Photoperioddependent floral reversion in the ... - Wiley Online Library

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Photoperiod-dependent floral reversion in the natural allopolyploid Arabidopsis suecica Erin McCullough1, Kirsten M. Wright1, Aurelia Alvarez1,2, Chanel P. Clark1, Wayne L. Rickoll1 and Andreas Madlung1 1

Department of Biology, University of Puget Sound, 1500 N. Warner St, Tacoma, WA 98416, USA; 2Sciences Department, 3240 Fort Rd, Heritage

University, Toppenish, WA 98948, USA

Summary Author for correspondence: Andreas Madlung Tel: +1 253 879 2712 Email: [email protected] Received: 18 October 2009 Accepted: 3 November 2009

New Phytologist (2010) 186: 239–250 doi: 10.1111/j.1469-8137.2009.03141.x

Key words: allopolyploid, Arabidopsis suecica, environmental effect on flowering, floral reversion, flower reversion, photoperiod, polyploidy.

• Flower reversion is the result of genetic or environmental effects that reverse developmental steps in the transition from the vegetative to the reproductive phase in plants. Here, we describe peculiar floral abnormalities, homeotic conversions, and flower reversion in several wild-type accessions of the natural allopolyploid Arabidopsis suecica. • Microscopy was used to illustrate the phenotype in detail and we experimented with varying photoperiod lengths to establish whether or not the phenotype was responsive to the environment. We also profiled the transcriptional activity of several floral regulator genes during flower reversion using real-time PCR. • We showed that the frequency of floral reversion was affected by day length and the position of the flower along the inflorescence axis. In reverting flowers we found unusual gene expression patterns of floral promoters and inflorescence maintenance genes, including lower mRNA levels of AGAMOUS-LIKE-24 (AGL24), APETALA1 (AP1), and SHORT VEGETATIVE PHASE (SVP), and higher mRNA levels of SUPRESSOR OF CONSTANS1 (SOC1) compared with normal flowers. • We conclude that the floral reversion frequency in A. suecica is susceptible to photoperiod changes, and that the floral abnormalities coincide with the competing expression of floral promoters and floral repressors in reverting floral tissue.

Introduction Patterning of the plant body in angiosperms is dependent on the development of groups of cells that are kept in an undifferentiated state, called the meristems. As long as the plant develops vegetatively, the shoot apical meristem continually adds leaves, stems, and axillary buds to the above-ground part of the plant, while the root apical meristem patterns the root development (Poethig, 2003). Genetic and environmental cues trigger changes in the meristem as the plant transitions from the juvenile to the reproductive phase. These changes cause a developmental switch from generating vegetative tissues to producing an inflorescence, which subsequently gives rise to flowers (Mouradov et al., 2002). Flowers are usually determinate structures of the angiosperm body plan that can result in the production of seeds to complete the plant’s life cycle, and lead in annuals to senescence of the entire plant body and ultimately to death

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(Nooden & Penney, 2001). In perennial plants, on the other hand, flowering and vegetative growth can alternate between seasons over many years after the plant has reached maturity. In many cases, perennial plants set aside vegetative meristems that do not undergo a change from the juvenile to the adult phase, allowing the plant to resume growth after flowering and restricting senescence locally within the plant body (Battey & Tooke, 2002). A reversal from determinate to indeterminate growth, called floral reversion, is a naturally occurring but relatively uncommon phenomenon, in which the inflorescence switches from terminal flower development back to a developmentally earlier phase, thus avoiding senescence (Battey & Lyndon, 1990). Two types of reversion can be distinguished: inflorescence reversion and flower reversion. In inflorescence reversion the development of inflorescences is followed by vegetative growth, rather than the production of terminal flowers. In flower reversion, incomplete or abnormal flowers are formed, which can bear either

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unusual numbers or types of floral organs, or resume indeterminate development after or during organ formation (Tooke et al., 2005). The role of flower reversion in the life history of a plant is still poorly understood. In Metrosideros excelsa (Sreekantan et al., 2001) or Cheiranthus cheiri (Diomaiuto, 1988), flower reversion might be a mechanism for the plant to pursue a perennial lifestyle. Alternatively, floral reversion may allow the plant to adjust expanding energy in flower production only during times when appropriate pollinators are present, presumably coinciding with circumstances the plant has evolved to use as cues to initiate flowering. When floral reversion occurs naturally, it is usually caused by a change in environmental conditions, such as temperature, light, or humidity. If such a change occurs at a time when the meristem is not yet fully committed to flowering, the removal of the cues normally leading to flower induction can lead to the reversion (Battey & Lyndon, 1990; Tooke & Battey, 2000; Tooke et al., 2005). Examples of environmentally caused floral reversions (Battey & Lyndon, 1990) have been documented in a number of species, including Impatiens balsamina (Battey & Lyndon, 1984, 1986), Glycine max (Washburn & Thomas, 2000; Wu et al., 2006), and Whytockia bijieensis (Wang, 2001). Although the molecular foundation of flower development in model plants, such as Arabidopsis thaliana, has been intensely studied, the molecular reasons for floral reversion are still not fully understood. In A. thaliana there are four major pathways involved in the induction of flowering, which are regulated by plant age (autonomous pathway), photoperiod, exposure to cold temperatures (vernalizaton), and the phytohormone gibberellin (Henderson et al., 2003; Ba¨urle & Dean, 2006). All four pathways converge on integrator proteins, including FLOWERING LOCUS C (FLC), CONSTANS (CO), SUPPRESSOR OF CONSTANS (SOC1), and LEAFY (LFY), which ultimately orchestrate the transition from the vegetative to the reproductive phase (Mouradov et al., 2002; Boss et al., 2004; Turck et al., 2008). SOC1 promotes flowering by activating LFY (Lee et al., 2008), but SOC1 is itself transcriptionally inhibited by FLC (Borner et al., 2000; Hepworth et al., 2002; Searle et al., 2006). SOC1 activation of LFY appears to be dependent on its co-localization with AGAMOUSLIKE 24 (AGL-24) (Lee et al., 2008). Paradoxically, AGL24 expression must be repressed in flower meristems or flowers will revert back to an inflorescence, even if floral organ identity genes have been activated (Yu et al., 2004). Repression of AGL24 in the appropriate tissues is achieved in a feedback loop by LFY and APETALA-1 (AP1) (Yu et al., 2002, 2004; Gregis et al., 2006). AGL24 therefore appears to be a central promoter of inflorescence fate by first promoting the transition from the vegetative to the inflorescence phase but then preventing its further transitioning to flowering (Yu et al., 2004).

