short title: 6 1,2,3,# 2,3 · 24. vegetative branches and a zone of dormant buds that . serve as...

1

Title 1

Flowering behaviour in Arabis alpina ensures the maintenance of a perennating dormant 2

bud bank 3

4

Short title: Perennial flowering and architecture 5

6

Alice Vayssières1,2,3, Priyanka Mishra1,2,3,#, Adrian Roggen1,2,3,#, Udhaya Ponraj2,3, Ulla 7

Neumann3, Klaus Theres2,3, Karin Ljung4, Maria C. Albani1,2,3,* 8

9

1 Botanical Institute, Cologne Biocenter, University of Cologne, Zülpicher Str. 47B, 50674 10

Cologne, Germany 11

2 Cluster of Excellence on Plant Sciences “From Complex Traits towards Synthetic Modules”, 12

40225 Düsseldorf, Germany 13

3 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, 14

Germany 15

4 Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish 16

University of Agricultural Sciences, Umeå, Sweden 17

18

# Equal contribution 19

* Materials & correspondence: [email protected] or [email protected] 20

21

ABSTRACT 22

Arabis alpina, similar to woody perennials, has a complex architecture with a zone of axillary 23

vegetative branches and a zone of dormant buds that serve as perennating organs. We show that 24

floral development during vernalization is the key for shaping the dormant bud zone by 25

facilitating a synchronized and rapid growth after vernalization and thereby causing an increase 26

in auxin response and transport and endogenous indole-3-acetic acid (IAA) levels in the stem. 27

Floral development during vernalization is associated with the development of axillary buds in 28

subapical nodes. Our transcriptome analysis indicated that these buds are not dormant during 29

vernalization but only attain sustained growth after the return to warm temperatures. Floral and 30

subapical vegetative branches grow after vernalization and inhibit the development of the buds 31

below. Dormancy in these buds is regulated across the A. alpina life cycle by low temperatures 32

and by apical dominance in a BRANCHED 1-dependent mechanism. 33

.CC-BY-ND 4.0 International licenseIt is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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INTRODUCTION 35

Bud dormancy plays an important role in survival through harsh environmental conditions and 36

long-term growth1. Thus, during the perennial life cycle, axillary and apical buds transition 37

through the various stages of dormancy before they resume active organogenesis and develop 38

into flowering or vegetative branches2. During development the outgrowth of axillary buds 39

close to the shoot apical meristem is repressed by apical dominance, a phenomenon also known 40

as correlative inhibition, latency or paradormancy, which occurs in both annual and perennial 41

species3,4. This form of dormancy is not definitive and paradormant buds can resume growth 42

when the main shoot apex is removed5. Buds in trees and herbaceous perennials also enter two 43

other forms of dormancy, endo- and eco- dormancy2. Endodormancy is regulated by 44

endogenous signals within the bud whereas ecodormancy is imposed by unfavorable 45

environmental conditions6. During the life cycle of a perennial plant, apical or axillary buds 46

experience winter in the endodormant state and later become ecodormant so that they will 47

actively grow only during favorable environmental conditions. It is however very common in 48

perennials for branches to have axillary buds during spring and summer, which later on will 49

stay dormant across multiple growing seasons. These dormant buds serve as a backup bud bank 50

and, in case of damage, are used as reservoirs for potential growth facilitating a bet-hedging 51

mechanism7,8. Interestingly, dormant buds and actively growing (vegetative or reproductive) 52

axillary branches are organized in zones in a species specific pattern9,10. 53

The outgrowth of an axillary bud after decapitation involves two phases, firstly the rapid release 54

from dormancy and secondly its sustained growth11. Auxin, strigolactones, cytokinin and sugar 55

fine-tune this process by regulating the expression of the TCP transcription factor 56

BRANCHED 1 (BRC1)12. Decapitation causes an elevation of sucrose levels followed by a 57

depletion of the endogenous indole-3-acetic acid (IAA) in the polar auxin transport stream, 58

which influences the auxin flux out of the axillary bud contributing to its sustainable growth13–59

17. Flowering transition triggers the activation of the upper most axillary buds in a basipetal 60

sequence having a similar effect to decapitation13,18. In an annual plant, the relation between 61

flowering and bud activation is easy to trace as its life cycle ends within one growing season. 62

However, many perennials spread the flowering process over several years and floral bud 63

initiation is temporally separated from anthesis. 64

Here we used the perennial Arabis alpina, a close relative of Arabidopsis thaliana, to 65

investigate the link between flowering and the maintenance of a dormant bud bank in 66



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perennials. The shoot architecture of A. alpina is polycarpic and consists of branches that 67

undergo flowering, others that remain vegetative and nodes with dormant axillary buds19. A. 68

alpina mutants or transgenic lines with reduced function in components of the age pathway 69

influence the fate of vegetative branches but still require exposure to prolonged cold to flower, 70

a process known as vernalization20–23. Interestingly, the maintenance of dormant buds is only 71

compromised in mutants that do not require vernalization19. The most described example is the 72

perpetual flowering 1 (pep1) mutant which carries lesions in the ortholog of the MADS box 73

transcription factor FLOWERING LOCUS C (FLC)19,24,25. Here we show that the requirement 74

of vernalization to flower is linked to the maintainance of dormant axillary buds. 75

76

RESULTS 77

Flowering during vernalization correlates with the formation of a perennial shoot 78

architecture including a zone of dormant axillary buds 79

To assess the relationship between flowering and plant architecture, we exposed the accession 80

Pajares to different durations of vernalization that influence flowering and we scored bud activity 81

and fate. As shown previously, in 8-week-old plants vernalized for 12 weeks the shoot apical 82

meristem (I zone, nodes 29–40) and the lower axillary branches (V1 zone, nodes 1–12) flowered 83

(Fig. 1a,e,f)10,19. The architecture of A. alpina plants was already established 3 weeks after 84

vernalization, showing a clear inhibition of axillary bud growth in nodes 12–21, with leaf axils 85

showing no visible outgrowth or buds that did not grow more than 0.5 cm (V2 zone, Fig. 86

1a,b,e,f)10. The nodes above (nodes 22–29) contained buds that actively grew after vernalization 87

and giving rise to axillary branches that remained vegetative (V3 zone, Fig. 1b,e, f)10,19,26. 88

When we compared the architecture of plants after vernalization with the one before 89

vernalization we observed a similar pattern of growth as described previously19,26,27. The V1 90

branches developed before vernalization, the V2 buds were present on the axils of leaves before 91

vernalization, and the V3 branches arose in the axils of leaves that grew during vernalization. 92

It has been recently shown that extended vernalization is required to ensure floral 93

commitment10. To test whether extended vernalization influences the size of the V2 zone, we 94

exposed 8-week-old plants to 12, 15, 18, 21 and 24 weeks of vernalization (Supplementary Fig. 95

1). The outgrowth of V3 branches and the inflorescence stem was accelerated after longer 96

durations of vernalization (Supplementary Fig. 1a,b)10. However, we observed no difference in 97

the number of dormant buds suggesting that extended vernalization does not have an impact on 98

the V2 buds (Supplementary Fig. 1c,d). We then exposed plants to durations of vernalization 99



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shorter than 12 weeks. As shown previously, plants grown continuously in LDs or vernalized 100

only for 3 weeks did not flower, whereas plants vernalized for 8 weeks showed extreme floral 101

reversion phenotypes (Supplementary Fig. 2)10,19. To follow bud activity, we measured the 102

length of axillary branches at each leaf axil 3 weeks after vernalization or after 11 weeks in LDs 103

for non-vernalized plants. Branch length in LD grown plants and in 3 week vernalized plants 104

was reduced acropetally (Fig. 1g,h). Interestingly, nodes 12–18 were completely inhibited only 105

in plants exposed to 12 weeks of vernalization (Fig. 1g,h). In plants vernalized for 8 weeks, the 106

zonation was visible by measuring the branch length (Fig. 1h) but no dormant bud zone was 107

observed (Fig. 1g,h). Nevertheless, in both 8 week and 12 week vernalized plants the nodes 108

occupied with actively growing branches were always just above the nodes with the inhibited 109

buds suggesting that the presence of V3 branches might be correlated with the stable inhibition 110

of bud growth in the V2 zone. 111

PEP1 determines the requirement for vernalization to flower and the fate of the V3 branches 112

after vernalization10,19. To test whether PEP1 plays a role in the zonation of A. alpina shoots, 113

we measured branch length and the fate of the axillary branches in the pep1-1 mutant compared 114

to the wild-type (Supplementary Fig. 3). pep1-1 flowered after 8 weeks in LDs and showed a 115

deviation in branching phenotype compared to the wild-type (Supplementary Fig. 3). 116

Nevertheless, both genotypes lacked the characteristic zonation observed in flowering plants 117

after vernalization suggesting that flowering in LDs does not ensure perennial plant architecture 118

in A. alpina. To test whether flowering during vernalization correlates with the repression of 119

V2 buds, we exposed plants of different ages to vernalization. Earlier reports showed that 3-120

week-old plants are not competent to flower in response to vernalization and remain vegetative, 121

whereas 5-week-old plants flower (Supplementary Fig. 4)20,21,26. Plants grown for 3 weeks prior 122

to vernalization did not show bud inhibition at any node below the newly formed branches, 123

whereas in 5-week-old plants they did in nodes 3–8 (Supplementary Fig. 4c). These plants also 124

contained V3 vegetative branches above the inhibited buds (nodes 8–19) (Supplementary Fig. 125

