Differential Expression of Genes Important forAdaptation in Capsella bursa-pastoris (Brassicaceae)1[W][OA]
Tanja Slotte*, Karl Holm, Lauren M. McIntyre, Ulf Lagercrantz, and Martin Lascoux
Department of Evolution, Genomics and Systematics, Uppsala University, SE–752 36 Uppsala,Sweden (T.S., K.H., U.L., M.L.); and Department of Molecular Genetics and Microbiology,University of Florida, Gainesville, Florida 32610–0266 (L.M.M.)
Understanding the genetic basis of natural variation is of primary interest for evolutionary studies of adaptation. In Capsellabursa-pastoris, a close relative of Arabidopsis (Arabidopsis thaliana), variation in flowering time is correlated with latitude,suggestive of an adaptation to photoperiod. To identify pathways regulating natural flowering time variation in C. bursa-pastoris, we have studied gene expression differences between two pairs of early- and late-flowering C. bursa-pastoris accessionsand compared their response to vernalization. Using Arabidopsis microarrays, we found a large number of significant dif-ferences in gene expression between flowering ecotypes. The key flowering time gene FLOWERING LOCUS C (FLC) was notdifferentially expressed prior to vernalization. This result is in contrast to those in Arabidopsis, where most natural floweringtime variation acts through FLC. However, the gibberellin and photoperiodic flowering pathways were significantly enrichedfor gene expression differences between early- and late-flowering C. bursa-pastoris. Gibberellin biosynthesis genes were down-regulated in late-flowering accessions, whereas circadian core genes in the photoperiodic pathway were differentiallyexpressed between early- and late-flowering accessions. Detailed time-series experiments clearly demonstrated that thediurnal rhythm of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1) expressiondiffered between flowering ecotypes, both under constant light and long-day conditions. Differential expression of floweringtime genes was biologically validated in an independent pair of flowering ecotypes, suggesting a shared genetic basis orparallel evolution of similar regulatory differences. We conclude that genes involved in regulation of the circadian clock, suchas CCA1 and TOC1, are strong candidates for the evolution of adaptive flowering time variation in C. bursa-pastoris.
Flowering time is a major life-history trait contrib-uting to reproduction and adaptation, especially inannual plants (Roux et al., 2006). The timing of flower-ing in relation to the environment is of crucial impor-tance for seed production, and different floweringstrategies may have evolved in response to local cli-matic conditions (Engelmann and Purugganan, 2006;Mitchell-Olds and Schmitt, 2006). The genetic basisof flowering time variation is well understood inArabidopsis thaliana. Four main pathways, the photo-period, vernalization, GA, and autonomous pathways,allow the plant to perceive and respond to changesin daylength, temperature, and hormonal status
(Mouradov et al., 2002; Simpson and Dean, 2002;Koornneef et al., 2004). Floral pathway integrator genesintegrate signals from these pathways and fine-tunethe transition from vegetative to reproductive devel-opment, although recent studies also indicate that thereis direct cross talk between pathways (Edwards et al.,2006; Gould et al., 2006; Salathia et al., 2007).
Understanding the genetic basis of natural variationis of primary interest for evolutionary studies of ad-aptation (Mitchell-Olds and Schmitt, 2006). The pre-cise role of flowering genes among and within speciescan vary significantly, and the effect of allelic variationfor these genes in natural populations is a focus ofcurrent research (Werner et al., 2005; Engelmann andPurugganan, 2006; Roux et al., 2006; Salathia et al.,2007). Recent studies demonstrate that the main genesresponsible for natural variation in flowering time candiffer between populations or species, reflecting dif-ferences in genetic architecture, ecological niche, andhistory. In A. thaliana, variation at the genes FRIGIDA(FRI) and FLOWERING LOCUS C (FLC), which areinvolved in the vernalization response, can explaina great deal of genetic variation in flowering time(Johanson et al., 2000; Caicedo et al., 2004; Zhao et al.,2007), and selection for earlier flowering appears tohave acted on FRI (Hagenblad and Nordborg, 2002;Le Corre et al., 2002; Toomajian et al., 2006). In Arabidopsissuecica allotetraploids, late flowering is accomplished
1 This work was supported by grants from the Swedish ResearchCouncil for Environment, Agricultural Sciences and Spatial Plan-ning (to M.L. and U.L.); a grant from the Swedish Research Council(to U.L.); and grants from the Nilsson-Ehle, Wallenberg, Sederholms,and Tullberg foundations (to T.S.).
* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to
the findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Martin Lascoux ([email protected]).
[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-
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by trans-activation of strong A. thaliana FLC by functionalFRI from Arabidopsis arenosa (Wang et al., 2006). Al-though most natural flowering time variation in A.thaliana seems to act through FLC, photoreceptor genessuch as CRYPTOCHROME2 and PHYTOCHROMEC have also been implicated (El-Assal et al., 2001;Balasubramanian et al., 2006). Findings from A. thali-ana have successfully been used to start to elucidatethe genetic basis of natural flowering time variation inother crucifer species (Brassica rapa: Schranz et al.,2002; Brassica nigra: Osterberg et al., 2002; Brassicaoleracea: Okazaki et al., 2007). However, despite theavailability of genomic tools, and although assessingthe generality of patterns seen in A. thaliana is clearlyimportant, there is a dearth of studies on the geneticcontrol of natural variation in flowering time in theclosest relatives of A. thaliana, such as Arabidopsis lyrataor Capsella.
Capsella bursa-pastoris L. Medik. is a predominantlyselfing, disomic tetraploid crucifer with a nearly world-wide distribution (Hurka and Neuffer, 1997). It is anannual plant species, characterized by great colonizingability. Within C. bursa-pastoris, there is considerablevariation for a range of life-history characteristics, in-cluding flowering time (Neuffer and Hurka, 1986;Paoletti et al., 1991; Ceplitis et al., 2005). As in A.thaliana, there is also variation in vernalization require-ment, with some late-flowering accessions having anobligate requirement for vernalization in order to flower(A. Ceplitis, unpublished data). Flowering time differ-ences are highly heritable (Linde et al., 2001), andcorrelation between flowering time and environmentalfactors indicates that flowering time may represent anadaptation to local climatic conditions (Neuffer andHurka, 1986; Neuffer and Bartelheim, 1989; Neuffer,1990). In C. bursa-pastoris, two to three major quantita-tive trait loci (QTL) for flowering time were found in
an F2 population derived from crosses of two NorthAmerican accessions (Linde et al., 2001; A. Ceplitis,B. Neuffer, M. Linde, T. Slotte, M. Kraft, and M.Lascoux, unpublished data), but so far little is knownabout the nature of the genetic differences underlyingthese QTL.
Changes in the balance between flowering time path-ways can result in dramatic differences in floweringtime (Lempe et al., 2005; Roux et al., 2006). To testwhether gene regulation differences in known flower-ing time genes in Arabidopsis are also responsible fornatural variation in flowering time in C. bursa-pastoris,we compare two accessions that differ widely in flower-ing time under a vernalization/nonvernalization re-gime for differences in gene expression and validatethese differences in two accessions with less extremedifferences in flowering time. This approach allowsus to both identify flowering pathways that are differ-entially regulated between C. bursa-pastoris floweringecotypes and to test whether these regulatory dif-ferences are shared across different early- and late-flowering ecotypes.
RESULTS
Flowering Time Variation in C. bursa-pastoris
Based on data from a survey of flowering timevariation in a worldwide sample of C. bursa-pastoris(Ceplitis et al., 2005), we found that there was a sig-nificant correlation between flowering time and lati-tude (Pearson P 5 0.64, P , 0.001; Fig. 1), but notbetween longitude and flowering time. This clinal var-iation could indicate that flowering time has evolvedas an adaptation to, for example, photoperiod. Al-though demographic processes can give rise to similar
Figure 1. A, Flowering time is correlated with lati-tude in C. bursa-pastoris. The number of days toflowering from germination is significantly correlatedwith latitude (Pearson P 5 0.64, P , 0.001), andlinear regression is also significant (P , 0.001, solidline). B, The distribution of flowering time of a setof natural accessions of C. bursa-pastoris. Floweringtime ranges from less than 35 d to more than 200 d(the y axis is truncated at 200 d), when plants aregrown without vernalization treatment. The blackbars indicate the flowering time of the accessionsused in this study, and their designations are givenunder the corresponding bar. Accessions PL and SE14were chosen to represent the extremes of the range ofvariation in flowering time variation, whereas acces-sions US721 and US740 were used for biologicalvalidation of gene expression differences.
