Intersubject Synchronizationof Cortical Activity During

Natural VisionUri Hasson,1* Yuval Nir,2 Ifat Levy,1,3 Galit Fuhrmann,1

Rafael Malach1†

To what extent do all brains work alike during natural conditions? We exploredthis question by letting five subjects freely view half an hour of a popular moviewhile undergoing functional brain imaging. Applying an unbiased analysis inwhich spatiotemporal activity patterns in one brain were used to “model”activity in another brain, we found a striking level of voxel-by-voxel synchro-nization between individuals, not only in primary and secondary visual andauditory areas but also in association cortices. The results reveal a surprisingtendency of individual brains to “tick collectively” during natural vision. Theintersubject synchronization consisted of a widespread cortical activation pat-tern correlated with emotionally arousing scenes and regionally selective com-ponents. The characteristics of these activations were revealed with the use ofan open-ended “reverse-correlation” approach, which inverts the conventionalanalysis by letting the brain signals themselves “pick up” the optimal stimulifor each specialized cortical area.

A fundamental question in neuroscience isto what extent brains of different humanindividuals operate in a similar manner.Although numerous neuroimaging studiesdemonstrated a substantial similarity acrossdifferent brains, these results have beenobtained in highly controlled experimentalsettings that constrain and remove all spon-taneous, individual variations. In the visualdomain, this question can be placed in thecontext of an even broader, fundamentalpuzzle: Do we all see the world in the sameway? In a typical visual mapping experi-ment, subjects are presented with simpli-fied visual stimuli and asked to maintainfixation and perform identical and particu-larly demanding attentional tasks. With theuse of such highly controlled conditions, aconsistent network of functionally distinctretinotopic areas (1, 2) and object-relatedregions has been described along the entireextent of human lateral-occipital and tem-poral cortices (3–5). However, natural vi-sion drastically differs from conventionalvisual mapping studies by relaxing at leastfour fundamental constraints: (i) Visualstimuli are not presented in isolation but are

embedded in a complex multiobject scene.(ii) Objects move in a complex mannerwithin the scene. (iii) Subjects freely movetheir eyes. (iv) Seeing usually interactswith additional modalities, as well as con-text and emotional valence. Thus, the worldseen in the controlled experimental settingbears little resemblance to our naturalviewing experience.

Recently, several studies have begun toinvestigate the functional architecture of ma-caque (6–8) and human brains (9–12) undermore naturalistic settings. However, becauseof the spatial and temporal complexity, mul-tidimensionality, and lack of any prefixedprotocol, it is difficult to use conventionalhypothesis-driven analysis methods in thenatural viewing setting [but see (11, 12)]. Toovercome this inherent limitation, we haveintroduced an unbiased type of analysis thatdoes not rely on predetermined stimulationprotocols. This was done in two ways: (i) inthe intersubject correlation analysis, we usedthe voxels’ time courses of one brain to pre-dict the activity in other brains. The strengthof this across-subject correlation measure isthat it allows the detection of all sensory-driven cortical areas without the need of anyprior design matrix or assumptions as to theirexact functional responses. We will refer tothis across-subject voxel-by-voxel synchroni-zation as the “intersubject dimension.” (ii) Inthe “reverse-correlation” analysis, we usedregionally specific brain responses to identifythe particular attributes or dimensions ofcomplex natural stimuli present at times of

peak responses. Put simply, we inverted theclassical approach, which uses a set of pre-defined stimuli to locate brain regions. In-stead, we used the brain activations them-selves to find the preferred stimuli embeddedin the complex stimulation sequence.

We implemented this approach in thestudy of the functional organization of humancortex under free viewing of a long (30 min)uninterrupted segment taken from an originalaudiovisual feature film (13). Subjects wereinstructed to freely view the movie segmentand report its plot at the end of the experi-ment (14). We reasoned that such rich andcomplex stimulation will be much closer toecological vision relative to the highly con-strained visual stimuli used in the laboratory.

