Action of GABAB receptor on local network oscillation in somatosensory cortex of oral part: focusing on NMDA receptor.

The balance of activity between glutamatergic and GABAergic networks is particularly important for oscillatory neural activities in the brain. Here, we investigated the roles of GABAB receptors in network oscillation in the oral somatosensory cortex (OSC), focusing on NMDA receptors. Neural oscillation at the frequency of 8-10 Hz was elicited in rat brain slices after caffeine application. Oscillations comprised a non-NMDA receptor-dependent initial phase and a later NMDA receptor-dependent oscillatory phase, with the oscillator located in the upper layer of the OSC. Baclofen was applied to investigate the actions of GABAB receptors. The later NMDA receptor-dependent oscillatory phase completely disappeared, but the initial phase did not. These results suggest that GABAB receptors mainly act on NMDA receptor, in which metabotropic actions of GABAB receptors may contribute to the attenuation of NMDA receptor activities. A regulatory system for network oscillation involving GABAB receptors may be present in the OSC.

Functional Dissection of Ipsilateral and Contralateral Neural Activity Propagation Using Voltage-Sensitive Dye Imaging in Mouse Prefrontal Cortex.

Prefrontal cortex (PFC) intrahemispheric activity and the interhemispheric connection have a significant impact on neuropsychiatric disorder pathology. This study aimed to generate a functional map of FC intrahemispheric and interhemispheric connections. Functional dissection of mouse PFCs was performed using the voltage-sensitive dye (VSD) imaging method with high speed (1 ms/frame), high resolution (256 × 256 pixels), and a large field of view (∼10 mm). Acute serial 350 µm slices were prepared from the bregma covering the PFC and numbered 1-5 based on their distance from the bregma (i.e., 1.70, 1.34, 0.98, 0.62, and 0.26 mm) with reference to the Mouse Brain Atlas (Paxinos and Franklin, 2008). The neural response to electrical stimulation was measured at nine sites and then averaged, and a functional map of the propagation patterns was created. Intracortical propagation was observed in slices 3-5, encompassing the anterior cingulate cortex (ACC) and corpus callosum (CC). The activity reached area 33 of the ACC. Direct white matter stimulation activated area 33 in both hemispheres. Similar findings were obtained via DiI staining of the CC. Imaging analysis revealed directional biases in neural signals traveling within the ACC, whereby the signal transmission speed and probability varied based on the signal direction. Specifically, the spread of neural signals from cg2 to cg1 was stronger than that from cingulate cortex area 1(cg1) to cingulate cortex area 2(cg2), which has implications for interhemispheric functional connections. These findings highlight the importance of understanding the PFC functional anatomy in evaluating neuromodulators like serotonin and dopamine, as well as other factors related to neuropsychiatric diseases.

Assessing seizure liability in vitro with voltage-sensitive dye imaging in mouse hippocampal slices.

Non-clinical toxicology is a major cause of drug candidate attrition during development. In particular, drug-induced seizures are the most common finding in central nervous system (CNS) toxicity. Current safety pharmacology tests for assessing CNS functions are often inadequate in detecting seizure-inducing compounds early in drug development, leading to significant delays. This paper presents an in vitro seizure liability assay using voltage-sensitive dye (VSD) imaging techniques in hippocampal brain slices, offering a powerful alternative to traditional electrophysiological methods. Hippocampal slices were isolated from mice, and VSD optical responses evoked by stimulating the Schaffer collateral pathway were recorded and analyzed in the stratum radiatum (SR) and stratum pyramidale (SP). VSDs allow for the comprehensive visualization of neuronal action potentials and postsynaptic potentials on a millisecond timescale. By employing this approach, we investigated the in vitro drug-induced seizure liability of representative pro-convulsant compounds. Picrotoxin (PiTX; 1-100 µM), gabazine (GZ; 0.1-10 µM), and 4-aminopyridine (4AP; 10-100 µM) exhibited seizure-like responses in the hippocampus, but pilocarpine hydrochloride (Pilo; 10-100 µM) did not. Our findings demonstrate the potential of VSD-based assays in identifying seizurogenic compounds during early drug discovery, thereby reducing delays in drug development and providing insights into the mechanisms underlying seizure induction and the associated risks of pro-convulsant compounds.

Stable wide-field voltage imaging for observing neuronal plasticity at the neuronal network level.