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patterns (Mitchell-Olds and Schmitt, 2006), the lack ofcorrelation to longitude suggests that at least part ofthe variation in flowering time in C. bursa-pastoris isadaptive. To accommodate the full range of floweringtime variation, we chose to include two ecotypes torepresent the extremes in flowering time variation (theearly-flowering accession PL from Puli, Taiwan, andthe late-flowering accession SE14 from Harnosand,Sweden; Fig. 1). Two less extreme ecotypes, the early-flowering US721 from Shafter, CA, and the late-floweringUS740, from Reno, NV, previously used as parents in aQTL-mapping cross (Linde et al., 2001; A. Ceplitis, B.Neuffer, M. Linde, T. Slotte, M. Kraft, and M. Lascoux,unpublished data), were selected for independent bio-logical validation of gene expression differences be-tween extreme flowering ecotypes (Fig. 1).
Flowering Time Is Affected by Vernalization
We assessed the flowering time of ecotypes PL andSE14, with and without vernalization, using survivalanalysis, an analysis method for time-dependent de-velopmental traits (see ‘‘Materials and Methods’’) suchas flowering time. We found that the survival function(i.e. the predicted probability of not flowering) wasdifferent across the four groups (P , 0.0001), and allpairwise comparisons, including that between vernal-ization treatments for the early-flowering accessionPL, exhibited significantly different median floweringtimes (P , 0.001; Table I; Fig. 2). Thus, vernalizationhad an effect on flowering time in both extreme flower-ing ecotypes, although the effect was greater for ac-cession SE14 than accession PL (Table I).
Characterization of Gene Expression Differencesbetween Flowering Ecotypes
To test whether genes involved in regulation offlowering time in A. thaliana were differentially ex-pressed between flowering ecotypes of C. bursa-pastoris,we used A. thaliana CATMA 25k (Complete Arabidop-sis Transcriptome Microarray; Allemeersch et al., 2005;www.catma.org) microarrays to assess genome-wide
differential gene expression. Gene expression wasmeasured in 1-week-old seedlings from each of thetwo extreme ecotypes, under a vernalization/non-vernalization regime (see ‘‘Materials and Methods’’).This assay allows us to identify both genes that aredifferentially expressed between accessions and thosethat are differentially expressed as a result of vernal-ization treatment.
We assembled a list of 214 genes that have beenidentified as involved in flowering time in A. thaliana,based on Gene Ontology (GO) annotation (see ‘‘Mate-rials and Methods’’; Supplemental Appendix S2). Ofthese, 112 probes were analyzed for differential expres-sion, and 21 were significantly differentially expressed(false discovery rate [FDR] # 0.1; Table II). Interest-ingly, all significant differences were between acces-sions (Table II). Key circadian clock genes, such asthe two myb-family transcription factor genes LATEELONGATED HYPOCOTYL (LHY; At1g01060) and CIR-CADIAN CLOCK-ASSOCIATED1 (CCA1; At2g46830)and TIMING OF CAB EXPRESSION1 (TOC1; At5g61380)involved in the core feedback loop of the circadianoscillator (Schaffer et al., 1998; Wang and Tobin, 1998;Alabadi et al., 2001; Mizoguchi et al., 2002), were dif-ferentially expressed, with LHY and CCA1 up-regulatedin the late-flowering accession SE14 and TOC1 down-regulated. A casein kinase II b-subunit-encoding gene(CKB4, At2g44680), involved in regulation of circa-dian rhythm (Perales et al., 2006), was also down-regulated in accession SE14 compared to PL (Table II;Fig. 3).
The expression of several genes in the GA pathwaydiffered between accessions (Table II; Fig. 2). Twogenes involved in GA biosynthesis, GA4 encodingGA 3-b-dioxygenase/GA 3-b-hydroxylase (At1g15550;
Table I. Flowering time of extreme flowering ecotypes with andwithout vernalization
The median, the third quartile (q3), and the first quartile (q1) of thenumber of days to flowering from germination, for each of the fourgroups studied (PLNV and PLV [nonvernalized and vernalized acces-sion PL, respectively], and SENV and SEV [nonvernalized and vernal-ized accession SE14, respectively]), are shown. The test for a differenceamong medians for the four groups was significant (P , 0.0001). If onlyPLNV and PLV were compared, the median flowering time was stillsignificantly different (P , 0.001).
Figure 2. Predicted probability of survival (not flowering) for each ofthe four strata, using a nonparametric Cox proportional hazards model.The horizontal axis displays the flowering time (in days after germina-tion), and the vertical axis displays the predicted probability of notflowering (a value of 1.0 indicates none of the plants are flowering).