Intersubject CorrelationTo examine the intersubject dimension, wenormalized all brains into a Talairach co-ordinate system, spatially smoothed thedata, and then used the time course of eachvoxel in a given source brain as a predictorof the activation in the corresponding voxelof the target brain (15). Overall, there were10 unique pairwise comparisons betweenthe five subjects watching the same movie.Despite the free viewing and complex na-ture of the movie, we found an extensiveand highly significant correlation acrossindividuals watching the same movie.Thus, on average over 29% ! 10 SD of thecortical surface showed a highly significantintersubject correlation during the movie(Fig. 1A). Figure 1B shows a representativecorrelation map between two subjects inwhom the percentage of functionally corre-lated cortical surface was around the mean(30%). All 10 pairwise comparisons, ar-ranged in a descending order according tothe fraction of correlated cortex, are pre-sented in fig. S1.

Close inspection of this across-subjectcorrelation revealed that the synchronizationwas far more extensive than the boundaries ofwell-known audiovisual sensory cortex de-fined with conventional mapping approach(4). This point is illustrated in Fig. 1B, wherethe borders of early retinotopic areas aremarked by black dotted lines, and color con-tours mark the high-order face-, building-,and common object-related regions. As canbe seen, the across-subject correlation cov-ered most of the visual system, includingearly retinotopic areas as well as high-orderobject areas within the occipitotemporal andintraparietal cortex. Moreover, the correlationextended far beyond the visual and auditorycortices [estimated border of auditory cortex(A1") is marked by black dotted line (16)] tothe entire superior temporal (STS) and lateral

1Department of Neurobiology, Weizmann Institute ofScience, Rehovot 76100, Israel. 2Department of Com-puter Science, Tel Aviv University, Tel Aviv 61390,Israel. 3Interdisciplinary Center for Neural Computa-tion, Hebrew University, Jerusalem 91904, Israel.

*Present address: Center for Neural Science, NewYork University, New York, NY 10003, USA.†To whom correspondence should be addressed. E-mail: rafi.malach@weizmann.ac.il

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sulcus (LS), retrosplenial gyrus, even second-ary somatosensory regions in the postcentralsulcus, as well as multimodal areas in theinferior frontal gyrus and parts of the limbicsystem in the cingulate gyrus.

This strong intersubject correlationshows that, despite the completely freeviewing of dynamical, complex scenes, in-dividual brains “tick together” in synchro-nized spatiotemporal patterns when ex-posed to the same visual environment.

In order to rule out the possibility that theacross-subject correlations were introducedby scanner noise or preprocessing proce-dures, we measured intersubject correlationsbetween five subjects scanned while lyingpassively in the dark with their eyes closedfor 10 min. The intersubject correlation dur-ing darkness was negligible compared to themovie condition (Fig. 1A and fig. S2).

Correlation across cortical space. Whatis the source of the strong intersubject corre-lation? We found two separate componentsthat underlie this correlation: (i) A wide-spread, spatially nonselective activation wavethat was apparent across cortical areas. (ii) Aselective, regionally distinct component, as-sociated with specific functional properties ofindividual cortical regions. First, we will dis-cuss the nonselective activation wave.

To assess the nonselective component wemapped, within each subject, the correlationbetween anatomically distinct cortical re-gions. Figure 2A demonstrates the correlationbetween the average time course of the entireventral occipito-temporal (VOT) cortex inone cortical hemisphere (red contour, Fig.2A), and the rest of the cortex, including theother hemisphere. As can be seen, the within-subject correlation to VOT activation wasextremely high and widespread and showedsimilarity to the intersubject correlation map(compare Figs. 1 and 2). Similar maps wereobtained with the use of the dorsal occipito-temporal (DOT) subdivision (17). Figure 2Bshows the nonselective time courses obtainedduring the first 10 min of the movie for allfive subjects. The striking similarity betweensubjects attests to the degree to which globalcortical activity was synchronized across in-dividuals watching the same movie.