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Once LFY and AP1 are active, they in turn activate floral homeotic genes, including AGAMOUS (AG), thus aiding in the transition from an inflorescence meristem to a floral meristem and specifying the floral organs inside the flower (Weigel & Meyerowitz, 1993; Jack, 2004; Turck et al., 2008). LFY and AP1 also promote floral transition by restricting the expression of TERMINAL FLOWER 1 (TFL1) to the center of the inflorescence meristem and thereby establishing floral meristem identity in the cells of the periphery of the apex (Blazquez et al., 1997; Hempel et al., 1997). Floral transition is repressed by SHORT VEGETATIVE PHASE (SVP) (Hartmann et al., 2000; Gregis et al., 2006), which can act redundantly with AGL24 (Gregis et al., 2006, 2008), and represses SOC1 transcription by binding to its promoter (Li et al., 2008). After the floral meristem identity is established, AG continues to be required to maintain the determinate and reproductive state of the meristem (Okamuro et al., 1996; Gregis et al., 2006). Mutations in AG, LFY, or AP1 can lead to partial or complete floral reversion (Okamuro et al., 1996; Mizukami & Ma, 1997; Parcy et al., 2002). Reversion phenotypes in wild-type accessions of A. thaliana are rare (Okamuro et al., 1996). Likely the most severe type of reversion reported in natural A. thaliana was observed in the ecotype Sy-0 from the Isle of Skye in Scotland (UK), where a gain-of-function variant of the RNA-binding protein HUA2 is responsible for late flowering via an unknown mechanism activating the floral repressor FLC and repressing AG (Poduska et al., 2003; Wang et al., 2007). Here, we describe, molecularly and morphologically, a new, remarkably plastic flower reversion phenotype in Arabidopsis suecica, an allopolyploid formed by the hybridization of A. thaliana and A. arenosa between 20 000 and 300 000 yr ago (Jakobsson et al., 2006). The plant produces both normal and reverting flowers. We report variation in the gene expression of several meristem identity or meristem maintenance genes between the two types of floral tissues in the plants, and show that environmental effects have profound consequences on the plasticity of the response in this species.

Materials and Methods Plant materials and growth Three different genetic accessions of A. suecica were used in this study: Sue1 (CS22505), Sue16 (CS22516), and Sue19 (CS22517). All lines can be obtained from the Arabidopsis Biological Resource Center (ABRC; http://www.arabidopsis.org). Plants were sown in soil (Sunshine Mix #4; Sungrow Horticulture, Vancouver, BC, Canada) in 10-cmdiameter pots and cold-treated for 3–4 d. Plants were fertilized approximately biweekly with equal amounts of


New Phytologist N-P-K (24-8-16) fertilizer according to the manufacturer’s recommendations (Miracle-Gro, Marysville, OH, USA). For day-length experiments, plants were grown in growth chambers (Percival, Perry, IO, USA) for long days with 16 h of light (20C) followed by 8 h of darkness (18C) or shortened days (12 h light, 20C: 12 h dark, 18C). Light intensities in the growth chambers were between 90 and 140 lmol m)2 s)1 at plant level. Flowering time To measure time to flowering, sets of plants were grown in four different plantings between 2007 and 2009, and the number of days between sowing and flowering was calculated. Seeds were planted directly in the soil in 20-cm-diameter pots and grown under glasshouse conditions as described earlier. Plants were considered to flower when the inflorescence shoot had reached a height of 1 cm or the first flower had opened, whichever occurred earlier. Statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) or VassarStats (http://faculty.vassar.edu/ lowry/VassarStats.html). Microscopy Flower phenotypes were photographed using a Zeiss dissecting microscope with a Nikon Coolpix 4200 digital camera mounted on a custom-made phototube attached to an ocular. All photographs were linearly adjusted for contrast and in most cases the background was blackened to eliminate plant tissues outside of the focal plane using Adobe Photoshop 7.0.1 software (Adobe, San Jose, CA, USA). For light microscopy of sectioned material, pre-anthesis flower buds were fixed under vacuum at room temperature in 2% glutaraldehyde, 2% paraformaldehyde, 2% acrolein, 2% dimethyl sulfoxide, and 1 mM calcium chloride in 0.05 M sodium phosphate buffer (pH 7.0) for 30 min, and then on ice for an additional 2 h. Buds were washed in 0.1 M sodium cacodylate buffer (pH 7.0) and post-fixed for 2 h on ice in 2% osmium tetroxide in 0.1 M sodium cacodylate. Subsequently, the buds were washed three times in distilled water and kept in 1% uranyl acetate at 5C overnight. The tissue was dehydrated through a graded series of ethanol to propylene oxide, and infiltrated with agitation over 2 d through a graded mixture of propylene oxide ⁄ Spurr’s resin to 100% resin. After 24 h in 100% resin, flowers were embedded in Spurr’s resin (hard mixture) and cured for 24 h at 70C. Sections were cut on a Leica Ultracut UCT at 800 nm thickness using a Diatome Histo diamond knife and stained with 1% toluidine blue in 1% sodium borate for light microscopy. All chemicals were purchased from Sigma-Aldrich. Slides were viewed using a Zeiss microscope. Digital images were acquired with a