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Table II. CATMA GST identifiers, locus tags, and annotation for the a priori flowering time genes that had significant expression differences,all of which were differences among accessions
Positive fold changes (FC) correspond to higher level of expression in accession SE14 than in accession PL. For each gene and contrast (SE14nonvernalized [SENV] versus PL nonvernalized [PLNV], and SE14 vernalized [SEV] versus PL vernalized [PLV]), the F value (F), P value, and FDR-corrected P value (FDR) are listed.
CATMA5a47752 AT5G51810 Encodes GA 20-oxidase(GA20OX2). Involvedin GA biosynthesis.Up-regulated by far-redlight in elongating petioles.Not regulated by acircadian clock.
Talon et al., 1990; Chiang et al., 1995) and a gene en-coding GA 20-oxidase (GA20OX2; At5g51810; Xuet al., 1995), were down-regulated in SE14 comparedto PL, whereas genes encoding RGL1, a GA regulatoryprotein that represses GA signaling (Wen and Chang,2002), and SLY1 (At4g24210), a gene involved in reg-ulation of GA signaling (Dill et al., 2004), were up-regulated in the late-flowering accession SE14compared to PL. In addition, six myb-family transcrip-tion factors whose expression is affected by GA (Chenet al., 2006) were differentially expressed across acces-sions. Three of these were up-regulated in accessionSE14 (At5g65790, At5g02840, and At5g37260), and threewere down-regulated (At3g46130, At5g67580, andAt5g47390).
Other differentially expressed candidate genes forflowering included two genes in the vernalizationpathway: VIP4 (At5g61150) and FRL1 (At5g16320),both involved in regulation of FLC expression (Zhangand van Nocker, 2002; Michaels et al., 2004), and thefloral repressors EMF (At5g11530; Moon et al., 2003)and SVP (At2g22540; Hartmann et al., 2000; Gregiset al., 2006; Table II; Fig. 3). The floral pathway inte-grator SOC1 (AGL20; At2g45660; Lee et al., 2000; Moonet al., 2003) was also differentially expressed, with alower level of expression in accession SE14 than in PL(Table II; Fig. 3).
Microarray data for an additional 10,859 probeswere also analyzed for differential expression. Theexpression of a total of 1,642 differed significantly be-tween groups at 10% FDR. The largest difference ingene expression was found between nonvernalizedseedlings of accessions PL and SE14 (PLNV versusSENV, 1,493 genes). Fewer genes were differentiallyexpressed between vernalized seedlings of the twoaccessions (PLV versus SEV, 874 genes), and very fewgene expression differences were found between ver-nalized and nonvernalized seedlings (PLV versusPLNV, and SEV versus SENV, two genes). However,GO annotation of the 1,642 genes indicates that mostof these genes function in various biological proces-ses with no obvious relation to control of floweringtime (Supplemental Appendix S3). Genes differen-tially expressed by vernalization encode a Gly-rich,endomembrane-located protein (At4g29030) and amicrotubule-associated protein (MAP70-1) that havenot been implicated previously in the vernalizationresponse.
List Enrichment Analysis
We used list enrichment analysis to assess whetherthere was an overrepresentation of differentially ex-pressed genes in GO categories of relevance to flower-ing time (see ‘‘Materials and Methods’’). We found asignificant overrepresentation of significantly differ-entially expressed genes in the category ‘‘circadianrhythm’’ (20 genes in category, seven significant, two-sided P 5 2.3 3 1022, Fisher’s exact test). There wasalso a significant overrepresentation of genes involved
in GA metabolism and signaling (49 genes in cate-gory, 13 significant at FDR 0.1, two-sided P 5 4.23 3 1022,Fisher’s exact test).
Chromosomal Clustering of Differentially Expressed
Genes on Ancestral Chromosome 4
To determine whether the positions of differentiallyexpressed genes were random or clustered, we exam-ined the chromosomal position of each differentiallytranscribed probe, based on the A. thaliana genomeannotation. We found that part of A. thaliana chromo-some 2, corresponding to ancestral chromosome 4(ak4) in Capsella (Schranz et al., 2006), had a signifi-cantly higher proportion of differentially expressedgenes in the PL-SE14 comparison than overall in thegenome (0.185 of genes significant for ak4, 0.152 sig-nificant for all detected genes, x2 5 9.00, d.f. 5 1, P 52.7 3 1023). This region of A. thaliana chromosome 2constitutes an entire, separate chromosome in bothA. lyrata and Capsella rubella. In A. thaliana, it corre-sponds to approximately 10 Mb of the lower part ofchromosome 2 (delimited by the loci At2g21160 andAt2g47730) containing a total of 2,867 annotated loci.In this study, 1,235 of these were labeled ‘‘present’’ and228 were differentially expressed. In the US721-US740comparison, we found no overrepresentation of dif-ferentially expressed genes for ak4.