The “reverse-correlation” approach. Inorder to identify the source of such powerfulcommon “consensus” between different indi-viduals watching the same movie, we adopt-ed an analysis approach loosely analogous tothe reverse-correlation method used for sin-gle unit mapping (6, 18). In this analysis, weused the peaks of activation in a given re-gion’s time course to recover the stimulus

events that evoked them, thus constructing aregionally specific “movie” based on the ap-pended sequence of all frames that evokedstrong activation in a particular region ofinterest (ROI) while skipping all weakly ac-tivating time points (19).

Applying the reverse correlation to theglobal nonselective time course (red line,Fig. 2B) revealed a substantial componentof emotionally charged and surprising mo-ments in the original movie (e.g., all gun-shots and explosion scenes, or surprisingshifts in the movie plot). The time codes ofall frames associated with the peaks ofactivation in the nonselective time courseare presented in table S1. Readers that canobtain the original digital video disc (DVD)version of the movie (13) are encouraged todownload the BrainShow.exe program (14 )to view the reverse-correlation movie of thenonselective component.

Category selectivity under natural viewingconditions. In addition to the global nonselec-tive activation, the movie evoked distinct ac-tivation patterns in different brain regions,which were nevertheless highly correlatedbetween individuals watching the same mov-ie. This was assessed by performing the sameacross-subject correlation to the movie dataset after the removal of the nonselective com-

Fig. 1. Intersubject correlation during free viewing of an uninterruptedmovie segment. (A) Average percentage of functionally correlated corticalsurface across all pairwise comparisons between subjects for the entiremovie time course (All), for the regionally specific movie time course(after the removal of the nonselective component, Regional) and for thedarkness control experiment (In darkness). (B) Voxel-by-voxel intersub-ject correlation between the source subject (ZO) and the target subject(SN). Correlation maps are shown on unfolded left and right hemispheres(LH and RH, respectively). Color indicates the significance level of theintersubject correlation in each voxel. Black dotted lines denote bordersof retinotopic visual areas V1, V2, V3, VP, V3A, V4/V8, and estimatedborder of auditory cortex (A1"). The face-, object-, and building-relatedborders (red, blue, and green rings, respectively) are also superimposed onthe map. Note the substantial extent of intersubject correlations and theextension of the correlations beyond visual and auditory cortices.

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ponent from each voxel’s time course (20).The extent of correlation between subjectswatching the movie remained high and large-ly unchanged (24% ! 8.5) when applied tothe residual data set (without the nonselectivecomponent) (Fig. 1A and fig. S3).

To assess the functionality of known areasduring natural viewing, we examined thetime course of two well-studied cortical re-gions: the face-related posterior fusiform gy-rus [pFs, also termed the FFA (21, 22)] andthe building-related collateral sulcus [CoS,also termed the PPA (23, 24)]. These regionswere defined independently with the use ofconventional static object images (ROIs areindicated by red and green for face and build-ing, respectively, on the inflated hemispheresshown from a ventral view, Fig. 3, A to B).We then extracted these regions’ time coursesof activation during the movie, subtracted thenonselective activation component, and aver-aged the signal across subjects (19). The levelof intersubject synchronization after the sub-traction of the nonselective activation wasquantified by performing a t test on each timepoint in the time course across subjects.Points that were significantly different fromthe mean value (P # 0.05) in the fusiformface-related region (Fig. 3A) and the collat-

eral building-related region (Fig. 3B) aremarked in red and green, respectively. As canbe seen, all selective peaks after subtractionwere significantly different from the meanvalue across subjects.

With the use of the reverse-correlationapproach, we could then ask what frames ofthe movie evoked the highest activation in theface-related and in the building-related re-gions. This approach was free from any pre-determined biases regarding the functionalproperties and thus could reveal for the firsttime their regional selectivities under naturalviewing conditions. Figure 3 shows sampledmovie frames from the highest five activationpeaks in the face-related (A) and building-related (B) regions. The time codes of allframes are presented in table S1. The activationpeaks are ordered according to descending sig-nal amplitude. The fusiform face-related regionwas activated mainly by close-ups of face im-ages, whereas the building-related region wasmostly activated by images of indoor (e.g.,peaks 1 and 5) and outdoor scenes, includingbuildings (e.g., peak 2) and open fields (e.g.,peaks 3 and 4) (25).