4c). Altogether, these results suggest that zones of differential bud activity in A. alpina are 126

formed only in genotypes that initiate flowering during vernalization. 127

128

V3 buds are not dormant at the end of vernalization, but attain sustained growth only 129

after the return to warm temperatures 130

In A. thaliana and other species, bud growth has been demonstrated using the auxin inducible 131

synthetic promoter DR513,17. To check whether V2 and V3 buds are active during or after 132

vernalization, we developed transgenic A. alpina plants carrying the DR5 promoter fused to the 133



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reporter gene β-glucuronidase (GUS). We observed the GUS signal only after vernalization in 134

the vasculature of V3 buds and in the main stem just below the leaf nodes (Fig. 2 a-d). These 135

data suggest that at 5 days after vernalization the V3 buds are growing whereas the V2 buds are 136

not. 137

To investigate the molecular mechanisms that lead to the activation of V3 buds and the 138

inhibition of growth in V2 buds, we performed a transcriptome profiling on dissected buds 139

directly at the end of vernalization and 5 days after vernalization. The transcriptome of V2 and 140

V3 buds at 5 days after vernalization was the most dissimilar (1984 genes; +5dV2 vs +5dV3; 141

Fig. 2e; Supplementary Table 1). However, the transcriptome of V2 and V3 buds differed also 142

at the end of vernalization (1128 genes for +0V2 vs +0V3; Fig. 2e; Supplementary Table 1). 143

This suggests that the V2 and V3 buds were differentiated already during vernalization although 144

active growth of the V3 buds was only observed after vernalization. Interestingly, 93% of the 145

genes differentially expressed between V2 and V3 buds at the end of vernalization were 146

differentially expressed between samples after vernalization or/and between developmental 147

stages (Fig. 2e). Likewise, Gene Ontology (GO) enrichment analysis indicated that all GOs 148

enriched for the differentially expressed genes between V2 and V3 buds at the end of 149

vernalization were also found in the other comparisons (Supplementary Fig. 5, Supplementary 150

Table 2). GO terms common to all comparisons were mainly associated with hormone 151

responses such as abscisic acid, ethylene and jasmonic acid (Supplementary Fig. 5). These 152

results suggest that hormones play an important role in the activation of V3 buds and the 153

repression of V2 buds. To identify genes that share similar expression patterns, we performed 154

hierarchical clustering analysis. We identified 34 co-expressed clusters, which were assigned 155

into two higher level clusters I and II (Fig. 2f). Interestingly, the separation of these higher level 156

clusters was shaped by the expression of genes in the V3 buds during vernalization. Genes in 157

Cluster I showed low and genes in Cluster II showed high transcript accumulation in V3 buds 158

at the end of vernalization (Fig. 2f). Genes in Cluster I3 and I6 showed higher transcript 159

accumulation in V3 buds after vernalization accounting for putative candidate genes involved 160

in the sustained growth of V3 buds (Fig. 2g,h). Specifically, in Cluster I3 most enriched GO 161

terms among the identified genes were associated with developmental processes including 162

genes involved in cell expansion (EXPB1) (Fig. 2g, I3, Supplementary Table 3). This result 163

clearly supports that V3 buds only grow after vernalization. The growth V3 buds was also 164

associated with the upregulation of transcript accumulation of the YUCCA2 homolog, a gene 165

coding for an enzyme that catabolizes the biosynthesis of IAA in A. thaliana, and the auxin 166

signaling factors IAA7 and IAA14, whose expression levels reflect also auxin levels, identified 167



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in cluster I6 (Fig. 2h)28,29. These results confirm our DR5 results that the activation of auxin 168

response occurs in V3 buds after vernalization. Clusters I14, I15, I20, I21, II7 and II8 showed 169

different transcript accumulation between buds already during vernalization (Fig. 2i,j). Genes 170

in these clusters also showed similar differences in transcript accumulation after vernalization 171

indicating that the majority of the differences observed during vernalization are also maintained 172

afterwards. Genes in Clusters II7 and II8 showed higher transcript accumulation in V3 buds 173

compared to V2 buds and were enriched for GO terms related to cell division, which included 174

homologs of cell cycle regulators, such as PCNA1, previously shown to be upregulated during 175

bud activation in other species (Fig. 2j, Supplementary Fig. 3)30,31. These results suggest that 176

genes involved in cell cycle and transcription machinery are upregulated in V3 buds during 177

vernalization. Clusters I14, I15, I20 and I21 showed lower transcript accumulation in V3 buds 178

compared to V2 buds. Interestingly, all these clusters were enriched for GOs related to response 179

to abscisic acid and water deprivation and contained genes related to ABA signaling (e.g. 180

AIRP2, ABF1, ABF2, ABI1, ABI5, KIN2, AFP1 AFP3, BLH1 and PP2CA) or ABA biosynthesis 181

(e.g. NCED3) shown to be associated with bud dormancy in several species (Fig. 3i)32–35. We 182

also detected the dehydrin coding genes ERD10, ERD14 and RAB18, which are induced by 183

ABA and suggested to prevent water dehydration during tree winter dormancy (Fig. 3i)36. 184

Transcript accumulation of homologs of genes associated with repression of the cytokinin level 185

and response such as CKX1 and KMD1–4 were also detected in these clusters (Fig. 3i)37,38. 186

These data suggest that V3 buds during vernalization might contain lower levels of ABA that 187

represses bud outgrowth and higher levels of cytokinin that promotes bud outgrowth compared 188

to V2 buds. We also identified the homologs of genes that have been previously shown in A. 189

thaliana to respond to conditions that trigger dormancy and therefore are considered as 190

dormancy markers39. This includes the A. alpina homologs of HB21, HB53, PSY1, NAC029, 191

SAG21 and HIS1-3 (Fig. 2i, Supplementary Table 5). Interestingly, genes in I21, in contrast to 192

clusters I14, I15 and I20, showed a high transcript upregulation in V2 buds after vernalization 193

(Fig. 2i). In addition to ABA signalling genes described before, we also detected the homologs 194

of the strigolactone signaling genes D14 and SMAX1 in this cluster. D14 in A. thaliana and its 195

homologs in other species regulate bud dormancy40,41. These results suggest that the dormancy 196

status of the V2 buds is enhanced after vernalization which correlates with the activation of 197

genes involved in ABA and SL signaling. Interestingly, we also identified homologs of the 198

floral repressors such as TEM1, TFL1 and SMZ that regulate flowering through the age pathway 199

(Fig. 2i)20,42–45. This result suggests that V3 buds are more competent to flower compared to V2 200



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buds during and after vernalization and may relate to the low dormancy status of these axillary 201

meristems. 202

203

Inhibition of V2 buds is controlled by paradormancy after vernalization and correlates 204

with the increase of auxin in the stem 205

To identify whether the outgrowth of V2 buds is determined by other parts of the plant or by 206

environmental conditions, we performed a series of decapitation and excision experiments (Fig. 207

3a). We first addressed the bud behavior in the V2 zone after vernalization by excising buds or 208

branches at the end of vernalization. Excision of inflorescence buds (I) or V1 branches 209

separately reduced the number of buds observed in the V2 zone (nodes 11–16; Fig. 3a, b). The 210

biggest effect on bud outgrowth in the V2 zone was observed when we excised both 211

inflorescence and V3 buds (I+V3) together. Removal of V3 buds alone was only feasible 2 212

weeks after vernalization when the V3 branches were already expanding and only had a slight 213

effect on the V2 zone (Supplementary Fig. 6). These results suggest that the outgrowth of buds 214

within the V2 zone is influenced by other parts of the plant after vernalization. We also tested 215

the effect of decapitation in plants exposed to different durations of vernalization 216

(Supplementary Fig. 7a). In all treatments, the branches in the V2 zone responded to 217

decapitation and were longer compared to non-decapitated controls (Supplementary Fig. 7b). 218

Interestingly, decapitation of plants vernalized for 12 weeks showed the biggest effect 219

compared to the controls suggesting that stable repression of V2 buds occurs only after 12 220

weeks of vernalization (Supplementary Fig. 7b). These results suggest that V2 buds are not 221

endodormant during vernalization. To assess the effect of the vernalization treatment on bud 222

outgrowth we decapitated plants and subsequently exposed them to 12 weeks of vernalization 223

(Supplementary Fig. 7c). Decapitated and intact vernalized plants showed a similar number of 224

buds in nodes 0–11 suggesting that cold during vernalization imposes an ecodormant state in 225

the V2 buds (Supplementary Fig. 7d,e). Overall, these results suggest that V2 buds are 226

ecodormant during vernalization and paradormant after vernalization. 227

To link these results to our transcriptome studies we tested the expression patterns of a set of 228

identified genes after decapitation (Fig. 3c). Most genes tested showed higher transcript 229

accumulation in V2 buds compared to V3 buds at the end and/or after vernalization and their 230

transcript accumulation was reduced in decapitated plants (Fig. 3c). Since auxin is a major 231

regulator of apical dominance, we measured auxin response in the DR5::GUS lines after 232

decapitation. At the end of vernalization, stems within the V2 zone did not show strong GUS 233

staining (Fig. 4a,d). A strong GUS signal was detected within 1 week after vernalization in the 234