Plasticity is the key feature of our brain function. Specifically, plasticity of hippocampal synapses is critical for learning and memory. The functional properties of the neuronal circuit change as a result of synaptic plasticity. This review summarizes the use of voltage-sensitive dyes (VSDs) to examine neuronal circuit plasticity. We will discuss the significance of plastic changes in circuit function as well as the technical issue of using VSDs. Further, we will discuss the neural circuit level plasticity of the hippocampus caused by long-term potentiation and the entorhinal-perirhinal connection. This review article is an extended version of the Japanese article, Membrane Potential Imaging with Voltage-sensitive Dye (VSD) for Long-term Recording, published in SEIBUTSU BUTSURI Vol. 61, p. 404-408 (2021).

Alternative strategy for driving voltage-oscillator in neocortex of rats.

Information integration in the brain requires functional connectivity between local neural networks. Here, we investigated the interregional coupling mechanism from the viewpoint of oscillations using optical recording methods. Low-frequency electrical stimulation of rat neocortical slices in a caffeine-containing medium induced oscillatory activity between the primary visual cortex (Oc1) and medial secondary visual cortex (Oc2M), in which the oscillation generator was located in the Oc2M and was triggered by a feedforward signal. During to-and-fro oscillatory activity, neural excitation was marked in layer II/III. When the upper layer was disrupted between Oc1 and Oc2M, feedforward signals could propagate through the deep layer and switch on the oscillator in the Oc2M. When the lower layer was disrupted between Oc1 and Oc2M, feedforward signals could propagate through the upper layer and switch on the oscillator in the Oc2M. In the backward direction, neither the upper layer cut nor the lower layer cut disrupted the propagation of the oscillations. In all cases, the horizontal and vertical pathways were used as needed. Fluctuations in the oscillatory waveforms of the local field potential at the upper and lower layers in the Oc2M were reversed, suggesting that the oscillation originated between the two layers. Thus, the neocortex may work as a safety device for interregional communications in an alternative way to drive voltage oscillators in the neocortex.

Transcriptome dynamics of rice in natura: Response of above and below ground organs to microclimate.

The long-term dynamics of the transcriptome under natural field conditions remain unclear. We conducted comprehensive gene expression analyses of rice leaves and roots grown under natural field conditions for a long period, from the tillering stage to the ripening stage. In this experiment, changes in the transcriptome were captured in relation to microclimatic parameters, particularly potential evaporation (Ep), which is a multiple meteorological factor and acts as an indicator of transpirational demand. The results indicated  that many genes were regulated by changes in temperature and Ep in both leaves and roots. Furthermore, the correlation between gene expression and meteorological factors differed significantly between the vegetative and reproductive stages. Since Ep triggers transpiration, we analyzed aquaporin gene expression, which is responsible for water transport, and found that many aquaporin genes in leaves were positively correlated with Ep throughout the growth period, whereas in roots, two plasma membrane intrinsic aquaporins, PIP2;4 and PIP2;5 were strongly correlated with Ep during reproductive growth. Other genes closely related to productivity, such as those involved in nutrient absorption and photosynthesis, exhibited different responses to meteorological factors at different growth stages. The stage-dependent shift in the microclimate response provides an important perspective on crop physiology in light of climate change.

A CCR5 antagonist, maraviroc, alleviates neural circuit dysfunction and behavioral disorders induced by prenatal valproate exposure.