It should be noted that the observed selec-tivity in the face and building-related regionswas maintained in most of the activations

peaks. Thus, in the face-related fusiform gy-rus, 15 our of the 16 highest peaks wereassociated with face images, whereas in thebuilding-related collateral sulcus 13 out ofthe 16 highest peaks were associated exclu-sively with scene images. Thus, it is clear thatthe fusiform face-related and collateralbuilding-related regions indeed maintainedtheir selectivity even under free viewing ofnatural and complex scenes.

Given that the intersubject correlation ac-tually extended far beyond the well-knownsensory regions (Fig. 1), we applied thereverse-correlation approach in the search foradditional functional preferences in regionsthat are typically not activated with the use ofthe conventional, discrete, object stimuli.

An example of such unexpected selec-tivity was a cortical region located in themiddle postcentral sulcus (PCS), in the vi-cinity of Brodmann area 5, which showed ahighly correlated activation across subjectsduring the movie (see PCS in Figs. 1 and4). Figure 4 shows the movie frames thatevoked the eight highest activation peaks inthis region. The time codes of all frames arepresented in table S1. Although on firstsight these frames do not seem to share acommon property, closer inspection reveals

Fig. 2. Nonselective activation across regions. (A) Correlation between theaveraged time course of the VOT cortex in one cortical hemisphere(correlation seed marked by the red contour) and the rest of the cortex,shown on unfolded left and right hemispheres. (B) The average nonselec-tive time course across all activated regions obtained during the first 10min of the movie for all five subjects. Red line represents the across-subject average time course. There is a striking degree of synchronizationamong different individuals watching the same movie.

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a consistent activation associated with theperformance of delicate hand movementsduring various motor tasks (white arrows,Fig. 4). Furthermore, 15 out of the 16 high-est peaks in this region were associatedwith hand-related movements (25).

Direct comparison between natural andcontrolled viewing conditions. The lack ofany predefined design matrix in the uned-ited movie prevented a direct comparison[e.g., using paradigm-based general linearmodel (GLM) analysis] between naturalis-

tic vision and the more conventional map-ping approach. To allow such comparison,we constructed a condition-based movie(experiment 3), which was composed ofconsecutive 15-s clips selected to containpreferentially one of four object categories:faces, buildings, open landscape scenes,and miscellaneous images of various ob-jects. Such object-selective movie seg-ments allowed conventional contrast-basedanalysis (14 ). We compared the selectivitymaps of the standard discrete images (ex-

periment 2, n $ 18, general-linear modelwith random-effect analysis, Fig. 5A) andthe movie clips (experiment 3, n $ 9, Fig.5B) for both hemispheres on a flattenedbrain format.

Recently, we identified seven category-related object regions in human occipito-temporal cortex (4), organized in a mirrorsymmetry structure. These include two face-related regions, three object-related regions,and two building-related regions. The catego-ry-related movie clips produced a map that

Fig. 3. Functional selectivityrevealed by the reverse-correlation method. The aver-aged time course of the re-gionally selective componentof the fusiform face-relatedregion (A) and collateralbuilding-related region (B)during the movie, definedwith the use of an externallocalizer (see inflated hemi-sphere from a central view).Time points that were signifi-cantly (P # 0.05) differentfrom baseline across subjectsare marked by red and green,respectively. Above each timecourse are the movie framesthat produced the highest ac-tivations in that region. Themovie frames are ordered ac-cording to descending signalamplitude. There is remark-able category selectivity re-vealed in the frames for facesin the fusiform face-relatedregion (A) and for indoor andoutdoor scenes in the build-ing-related region (B). This se-lectivity was apparent in 16 outof the 16 marked peaks in thefusiform face-related regionand in 12 out of the16 markedpeaks in the collateral building-related region. [Movie stillscourtesy of MGM CLIP"STILL.The Good, The Bad, and TheUgly. ©1966 Alberto GrimaldiProductions S.A. All RightsReserved.]