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epidermis and in the vasculature of V2 stems and was reduced in decapitated plants (Fig. 4c,f, 235

Supplementary Fig. 8). We also measured the levels of endogenous IAA in V2 stems of intact 236

plants during the A. alpina life cycle. Similar to DR5::GUS results, IAA levels in V2 stems 237

were reduced during vernalization and transiently increased again after vernalization (Fig. 4g). 238

Interestingly, IAA levels at 2 weeks after vernalization were very high, which correlated with 239

high IAA levels in the stems of the rapidly growing tissues (inflorescence and V3 branches) 240

above the V2 zone (Fig. 4h,i). Decapitation at the end of vernalization also resulted in a decrease 241

of the endogenous IAA levels in V2 stems (Fig. 4j). This result suggests that the faster 242

outgrowth of the inflorescence and V3 branches after vernalization induces a higher IAA in the 243

V2 stem. To assess the role of auxin and auxin transport in the inhibition of the V2 buds we 244

applied the synthetic auxin NAA and the transport inhibitor NPA in vernalized plants before 245

transferring them to normal greenhouse conditions. NAA treatment increased the number of 246

inhibited buds (Fig. 4k,l) whereas NPA treatment reduced the number of inhibited buds in the 247

V2 zone compared to mock-treated plants (Fig. 4l). In addition, NPA strongly impaired the 248

development of the inflorescence and V1 branches (Fig. 4k). These results confirmed the 249

importance of auxin levels and auxin transport for the inhibition of the buds in the V2 zone after 250

vernalization. We subsequently measured the IAA transport capacity using acropetal 3H-IAA 251

treatment in excised V2 stem segments from 8-week-old plants in LDs (8wLD), plants 252

vernalized for 12 weeks (+0) and 5 days after vernalization (+5d). 3H-IAA levels in V2 stems 253

were higher before and after vernalization, compared to at the end of vernalization (Fig. 4m,n). 254

Our results suggest that vernalization leads to a decrease in auxin transport in V2 stems (Fig. 255

4o). Altogether we conclude that the outgrowth of the inflorescence and the vegetative branches 256

after vernalization correlates with an enhancement of endogenous IAA levels, auxin response 257

and transport in the V2 zone after vernalization which may stably repress the outgrowth of V2 258

buds (Fig. 4o). During vernalization, although auxin transport is low, the development of V2 259

axillary buds is inhibited (Fig. 4o). 260

261

AaBRC1 ensures the maintenance of the dormant V2 buds after vernalization 262

We subsequently tested the expression of genes differentially expressed in our transcriptome 263

analysis on V2 buds across the A. alpina life cycle, before, at the end and after vernalization. 264

In addition to our candidate genes we followed the expression patterns of the A. alpina homolog 265

of BRC1 (AaBRC1) and its paralog BRC2 (AaBRC2), as in A. thaliana the majority of our 266

candidate genes are misexpressed in the brc1 mutant (Supplementary Fig. 9a)46. Compared to 267

before vernalization, AaBRC1 transcript levels in the V2 buds were increased after prolonged 268



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exposure to cold and were maintained in buds harvested 5 days after vernalization (Fig. 5a). 269

Transcript accumulation of the HB53 homolog (AaHB53), a direct target of BRC147, and other 270

BRC1-downstream genes also showed an increase of expression in vernalization (Fig. 5a). 271

Interestingly, transcript accumulation of AaHIS1-3 only increased after vernalization, 272

suggesting a BRC1-independent regulation of bud dormancy in A. alpina (Fig. 5a). We also 273

created transgenic lines with reduced expression of AaBRC1. Plants of 35S:AaBRC1dsRNAi 274

lines 1 and 2 showed significant downregulation of AaBRC1 expression whereas line 3 did not 275

(Supplementary Fig. 9b). We characterized the 35S:AaBRC1dsRNAi lines in the same 276

conditions (before, at the end and after vernalization). Before and at the end of vernalization we 277

observed no major difference in branch number and branch length (Fig. 5b,c, Supplementary 278

Fig. 10a,b). Transgenic lines also flowered with a similar total number of leaves compared to 279

control plants (Supplementary Fig. 10 d-h). We observed a phenotype only after vernalization, 280

and nodes 12-16 corresponding to the V2 zone in 35S:AaBRC1dsRNAi lines 1 and 2 were 281

occupied by a branch (Fig. 5b,e,f, Supplementary Fig. 10c). In these lines, transcript levels of 282

AaHB53 were reduced in V2 buds of 35S:AaBRC1dsRNAi lines 1 and 2 at the end and after 283

vernalization but not before vernalization (Supplementary Fig. 10). Interestingly, the transcript 284

accumulation of AaHIS1-3 and AaAIRP2 was not misregulated in the 35S:AaBRC1dsRNAi lines 285

in all developmental stages (Supplementary Fig. 10). These results suggest that the activity of 286

V2 buds is regulated by AaBRC1 after vernalization, although an AaBRC1-independent 287

pathway might exist. 288

289

DISCUSSION 290

The induction of flowering in temperate perennials is uncoupled from anthesis so that the 291

flowering process takes place for several years8,10,48,49. In the alpine perennial A. alpina, flower 292

buds are formed during vernalization and emerge when plants experience favorable 293

environmental conditions. Despite the differences in flowering behavior, similar principles of 294

bud activation described in A. thaliana also apply in A. alpina. For example, axillary buds close 295

to the shoot apical meristem are temporarily inhibited during vegetative development and the 296

initiation of flowering results in the activation of the upper most axillary buds13. However, floral 297

development prior to anthesis is the key for the establishment of a zone of dormant axillary 298

buds. During vernalization, flowering in the main shoot apex always correlates with the 299

presence of V3 buds19. Our transcriptome analysis suggests that at the end of vernalization V3 300

buds are not dormant and might not be dormant throughout the vernalization period. In tulip 301

bulbs, which show a similar pattern of axillary bud activity, the axillary buds located close to 302



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the flowering shoot apex never arrest growth50. However, exposure to warm temperatures is 303

still required to achieve sustained growth of the V3 buds. This suggests that, in contrast to other 304

species, the phases of bud activation and sustained growth are temporarily separated in A. 305

alpina. The link between flowering and paradormancy has been demonstrated in A. thaliana 306

and rice with the flowering time regulator FLOWERING LOCUS T/Heading date 3a acting as 307

the systemic signal for flowering and axillary bud activation51–53. In A. alpina, the pep1-1 308

mutant has a clear branching phenotype suggesting that PEP1 might regulate the crosstalk 309

between flowering and bud activity. Other MADS box proteins have been reported in woody 310

perennials to regulate endodormancy and the requirement of prolonged exposure to cold to 311

break dormancy54. 312

Buds in the V2 zone probably transition between different forms of dormancy. Before 313

vernalization, V2 buds are only temporarily dormant due to apical dominance and during 314

exposure to vernalization become ecodormant. This is in contrast to studies in woody 315

perennials, in which buds enter a deeper form of dormancy – endodormancy – during the winter. 316

From the transcript levels of dormancy marker genes we can conclude that the dormancy status 317

of V2 buds is enhanced during vernalization. We detected genes associated with cell cycle and 318

cell division, ABA biosynthesis and signaling to be differentially expressed between dormant 319

(V2) and non-dormant buds (V3). The expression of many of the identified genes in our study 320

has been previously reported to be dependent on BRC146 and is induced under carbon limiting 321

conditions and bud dormancy in A. thaliana, grapevine and poplar buds39. In our system, the 322

enhancement of bud dormancy during cold correlates with the development of the inflorescence 323

meristem10,19. This suggests that sugar demand for inflorescence development during 324

vernalization might be responsible for the carbon starvation response observed in axillary buds. 325

The timing of bud initiation, whether it occurs before or during vernalization might also be an 326

important factor as the carbon starvation response might explain the dormancy status of V2, but 327

not the activation of V3 buds. The link between flowering and bud activity has also been 328

explained by the release from apical dominance due to changes in polar auxin transport13. At 329

the end of vernalization, the low IAA levels indicate either that after prolonged exposure to cold 330

the levels of auxin transported basipetally is diminished probably due to a generalized 331

slowdown of growth or that cold influences the auxin metabolism. After the return to warm 332

temperatures, V2 buds once more enter a paradormant state being dominated by the 333

inflorescence and V3 or V1 branches. The transition from the ecodormant back to the 334

paradormant state involves the enhancement of basipetal auxin transport after vernalization and 335

increased activity of AaBRC1. 336



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Overall, we conclude that A. alpina is a good model system to follow different stages of 337

dormancy across the perennial life cycle and to dissect the phases of bud outgrowth, release of 338

dormancy and sustained branch growth. 339

340

Methods 341

Plant material, growth conditions and phenotyping. All our experiments were performed 342

with the Arabis alpina accession Pajares or the pep1-1 mutant described by Wang et al.19. Seeds 343

were stratified on humidified paper at 4°C in the dark for 4 days prior to germination on soil 344

under temperatures ranging from 20 °C during the day to 18 °C during the night in a long day 345

(LD, 16 hours light, 8 hours dark) greenhouse. After 8 weeks of growth, vernalization 346

treatments were carried out in a short day (SD, 8 hours light, 16 hours dark) growth chamber at 347