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MicroPublisher 3.3 digital camera and QCapure software 2.71 (both from Quantitative Imaging Corporation, Surrey, BC, Canada). Whole images were cropped if needed and linearly adjusted for contrast, brightness, and color using Adobe Photoshop 7.0.1. Analysis of frequency and day-length dependence of floral abnormalities To determine if floral abnormalities occurred preferentially during a certain developmental period and whether or not day length was influential in this process, we evaluated each silique along the main inflorescence axis from the oldest (most basipetal) to the 40th silique in plants grown in either long days or short days. We classified each silique as sterile (no seeds and shorter than 5 mm), normal (containing seeds), or having undergone any degree of reversion (receptacle resuming elongation beyond the floral scar leading to a new inflorescence or swollen ‘bud’; see arrowheads in Figs 1j and 4b). In all analyses we compared reverting flowers against the sum of sterile and normal siliques. Statistical significance of overall reversion frequencies was determined using two-tailed t-tests (GraphPad, La Jolla, CA, USA). Reverse transcription and real-time PCR To assess transcriptional activity of flower development genes, we performed real-time and reverse transcription PCR. We chose uniform tissues of reverting flowers where new buds developing within an original flower were about to break through (Fig. 4b). Tissues that were not used at this stage and that were allowed to develop further went through the phases pictured in Fig. 4c,d. For the wild-type comparison, flower buds just about to open were used (Fig 4e). Both types of tissue were harvested from the same individuals of accession Sue1. RNA was extracted from two pools each of either normal flower buds or reverting flowers using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and treated with 2 units of DNAse (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Three micrograms of the treated RNA was reverse-transcribed with 4.8 lM random hexamer primers (Amersham, Piscataway, NJ, USA), 5· firststrand buffer, 100 mM dithiothreitol (DTT), 2.5 mM deoxyribonucleotide triphosphates (dNTPs), and 200 units of Moloney Murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). The 25 ll volume mixture was incubated for 10 min at 25C, 45 min at 39C, and 5 min at 85C. Real-time quantitative PCR was performed using a MiniOpticon Real-Time PCR Detection System (BioRad, Hercules, CA, USA). We examined the relative expression of candidate genes AG, AGL-24, AP1, FLC, LFY, SOC1,


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Fig. 1 Floral mutations in natural Arabidopsis suecica. Flower buds from A. suecica show either a normal phenotype (a), or phenotypes resembling homeotic mutations in c. 1–2% of flowers on every plant: reversion from floral to inflorescence meristem (b, d, h, i); duplication of floral organs (c); fusion of anther and petal (e); open carpels (f, arrowhead points to an uncovered ovule); extra petals (c, d, g–i); formation of a new inflorescence inside a developing, fertile carpel (j, k); inflorescence stem fasciation (m vs normal in l). Arrowheads in (j) indicate the scar of the floral bud in normal (top) vs reverting flower (bottom). Bars, 1 mm (a–i); 1 cm (j, l, m); 0.5 cm (k).

and SVP using 2 ll of cDNA in 20 ll PCR reactions of a ready-mix PCR cocktail (iQ SYBR Green Supermix; BioRad) and 6 pmol of each primer. PCR cycling conditions after an initial 3 min at 95C were set at 94C (30 s), 59C (45 s), 72C (60 s) and repeated 40 times for all genes. Primers were designed to the published A. thaliana genome sequence using Primer3 software (http://www. primer3.sourceforge.net) to detect the combined transcripts of the A. thaliana and A. arenosa parents in the allopolyploid genome (for primer sequences, see Supporting Information, Table S1). As internal control, 18S rDNA was used (Davison et al., 2007). Reactions were performed twice in triplicate for every gene using two reverting or wild-type biological replicates each. Melting curve analysis was performed on all reactions to verify the quality of the desired gene product. Fold difference in relative gene expression between normal and reverting flowers was calculated using the DDCt method (Livak & Schmittgen, 2001). The fold difference in gene expression was averaged across all


biological and technical replications per gene (n = 12 for AG, AGL-24, AP1, FLC, LFY, and SOC1, and n = 10 for SVP). To determine if the intensity of gene expression of normal buds was statistically significantly different from gene expression of reverting flowers, we performed paired t-tests on the normalized cycle number at the threshold level of each gene between normal and reverting flowers with a = 0.05 (SPSS 13.0; SPSS Inc.).

Results Natural A. suecica displays multiple abnormal floral phenotypes Plants grown under controlled long-day or glasshouse conditions produced flowers with a wide range of phenotypic abnormalities that were never observed in hundreds of plants of the original parental species in various ecotypes of A. thaliana and A. arenosa raised in the same growth


New Phytologist facilities (A.M., unpublished observations). Abnormal flowers occurred at variable frequencies among different accessions and individual plants. The most frequent phenotypic abnormality was a reversion of self-terminating flower development into an indeterminate inflorescence (Fig. 1). In some cases, a new inflorescence replaced the entire carpel, leading to flowers with sepals, petals, stamens and a central multibud inflorescence (Fig. 1b). This reversion usually gave rise to an additional two to 10 flowers, approximately, which were fertile and produced seed. Offspring from this seed were not any more or less likely to produce abnormal floral phenotypes (data not shown). Floral homeotic phenotypes (Fig. 1c–h) were observed in c. 2–5% of abnormal flowers. The most frequent observation was an increased number of petals (Figs 1c,d,h,i, 2c–e), while duplicated carpels (Fig. 1c) were extremely rare. Combinations of phenotypes, such as additional petals and replacement of the carpel with a new inflorescence, were more frequent (Figs 1d,g–i, 2c,f). In some flowers, organ fusions occurred between petals and stamens (Figs 1e, 2c–e). In some cases, these fused organs resembled a petal more than a stamen, while in other cases the stamen consisted of pollen-bearing anthers on top of filaments fused to white petal tissue, like a flag on a flagpole (not shown). Carpel phenotypes, when not replaced by inflorescences, ranged from normal to an apetala-like appearance. In these carpels, the ovary walls occasionally split open, allowing a view of the ovules inside (Fig. 1f). In some cases, the carpel was split and an ectopic inflorescence emerged from within (Fig. 1g,h). The most common phenotype associated with flower reversion was a swelling of the carpel (Fig. 1i–k). Such swelling usually developed inside an initially normal looking carpel and increased after petal fall (Fig. 1i shows a transition state), with the receptacle resuming elongation growth resembling a new peduncle. Figure 1j shows a normal developing silique after pollination next to a swollen carpel of similar age. Arrowheads indicate the floral scar where sepals, petals, and stamens originated. Some swollen carpels eventually stopped developing further and dried up, while others burst through the ovary wall, giving rise to a new inflorescence. Most often, these new inflorescences developed ectopically at the most proximal end of the carpel. Figure 1k shows a swollen carpel sliced open from the proximal (top in Fig. 1k) to the distal (bottom in Fig. 1k) end. An inflorescence can be seen at the proximal end, while the distal end displays several ovules. Swollen carpels like this one gave rise to new fertile inflorescences and produced fertile seeds themselves. Floral meristem reversion leading to swollen carpels was by far the most frequent abnormal phenotype, with c. 95– 98% of all abnormal flowers observed showing reversion, and the rest displaying either additional petals, organ fusions, or homeotic conversions.