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nicely agrees with the conventionally pro-duced selectivity map within the VOT andDOT subdivisions (dashed rectangles in Fig.5, A and B). White arrows point to corticalregions showing similar object selectivity un-der the conventional and movie clips condi-tions. However, the movie clips of faces in-duced additional activations, which extendedfurther anteriorly in the occipitotemporal cor-tex, including regions anterior to area MT(V5) and a region in the vicinity of the STS(black arrows, Fig. 5B). These regions areknown to be sensitive to movements of bodyparts (26, 27) and shifts in eye gaze (28, 29)and were probably strongly affected by thedynamic human-related motion present in themovie clips.

Selectivity index. The results so far indicatethat, qualitatively, the object selectivity appearedto be maintained under free viewing of complexnatural stimuli. To obtain a more quantitativeindex of such selectivity, we examined the cor-relations between cortical regions that are knownto have either similar or different functional pref-erences. In this analysis, we compared the cor-relations within face-related regions (pFs andinferior occipital gyrus, i.e., F-F), within build-ing-related regions (CoS and transverse occipitalgyrus, B-B), as well as correlations across theseregions (F-B). “Within” (F-F and BB) correla-tions were significantly higher than the “across”(F-B) correlations in all three presentation meth-ods (one tail, paired t test; P # 0.01) (Fig. 5C).

Thus, the selectivity of the face- and building-related regions was maintained under natural andopen-ended viewing conditions.

DiscussionIn this study, we report the unexpected find-ing that brains of different individuals show ahighly significant tendency to act in unisonduring free viewing of a complex scene suchas a movie sequence (Fig. 1). Such responsesimply that a large extent of the human cortexis stereotypically responsive to naturalisticaudiovisual stimuli. These intersubject corre-lations extended beyond the well-known vi-sual and auditory cortices into high-order as-sociation areas, e.g., along the STS, LS, andretrosplenial and cingulate cortex, whichhave not been previously associated with sen-sory processing. Thus, the “collective” di-mension, i.e., the level of intersubjectsynchronization, appears to provide a newsensitive and quantifiable measure of theinvolvement of cortical areas with externalsensory stimuli. Critically, this measure doesnot depend on any prior knowledge or as-sumptions regarding the functionality ofthese areas.

In addition to the highly synchronizedcortex, we also found a pattern of areas whichconsistently failed to show intersubject co-herence. These areas included the supramar-ginal gyrus, angular gyrus, and prefrontalareas. Thus, the “collective” coherence effect

naturally divides the cortex into a system ofareas that manifest an across-subject, stereo-typical response to external world stimuliversus regions that are linked to unique, in-dividual variations (30).

The intersubject correlation was composedof two unrelated components: a spatially non-selective component and a regionally selectiveone. Below, we discuss these separately.

Nonselective activation component. Thespatially nonselective interarea response couldresult from several factors. First, it could reflectmodulations in the feed-forward processingload imposed by variations in the visual andcontextual complexity of the movie scenes.Such global variations are expected particularlyif the object representations have a widely dis-tributed nature (31). Second, the nonselectivecomponent might reflect the global attentionaland arousal impact of the scenes, as indeed wassuggested by the highly emotional activationclips generated through reverse correlation ofthe nonselective component with the movie(25). Although such arousal effects might alsoproduce global autonomic responses, this isunlikely to explain the results, which show ahighly stereotyped and heterogeneous neuro-anatomical distribution (Fig. 2; also see similarconclusion in (32)].

Selective activation component. Given thecomplete lack of control over the stimuli, therecovery of the known functional selectivityof cortical areas using the reverse-correlation

Fig. 4. Selectivity preference of themid-postcentral sulcus. Averagedtime course of the mid-postcentralsulcus region; coloring and frameselection as in Fig. 3. A commontheme across all frames, revealedwithout any prior experimental de-sign, is the usage of hands for per-formance of various motor tasks, asdenoted by the white arrow in eachframe. This selectivity was appar-ent in 15 out of the 16 markedpeaks. [Movie stills courtesy ofMGM CLIP"STILL. The Good, TheBad, and The Ugly. ©1966 AlbertoGrimaldi Productions S.A. AllRights Reserved.]