4°C for different durations of vernalization (depending on the experiment) before the plants 348

were moved back to a LD (16 hours light, 8 hours dark) greenhouse at 20°C. For the juvenility 349

experiment plants were grown for 3 weeks (juvenile) or 5 weeks (adult) before being vernalized. 350

For 1-naphthaleneacetic acid (NAA) and 1-N-naphthylphthalamic acid (NPA) treatments at the 351

end of vernalization and one week after vernalization, plants were sprayed with 100 µM NAA 352

(Sigma-Aldrich), 100 µM NPA (Chem Service) or DMSO as a mock treatment with 0.2% (v/v) 353

Tween-20 immediately after being transferred back to a LD greenhouse. Excision of 354

inflorescence and/or V3 buds at the end of vernalization was performed under a 355

stereomicroscope by removing the eight nodes below of the lowest flowering bud. In the simple 356

decapitation method (Supplementary Fig. 7), the apex was cut off directly above the point where 357

no stem elongation was observed, corresponding to nodes 11–14 within the V2 zone. The shoot 358

architecture at different time points was recorded by observing the fate and the length of the 359

branch at each node of the plant, and the number and type of branches per zone. All experiments 360

were performed with 10 to 12 plants. 361

362

Plasmid constructs. The DR5::GUS fragment (excised from plasmid DR5::GUS, kindly 363

provided by Tom Guilfoyle) was introduced into the GATEWAY-compatible pEarleyGate 301 364

plasmid containing the BASTA resistance gene using site-directed recombination. For the 365

35S:AaBRC1dsRNAi constructs, three cDNA fragments of AaBRC1 (Fragment 1-3; see 366

Supplementary Table 5) were amplified and introduced into the GATEWAY-compatible 367

pJawohl-8-RNAi plasmid using site-directed recombination. The names of the A. alpina 368

35S:AaBRC1dsRNAi lines correspond to the fragments introduced. For each construct, 369



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homozygous transgenic A. alpina lines carrying single-copy transgenes were generated using 370

the floral dip method55. 371

372

GUS staining. For GUS staining assays, stems within the V2 zone below the point where no 373

stem elongation was observed were harvested and leaf axils carrying V2 and V3 buds of 374

DR5::GUS plants were excised and placed directly into 90% ice-cold acetone and incubated for 375

1 h on ice. V3 buds were identified as the 8 nodes directly below the flowering buds. V2 buds 376

were identified below the V3 buds where no stem elongation was observed. The samples were 377

washed in 50 mM phosphate buffer (pH 7.0) and submerged in 2.5 mL GUS staining solution 378

under moderate vacuum for 20 min56. After incubation at 37°C in the dark for a maximum of 379

16 h, chlorophyll was removed by transferring the samples through an ethanol series. GUS 380

activity was observed in whole stem tissues, transverse stem sections or longitudinal leaf axil 381

sections. We prepared 50–60 µm sections on a Leica VT1000S vibratome from samples 382

immobilized on 6% (w/v) agarose. Representative photographs from two different biological 383

experiments were taken using the stereomicroscope Nikon SMZ18 and Nikon Digital Sight 384

camera (DS-Fi2) for whole stem segments, and the Zeiss Axio Imager microscope with the 385

Zeiss Axio Cam 105 color camera for cuttings. 386

387

RNA extraction and cDNA synthesis. For RNA-Seq transcript profiling and quantitative RT-388

PCR analysis, V2 and V3 buds were specifically harvested under a stereomicroscope 389

immediately after vernalization and 5 days after vernalization. In all experiments, V2 buds 390

corresponded to the leaf axils 16–19, and V3 buds corresponded to the leaf axils 23–26. For 391

quantification of the GUS expression of the DR5::GUS lines, the stem was cut off directly 392

below the point where stem elongation was observed, axillary buds were removed so that RNA 393

was isolated only from three internodes within the V2 zone. Each experiment comprised three 394

independent biological replicates. Samples were stored at –80°C prior to RNA extraction. Total 395

RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s 396

instructions, followed by a DNase treatment with Ambion DNA free-kit DNase treatment and 397

removal (Invitrogen). Total RNA (2 µg) was used for the synthesis of cDNA by reverse 398

transcription with SuperScript II Reverse Transcriptase (Invitrogen) and oligo dT (18) primers. 399

400

RNA sequencing transcript profiling. Poly(A) RNA enrichment, library preparation and 401

sequencing were carried out at the Max Planck Genome Center, Cologne, Germany 402

(https://mpgc.mpipz.mpg.de/home/) using 1 µg total RNA. Poly(A) RNA was isolated using 403



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the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and the 404

library was prepared using the NEBNext Ultra Directional II RNA Library Prep Kit for Illumina 405

(New England Biolabs). RNA quality and quantity were checked by capillary electrophoresis 406

(TapeStation, Agilent Technologies) and fluorometry (Qubit, Thermo Fisher Scientific) after 407

each step. Sequencing was carried out using a HiSeq3000 (Illumina) with 1 x 150 bp single 408

reads. 409

Reads derived from the Illumina library were mapped and aligned to the reference genome 410

using HISAT2 followed by assembly and quantification of expression levels in different 411

samples using STRINGTIE. The gene counts of all samples were obtained by using a Python 412

script (http://ccb.jhu.edu/software/stringtie/dl/prepDE.py). The quality of the samples was 413

assessed by producing dispersion plots among replicates. The differentially expressed genes 414

with more than 2-fold change and a corrected p-value below 0.05 were obtained using DESeq2 415

and selected for further analysis. We focused on genes with a greater than 2-fold change in 416

transcript abundance. The complete transcriptome data set is available as series GSE126944 at 417

the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). The Database for 418

Annotation, Visualization and Integrated Discovery (DAVID; Huang et al., 2009) was used for 419

Gene Ontology enrichment analysis for biological processes 5, with Benjamini corrected 420

P0.05. Only Arabidopsis annotated orthologues were included for GO analysis. Data were 421

clustered using Cluster 3.0 and visualized using Java TreeView 422

(http://doi.org/10.5281/zenodo.1303402). From the 25,817 genes which showed transcript 423

accumulation in at least one of the conditions tested, 4983 participated in the hierarchical 424

clustering. Only 11% of genes in the cluster did not behave similarly between replicates 425

(Supplementary Table S2; clusters I0 and II0). Venn diagrams were constructed using Venny 426

v2.1 (http://bioinfogp.cnb.csic.es/tools/venny/). 427

Quantitative real-time PCR. Three technical replicates were prepared using 26 ng of cDNA 428

for each reaction. The relative gene expression values were based on ΔCt calculations using the 429

mean of the two reference gene expression values, according to Pfaffl et al.,58. AaUBI10 and 430

AaPP2A were used for expression data normalization. The ΔCt values were scaled to the 431

average for the control condition. Primers used can be found in Supplementary Table 6. 432

433

Quantification of IAA. For free IAA quantification, V2 stems were cut off directly below the 434

point where stem elongation was observed, axillary buds were removed so that IAA was 435

measured only from three internodes within the V2 zone. Inflorescence (I) and V3 stems 2cm 436



http://ccb.jhu.edu/software/stringtie/dl/prepDE.py

http://www.ncbi.nlm.nih.gov/geo/

http://doi.org/10.5281/zenodo.1303402

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from the base of stems were harvested. Plant material (around 15 mg fresh weight) was purified 437

as previously described in Andersen et al.59, and 500 pg 13C6-IAA internal standard was added 438

to each sample before homogenisation and extraction. Free IAA was quantified in the purified 439

samples using combined gas chromatography - tandem mass spectrometry. 440

441

3H-IAA transport assay. Stem segments (21 mm) from 8-week-old plants (8wLD), from plants 442

at the end of vernalization (+0) and from plants 5 days after vernalization (+5) were cut off 443

directly above the point where no stem elongation was observed. The segments were placed on 444

wet paper and transferred to 30 μl 0.05% MES (pH 5.5–5.7) containing 100 nM 3H-IAA 445

(Hartmann Analytic). After incubation for 10 min, the stem segments were transferred to fresh 446

0.05% MES containing 1 µM IAA for 90 min60. Incubation was performed at 4°C for the stem 447

segment of samples harvested at the end of the vernalization. The stems were then cut into 3 448

mm segments and immersed in Rotiszint eco plus (Roth) for 16 h before the radiolabel was 449

quantified by scintillation for 2 min using a LS6500 Multi-Purpose Scintillation Counter 450

(Beckman Coulter). CPM values were scaled to the average for the 8 week long day plant 451

sample at 6-9 mm or 8 week long day total samples. 452

453

Statistical analysis. Data were processed by analysis of variance (ANOVA) followed by a post 454

hoc test for pairwise multiple comparisons using Tukey post hoc test using the R platform 455

(http://r-project.org/). Pairwise comparisons were analyzed using Student’s t test. 456

457

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606

Acknowledgements 607

M.C.A. and K.T. acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, 608

German Research Foundation) under Germany´s Excellence Strategy – EXC 2048/1 – Project 609



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ID: 390686111. K.L. acknowledges support from the Knut and Alice Wallenberg Foundation 610

(KAW), the Swedish Research Council (VR) and the Swedish Governmental Agency for 611

Innovation Systems (VINNOVA). The authors would like to thank Roger Granbom, Lisa Clark, 612

Julia Benecke, Vicky Tilmes, Kirstin Krause and Panpan Jiang for technical assistance or for 613

sharing primers and Margaret Kox for the critical reading of the manuscript. 614

615

Contributions 616

A.V., P.M., K.T. and M.C.A. conceived the study and designed the experiments; A.V., A.R., 617

K.L., U.N., U.P. performed the experiments; A.V., P.M., A.R., and M.C.A. analysed the data; 618

A.V. and M.C.A. wrote the manuscript with the contributions from all authors. 619

620

Competing interests 621

The authors declare no competing interests 622

623

Figure Legends: 624

Fig. 1: Flowering in the main shoot correlates with perennial shoot architecture in A. 625

alpina. a-e, Analysis of branch formation in plants vernalized for 12 weeks which is sufficient 626

for flowering in A. alpina. Plants were grown in long days (LDs) for 8 weeks (8wLD), 627

vernalized for 12 weeks (+0) and transferred back to LDs for 1, 2, 3 and 9 weeks (+1w, +2w, 628

+3w, +9w). Flowering plants have a complex plant architecture and some axillary branches 629

flower (a) or grow vegetatively (b). Flowering plants also have leaf axils that are empty (c) or 630

contain a branch smaller than 0.5 cm (d). In (e) each column represents a single plant and each 631

square within a column indicates an individual leaf axil. The bottom row represents the oldest 632

leaf axil. Yellow denotes the presence of a flowering branch (a). Green denotes the presence of 633

a vegetative axillary branch (b). Grey denotes an empty leaf axil (c). Brown denotes a leaf axil 634

with a branch smaller than 0.5 cm (d). f, This plant architecture is organized in zones described 635

as V1, V2, V3 and I in Lazaro et al.10. Yellow circle denotes flowering of the main or side 636

shoots, grey and brown circle indicate the presence of dormant buds (V2), green triangle the 637

presence of a vegetative branch. g, Analysis of branch formation and h, branch length in A. 638

alpina plants exposed to shorter lengths of vernalization that do not secure flowering. Plants 639

were grown for 11 weeks in LDs (0), or for 8 weeks in LDs and subsequently vernalized for 3 640

(3w), 8 (8w) or 12 (12w) weeks. Plants were scored 3 weeks after they were returned to a LD 641

greenhouse, n=12. Bar size indicates 1 cm. 642



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643

Fig. 2: The transcriptome of buds that will develop into axillary vegetative branches or 644

stay dormant is already differentiated during vernalization. a-d, GUS activity in 645

longitudinal sections of axillary buds harvested from the nodes that will stay dormant (V2; a 646

and b) or give rise to axillary vegetative branches (V3; c and d) at the end of vernalization (+0; 647

a and c) and five days after vernalization (+5d; b and d) in DR5::GUS A. alpina plants. Arrows 648

indicate GUS signal. e, Venn diagram represents the overlap of genes significantly regulated in 649

the four comparisons tested. Percentages indicate overlap with differentially expressed genes 650

between comparisons. f, Heat map representing the hierarchical clustering of 4983 coexpressed 651

transcripts between V2 and V3 buds at the end of vernalization (+0) and 5 days after 652

vernalization (+5d). Coexpressed clusters were assigned into two higher level clusters (I and 653

II). g-j Selected clusters with common GOs are shown as FPKM (+/- SE) values. Colors 654

indicate specific pattern of interest; orange: genes upregulated specifically in V3 buds after 655

vernalization, blue: genes upregulated specifically in V2 buds after vernalization, green: genes 656

differentially expressed between V2 and V3 buds at the end of vernalization. Numbers in 657

brackets indicate the number of genes present in each cluster. Gene names indicated in bold 658

have been annotated as “ bud dormancy” genes in Tarancon et al.39. 659

660

Fig. 3: Dormancy of V2 buds is regulated by an apical signal after vernalization. a, 661

Analysis of branch formation after excision of axillary branches or buds belonging to different 662

zones in vernalized plants. Control indicates intact plants, I indicates plants in which the 663

inflorescence buds have been dissected, V1 indicates plants in which V1 branches have been 664

removed, I+V3 indicates plants in which the inflorescence and V3 buds have been dissected. 665

As in Fig. 1, each column represents a single plant and each square within a column indicates 666

an individual leaf axil. The bottom row represents the oldest leaf axil. grey denotes an empty 667

leaf axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the 668

presence of a vegetative axillary branch and yellow denotes the presence of a flowering branch. 669

(n= 10 or 12). b, Number of buds per plant after the excision of buds or branches in different 670

zones. c, Relative transcript accumulation of AaHB53, AaDRM2, AaNAC029, AaAIRP2, 671

AaABI1, AaHIS1-3, AaKMD1 and AaSAG21, AaD14 and AaSMAX1 in V2 (light grey) and V3 672

buds (dark grey) at end of vernalization (+0) and five days after vernalization (+5d) in control 673

(V2) and decapitated plants (V2 decapitated, black). Expression levels of all genes was 674

normalized with AaPP2A and AaUBI10. Letters show significant differences between 675



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conditions (P<0.05, n=3) using ANOVA followed by pairwise multiple comparison using 676

Tukey. Errors indicate SD. 677

678

Fig. 4: Endogenous IAA levels and auxin response in the stem within the dormant bud 679

zone increase transiently after vernalization. a-f, GUS activity in stems within the V2 zone 680

a-c, and transversal sections of V2 stem (d-f) at end of vernalization (a,d) and five days after 681

vernalization (b,c,e,f) in control (b,e) and decapitated (c,f) DR5::GUS plants. g-j, IAA level in 682

pg/mg of Fresh Weight (FW) before vernalization (8wLD), end of vernalization (+0), 3 and 5 683

days after vernalization (+3d and +5d), and 1, 2, 3 and 9 weeks after vernalization (+1w, +2w, 684

+3w, and +9w) measured at the V2 stems (g) at the base of the inflorescence stem (h), at the 685

base of the V3 axillary vegetative branches (i) and at V2 stems 5 days after decapitation (j). 686

Letters indicate significant differences between conditions (P<0.05, n=3) using ANOVA 687

followed by pairwise multiple comparison using Tukey. Asterisks in (j) indicate significant 688

differences between control and decapitated plants using student’s t test (P<0.05, n=3). Errors 689

indicate SD. k-l, Analysis of branch formation in A. alpina plants vernalized for 12 weeks and 690

subsequently sprayed with mock or 100μM NAA or 100μM NPA at the end of vernalization 691

and one week after vernalization. Plants were scored 5 weeks after vernalization. As in Fig. 1, 692

each column represents a single plant and each square within a column indicates an individual 693

leaf axil. The bottom row represents the oldest leaf axil. grey denotes an empty leaf axil, brown 694

denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence of a vegetative 695

axillary branch and yellow denotes the presence of a flowering branch. (l) Number of buds per 696

plant (represented with brown and grey boxes) after mock, NAA or NPA treatment. Letters 697

indicate significant differences between conditions (P<0.05, n=12) using ANOVA followed by 698

pairwise multiple comparison using Tukey. m-n, IAA transport capacity in V2 stems in 8 week 699

old plants grown in LD (8wLD), at end of vernalization (+0) and 5 days after vernalization 700

(+5d). 3H-IAA measured in (m) mm of stem from the 3H-IAA source and (n) total 3H-IAA in 701

stem. Errors indicate SE. o, Working model illustrating the reduction of the auxin transport in 702

the V2 zone compared to before (8wLD), at the end of (+0) and 5 days (+5d) after vernalization. 703

Yellow circle denotes flowering of the main or side shoots, grey circle indicates the presence 704

of dormant buds, green triangle the presence of vegetative growth. Red arrow indicates auxin 705

flow in stem. 706

707

Fig. 5: AaBRC1 ensures the maintenance of the dormant V2 buds after vernalization. a, 708

Relative transcript accumulation of AaBRC1, AaBRC2, AaHB53, AaDRM2, AaNAC029, 709



http://dx.doi.org/10.1101/562868


23

AaAIRP2, AaABI1, AaHIS1-3, AaKMD1 and AaSAG21 in 8-week-old plants grown in LD 710

(8wLD; light grey), at the end of vernalization (+0; grey) and five days after vernalization (+5d, 711

dark grey). Transcript levels of all genes are normalized with AaPP2A and AaUBI10. (n=3). 712

Errors indicate SD. b, Analysis of branch formation in wild-type (wt) plants and in 713

35S:AaBRC1dsRNAi lines 1 to 3 in 8-week-old plants grown in LD (8wLD), at the end of 714

vernalization (+0) and five weeks after vernalization (+5w). As in Fig. 1, each column 715

represents a single plant and each square within a column indicates an individual leaf axil. The 716

bottom row represents the oldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf 717

axil with a branch smaller than 0.5 cm, green denotes the presence of a vegetative axillary 718

branch and yellow denotes the presence of a flowering branch. n=12. c-e, Branch number per 719

plant in wt plants and in 35S:AaBRC1dsRNAi lines 1 to 3 measured in 8-week-old plants grown 720

in LDs (c), at the end of the vernalization period (d) and 5 weeks after vernalization (e). f, Bud 721

number per plant 5 weeks after vernalization. Letters indicate significant differences between 722

conditions (P<0.05) using ANOVA followed by pairwise multiple comparison using Tukey. 723

724

Supplementary Figures: 725

Supplementary Fig. 1: Extended vernalization accelerates the outgrowth of the vegetative 726

branches (V3) and the inflorescence but does not influence the final shoot architecture. a-727

b, Length of the vegetative branches (V3) (a) and inflorescence (b) at 1, 2 and 3 weeks (+1w, 728