BACKGROUND: Valproic acid (VPA) is a clinically used antiepileptic drug, but it is associated with a significant risk of a low verbal intelligence quotient (IQ) score, attention-deficit hyperactivity disorder and autism spectrum disorder in children when it is administered during pregnancy. Prenatal VPA exposure has been reported to affect neurogenesis and neuronal migration and differentiation. In addition, growing evidence has shown that microglia and brain immune cells are activated by VPA treatment. However, the role of VPA-activated microglia remains unclear. METHODS: Pregnant female mice received sodium valproate on E11.5. A microglial activation inhibitor, minocycline or a CCR5 antagonist, maraviroc was dissolved in drinking water and administered to dams from P1 to P21. Measurement of microglial activity, evaluation of neural circuit function and expression analysis were performed on P10. Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test and marble burying test from the age of 6 weeks. RESULTS: Prenatal exposure of mice to VPA induced microglial activation and neural circuit dysfunction in the CA1 region of the hippocampus during the early postnatal periods and post-developmental defects in working memory and social interaction and repetitive behaviors. Minocycline, a microglial activation inhibitor, clearly suppressed the above effects, suggesting that microglia elicit neural dysfunction and behavioral disorders. Next-generation sequencing analysis revealed that the expression of a chemokine, C-C motif chemokine ligand 3 (CCL3), was upregulated in the hippocampi of VPA-treated mice. CCL3 expression increased in microglia during the early postnatal periods via an epigenetic mechanism. The CCR5 antagonist maraviroc significantly suppressed neural circuit dysfunction and post-developmental behavioral disorders induced by prenatal VPA exposure. CONCLUSION: These findings suggest that microglial CCL3 might act during development to contribute to VPA-induced post-developmental behavioral abnormalities. CCR5-targeting compounds such as maraviroc might alleviate behavioral disorders when administered early.

In Planta Monitoring of Cold-Responsive Promoter Activity Reveals a Distinctive Photoperiodic Response in Cold Acclimation.

Plant cold acclimation involves complicated pathways that integrate signals from temperature changes and light conditions. To understand plant responses to environmental signals in detail, molecular events that are regulated by temperature and light must be investigated at the whole-plant level in a nondestructive way. Using the promoter of COR15A connected to the luciferase reporter gene as a cold-responsive indicator, we developed an in planta monitoring system for gene expression under controlled temperature and photoperiod conditions. COR15A promoter activity was intensified by day-night cycles at 2�C, while its induction was abruptly suppressed in the dark at 8�C or higher, indicating a difference in responsiveness to photocycle between these two acclimation conditions. Freeze-thawing tests of whole plants proved that lower acclimation temperature resulted in higher tolerance to freezing, consistent with the temperature-dependent induction of COR15A. Inhibition of photosynthetic electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea eliminated the responsiveness to the day-night cycles at 2�C, indicating a possibility that the photosynthetic redox and/or the accumulation of photosynthates modulate COR15A responsiveness to photoperiod during cold acclimation, in addition to the well-known regulation by CBF (C-repeat binding factor) genes. These findings indicate that the cold-responsive promoter is regulated by distinctive mechanisms dependent on temperature and simultaneously affected by photocycle and photosynthesis.

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

Network Plasticity Involved in the Spread of Neural Activity Within the Rhinal Cortices as Revealed by Voltage-Sensitive Dye Imaging in Mouse Brain Slices.

The rhinal cortices, such as the perirhinal cortex (PC) and the entorhinal cortex (EC), are located within the bidirectional pathway between the neocortex and the hippocampus. Physiological studies indicate that the perirhinal transmission of neocortical inputs to the EC occurs at an extremely low probability, though many anatomical studies indicated strong connections exist in the pathway. Our previous study in rat brain slices indicated that an increase in excitability in deep layers of the PC/EC border initiated the neural activity transfer from the PC to the EC. In the present study, we hypothesized that such changes in network dynamics are not incidental observations but rather due to the plastic features of the perirhinal network, which links with the EC. To confirm this idea, we analyzed the network properties of neural transmission throughout the rhinal cortices and the plastic behavior of the network by performing a single-photon wide-field optical recording technique with a voltage-sensitive dye (VSD) in mouse brain slices of the PC, the EC, and the hippocampus. The low concentration of 4-aminopyridine (4-AP; 40 µM) enhanced neural activity in the PC, which eventually propagated to the EC via the deep layers of the PC/EC border. Interestingly, washout of 4-AP was unable to reverse entorhinal activation to the previous state. This change in the network property persisted for more than 1 h. This observation was not limited to the application of 4-AP. Burst stimulation to neurons in the perirhinal deep layers also induced the same change of network property. These results indicate the long-lasting modification of physiological connection between the PC and the EC, suggesting the existence of plasticity in the perirhinal-entorhinal network.

Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging.