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method has important implications to the na-ture of object representations. These becomeapparent when considering which constraintshave been relaxed during the movie. First,subjects’ eye movements were completelyuncontrolled, which allowed subjects to viewthe stimuli at different retinal locations. Theconsistency of the results despite the sponta-neous nature of eye movement is compatiblewith the notion that high-order object areasare not very sensitive to changes in retino-topic position (33–35).

Second, the finding that selectivity did notdecrease with the movie’s spatiotemporalcomplexity (Fig. 5C) points to the efficientoperation of selection mechanisms, whichgovern subjects’ attention (36–38). Thus, giv-en that several objects were embedded withina complex background in each scene, it seemsthat object-based attention (8, 35) was capa-ble of isolating the object of choice within thecomplex frame.

Our results demonstrate that the unifiednature of conscious experience in fact con-sists of temporally interleaved and highlyselective activations in an ensemble of spe-cialized regions, each of which “picks up”and analyzes its own unique subset of stimuliaccording to its functional specialization. Fi-nally, the collective correlation attests to theengaging power of the movie to evoke aremarkably similar activation across subjects.

In that sense, the across-subject correlationmay serve as a potential measure for tracingcultural and attentional differences amongvarious populations. An interesting, relatedissue is the potential impact of prior experi-ence on the responses to the movie. For ex-ample, might the viewer’s background (e.g.,combat experience or prior exposure to themovie) be a factor in modulating the inter-subject synchronization? Future studies in-volving carefully selected target groups willbe needed to further examine these questions.

The reverse-correlation method. Thereverse-correlation method has proven tobe very effective in uncovering both knownand unexpected functional specializations.Thus, the approach could serve as a prom-ising unbiased tool for probing functionalcharacteristics of new brain areas. This wasmost evident in the unexpected finding thatthe hand-related somatosensory region inthe postcentral sulcus was activated by im-ages of delicate hand movements. It ap-pears likely that this activation is part of thevisuo-somato-motor “mirror” system origi-nally reported in macaque monkeys (39,40) and more recently extended to the hu-man cortex (41). Recently, social psychol-ogists have stressed the role of such amirror system directly linking perceptionand behavior as one of the fundamentalbases of social cognition (42). Moreover, a

recent study of this system has stressed,similar to the present work, the advantageinherent in the use of complex natural stim-ulation to discover the underlying functionsof different brain regions (43).

The reverse-correlation method has sever-al limiting factors as well. First, the rathersluggish nature of the hemodynamic responsecan make this method unsuitable when pre-senting a particularly rapid movie sequence.However, it seems that the natural temporalflow of visual events tends to be rather slow,and sufficient for blood oxygenation level–dependent (BOLD) resolution. Thus, al-though no “blank” periods were introducedto segregate BOLD responses in the presentstudy, our results show that the BOLDsignal was able to “pick” quite successfullythe object-selective frames appropriate foreach cortical area despite the continuousflow of the movie.

Second, given the complexity and multi-dimensionality of each frame, it will obvious-ly be impossible to isolate the appropriatefunctional dimensions solely on the basis ofthe reverse-correlation method. Thus, thereverse-correlation should be viewed as acomplementary tool for evaluating putativeselectivities found under natural vision andfor “pilot” searches, both for normal andpathological cases, which can suggest prelim-inary functional specializations to be fol-

Fig. 5. Comparison of free viewing and controlled viewing: activationmaps for conventional mapping using line drawings of static objects (A)and free viewing of preedited, category-specific movie clips (B) of faces(red), objects (blue), and buildings (green). The DOT and VOT subdivi-sions are indicated by dashed rectangles. Colors indicate contrast ofeach category with the other two (e.g., faces versus buildings andobjects). There is a clear similarity between the maps generated byconventional and category-specific movie clips within the occipito-temporal cortex (see small white arrows within the DOT and VOTsubdivisions). (C) Correlation level between VOT and DOT subregionssharing similar object preference for faces (F-F) and buildings (B-B), aswell as across different object preference (F-B). All data were obtained

from ROIs defined by the conventional mapping after removal of the nonselective component. In all three presentation methods, the within-categorycorrelation was dramatically higher than the across-category correlation, indicating that object selectivity was maintained despite the free viewing ofcomplex stimuli. Asterisks indicate P # 0.05.

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lowed by a more controlled set of stimulationconditions. The reverse-correlation methodcould be further supported by a more quanti-tative analysis, e.g., by a binomial probabilityestimation using an objective, binary ratingof the presence of specific stimuli (e.g., faces)in each frame of the various movies.

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44. This study was funded by the Benoziyo Center. Wethank the Wohl Institute for Advanced Imaging inthe Tel-Aviv Sourasky Medical Center; M. Behr-mann, K. Grill-Spector, G. Avidan, M. Peleg, and S.Gilaie-Dotan for fruitful discussions and com-ments; R. Goebel and D. Paserman for helping withthe intersubject analysis; M. Harel for three-dimensional brain reconstructions; E. Okon fortechnical assistance; E. Malach for programmingthe “brain-show” reverse-correlation analysis tool;and R. Mukamel for software development. Wethank C. Eastwood and H. Grimaldi for kindly pro-viding us with permission to use the frames.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/303/5664/1634/DC1Materials and MethodsFigs. S1 to S3Table S1

22 July 2003; accepted 15 January 2004

The Wheat VRN2 Gene Is aFlowering Repressor

Down-Regulated by VernalizationLiuling Yan,1* Artem Loukoianov,1 Ann Blechl,2

Gabriela Tranquilli,1† Wusirika Ramakrishna,3 Phillip SanMiguel,4

Jeffrey L. Bennetzen,5 Viviana Echenique,1‡ Jorge Dubcovsky1*§

Plants with a winter growth habit flower earlier when exposed for several weeksto cold temperatures, a process called vernalization. We report here the po-sitional cloning of the wheat vernalization gene VRN2, a dominant repressor offlowering that is down-regulated by vernalization. Loss of function of VRN2,whether by natural mutations or deletions, resulted in spring lines, which donot require vernalization to flower. Reduction of the RNA level of VRN2 by RNAinterference accelerated the flowering time of transgenic winter-wheat plantsby more than a month.

Common wheat (Triticum aestivum L.) is oneof the primary grains consumed by humansand is grown in very different environments.

This wide adaptability has been favored bythe existence of wheat varieties with differentgrowth habits. Winter wheats require a longexposure to low temperatures in order toflower (vernalization) and are sown in thefall, whereas spring wheats do not requirevernalization and can be planted in the springor fall. The genes from the vernalization path-way prevent flower development during thewinter, providing protection for the tempera-ture-sensitive floral organs against the cold.VRN1 and VRN2 are the central genes in thevernalization pathway in wheat, barley, andother temperate cereals. These two geneshave strong epistatic interactions and are like-ly part of the same regulatory pathway (1, 2).In both diploid wheat (T. monococcum L.)and barley, VRN1 is dominant for springgrowth habit, whereas VRN2 is dominant for

1Department of Agronomy and Range Science, Uni-versity of California, Davis, CA 95616, USA. 2U.S.Department of Agriculture–Agricultural Research Ser-vice, Albany, CA 94710, USA. 3Department of Biolog-ical Sciences, Michigan Technological University,Houghton, MI 49931, USA. 4Purdue UniversityGenomics Core, Purdue University, West Lafayette, IN47907, USA. 5Department of Genetics, University ofGeorgia, Athens, GA 30602, USA.

*These authors contributed equally to this work.†Present address: Instituto Recursos Biologicos, Insti-tuto Nacional de Tecnologia Agropecuaria, (1712)Castelar, Buenos Aires, Argentina.‡Present address: Consejo Nacional Investigaciones Ci-entificas y Tecnicas Departamento de Agronomıa, Uni-versidad Nacional del Sur, 8000 Bahıa Blanca, Argentina.§To whom correspondence should be addressed. E-mail: jdubcovsky@ucdavis.edu

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