+2w, +3w) after vernalization measured in plants grown for 8 weeks in LDs and vernalized for 729

12, 15, 18, 21 and 24 weeks. (c) Analysis of branch formation in plants vernalized for 12, 15, 730

18, 21 and 24 weeks, measured 3 weeks after vernalization. As in Fig. 1, each column represents 731

a single plant and each square within a column indicates an individual leaf axil. The bottom 732

row represents the oldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf axil 733

with a branch smaller than 0.5 cm, green denotes the presence of a vegetative axillary branch 734

and yellow denotes the presence of a flowering branch. d, Number of buds per plant (represented 735

with brown and grey boxes) in plants vernalized for 12, 15, 18, 21 and 24 weeks. Letters show 736

significant differences between conditions (P<0.05) using ANOVA followed by pairwise 737

multiple comparison using Tukey. n=9-12. 738

739

Supplementary Fig. 2: Duration of vernalization influences floral commitment in the 740

shoot apical meristem. Percentage of vegetative plants, plants with floral reversion and 741

flowering plants after different durations of vernalization. Plants were grown for 8 weeks in 742



http://dx.doi.org/10.1101/562868


24

LDs (0), or vernalized for 3 (3), 8 (8) or 12 (12) weeks. Plants were scored 3 weeks after they 743

were returned to a LD greenhouse, n=12. 744

745

Supplementary Fig. 3: All axils in the A. alpina pep1-1 mutant develop a flowering axillary 746

branch. a-b, Analysis of branch formation in wild type (wt) (a) and pep1-1 mutant (b) and c, 747

Branch length according to the node position in wt and pep1-1 mutant growing for 5, 7, 9, 10 748

and 13 weeks in a long day greenhouse (LD). As in Fig. 1, each column represents a single 749

plant and each square within a column indicates an individual leaf axil. The bottom row 750

represents the oldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf axil with a 751

branch smaller than 0.5 cm, green denotes the presence of a vegetative axillary branch and 752

yellow denotes the presence of a flowering branch. I indicates the inflorescence branches in 753

pep1-1. n=12. 754

755

Supplementary Fig. 4: Only flowering plants have a zone with inhibited buds after 756

vernalization. a-c, Analysis of branch formation in juvenile and adult plants after 757

vernalization. Plants were grown for 3 weeks (juvenile) or 5 weeks (adult) in long days (LDs) 758

before being vernalized for 12 weeks. Only 5-week-old vernalized plants will initiate flowering 759

during vernalization whereas 3-week-old vernalized plants continue vegetative growth. Pictures 760

of an adult (a) and a juvenile (b) vernalized plant after being returned for 2 weeks in LDs. For 761

(c), similar to Fig. 1, each column represents a single plant and each square within a column 762

indicates an individual leaf axil. The bottom row represents the oldest leaf axil. grey denotes an 763

empty leaf axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the 764

presence of a vegetative axillary branch and yellow denotes the presence of a flowering branch. 765

766

Supplementary Fig. 5: GO enrichment analysis in differentially regulated genes in V2 and V3 767

buds at the end of vernalization (+0) and five days after vernalization (+5d). Circle size indicats 768

the number of genes in the GO category. 769

770

Supplementary Fig. 6: Dormancy of V2 buds is slightly affected by the removal of V3 771

branches 2 weeks after vernalization. a, Analysis of branch formation and b, number of buds 772

in plants after removal of axillary vegetative branches in the V3 zone 2 weeks after 773

vernalization. Plants were grown for 8 weeks in long days (LDs) and vernalized for 12 weeks. 774

Scoring of the branching pattern in each node was performed 2 weeks after treatment. As in 775

Fig. 1, each column represents a single plant and each square within a column indicates an 776



http://dx.doi.org/10.1101/562868


25

individual leaf axil. The bottom row represents the oldest leaf axil. grey denotes an empty leaf 777

axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence 778

of a vegetative axillary branch and yellow denotes the presence of a flowering branch. Asterisks 779

indicate significant differences using student’s t test, P<0.05 (n= 10). 780

781

Supplementary Fig. 7: Buds in the V2 zone respond to decapitation before or after 782

different vernalization durations after being returned to LD greenhouse conditions but 783

not when remained in vernalization a, Schematic representation of the experimental design 784

of b. Plants were grown for 8 weeks in long days (8wLD) and subsequently vernalized for 0, 3, 785

8 and 12 weeks. Prior to being returned to warm temperatures, plants were decapitated. b, 786

Length of new branches of control (white) or decapitated plants (grey) at 0, 3, 8 and 12 weeks 787

of vernalization. Branch length was scored 2 weeks after decapitation. Control plants are the 788

same as in Fig. 1g and h. c, Schematic representation of the experimental design of d and e. 789

Plants were grown for 8 weeks in long days (8wLD), decapitated and subsequently vernalized 790

for 12 weeks. d, Analysis of branch formation in plants at the end of the 12 week vernalization 791

period in control plants and decapitated plants. As in Fig. 1, each column represents a single 792

plant and each square within a column indicates an individual leaf axil. The bottom row 793

represents the oldest leaf axil.grey denotes an empty leaf axil, brown denotes a leaf axil with a 794

branch smaller than 0.5 cm and green denotes the presence of a vegetative axillary branch . e, 795

Number of buds in nodes 1-11 after 12 weeks of vernalization in control plants or decapitated 796

plants. Asterisks indicate significant differences using student’s t test P<0.05 between control 797

and decapitation. n=12. 798

799

Supplementary Fig. 8: GUS transcript accumulation in DR5::GUS A. alpina lines 4, 6 and 15. 800

GUS transcript accumulation was tested in V2 stems of plants grown for 8 weeks in LDs and 801

vernalized for 12 weeks at the end of vernalization (+0) and 5 days after in control plants (+5d) 802

and decapitated plants (+5d+decap). Samples were normalized with AaPP2A and AaUBI10. 803

Letters show significant differences between conditions (P<0.05, n=3) using ANOVA followed 804

by pairwise multiple comparison using Tukey. 805

806

Supplementary Fig. 9: Transcript accumulation of dormancy-associated genes was 807

reduced after vernalization in 35S:AaBRC1dsRNAi lines. a, Phylogenetic tree showing 808

relationship between A. alpina BRC1 and BRC2 homologues. b, Relative transcript 809

accumulation of AaBRC1, AaBRC2, AaHB53, AaDRM2, AaHIS1-3 and AaAIRP2 in V2 buds 810



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26

in 8-week-old plants grown in LD (8wLD; light grey), at end of vernalization (+0; grey) and 811

five days after vernalization (+5d, dark grey) in wt plants and in 35S:AaBRC1dsRNAi lines 1 812

to 3. Expression of all genes was normalized with AaPP2A and AaUBI10. Asterisks indicate 813

significant differences between the tested conditions and the wt using student’s t test (P<0.05, 814

n=3). Errors indicate SD. 815

816

Supplementary Fig. 10: 35S:AaBRC1dsRNAi lines do not show major differences in 817

branch length and total leaf number. a-c, Branch length according to the node position in wt 818

and 35S:AaBRC1dsRNAi lines 1-3 before- (a), at the end- (b) and 2 weeks after- (c) 819

vernalization. d-g, Pictures of the wt (d), the 35S:AaBRC1dsRNAi lines 1 (e), line 2 (f) and line 820

3 (g) 3 weeks after vernalization. h, Total leaf number at flowering in wt and the 821

35S:AaBRC1dsRNAi lines 1 -3. Asterisks indicate significant differences between the tested 822

condition and the wt using student’s t test (P<0.05, n=10-12). Hashtags indicate nodes where 823

less than 3 branches could be measured for the wt plants. 824

825

Supplementary Tables 826

Supplementary Table 1. List of genes whose transcript levels have been identified to be 827

differentially expressed between V2 and V3 buds at the end of vernalization and 5 days later. 828

829

Supplementary Table 2. GO enrichment categories identified in genes whose transcript levels 830

have been identified to be differentially expressed between V2 and V3 buds at the end of 831

vernalization and 5 days later. 832

833

Supplementary Table 3. List of coexpressed clusters obtained after hierarchical clustering of 834

the transcript accumulated in V2 and V3 buds at the end of vernalization and 5 days later. 835

836

Supplementary Table 4. GO enrichment categories identified in the different coexpressed 837

clusters. 838

839

Supplementary Table 5. Genes differentially regulated in at least one of the conditions, 840

homologues to A. thaliana gene identified as “bud dormancy” marker genes in Tarancón et al39 841

842

Supplementary Table 6: Primers used in this article. 843

844



http://dx.doi.org/10.1101/562868


h

0 3 8 12

0 3 8 12

V3

V2Node p

ositio

n

Branch length (cm)

8wLD +0 +1w +2w +3w +9w

e

G

d

a

c

b

Time after vernalization

I

V3

V2

V1

f

g

Fig. 1: Flowering in the main shoot correlates with perennial shoot architecture in A. alpina. a-e, Analysis of branch formation in plants vernalized for 12weeks which is sufficient for flowering in A. alpina. Plants were grown in long days (LDs) for 8 weeks (8wLD), vernalized for 12 weeks (+0) and transferredback to LDs for 1, 2, 3 and 9 weeks (+1w, +2w, +3w, +9w). Flowering plants have a complex plant architecture and some axillary branches flower (a) or growvegetatively (b). Flowering plants also have leaf axils that are empty (c) or contain a branch smaller than 0.5 cm (d). In (e) each column represents a singleplant and each square within a column indicates an individual leaf axil. The bottom row represents the oldest leaf axil. Yellow denotes the presence of aflowering branch (a). Green denotes the presence of a vegetative axillary branch (b). Grey denotes an empty leaf axil (c). Brown denotes a leaf axil with abranch smaller than 0.5 cm (d). f, This plant architecture is organized in zones described as V1, V2, V3 and I in Lazaro et al.10. Yellow circle denotes floweringof the main or side shoots, grey and brown circle indicate the presence of dormant buds (V2), green triangle the presence of a vegetative branch. g,Analysis of branch formation and h, branch length in A. alpina plants exposed to shorter lengths of vernalization that do not secure flowering. Plants weregrown for 11 weeks in LDs (0), or for 8 weeks in LDs and subsequently vernalized for 3 (3w), 8 (8w) or 12 (12w) weeks. Plants were scored 3 weeks afterthey were returned to a LD greenhouse, n=12. Bar size indicates 1 cm.