Verification of Differential Expression
We selected four genes for verification of the micro-array results (SUPPRESSOR OF OVEREXPRESSIONOF CONSTANS1 [SOC1], TOC1, CCA1, and FLC).Although FLC was not differentially expressed aftercorrection for multiple testing, there was some evi-dence for differential expression (P 5 0.03), and theliterature on this gene as well as the vernalizationresponse led us to include it in our panel. Real-timereverse transcription (RT)-PCR DCT values for dif-ferentially expressed candidate genes (SOC1, TOC1,CCA1) were consistent with array results (Supplemen-tal Appendix S4). Thus, we did not identify any falsepositives among the genes assessed. Analysis of real-time RT-PCR gene expression measurements indicatedthat FLC expression did not differ between accessionsprior to vernalization and was diminished after ver-nalization in both accessions, but to a greater extentin SE14.
Flowering Time Ecotypes Differ in Rhythmic Expressionof CCA1 and TOC1
Because the microarray data analysis indicated thatcircadian core genes were differentially expressed, weset up two experiments to assess differences in theexpression of circadian genes over time. The rhythmicexpression of the circadian core oscillator genes TOC1and CCA1 differed between accessions PL and SE14
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Independent biological validation of differentiallyexpressed flowering time genes was obtained in a pairof less extreme flowering ecotypes, representative ofthe average part of the flowering time distribution(Fig. 1; accessions US721 and US740). Gene expressionmicroarray analysis (see ‘‘Materials and Methods’’)indicated that a total of 97 probes for flowering timegenes, including probes for 18 of the 21 differentiallyexpressed flowering time genes in the PL-SE14 com-parison, were in common between experiments (Sup-plemental Appendix S5). As an independent biologicalvalidation, we asked whether the set of 18 floweringtime genes that were differentially expressed betweenthe extreme flowering ecotypes also had evidence fordifferential expression in the US721-US740 compari-son. Out of 33 significant contrasts between accessions
for these genes in the PL-SE14 comparison, 12 con-trasts corresponding to eight different genes were alsosignificant in the US721-US740 comparison (Table III).These genes included circadian core genes such asLHY and TOC1, as well genes involved in GA biosyn-thesis and response (e.g. GA4, RGL1, MYB48, and themyb-family transcription factor At5g02840); FRL1, agene involved in the vernalization response; and SVP,a floral repressor (Table III). Overall, this constitutesgood agreement between experiments and indicatesthat flowering ecotypes with intermediate differencesin flowering time also differ in the expression of genesregulating circadian rhythm and GA biosynthesis andresponse.
DISCUSSION
In this study we have characterized differential geneexpression between flowering ecotypes of C. bursa-pastoris, to test whether gene regulation differences inknown flowering time genes in Arabidopsis are also
Figure 3. Overview of flowering time pathways in A. thaliana. Figures are adapted from Mouradov et al. (2002), He and Amasino(2005), and Roux et al. (2006). Genes that were significantly differentially expressed between accessions are in boldface. Genesthat were up-regulated in SE14 compared to PL are marked in green, and genes down-regulated in SE14 are marked in magenta.
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responsible for natural variation in flowering time inC. bursa-pastoris. In the close relative A. thaliana, amajor part of natural flowering time variation is due tomultiple independent mutations in the FRI gene, thefunction of which is to induce FLC expression that inturn represses the transition to flowering (Johansonet al., 2000). In our experiment, quantitative RT-PCRanalysis of FLC expression showed that FLC was in-deed down-regulated in both early- and late-floweringaccessions as a result of vernalization, but did notdiffer significantly in expression between accessionsbefore vernalization. Thus, although it seems likelythat the function of FLC as an important mediatorof the vernalization response is conserved acrossA. thaliana and C. bursa-pastoris, our data shows thatsimilar mutations as those found in A. thaliana FRIhave not been important in generating natural flower-ing time variation in the C. bursa-pastoris accessions wehave studied. Other pathways and genes are morelikely responsible for natural variation in floweringtime in this species. Microarray analysis of differentialexpression between early- and late-flowering C. bursa-pastoris offered some insight as to which pathwaysthese may be. Indeed, we found a significant enrich-ment of differentially expressed genes in two of themain A. thaliana flowering time pathways, the GApathway and the photoperiodic pathway, and, morespecifically, circadian clock-related genes in the latter.The fact that different pathways seem responsible fornatural flowering time variation in A. thaliana and thestudied accessions of C. bursa-pastoris could suggestthat these species have experienced different selectiveconstraints on flowering time, or that genetic variationat flowering time genes differed between species, pro-viding different avenues to variation in flowering time.