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In addition to floral abnormalities, we observed fasciation (Fig. 1m). A fasciated inflorescence is characterized by a laterally enlarged meristem that produces ribbon-like inflorescence stems. In A. suecica these stems can vary in width from barely wider than normal to more than 10 times the width of an ordinary inflorescence stem. Fasciated stems may produce flowers that can develop normally and often bear fertile seeds. While more than half of all individuals of accession Sue16 were affected by fasciation to some degree, fasciated inflorescences, ranging from mild to severe in phenotype, were observed in only c. 15% of all Sue1 individuals (A.M., unpublished). Light microscopy revealed that, occasionally, multiple phenotypes occurred in the same flower, such as floral reversion and petal–anther fusions (Fig. 2c) or supernumerary petals combined with petal–anther fusions (Fig. 2c–f). Microscopy showed that flowers with abnormal petals either added additional normal petals or produced petal-like tissues that were wider than normal or displayed extra folds and additional vascular bundles (Fig. 2f). Although reverting inflorescences usually developed flowers bearing fertile seeds, we frequently observed misshaped pollen grains, or pollen that appeared empty in abnormal flowers (Fig. 2f). Floral reversion often began at the proximal end of the carpel, with ectopic shoots developing from underneath the fully developed and usually fertile carpel (Fig. 1k). Occasionally, however, flower reversion appeared to occur before the carpel was fully developed, replacing all ovaries with new floral tissue (Fig. 2g). Here, the new inflorescence developed a new peduncle (Fig. 2g). In some cases, flower reversion did not seem to proceed in an organized fashion. In those cases a mixture of floral tissues was produced that did not result in separate whorls of organs and usually did not produce a mature flower but instead dried up and died before breaking through the carpel walls (Fig. 2h). A likely extension of this phenotype was seen in swollen carpels that did not contain any healthy tissue. These structures were characterized by either underdeveloped or deteriorated tissue that was organized in separate new flower buds but only showed rudimentary similarity to normal flower organs (Fig. 2i). The effect of day length on flowering Initial phenotypic observations were done on plants that were raised in the glasshouse under varying light conditions throughout the year. Some plants displayed almost exclusively normal flowers, while other individuals, particularly among accession Sue1, showed a high degree of floral reversion or homeotic floral phenotypes. Flowering time measurements in these plants showed that Sue1 flowered statistically significantly later than Sue16 and Sue19 (Table 1). To test if flower reversion was affected by day length or genetic background, we grew plants of the three


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Fig. 2 Meristem reversion and organ malformation in natural Arabidopsis suecica. Flower buds were fixed in glutaraldehyde, embedded in plastic, sectioned, and stained with toluidine blue. (a, b) Normal flowers: longitudinal (a); cross-section (b). (c, h) Developing inflorescence in place of the carpel; (c–e) fusions of petals and anthers; (d) extra petals; (f) malformed pollen; (g, h) replacement of carpel with inflorescence tissue on a new peduncle; mildly (h) to severely (i) deteriorated floral tissue inside developing carpel. ap, anther–petal fusion; cw, carpel wall; dft, deteriorated floral tissue; mp, malformed pollen; pd, peduncle; pxf, petals with extra folds; tr, tissue reverted to new inflorescence. Bars, 0.8 mm.

accessions in short- or long-day conditions and determined the frequency of flower reversion on their first 40 flowers. We observed an earlier onset and greater frequency of flower reversion in short days in all three accessions both when each accession was compared individually and when all data from the three accessions were combined (not shown). The effect was strongest in Sue1 (Fig. 3, Table 2). Plants kept in short-day conditions showed floral reversion


in c. 5% of flowers at position two (i.e. the second oldest flower), which increased in frequency to 79% in position 12, tapering off towards more acropetal flowers (7% at position 40). A similar trend was seen in long-day plants, although onset of floral reversions was not noticeable before the fifth flower and the frequency of reversion reached its maximum of 71% at position 18 before also tapering off as the plants matured. The cut-off at position 40 was chosen


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Table 1 Flowering time variation in accessions of Arabidopsis suecica Accession

Days to floweringa

Significance groupsb

Sue1 Sue16 Sue19

72 ± 2.1 59 ± 1.3 61.9 ± 2.2

A B B

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Averages are shown ± SE. n = 27, 40, 34, for Sue1, Sue16, and Sue19, respectively. b A one-way ANOVA (F = 12.78; P < 0.0001), and a Tukey HSD post hoc test were performed. Accessions were grouped by significance using the Tukey HSD test (P < 0.01).

as the oldest flower common to all individuals tested. In some cases, plants grew considerably longer, producing additional flowers that were almost exclusively normal. Had we included all additional information for flowers at position 41 or higher, the overall frequency of reversion would thus have been considerably smaller. To assess the overall frequency of reversion in each of the three accessions tested, we determined the average number of reverting flowers per plant. Reversion frequency in all three accessions was statistically significantly higher in short-day plants compared with long-day plants (Table 2). Reverting floral buds exhibit altered expression levels of key floral regulators To test whether or not flower reversion in A. suecica is dependent on meristem identity or meristem maintenance genes, we measured the transcriptional activity of the floral repressors FLC, TFL1, and SVP; pathway integrators, such as LFY, AGL24, and SOC1; and floral patterning genes, including AP1 and AG. We defined a narrow range of phenotypes to be used for this study (Fig. 4) to ensure that changes in gene expression were not the result of the

Fig. 3 Position of reverting flowers along the inflorescence axis during development. Arabidopsis suecica plants (accession Sue1) were grown under 16 h (long day) or 12 h (short day) conditions. Flowers along the main inflorescence were visually inspected for exhibition of the reversion phenotype from the oldest (most basipetal) flower up to the 40th flower. n = 19 (short days); n = 14 (long days).


Table 2 Statistical analysis of day-length effect on floral reversion No. mutant % mutant flowers ⁄ plant flowers ⁄ plant df Sue1, SD Sue1, LD Sue16, SD Sue16, LD Sue19, SD Sue19, LD

17.6 13.4 6.0 4.4 4.8 2.9

44.1 33.6 15.0 10.9 11.9 7.4

t-statistic P-value

78 2.1302

0.0363

78 2.131

0.0362

78 2.408

0.0183

SD, short-day; LD, long-day.

breadth of abnormal phenotypes described in Fig. 1. We used both semiquantitative reverse transcription (data not shown) and real-time PCR to compare gene expression between normal flower buds and reverting flowers. Both types of PCR analysis showed similar results. Reverting flowers expressed several inflorescence and floral meristem identity genes at statistically significantly different levels than did normal flower buds (Fig. 5, Table S2), while other genes were only marginally or not at all affected. Specifically, we found that the expression of three genes, AGL-24, SVP, and AP1, was lower in reverting flowers. Expression of FLC, AG, and LFY was barely affected in reverting flowers compared with wild-type buds, and expression of SOC1 was higher in reverting flowers compared with normal buds. TFL1 expression was not detected in either of the two tissues using reverse transcription PCR (data not shown).