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A

GO:0009414~response to water deprivation

GO:0009737~response to abscisic acid

GO:0006259~DNA metabolic process

GO:0006260~DNA replication

GO:0080027~response to herbivore

GO:0010026~trichome differentiation

GO:0090626~plant epidermis morphogenesis

GO:0045165~cell fate commitment

I3 (248)

CYCLIN D6;1, EXPB1

I6 (312)

YUCCA2, IAA7, IAA14, PILS3,

DAO2

I14 (115)

AIRP2, PP2CA, NCED3, KMD1, CKX1,

ERD10

I15 (182)

ABI5, ABF1, AFP3, AFP1, KMD2,

KMD3, HB53, DRM2, SUS3, TFL1

I20 (355)

RAB18, PYL7, KMD4, SAG21, HB21, PSY1,

NAC029, TPS11,TEM1

I21 (932)

ABI1, KIN2, BLH1, ABF2, ERD14, D14,

SMAX1, HIS1-3, EBF2, SMZ

II7 (397)

CYCB2;2, CYCA3;2, PCNA1

II8 (377)

RPL24A, CYCB1;3

e

+5dV2 vs +5dV3

61%

+0V2 vs +0V3

93%

+0V2 vs +5dV2

73%

+0V3 vs +5dV3

72%

a

c

b

d

I

II

I3

I6

I21

I20

II8

II7

I14

I15

+0V2 +0V3 +5dV3+5dV2

1 2 3 1 2 3 1 2 3 1 2

3

3

f

g h

i

j

Fig. 2: The transcriptome of buds that will develop into axillary vegetative branches or stay dormant is alreadydifferentiated during vernalization. a-d, GUS activity in longitudinal sections of axillary buds harvested from the nodes thatwill stay dormant (V2; a and b) or give rise to axillary vegetative branches (V3; c and d) at the end of vernalization (+0; a andc) and five days after vernalization (+5d; b and d) in DR5::GUS A. alpina plants. Arrows indicate GUS signal. e, Venn diagramrepresents the overlap of genes significantly regulated in the four comparisons tested. Percentages indicate overlap withdifferentially expressed genes between comparisons. f, Heat map representing the hierarchical clustering of 4983coexpressed transcripts between V2 and V3 buds at the end of vernalization (+0) and 5 days after vernalization (+5d).Coexpressed clusters were assigned into two higher level clusters (I and II). g-j Selected clusters with common GOs areshown as FPKM (+/- SE) values. Colors indicate specific pattern of interest; orange: genes upregulated specifically in V3 budsafter vernalization, blue: genes upregulated specifically in V2 buds after vernalization, green: genes differentially expressedbetween V2 and V3 buds at the end of vernalization. Numbers in brackets indicate the number of genes present in eachcluster. Gene names indicated in bold have been annotated as “ bud dormancy” genes in Tarancon et al.39.

0

50

100

150

200

+0V2 +0V3 +5dV2 +5dV3

FP

KM

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FP

KM

0

50

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+0V2 +0V3 +5dV2 +5dV3

FP

KM

0

50

100

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+0V2 +0V3 +5dV2 +5dV3

FP

KM

0

50

100

+0V2 +0V3 +5dV2 +5dV3

FP

KM

0

50

100

+0V2 +0V3 +5dV2 +5dV3

FP

KM

0

50

100

150

+0V2 +0V3 +5dV2 +5dV3

FP

KM

0

100

200

300

400

+0V2 +0V3 +5dV2 +5dV3

FP

KM

GO:0000280~nuclear division

GO:0065004~protein-DNA complex assembly

+5dV2

+5dV3+0dV3

+0dV2



http://dx.doi.org/10.1101/562868


0

2

4

6

+0 +5d

SAG21

0

1

2

3

4

+0 +5d

AaNAC029

a

b

a

b b

0

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+0 +5d

AaSMAX1

ab

bcc

d

0

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1

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AaHB53

a

c

b

c c

0

1

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3

4

+0 +5d

AaKMD1

abab

bb

a

0

1

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+0 +5d

AaHIS1-3

a

ab

bb b

0

0,5

1

1,5

+0 +5d

AaD14

bc

d

a

c

ab

0

1

2

3

4

5

+0 +5d

AaAIRP2

ab

b

a

b b

a control I V1 I+V3

V1

V2

V3

I

Fig. 3: Dormancy of V2 buds is regulated by an apical signal after vernalization. a, Analysis of branch formation afterexcision of axillary branches or buds belonging to different zones in vernalized plants. Control indicates intact plants, Iindicates plants in which the inflorescence buds have been dissected, V1 indicates plants in which V1 branches havebeen removed, I+V3 indicates plants in which the inflorescence and V3 buds have been dissected. As in Fig. 1, eachcolumn represents a single plant and each square within a column indicates an individual leaf axil. The bottom rowrepresents the oldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf axil with a branch smaller than 0.5cm, green denotes the presence of a vegetative axillary branch and yellow denotes the presence of a flowering branch.(n= 10 or 12). b, Number of buds per plant after the excision of buds or branches in different zones. c, Relative transcriptaccumulation of AaHB53, AaDRM2, AaNAC029, AaAIRP2, AaABI1, AaHIS1-3, AaKMD1 and AaSAG21, AaD14 andAaSMAX1 in V2 (light grey) and V3 buds (dark grey) at end of vernalization (+0) and five days after vernalization (+5d) incontrol (V2) and decapitated plants (V2 decapitated, black). Expression levels of all genes was normalized with AaPP2Aand AaUBI10. Letters show significant differences between conditions (P<0.05, n=3) using ANOVA followed by pairwisemultiple comparison using Tukey. Errors indicate SD.

0

1

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+0 +5d

AaABI1

a

b

b b b

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AaDRM2

a

bbc

c c

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b b



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+2w +3w +9w

IAA

(p

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30

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45

Control DecapI1+V3

IAA

(p

g/m

g F

W)

i j

a b c

d e f

*

a a

a

0

10

20

30

40

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60

70

8wLD +0 +3d +5d +1w +2w +3w +9w

IAA

(p

g/m

g F

W)

g

b

c

bcbc

bc

bc ab

a

after vernalization

0

10

20

30

40

50

60

70

+2w +3w +9w

IAA

(p

g/m

g F

W)

after vernalizationafter vernalization

Fig. 4: Endogenous IAA levels and auxin response in the stem within the dormant bud zone increase transiently after vernalization. a-f, GUSactivity in stems within the V2 zone a-c, and transversal sections of V2 stem (d-f) at end of vernalization (a,d) and five days after vernalization(b,c,e,f) in control (b,e) and decapitated (c,f) DR5::GUS plants. g-j, IAA level in pg/mg of Fresh Weight (FW) before vernalization (8wLD), end ofvernalization (+0), 3 and 5 days after vernalization (+3d and +5d), and 1, 2, 3 and 9 weeks after vernalization (+1w, +2w, +3w, and +9w)measured at the V2 stems (g) at the base of the inflorescence stem (h), at the base of the V3 axillary vegetative branches (i) and at V2 stems 5days after decapitation (j). Letters indicate significant differences between conditions (P<0.05, n=3) using ANOVA followed by pairwise multiplecomparison using Tukey. Asterisks in (j) indicate significant differences between control and decapitated plants using student’s t test (P<0.05,n=3). Errors indicate SD. k-l, Analysis of branch formation in A. alpina plants vernalized for 12 weeks and subsequently sprayed with mock or100μM NAA or 100μM NPA at the end of vernalization and one week after vernalization. Plants were scored 5 weeks after vernalization. As inFig. 1, each column represents a single plant and each square within a column indicates an individual leaf axil. The bottom row represents theoldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence of avegetative axillary branch and yellow denotes the presence of a flowering branch. (l) Number of buds per plant (represented with brown andgrey boxes) after mock, NAA or NPA treatment. Letters indicate significant differences between conditions (P<0.05, n=12) using ANOVAfollowed by pairwise multiple comparison using Tukey.m-n, IAA transport capacity in V2 stems in 8 week old plants grown in LD (8wLD), at endof vernalization (+0) and 5 days after vernalization (+5d). 3H-IAA measured in (m) mm of stem from the 3H-IAA source and (n) total 3H-IAA instem. Errors indicate SE. o, Working model illustrating the reduction of the auxin transport in the V2 zone compared to before (8wLD), at theend of (+0) and 5 days (+5d) after vernalization. Yellow circle denotes flowering of the main or side shoots, grey circle indicates the presence ofdormant buds, green triangle the presence of vegetative growth. Red arrow indicates auxin flow in stem.

I+V3Mock NAA NPA

k

I

V3

V2

V1

+5d

l

a

c

b

mock NAA NPA

Bu

d n

um

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V3

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V1

+0

n

0

0,2

0,4

0,6

0,8

1

1,2

1,4

6-9 9-12 12-15 15-18 18-21

Re

lative

3H

-IA

Ab

asip

eta

ltra

nsp

ort

Distance from the 3H-IAA source (mm)

8wLD

0

+5d

0

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tive

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A b

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a

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http://dx.doi.org/10.1101/562868


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AaHB53

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AaNAC029

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AaKMD1

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AaBRC1

aa

b

0

0,5

1

8wLD +0 +5d

AaBRC2

a a

a

8wLD

+0

+5w

RNAi-BRC1 lines

Rela

tive

tra

nscri

pt

accu

mu

latio

n

Rela

tive

tra

nscri

pt

accu

mu

latio

n

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6

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10

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AaSAG21

a

ab

b

wt RNAi1 RNAi2 RNAi3a b

c d

e fB

ud

nu

mb

er

Bra

nch

nu

mb

er

Bra

nch

nu

mb

er

Bra

nch

nu

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Fig. 5: AaBRC1 ensures the maintenance of the dormant V2 buds after vernalization. a, Relative transcript accumulation ofAaBRC1, AaBRC2, AaHB53, AaDRM2, AaNAC029, AaAIRP2, AaABI1, AaHIS1-3, AaKMD1 and AaSAG21 in 8-week-old plants grown inLD (8wLD; light grey), at the end of vernalization (+0; grey) and five days after vernalization (+5d, dark grey). Transcript levels of allgenes are normalized with AaPP2A and AaUBI10. (n=3). Errors indicate SD. b, Analysis of branch formation in wild-type (wt) plantsand in 35S:AaBRC1dsRNAi lines 1 to 3 in 8-week-old plants grown in LD (8wLD), at the end of vernalization (+0) and five weeks aftervernalization (+5w). As in Fig. 1, each column represents a single plant and each square within a column indicates an individual leafaxil. The bottom row represents the oldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf axil with a branchsmaller than 0.5 cm, green denotes the presence of a vegetative axillary branch and yellow denotes the presence of a floweringbranch. n=12. c-e, Branch number per plant in wt plants and in 35S:AaBRC1dsRNAi lines 1 to 3 measured in 8-week-old plantsgrown in LDs (c), at the end of the vernalization period (d) and 5 weeks after vernalization (e). f, Bud number per plant 5 weeksafter vernalization. Letters indicate significant differences between conditions (P<0.05) using ANOVA followed by pairwise multiplecomparison using Tukey.

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Supplementary Fig. 1: Extended vernalization accelerates the outgrowth of the vegetative branches (V3) and theinflorescence but does not influence the final shoot architecture. a-b, Length of the vegetative branches (V3) (a) andinflorescence (b) at 1, 2 and 3 weeks (+1w, +2w, +3w) after vernalization measured in plants grown for 8 weeks in LDsand vernalized for 12, 15, 18, 21 and 24 weeks. (c) Analysis of branch formation in plants vernalized for 12, 15, 18, 21and 24 weeks, measured 3 weeks after vernalization. As in Fig. 1, each column represents a single plant and each squarewithin a column indicates an individual leaf axil. The bottom row represents the oldest leaf axil. grey denotes an emptyleaf axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence of a vegetativeaxillary branch and yellow denotes the presence of a flowering branch. d, Number of buds per plant (represented withbrown and grey boxes) in plants vernalized for 12, 15, 18, 21 and 24 weeks. Letters show significant differences betweenconditions (P<0.05) using ANOVA followed by pairwise multiple comparison using Tukey. n=9-12.

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Supplementary Fig. 2: Duration of vernalization influences floral commitment in the shoot apical meristem. Percentage of vegetative plants,plants with floral reversion and flowering plants after different durations of vernalization. Plants were grown for 8 weeks in LDs (0), orvernalized for 3 (3), 8 (8) or 12 (12) weeks. Plants were scored 3 weeks after they were returned to a LD greenhouse, n=12.

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Supplementary Fig. 3: All axils in the A. alpina pep1-1 mutant develop a flowering axillary branch. a-b, Analysis of branch formation in wild type (wt) (a)and pep1-1 mutant (b) and c, Branch length according to the node position in wt and pep1-1 mutant growing for 5, 7, 9, 10 and 13 weeks in a long daygreenhouse (LD). As in Fig. 1, each column represents a single plant and each square within a column indicates an individual leaf axil. The bottom rowrepresents the oldest leaf axil. grey denotes an empty leaf axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence ofa vegetative axillary branch and yellow denotes the presence of a flowering branch. I indicates the inflorescence branches in pep1-1. n=12.

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Supplementary Fig. 4: Only flowering plants have a zone with inhibited buds after vernalization. a-c, Analysis of branchformation in juvenile and adult plants after vernalization. Plants were grown for 3 weeks (juvenile) or 5 weeks (adult) in longdays (LDs) before being vernalized for 12 weeks. Only 5-week-old vernalized plants will initiate flowering duringvernalization whereas 3-week-old vernalized plants continue vegetative growth. Pictures of an adult (a) and a juvenile (b)vernalized plant after being returned for 2 weeks in LDs. For (c), similar to Fig. 1, each column represents a single plant andeach square within a column indicates an individual leaf axil. The bottom row represents the oldest leaf axil. grey denotes anempty leaf axil, brown denotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence of a vegetativeaxillary branch and yellow denotes the presence of a flowering branch.

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Supplementary Fig. 5: GO enrichment analysis in differentially regulated genes in V2 and V3 buds at the end of vernalization (+0) and five daysafter vernalization (+5d). Circle size indicats the number of genes in the GO category.



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Supplementary Fig. 7: Buds in the V2 zone respond to decapitation before or after different vernalization durationsafter being returned to LD greenhouse conditions but not when remained in vernalization a, Schematic representationof the experimental design of b. Plants were grown for 8 weeks in long days (8wLD) and subsequently vernalized for 0, 3,8 and 12 weeks. Prior to being returned to warm temperatures, plants were decapitated. b, Length of new branches ofcontrol (white) or decapitated plants (grey) at 0, 3, 8 and 12 weeks of vernalization. Branch length was scored 2 weeksafter decapitation. Control plants are the same as in Fig. 1g and h. c, Schematic representation of the experimentaldesign of d and e. Plants were grown for 8 weeks in long days (8wLD), decapitated and subsequently vernalized for 12weeks. d, Analysis of branch formation in plants at the end of the 12 week vernalization period in control plants anddecapitated plants. As in Fig. 1, each column represents a single plant and each square within a column indicates anindividual leaf axil. The bottom row represents the oldest leaf axil.grey denotes an empty leaf axil, brown denotes a leafaxil with a branch smaller than 0.5 cm and green denotes the presence of a vegetative axillary branch . e, Number ofbuds in nodes 1-11 after 12 weeks of vernalization in control plants or decapitated plants. Asterisks indicate significantdifferences using student’s t test P<0.05 between control and decapitation. n=12.

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Supplementary Fig. 6: Dormancy of V2 buds is slightly affected by the removal of V3 branches 2 weeks aftervernalization. a, Analysis of branch formation and b, number of buds in plants after removal of axillaryvegetative branches in the V3 zone 2 weeks after vernalization. Plants were grown for 8 weeks in long days (LDs)and vernalized for 12 weeks. Scoring of the branching pattern in each node was performed 2 weeks aftertreatment. As in Fig. 1, each column represents a single plant and each square within a column indicates anindividual leaf axil. The bottom row represents the oldest leaf axil. grey denotes an empty leaf axil, browndenotes a leaf axil with a branch smaller than 0.5 cm, green denotes the presence of a vegetative axillarybranch and yellow denotes the presence of a flowering branch. Asterisks indicate significant differences usingstudent’s t test, P<0.05 (n= 10).

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Supplementary Fig. 9: Transcript accumulation of dormancy-associated genes was reduced aftervernalization in 35S:AaBRC1dsRNAi lines. a, Phylogenetic tree showing relationship between A. alpinaBRC1 and BRC2 homologues. b, Relative transcript accumulation of AaBRC1, AaBRC2, AaHB53, AaDRM2,AaHIS1-3 and AaAIRP2 in V2 buds in 8-week-old plants grown in LD (8wLD; light grey), at end ofvernalization (+0; grey) and five days after vernalization (+5d, dark grey) in wt plants and in35S:AaBRC1dsRNAi lines 1 to 3. Expression of all genes was normalized with AaPP2A and AaUBI10.Asterisks indicate significant differences between the tested conditions and the wt using student’s t test(P<0.05, n=3). Errors indicate SD.

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http://dx.doi.org/10.1101/562868


short title: 6 1,2,3,# 2,3 · 24. vegetative branches and a zone of dormant buds that . serve as...

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