In A. thaliana, variation in circadian rhythm amongnatural accessions contributes to fitness (Dodd et al.,2005), is correlated with latitude of origin (Michaelet al., 2003), and can cause variation in flowering time(Imaizumi and Kay, 2006). Because differences in geneexpression, especially for genes with circadian expres-sion, may be difficult to interpret based on data from asingle time point (Michael et al., 2003; Darrah et al.,2006; Keurentjes et al., 2007), we conducted a time-series study of gene expression for two core circadiangenes, CCA1 and TOC1. Both of these genes differed indiurnal expression between the early-flowering PLand the late-flowering SE14 ecotype. In A. thaliana,changes in rhythmic expression of CCA1 or TOC1 haveeffects on flowering time (Strayer et al., 2000; Alabadiet al., 2001; Mizoguchi et al., 2002). Interestingly, in ourmicroarray experiment, CKB4, which encodes a regu-latory subunit of casein kinase II and leads to changesin circadian period and phase in A. thaliana whenoverexpressed (Perales et al., 2006), was up-regulatedin the early-flowering accession PL. Circadian rhythmis a crucial component in the now generally acceptedexternal coincidence model (Bunning, 1936). In a mo-lecular version of this model, the circadian clock gen-erates daily oscillation of CONSTANS (CO) mRNA. Asprotein stability of CO is controlled by light, the coin-cidence of light and high CO expression that onlyoccurs in long days induce the pathway integrator FTand thereby flowering (Valverde et al., 2004; Corbesieret al., 2007). Thus, alterations in genes controlling thecircadian clock are attractive candidates for the evo-lution of flowering time differences in C. bursa-pastoris.
The GA pathway was also enriched for differentiallyexpressed genes among the early-flowering accessionPL and the late-flowering accession SE14. In A. thaliana,
Figure 4. Normalized expressionlevels (CTtarget 2 CTreference) for CCA1and TOC1 at 12 time points fol-lowing entrainment. Mean expres-sion levels are indicated by blacksymbols for accession PL and whitesymbols for accession SE14. Errorbars indicate SE of the mean. Thetop pair of plots shows CCA1 andTOC1 expression in constant light,and the bottom pair shows theirexpression under long-day condi-tions.
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the GA pathway is generally considered as a defaultpathway acting mainly when flowering is not inducedby long days. Although gene expression differencesfor genes in the GA pathway might be important forflowering time variation in C. bursa-pastoris, an attrac-tive alternative hypothesis is that these expressiondifferences are a secondary effect of altered circadianclock function, and that this altered clock functionaffects flowering time mainly through other pathways(e.g. through CO and FT). In this study, two GAbiosynthesis genes displayed a higher expression inearly flowering accession PL as compared to SE14,which might well be an effect of altered clock function(Blazquez et al., 2002). Blazquez et al. (2002) furtherconcluded that GA contribution is not quantitativelyimportant in the determination of flowering time bythe photoperiod pathway in A. thaliana. Rather, theincrease in GA concentration induced by long daysmight be relevant for cell expansion required duringstem elongation, rather than the determination offlowering time.
Differential expression of flowering time genes wasbiologically validated in a pair of less extreme flower-ing ecotypes from North America. The good agree-ment of flowering time gene expression differencesbetween both pairs of accessions could indicate thatthe genetic basis of expression differences is shared bycommon ancestry, or that similar regulatory differ-ences have evolved in parallel. Although the two pairsof accessions were sampled in widely different geo-graphical regions (the extreme flowering ecotypes PLand SE14 from Taiwan and Sweden, respectively, and
the less extreme flowering accessions US721 andUS740 from the United States), a shared genetic back-ground is not unlikely, as the species has apparentlyattained its present distribution recently (Ceplitiset al., 2005). Indeed, both early- and late-floweringC. bursa-pastoris accessions were introduced into NorthAmerica by European settlers (Neuffer and Hurka,1999). To resolve the genetic basis of gene expressiondifferences, a natural extension of this study is to mapgene expression as a quantitative trait, as has been donee.g. in yeast (Brem et al., 2002), maize (Zea mays),humans, and mice (Schadt et al., 2003) and in A. thaliana(Keurentjes et al., 2007).