Activity-dependent changes in the input-output (I-O) relationship of a neural circuit are central in the learning and memory function of the brain. To understand circuit-wide adjustments, optical imaging techniques to probe the membrane potential at every component of neurons, such as dendrites, axons and somas, in the circuit are essential. We have been developing fast voltage-sensitive dye (VSD) imaging methods for quantitative measurements, especially for single-photon wide-field optical imaging. The long-term continuous measurements needed to evaluate circuit-wide modifications require stable and quantitative long-term recordings. Here, we show that VSD imaging (VSDI) can be used to record changes in circuit activity in association with theta-burst stimulation (TBS)-induced long-term potentiation (LTP) of synaptic strength in the CA1 area. Our optics, together with the fast imaging system, enabled us to measure neuronal signals from the entire CA1 area at a maximum frame speed of 0.1 ms/frame every 60 s for over 12 h. We also introduced a method to evaluate circuit activity changes by mapping the variation in recordings from the CA1 area to coordinates defined by the morphology of CA1 pyramidal cells. The results clearly showed two types of spatial heterogeneity in LTP induction. The first heterogeneity is that LTP increased with distance from the stimulation site. The second heterogeneity is that LTP is higher in the stratum pyramidale (SP)-oriens region than in the stratum radiatum (SR). We also showed that the pattern of the heterogeneity changed according to the induction protocol, such as induction by TBS or high-frequency stimulation (HFS). We further demonstrated that part of the heterogeneity depends on the I-O response of the circuit elements. The results show the usefulness of VSDI in probing the function of hippocampal circuits.

Interplay between non-NMDA and NMDA receptor activation during oscillatory wave propagation: Analyses of caffeine-induced oscillations in the visual cortex of rats.

Generation and propagation of oscillatory activities in cortical networks are important features of the brain. However, many issues related to oscillatory phenomena are unclear. We previously reported neocortical oscillation following caffeine treatment of rat brain slices. Input to the primary visual cortex (Oc1) generates N-methyl-d-aspartate (NMDA) receptor-dependent oscillations, and we proposed that the oscillatory signals originate in the secondary visual cortex (Oc2). Because non-NMDA and NMDA receptors cooperate in synaptic transmission, non-NMDA receptors may also play an important role in oscillatory activities. Here we investigated how non-NMDA receptor activities contribute to NMDA receptor-dependent oscillations by using optical recording methods. After induction of stable oscillations with caffeine application, blockade of NMDA receptors abolished the late stable oscillatory phase, but elicited 'hidden' non-NMDA receptor-dependent oscillation during the early depolarizing phase. An interesting finding is that the origin of the non-NMDA receptor-dependent oscillation moved from the Oc1, during the early phase, toward the origin of the NMDA receptor-dependent oscillation that is fixed in the Oc2. In addition, the frequency of the non-NMDA receptor-dependent oscillation was higher than that of the NMDA receptor-dependent oscillation. Thus, in one course of spatiotemporal oscillatory activities, the relative balance in receptor activities between non-NMDA and NMDA receptors gradually changes, and this may be due to the different kinetics of the two receptor types. These results suggest that interplay between the two receptor types in the areas of Oc1 and Oc2 may play an important role in oscillatory signal communication.

Paired Burst Stimulation Causes GABAA Receptor-Dependent Spike Firing Facilitation in CA1 of Rat Hippocampal Slices.

The theta oscillation (4-8 Hz) is a pivotal form of oscillatory activity in the hippocampus that is intermittently concurrent with gamma (25-100 Hz) burst events. In in vitro preparation, a stimulation protocol that mimics the theta oscillation, theta burst stimulation (TBS), is used to induce long-term potentiation. Thus, TBS is thought to have a distinct role in the neural network of the hippocampal slice preparation. However, the specific mechanisms that make TBS induce such neural circuit modifications are still unknown. Using electrophysiology and voltage-sensitive dye imaging (VSDI), we have found that TBS induces augmentation of spike firing. The augmentation was apparent in the first couple of brief burst stimulation (100 Hz four pulses) on a TBS-train in a presence of NMDA receptor blocker (APV 50 µM). In this study, we focused on the characterizes of the NMDA independent augmentation caused by a pair of the brief burst stimulation (the first pair of the TBS; paired burst stimulation-PBS). We found that PBS enhanced membrane potential responses on VSDI signal and intracellular recordings while it was absent in the current recording under whole-cell clamp condition. The enhancement of the response accompanied the augmentation of excitatory postsynaptic potential (EPSP) to spike firing (E-S) coupling. The paired burst facilitation (PBF) reached a plateau when the number of the first burst stimulation (priming burst) exceeds three. The interval between the bursts of 150 ms resulted in the maximum PBF. Gabazine (a GABAA receptor antagonist) abolished PBF. The threshold for spike generation of the postsynaptic cells measured with a current injection to cells was not lowered by the priming burst of PBS. These results indicate that PBS activates the GABAergic system to cause short-term E-S augmentation without raising postsynaptic excitability. We propose that a GABAergic system of area CA1 of the hippocampus produce the short-term E-S plasticity that could cause exaggerated spike-firing upon a theta-gamma activity distinctively, thus making the neural circuit of the CA1 act as a specific amplifier of the oscillation signal.

Arabidopsis dynamin-related protein 1E in sphingolipid-enriched plasma membrane domains is associated with the development of freezing tolerance.

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin-related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol-enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye-labelled plasma membrane, providing evidence that DRP1E localizes non-uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol-enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.

A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity.

Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca(2+) dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-µm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca(2+) imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame.

GABAA receptor-mediated modulation of neuronal activity propagation upon tetanic stimulation in rat hippocampal slices.

Tetanic stimulation (100 Hz), which can induce long-term potentiation in synaptic connections in the hippocampal CA1 region, causes γ-aminobutyric acid (GABA)(A) receptor-mediated long-lasting depolarization of postsynaptic neurons. However, it is not clear how this stimulation modulates neuronal activity propagation. We studied tetanic burst-induced neuronal responses in the hippocampal CA1 region by using optical-recording methods employing a voltage-sensitive dye and focused on GABA(A) receptor-mediated modulation. We observed that burst stimulation induced long-lasting depolarization and progressive decrease in individual excitatory postsynaptic potentials (EPSPs). Both these effects were suppressed by picrotoxin, a GABA(A) receptor antagonist. Under whole-cell voltage-clamp conditions, we observed a long-lasting inhibitory current (IPSC) and a prominent progressive decrease in the amplitude of the excitatory postsynaptic current (EPSC). Further, picrotoxin inhibited the IPSC and the progressive decrease in EPSC. The optically recorded long-lasting depolarization and progressive decrease of EPSPs were strongly dependent on the distance between the recording electrode and the stimulation site. Optical recordings performed across a wide swatch of CA1 revealed that the decrease in activity propagation was followed by facilitation of propagation after recovery and that this facilitation also depended on GABA(A) receptors. Intense activation of GABA(A) receptors is a key factor shaping the spatiotemporal patterns of high-frequency stimulation-induced responses in the CA1 region.

Membrane potential response profiles of CA1 pyramidal cells probed with voltage-sensitive dye optical imaging in rat hippocampal slices reveal the impact of GABA(A)-mediated feed-forward inhibition in signal propagation.

The spatial and temporal distribution of excitatory and inhibitory membrane potential responses on a cell plays an important role in neuronal calculations in local neuronal circuits in the brain. The electrical dynamics of excitatory and inhibitory inputs along the somatodendritic extent of CA1 pyramidal cells during circuit activation were examined by stimulating strata radiatum (SR), oriens (SO), and lacunosum-moleculare (SLM) and measuring laminar responses with voltage-sensitive dye (VSD) optical recording methods. We first confirmed the linearity of the optical signal by comparing fluorescence changes in CA1 to global membrane potential changes when slices were bathed in high-potassium ([K+](O)=25 mM) solution. Except for a TTX-sensitive component in stratum pyramidale, fluorescence changes were equal in all strata, indicating that VSD sensitivity had reasonable linearity across layers. We then compared membrane potential profiles in slices exposed to picrotoxin, a GABA(A) receptor antagonist. We attributed the picrotoxin-induced changes in the first peak of the excitatory membrane potential to feed-forward inhibition and the later response (appearing 30 ms after stimulation) to feedback inhibition. A difference in feed-forward components was observed in perisomatic and distal apical dendritic regions after SR stimulation. SLM stimulation produced large differences in perisomatic and apical dendritic regions. SO stimulation, however, produced no feed-forward inhibition at the perisomatic region, but produces feed-forward inhibition in distal dendritic regions. These results suggest that actual inhibition of membrane potential response by feed-forward inhibition is greater at perisomatic regions after SR or SLM stimulation but is smaller at distal dendritic regions after SR, SO, and SLM stimulation.

Structure and functional analysis of wheat ICE (inducer of CBF expression) genes.

Two different inducers of CBF expression (ICE1-like genes), TaICE41 and TaICE87, were isolated from a cDNA library prepared from cold-treated wheat aerial tissues. TaICE41 encodes a protein of 381 aa with a predicted MW of 39.5 kDa while TaICE87 encodes a protein of 443 aa with a predicted MW of 46.5 kDa. TaICE41 and TaICE87 share 46% identity while they share 50 and 47% identity with Arabidopsis AtICE1 respectively. Expression analysis revealed that mRNA accumulation was not altered by cold treatment suggesting that both genes are expressed constitutively. Gel mobility shift analysis showed that TaICE41 and TaICE87 bind to different MYC elements in the wheat TaCBFIVd-B9 promoter. Transient expression assays in Nicotiana benthamiana, showed that both TaICE proteins can activate TaCBFIVd-B9 transcription. The different affinities of TaICE41 and TaICE87 for MYC variants suggest that ICE binding specificity may be involved in the differential expression of wheat CBF genes. Furthermore, analysis of MYC elements demonstrates that a specific variant is present in the wheat CBF group IV that is associated with freezing tolerance. Overexpression of either TaICE41 or TaICE87 genes in Arabidopsis enhanced freezing tolerance only upon cold acclimation suggesting that other factors induced by low temperature are required for their activity. The increased freezing tolerance in transgenic Arabidopsis is associated with a higher expression of the cold responsive activators AtCBF2, AtCBF3, and of several cold-regulated genes.

Overexpression of TaVRN1 in Arabidopsis promotes early flowering and alters development.

TaVRN1, a member of the APETALA1 (AP1) subfamily of MADS-box transcription factors, is a key gene that controls transition from vegetative to reproductive phase in wheat. The accumulation of TaVRN1 transcripts in winter wheat probably requires the down-regulation of TaVRT2, a MADS-box factor that binds and represses the TaVRN1 promoter, and of the flowering repressor TaVRN2. However, the molecular mechanisms by which TaVRN1 functions as an activator of phase transition is unknown. To address this, a combination of gene expression and functional studies was used. RNA in situ hybridization studies showed that TaVRN1 transcripts accumulate in all meristems and primordia associated with flower development. An interaction screen in yeast revealed that TaVRN1 interacts with several proteins involved in different processes of plant development such as transcription factors, kinases and a cyclophilin. Arabidopsis plants overexpressing TaVRN1 flower early and show various levels of modified plant architecture. The ectopic expression causes an overexpression of the AP1 and MAX4 genes, which are associated with flowering and auxin regulation, respectively. The induction of gene expression probably results from the binding of TaVRN1 to CArG motifs present on the AP1 and MAX4 promoters. In contrast, Arabidopsis plants that overexpress TaVRT2, which encodes a putative flowering repressor, show an opposite late flowering phenotype. Together, the data provide molecular evidence that TaVRN1 may have pleiotropic effects in various processes such as control of axillary bud growth, transition to flowering and development of floral organs.

TaVRT2 represses transcription of the wheat vernalization gene TaVRN1.

In wheat, VRN1/TaVRN1 and VRN2/TaVRN2 determine the growth habit and flowering time. In addition, the MADS box transcription factor VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (TaVRT2) is also associated with the vernalization response in a manner similar to TaVRN2. However, the molecular relationship between these three genes and their products is unknown. Using transient expression assays in Nicotiana benthamiana, we show that TaVRT2 acts as a repressor of TaVRN1 transcription. TaVRT2 binds the CArG motif in the TaVRN1 promoter and represses its activity in vivo. In contrast, TaVRN2 does not bind the TaVRN1 promoter and has no direct effect on its activity, but it can enhance the repression effect of TaVRT2. This suggests that a repressor complex regulates the expression of TaVRN1. In winter wheat, TaVRT2, TaVRN2 and TaVRN1 transcripts accumulate in the shoot apical meristem and young leaves, and temporal expression is consistent with TaVRT2 and TaVRN2 being repressors of floral transition, whereas TaVRN1 is an activator. Non-vernalized spring wheat grown under a short-day photoperiod accumulates TaVRT2 and shows a delay in flowering, suggesting that TaVRT2 is regulated independently by photoperiod and low temperature. The data presented suggest that TaVRT2, in association with TaVRN2, represses the transcription of TaVRN1.