Discussion We have described novel, plastic reversion phenotypes in the natural allopolyploid species A. suecica at frequencies and in severity that have to our knowledge not been described for wild-type plants in the genus Arabidopsis. Some aspects of the phenotype described here for A. suecica resemble the short photoperiod-dependent floral reversion previously observed in A. thaliana homozygous ag-1 or heterozygous lfy-6 mutants (Okamuro et al., 1996; Parcy et al., 2002). The A. thaliana ecotype Sy-0 also exhibits infrequent floral reversions as a result of a genetic mutation in the AERIAL ROSETTE-1 (ART-1) gene, which activates FLC in Sy0 (Poduska et al., 2003; Wang et al., 2007). The unstable homeotic phenotypes and floral reversions described here for A. suecica are, by contrast, were frequent even under long-day conditions, and were not caused by a known genetic lesion. Flower reversion increases in short days Floral reversions have often been observed as a result of environmental changes that cause the removal of inductive conditions, leading to a reversal of flowering (reviewed in Battey & Lyndon, 1990; Tooke et al., 2005). This and the


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Fig. 5 Fold difference in relative expression of genes in reverting floral buds compared with nonreverting buds of Arabidopsis suecica. Real-time PCR was performed on cDNA from wild-type and reverting buds. Expression of AGL-24, AP1, and SVP was lower, while expression of SOC1 was greater in reverting floral buds as compared with normal buds (P < 0.005). Changes in LFY, and FLC were very small (although statistically significant, P < 0.05). Transcription in AG was not statistically significantly different between the two tissue types (see Supporting Information, Table S2, for complete statistics). Error bars, ± SE.

short-day dependency for floral reversion in ag-1 and heterozygous lfy-6 A. thaliana led us to test the hypothesis that floral abnormalities in A. suecica in long-day conditions might even be exacerbated in short-day conditions. We show that floral reversion was statistically significantly more


Fig. 4 Phenotypes of flowers used in transcriptional analysis. (a) Arabidopsis suecica wild-type inflorescence; (b) example of a reverting flower bud used for transcriptional analysis; (c) reverting flower bud at advanced developmental stage. Numerous flowers open up from a new inflorescence that has developed from a new branch below the original carpel. Additional carpels, petals and stamens are clearly distinguishable. (d) Fully reverted flower bud with mature inflorescence and fully developed siliques. The original carpel walls are still visible at the basipetal end. (e) Representative flower bud of normal inflorescence at the time of harvest for wildtype comparison in expression analysis. Bars, 0.25 cm.

common for plants grown in short-day conditions (Fig. 3, Table 2), but did not affect the frequency of homeotic mutations that we described in Fig. 1b–h (data not shown). A. suecica is native to central and northern Scandinavia and flowers in its natural habitat from May to June (Andersberg, 2009), where photoperiods range, depending on latitude, from 16–19 h of daylight in May, to 19–24 h in June (http://www.usno.navy.mil/). It may therefore be possible that 16 h day-length requirements for A. suecica are adequate in the wild during its early inflorescence development, while later during the bloom season the plant is adapted to increasing photoperiods and thus its floral meristems do not become fully committed to flowering under the growthroom or glasshouse conditions used in our experiments. A requirement for an increase to photoperiods greater than 16 h during mid-bloom might also contribute to flower reversion. This requirement may not be necessary during late-season flowering when day length in its natural habitat has returned to 16 h, explaining why flower reversion occurred most frequently during peak bloom, and less frequently in the youngest, most basipetal, and oldest, most acropetal flowers (Fig. 3). Plants grown in short-day conditions generally started flowering later than those grown in long-day conditions (data not shown). This indicates that flower initiation in A. suecica is not dependent on long-day conditions, but suggests that a threshold of signal accumulation has to be passed before flowering can commence. Our data also show that once flowering starts in weak inductive short-day conditions, flower reversion becomes more likely even in the most basipetal flowers, suggesting that the


New Phytologist flower-inducing signal never reaches optimal intensity inside the floral meristem (Fig. 3). Since the flower-promoting gene SOC1 is responsive to the photoperiod pathway, showing increasing transcript abundance in longer days in A. thaliana (Samach et al., 2000), one might assume that its expression levels could present a connection between photoperiod and reversion frequency. While all our transcript analyses (Fig. 5) were performed only in long-day grown plants and lack the direct comparison, our data do suggest that such a connection between photoperiod and reversion frequency is unlikely, given that SOC1 expression levels are high in reverting buds of plants grown in long-day conditions, which displayed less frequent reversion than plants grown in shorterday conditions (Fig. 3). Accession Sue1 displayed approximately three times as many floral reversion events as accessions Sue16 and Sue19, suggesting that, similar to A. thaliana ecotypes, the genetic background plays a role in the variability of the phenotypic expression. Furthermore, the high frequency of reversion in Sue1 is correlated with its overall later flowering time when compared with accessions Sue16 and Sue19 (Table 1). Since the exact collection locations for the three accessions are not known, it remains unknown if the difference in reversion response reflects a difference in geographic or environmental adaptation. Taken together, our data show that day length plays a role in floral reversion in A. suecica, and suggest the possibility that adaptation to transiently longer photoperiods may require continuously increasing flower-inducing signals to maintain normal flowering. Flower reversion coincides with opposing signals from floral repressors and floral activators The underlying physiological or molecular mechanisms for floral reversion seem to vary between species. In Impatiens, removal of a leaf-borne flower-inducing signal leads to reversion back to the vegetative state (Tooke & Battey, 2000), suggesting that the meristem in Impatiens does not become irreversibly committed even under optimal environmental conditions. By contrast, flower reversion in many other species, including A. thaliana, never results in a reversal back to the vegetative phase but only in the resumption of inflorescence production (Parcy et al., 2002; Wang et al., 2007). Because the homeotic mutations and floral reversion phenotypes in A. suecica (Figs 1, 4) were not caused by any known or obvious genetic mutations, yet resembled known mutant floral phenotypes in A. thaliana, we reasoned that the activity of genes involved in the regulation of the transition from inflorescence to floral meristem might be altered or that gene expression patterns of floral gene networks might be deregulated leading to intermediate developmental phenotypes.


Research

We focused our molecular analysis exclusively on inflorescence reversion. Using real-time PCR, we assessed the transcriptional activity of eight key regulators in floral development (Table S1, Fig. 5). One difficulty in comparing normal and reverting flowers was to find a suitable control tissue with which to compare the reverting flowers. Ideally, tissues of flowers would be compared that are either clearly wild-type or can be predicted with high certainty to become reverting flowers later in their development. Since reverting flowers show no clear macroscopic sign of reversion until after initial flower development, we chose the developmental stage at which a reverting flower most resembled the developmental stage of normal flowers. This stage was the production of unopened buds in wild-type flowers that formed within a cluster of other normal flowers of various stages in development (Fig. 4a), or unopened buds forming from within a reverting structure (Fig. 4b). Interestingly, our molecular analysis showed unexpected gene expression patterns in reverting flowers that resembled neither committed flower development nor the maintenance of an uncommitted inflorescence. Low expression of floral repressors AGL-24, and SVP, and floral promoter AP1, concomitant with high expression of floral promoter SOC1 in reverting flowers compared with normal flower buds, creates opposing signals within the network of flower developmental genes that both promote and repress the transition to flowering. Low expression levels of SVP should release repression of SOC1 (Li et al., 2008), which was indeed observed in reverting flower buds (Fig. 5). The high expression levels of SOC1 would be expected to push development towards a floral meristem via the activation of LFY (Lee et al., 2008). However, LFY levels in the reverting buds are essentially unchanged compared with normal buds, suggesting that almost threefold higher levels of SOC1 in the reverting buds have no effect on committing the meristem to flowering. One reason for this may be the extremely low expression levels of AGL24 in this tissue (Fig. 5). Co-expression of AGL24 has been shown to be required for the flower-promoting activity of SOC1 (Lee et al., 2008), suggesting that SOC1 alone cannot activate enough LFY to commit the inflorescence to flowering, thus explaining the essentially flat expression levels of LFY (Fig. 5). On the other hand, LFY activation may be sufficient to keep AG expression levels unchanged in normal vs reverting flowers. The reversions observed therefore do not appear to be the result of insufficient amounts of AG, a condition that has been shown to result in flower reversion in A. thaliana (Okamuro et al., 1996; Parcy et al., 2002). Expression levels of AP1 were also lower in reverting vs normal flowers (Fig. 5). Since AP1 has been shown to be a floral promoter (Irish & Sussex, 1990; Bowman et al., 1993; Litt, 2007) it is possible that its relatively low expression levels contribute to the lack of floral meristem commitment. Further, during the transition from an inflorescence


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to a floral meristem, AP1 together with LFY is required to restrict AGL24 expression to the meristem’s tunica and innermost two whorls of floral meristems (Yu et al., 2004). Absence of this restriction in ap1-1 mutants leads to expression of AGL24 throughout the floral meristem and its expression in supernumerary floral meristems in ap1-1 ⁄ cal1 double mutants (Yu et al., 2004). In the absence of tissuespecific expression data, we speculate that the relatively low expression of AP1 in reverting flower buds may thus contribute to ectopic expression of the remaining AGL24, leading to initiation but only partial commitment to flowering. Taken together, we propose that opposing developmental signals, which both promote floral determination and also maintain inflorescence indeterminancy, may lead to a developmental conflict inside the meristem. If this conflict is not resolved by unequivocally tipping the developmental response towards one side, the result may be tissue death, as was seen in the dried flowers or deteriorated tissues inside mutant flowers (Fig. 2h,i). It is important to note that the unusual expression patterns of floral network genes relative to each other have, to our knowledge, not been seen at any stage in normal flower development in A. thaliana. Therefore, the fact that an ideal control tissue could not be used here, or that our experiments provide transcriptional analysis on the whole tissue, rather than the individual organ level, cannot detract from our observation that the transcription of major floral regulators is disrupted in the reverting flowers. Genomic instability resulting from allopolyploidization may contribute to improper cross-talk within genetic networks in A. suecica flowers Arabidopsis suecica is an allopolyploid plant derived from the concomitant hybridization and genome duplication of its parental species A. thaliana and A. arenonsa (Mummenhoff & Hurka, 1995). While established allopolyploids in nature are frequently vigorous, fit, and well adapted, newly formed allopolyploids (neoallopolyploids) often exhibit a high degree of infertility, and genotypic and phenotypic instability (Ramsey & Schemske, 2002). Genome and transcriptome changes in response to allopolyploidization in Arabidopsis have been linked to alterations in phenotype and morphology in the allopolyploid offspring (reviewed in Chen, 2007). These studies show that the formation of a hybrid genome in a new allopolyploid can have profound effects on the timing of developmental programs. Recent cytogenetic analysis uncovered chromosomal instability and variation in somatic cells also for several different accessions in established A. suecica (Wright et al., 2009), suggesting that evolution and adaptation since the inception of this species have not led to a fully stabilized genomic constitution. An attractive hypothesis to explain the observed changes in the transcriptional activity of the floral network genes


might therefore be to invoke genomic instability in A. suecica as a result of genomic conflicts within the allopolyploid genome. Genomic instability in A. suecica-like allopolyploids arises from chromosomal rearrangements, transposon activation, and epigenetic (Madlung et al., 2002, 2005), transcriptional (Wang et al., 2006), and cytogenetic changes (Comai et al., 2000, 2003b; Pontes et al., 2004; Madlung et al., 2005; Wright et al., 2009). Most of the genes that regulate the floral transition belong to the large family of MADS box transcription factors that are characterized by their ability to bind DNA and to function as dimers or larger protein complexes (Davies et al., 1996; Riechmann et al., 1996; Riechmann & Meyerowitz, 1997; Jack, 2004; de Folter et al., 2005). The nonMADS box genes LFY and TFL1 may also function as part of protein complexes (Weigel, 1995). The combination of slightly mismatched proteins in complexes contributed from two divergent genomes in allopolyploids could therefore lead to protein complexes with altered or attenuated activity (Comai et al., 2003a; Osborn et al., 2003). However, whether or not mismatched protein complexes or altered gene transcription from the two progenitor genomes contribute to the occurrence of opposing signals in reverting flowers remains, at this point, unknown. Our transcriptional analysis did not attempt to test for the contribution of each species-specific homolog, but rather detected the sum of both genomes. In future studies it would be interesting to measure the species-specific relative contribution of the A. arenosa and A. thaliana genes in the floral transition pathway of reverting flowers. It would also be interesting to determine to what degree the proteins from both parental genomes are able to interact to form functional protein complexes, or if the protein complexes in the allopolyploid comprise entirely proteins from a single genome. Conclusions We have described a highly plastic reversion phenotype as well as floral homeotic changes in the established allopolyploid A. suecica. Our analysis shows that this phenotype is sensitive to changes in photoperiod. It appears likely that suboptimal environmental conditions prevent the floral meristem from becoming fully committed to flowering, possibly because of mixed signals from several key floral regulators. It is not clear if genomic instabilities resulting from the allopolyploid background of A. suecica contribute to the abnormal transcriptional patterns in the floral meristem, but it is at least formally possible that they exacerbate the environmental effects that lead to flower reversion in this species.

Acknowledgements The authors thank M. Morrison, S. Bennett, and A. Vallecorsa for technical and logistical help, M. Martin for use of


New Phytologist his MiniOpticon, B. Rowan for advice on real-time PCR, and L. Comai, B. Dilkes, and the Polyploidy Consortium for stimulating discussions. We thank M. Jost for help with the figures. We also thank two anonymous reviewers for helpful comments. This study was supported by an NSF Plant Genome grant (DBI-0501712, to AM), an NSF Major Research Instrumentation grant (MRI-0619009 to AM), and funds from the University of Puget Sound Enrichment Committee (to AM and WLR).

References Andersberg A. 2009. Den virtuella floran. In Naturhistoriska riksmuseet: http://linnaeus.nrm.se/flora/welcome.html. Battey NH, Lyndon RF. 1984. Changes in apical growth and phyllotaxis on flowering and reversion in Impatiens balsamina L. Annals of Botany 54: 553–567. Battey NH, Lyndon RF. 1986. Apical growth and modification of the development of primordia during re-flowering of reverted plants of Impatiens balsamina L. Annals of Botany 58: 333–341. Battey NH, Lyndon RF. 1990. Reversion of flowering. Botanical Review 56: 162–189. Battey NH, Tooke F. 2002. Molecular control and variation in the floral transition. Current Opinion in Plant Biology 5: 62–68. Ba¨urle I, Dean C. 2006. The timing of developmental transitions in plants. Cell 125: 655–664. Blazquez MA, Soowal LN, Lee I, Weigel D. 1997. LEAFY expression and flower initiation in Arabidopsis. Development 124: 3835– 3844. Borner R, Kampmann G, Chandler J, Gleissner R, Wisman E, Apel K, Melzer S. 2000. A MADS domain gene involved in the transition to flowering in Arabidopsis. Plant Journal 24: 591–599. Boss PK, Bastow RM, Mylne JS, Dean C. 2004. Multiple pathways in the decision to flower: enabling, promoting, and resetting. Plant Cell 16(Suppl.): S18–S31. Bowman JL, Alvarez J, Weigel D, Meyerowitz EM, Smyth DR. 1993. Control of flower development in Arabidopsis thaliana by APETALA1 and interacting genes. Development 119: 721–743. Chen ZJ. 2007. Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annual Review of Plant Biology 58: 377–406. Comai L, Tyagi AP, Winter K, Holmes-Davis R, Reynolds SH, Stevens Y, Byers B. 2000. Phenotypic instability and rapid gene silencing in newly formed Arabidopsis allotetraploids. Plant Cell 12: 1551–1568. Comai L, Madlung A, Josefsson C, Tyagi A. 2003a. Do the different parental ‘heteromes’ cause genomic shock in newly formed allopolyploids? Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 358: 1149–1155. Comai L, Tyagi AP, Lysak MA. 2003b. FISH analysis of meiosis in Arabidopsis allopolyploids. Chromosome Research 11: 217–226. Davies B, Egea-Cortines M, de Andrade Silva E, Saedler H, Sommer H. 1996. Multiple interactions amongst floral homeotic MADS box proteins. EMBO Journal 15: 4330–4343. Davison J, Tyagi A, Comai L. 2007. Large-scale polymorphism of heterochromatic repeats in the DNA of Arabidopsis thaliana. BMC Plant Biology 7: 44. Diomaiuto J. 1988. Periodic flowering or continual flowering as a function of temperature in a perennial species: the Ravenelle wallflower (Cheiranthus cheiri). Phytomorphology 38: 163–171. de Folter S, Immink RG, Kieffer M, Parenicova L, Henz SR, Weigel D, Busscher M, Kooiker M, Colombo L, Kater MM et al. 2005.


Research Comprehensive interaction map of the Arabidopsis MADS box transcription factors. Plant Cell 17: 1424–1433. Gregis V, Sessa A, Colombo L, Kater MM. 2006. AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis. Plant Cell 18: 1373–1382. Gregis V, Sessa A, Colombo L, Kater MM. 2008. AGAMOUS-LIKE24 and SHORT VEGETATIVE PHASE determine floral meristem identity in Arabidopsis. The Plant Journal 56: 891–902. Hartmann U, Hohmann S, Nettesheim K, Wisman E, Saedler H, Huijser P. 2000. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant Journal 21: 351–360. Hempel FD, Weigel D, Mandel MA, Ditta G, Zambryski PC, Feldman LJ, Yanofsky MF. 1997. Floral determination and expression of floral regulatory genes in Arabidopsis. Development 124: 3845–3853. Henderson IR, Shindo C, Dean C. 2003. The need for winter in the switch to flowering. Annual Review of Genetics 37: 371–392. Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G. 2002. Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO Journal 21: 4327–4337. Irish VF, Sussex IM. 1990. Function of the apetala-1 gene during Arabidopsis floral development. Plant Cell 2: 741–753. Jack T. 2004. Molecular and genetic mechanisms of floral control. Plant Cell 16(Suppl.): S1–S17. Jakobsson M, Hagenblad J, Tavare S, Sall T, Hallden C, Lind-Hallden C, Nordborg M. 2006. A unique recent origin of the allotetraploid species Arabidopsis suecica: evidence from nuclear DNA markers. Molecular Biology and Evolution 23: 1217–1231. Lee J, Oh M, Park H, Lee I. 2008. SOC1 translocated to the nucleus by interaction with AGL24 directly regulates LEAFY. Plant Journal 55: 832–843. Li D, Liu C, Shen L, Wu Y, Chen H, Robertson M, Helliwell CA, Ito T, Meyerowitz E, Yu H. 2008. A repressor complex governs the integration of flowering signals in Arabidopsis. Developmental Cell 15: 110–120. Litt A. 2007. An evaluation of A-function: evidence from the APETALA1 and APETALA2 gene lineages. International Journal of Plant Sciences 168: 73–91. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. Madlung A, Masuelli RW, Watson B, Reynolds SH, Davison J, Comai L. 2002. Remodeling of DNA methylation and phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids. Plant Physiology 129: 733–746. Madlung A, Tyagi AP, Watson B, Jiang H, Kagochi T, Doerge RW, Martienssen R, Comai L. 2005. Genomic changes in synthetic Arabidopsis polyploids. Plant Journal 41: 221–230. Mizukami Y, Ma H. 1997. Determination of Arabidopsis floral meristem identity by AGAMOUS. Plant Cell 9: 393–408. Mouradov A, Cremer F, Coupland G. 2002. Control of flowering time: interacting pathways as a basis for diversity. Plant Cell 14: S111–S130. Mummenhoff K, Hurka H. 1995. Allopolyploid origin of Arabidopsis suecica (Fries) Norrlin: evidence from chloroplast and nuclear genome markers. Botanica Acta 108: 449–456. Nooden LD, Penney JP. 2001. Correlative controls of senescence and plant death in Arabidopsis thaliana (Brassicaceae). Journal of Experimental Botany 52: 2151–2159. Okamuro JK, den Boer BG, Lotys-Prass C, Szeto W, Jofuku KD. 1996. Flowers into shoots: photo and hormonal control of a meristem identity switch in Arabidopsis. Proceedings of the National Academy of Sciences, USA 93: 13831–13836. Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee HS, Comai L, Madlung A, Doerge RW, Colot V et al. 2003. Understanding mech-


249

New Phytologist

250 Research anisms of novel gene expression in polyploids. Trends in Genetics 19: 141–147. Parcy F, Bomblies K, Weigel D. 2002. Interaction of LEAFY, AGAMOUS and TERMINAL FLOWER1 in maintaining floral meristem identity in Arabidopsis. Development 129: 2519–2527. Poduska B, Humphrey T, Redweik A, Grbic V. 2003. The synergistic activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis. Genetics 163: 1457–1465. Poethig RS. 2003. Phase change and the regulation of developmental timing in plants. Science 301: 334–336. Pontes O, Neves N, Silva M, Lewis MS, Madlung A, Comai L, Viegas W, Pikaard CS. 2004. Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proceedings of the National Academy of Sciences, USA 101: 18240–18245. Ramsey J, Schemske D. 2002. Neopolyploidy in flowering plants. Annual Review of Ecology and Systematics 33: 589–639. Riechmann JL, Meyerowitz EM. 1997. MADS domain proteins in plant development. Biological Chemistry 378: 1079–1101. Riechmann JL, Krizek BA, Meyerowitz EM. 1996. Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Proceedings of the National Academy of Sciences, USA 93: 4793–4798. Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G. 2000. Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288: 1613–1616. Searle I, He Y, Turck F, Vincent C, Fornara F, Krober S, Amasino RA, Coupland G. 2006. The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes and Development 20: 898–912. Sreekantan L, McKenzie MJ, Jameson PE, Clemens J. 2001. Cycles of floral and vegetative development in Metrosideros excelsa (Myrtaceae). International Journal of Plant Sciences 162: 719–727. Tooke F, Battey NH. 2000. A leaf-derived signal is a quantitative determinant of floral form in Impatiens. Plant Cell 12: 1837–1848. Tooke F, Ordidge M, Chiurugwi T, Battey N. 2005. Mechanisms and function of flower and inflorescence reversion. Journal of Experimental Botany 56: 2587–2599. Turck F, Fornara F, Coupland G. 2008. Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annual Review of Plant Biology 59: 573–594. Wang YZ. 2001. Reversion of floral development under adverse ecological conditions in Whytockia bijieensis (Gesneriaceae). Australian Journal of Botany 49: 253–258. Wang J, Tian L, Lee HS, Wei NE, Jiang H, Watson B, Madlung A, Osborn TC, Doerge RW, Comai L et al. 2006. Genomewide


nonadditive gene regulation in Arabidopsis allotetraploids. Genetics 172: 507–517. Wang Q, Sajja U, Rosloski S, Humphrey T, Kim MC, Bomblies K, Weigel D, Grbic V. 2007. HUA2 caused natural variation in shoot morphology of A. thaliana. Current Biology 17: 1513–1519. Washburn CF, Thomas JF. 2000. Reversion of flowering in Glycine max (Fabaceae). American Journal of Botany 87: 1425–1438. Weigel D. 1995. The genetics of flower development: from floral induction to ovule morphogenesis. Annual Review of Genetics 29: 19–39. Weigel D, Meyerowitz EM. 1993. Activation of floral homeotic genes in Arabidopsis. Science 261: 1723–1726. Wright KM, Pires JC, Madlung A. 2009. Mitotic instability in resynthesized and natural polyploids of the genus Arabidopsis (Brassicaceae). American Journal of Botany 96: 1656–1664. Wu C, Ma Q, Yam K-M, Cheung M-Y, Xu Y, Han T, Lam H-M, Chong K. 2006. In situ expression of the GmNMH7 gene is photoperioddependent in a unique soybean (Glycine max (L.) Merr.) flowering reversion system. Planta 223: 725–735. Yu H, Xu Y, Tan EL, Kumar PP. 2002. AGAMOUS-LIKE 24, a dosagedependent mediator of the flowering signals. Proceedings of the National Academy of Sciences, USA 99: 16336–16341. Yu H, Ito T, Wellmer F, Meyerowitz EM. 2004. Repression of AGAMOUS-LIKE 24 is a crucial step in promoting flower development. Nature Genetics 36: 157–161.

Supporting Information Additional supporting Information may be found in the online version of this article. Table S1 Primer sequences used to amplify candidate gene products Table S2 Statistical analysis of real-time PCR gene products from Fig. 5 Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.