Overall, most genes differed in expression acrossaccessions, and not as a result of the vernalizationtreatment, although vernalization had an effect onflowering time. This could indicate that vernalizationaffected the expression of very few genes, or that theeffect on gene expression was generally small so thatwe had limited power to detect these differences.Similar results have been obtained in other species,for example, in Lolium perenne, where cDNA micro-array analysis identified only a handful of genes dif-ferentially expressed as a result of vernalizationtreatment (Ciannamea et al., 2006). In A. thaliana, sev-eral known components of the vernalization pathwayare not themselves regulated by vernalization (VRN1,VRN2) or regain their normal level of expression uponreturn to warmer temperatures (VIN3; Levy et al., 2002;Wood et al., 2006). Indeed, localized modification ofFLC chromatin may be the main underlying mechanismfor vernalization response in A. thaliana (Bastow et al.,
Table III. Independent biological validation of differentially expressed flowering time genes
The table shows the contrast P values for the independent biological validation of differentially expressedflowering time genes, with contrasts significant in the PL-SE14 comparison written in italics. The groupdesignations are as follows: nonvernalized accession US721 (721NV), nonvernalized accession US740(740NV), vernalized accession US721 (721V), and vernalized accession US740 (740V).
2004; He et al., 2004; Sung and Amasino, 2004; Shindoet al., 2006; Swiezewski et al., 2007). Interestingly, weidentified two novel vernalization-responsive genes,a cortical microtubule-associated protein (MAP70-1;Korolev et al., 2005) and a Gly-rich, endomembrane-located protein (At4g29030). Whether these expressionchanges are involved in vernalization is unclear, butthey could be related to cold acclimatization becausechanges in membrane composition and cytoskeletal or-ganization are both believed to play a role in this pro-cess (Browse and Xin, 2001).
Most of the differentially expressed genes werescattered across different chromosomal regions. How-ever, the proportion of significant genes (out of alldetected genes) was higher than expected for ancestralchromosome 4, which corresponds to the lower partof A. thaliana chromosome 2 (Schranz et al., 2006). Noclear signs of amplification or deletion of specific chro-mosomal regions were observed, with approximatelyequal numbers of genes up- and down-regulated ineach flowering ecotype. Chromosome-scale transcrip-tional profiling in rice (Oryza sativa) and Arabidopsishas identified variation in transcriptional activity acrosschromosomes (Li et al., 2005; Schmid et al., 2005). Suchvariation has been shown to be correlated with tissueand developmental stage as well as external factorssuch as cold stress (Yamada et al., 2003). A recentstudy on gene expression diversity among genotypesin A. thaliana (Kliebenstein et al., 2006) also reported acorrelated variation of DNA sequence divergence andexpression variation along chromosomes. In C. bursa-pastoris, increased localized sequence divergence be-tween extreme flowering ecotypes or differences inchromatin structure between these accessions couldexplain the observed clustering of differentially ex-pressed genes.
In this study we have characterized gene expressiondifferences between early- and late-flowering acces-sions of C. bursa-pastoris. Flowering time variation mayhave evolved rapidly in this species and is probably ofadaptive importance (Ceplitis et al., 2005). We haveshown that natural variation in the C. bursa-pastorisflowering time ecotypes we have studied is likely notcaused by variation at the FRI gene, as in A. thaliana.Instead, the evolution of flowering time variationappears to have involved changes in the expressionof genes regulating the circadian rhythm, and possiblyalso regulatory changes in the GA pathway. Whilefurther study is needed to elucidate the full pathwayand mechanisms involved, genes involved in regula-tion of the circadian clock, such as CCA1 and TOC1,clearly constitute strong candidates for adaptive evo-lution in C. bursa-pastoris.
MATERIALS AND METHODS
Flowering Time
We compared vernalized and nonvernalized plants for each of the two
accessions (PL and SE14). Thus, for this experiment there were four groups:
