Riese et al. 2007) it will be interesting to learn to what extent their molecular interplay is of importance to developmental phase transitions in plants in general.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Fig. 1
Expression of SPL15 as detected with a GUS reporter gene construct. A pSPL15::GUS:SPL15 reporter was constructed by subcloning a 2,793 bp genomic fragment of SPL15 beginning 1,260 bp upstream of the SPL15-ATG start codon and extending to 132 bp downstream of the SPL15-stop codon into the binary vector pGJ2148 (kindly provided by Guido Jach, MPIZ Cologne). A ß-glucuronidase (GUS) reporter was subsequently cloned in frame shortly downstream of the SPL15-ATG start codon and the whole construct stably transformed into Arabidopsis Col-0 background. (A–B) GUS staining of mature embryo gently squeezed out of the imbibed seed (A) and of a ca. 10 day old seedling grown in LD (B). Bars in A and B represent respectively 200 μm and 2 mm. (TIF 26,178 KB)
Supplementary Fig. 2
Phenotypes of spl9 and spl15 related mutants. (A) Flowering plants homozygous for spl9-1, -2 and -3 alleles in Col-0 background next to wild type. (B) Flowering plants homozygous for spl15-1, -2 and -3 alleles in Col-0 background next to wild type. (C) Flowering plants, from left to right, of Col-0 wild type, the double mutants spl9-1 spl15-1 and spl9-2 spl15-2, the triple mutant spl2-1 spl9-1 spl15-1 and the MIR156b overexpressor. (TIF 32,914 KB)

sbp-box genes
shoot maturation
arabidopsis
phase change
juvenile phase
mirna