2 3 corresponding author - plant physiology83 sgh1, and sgh3, respectively). vrn-h1 encodes a...

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Running head: Barley Phytochrome C controls long-day flowering 1

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Corresponding author: Kenji Kato 3

Graduate School of Environmental and Life Science, Okayama University, 1-1-1, Tsushima-Naka, 4

Kita-Ku, Okayama 700-8530, Japan 5

Phone: +81-86-251-8323 6

e-mail: [email protected] 7

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Research area: Genes, Development and Evolution 9

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Plant Physiology Preview. Published on September 6, 2013, as DOI:10.1104/pp.113.222570

Copyright 2013 by the American Society of Plant Biologists

www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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Title of article: Phytochrome C Is A Key Factor Controlling Long-day Flowering in Barley 12 (Hordeum vulgare L.) 13

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All authors’ full names: Hidetaka Nishida, Daisuke Ishihara, Makoto Ishii, Takuma Kaneko, 15

Hiroyuki Kawahigashi, Yukari Akashi, Daisuke Saisho, Katsunori Tanaka, Hirokazu Handa, 16

Kazuyoshi Takeda, Kenji Kato 17

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Institution addresses (authors’ initials): 19

Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, 20

Japan (H. N., D. I., T. K., Y. A., K. K.) 21

Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046 Japan (M. I., D. 22

S., K. T.) 23

Faculty of Humanities, Hirosaki University, Hirosaki 036-8560, Japan (K. T.) 24

National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan (H. K., H. H.) 25

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One-sentence summary: In a long-day plant barley, a red/far-red light photoreceptor gene HvPhyC 27

controls flowering time under long photoperiods by stimulating a flowering promoter gene HvFT1 in 28

the most downstream part of the genetic pathways for flowering. The results showed that HvPhyC is 29

a key factor for long-day flowering. 30

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Financial sources: This work was partly supported by a grant from the Ministry of Agriculture, 34

Forestry and Fisheries of Japan (Integrated research project for plant, insect, and animal using 35

genome technology GD-3005) to Hirokazu Handa and a grant from Ministry of Agriculture, Forestry 36

and Fisheries of Japan (Genomics for Agricultural Innovation, FBW-1201) to Hidetaka Nishida. 37

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Corresponding autor (e-mail): Kenji Kato ([email protected]) 39

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The author(s) responsible for the distribution of materials integral to the findings presented in this 41

article in accordance with the Journal policy described in the Instructions for Authors 42

(http://www.plantphysiol.org) are: Kenji Kato ([email protected]) and Hirokazu Handa 43

([email protected]) 44

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ABSTRACT 47

The spring-type near isogenic line (NIL) of the winter-type barley (Hordeum 48

vulgare ssp. vulgare L.) variety Hayakiso 2 (HK2) was developed by introducing 49

Vrn-H1 for spring growth habit from a spring-type variety Indo Omugi. Contrary to 50

expectations, the spring-type NIL flowered later than winter-type HK2. This phenotypic 51

difference was controlled by a single gene which co-segregated only with phytochrome 52

C (HvPhyC) among three candidates around the Vrn-H1 region (Vrn-H1, HvPhyC, and 53

HvCK2α), indicating that HvPhyC was the most likely candidate gene. Compared to the 54

late-flowering allele HvPhyC-l from the NIL, the early-flowering allele HvPhyC-e from 55

HK2 had a single nucleotide polymorphism T1139C in exon 1, which caused a 56

non-synonymous amino acid substitution Phe380Ser in the functionally essential GAF 57

domain. Functional assay using a rice (Oryza sativa L.) phyA phyC double mutant line 58

showed that both of the HvPhyC alleles are functional but HvPhyC-e may have a 59

hyperfunction. Expression analysis using NILs carrying HvPhyC-e and HvPhyC-l (NIL 60

(HvPhyC-e) and NIL (HvPhyC-l), respectively) showed that HvPhyC-e up-regulated 61

only the flowering promoter HvFT1 by bypassing the circadian clock genes and 62

flowering integrator HvCO1 under a long photoperiod. Consistent with the 63

up-regulation, NIL (HvPhyC-e) flowered earlier than NIL (HvPhyC-l) under long 64

photoperiods. These results implied that HvPhyC is a key factor to control long-day 65

flowering directly. 66

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INTRODUCTION 69

The flowering time of barley (Hordeum vulgare ssp. vulgare L.) is a complex 70

character that is composed of three different sub-characters: earliness per se, 71

vernalization requirement, and photoperiod sensitivity (Takahashi and Yasuda, 1970). 72

The first character determines flowering time alone, while the latter two modify it in 73

response to environmental signals including temperature and photoperiod. These are 74

essential for successful seed set and maximizing yield by contributing greatly to 75

adaptation to different climatic regions (Knüpffer et al., 2003; von Bothmer et al., 76

2003). 77

In winter-type barley, the vernalization requirement avoids frost injury by delaying 78

flower induction until the extended period of cold temperature during winter satisfies 79

the requirement. On the other hand, spring-type barley does not have such a requirement. 80

The vernalization requirement is controlled by three major genes: VERNALIZATION-H1, 81

2, and 3 (Vrn-H1, 2, and 3, respectively; former SPRING GROWTH HABIT2 (Sgh2), 82

Sgh1, and Sgh3, respectively). Vrn-H1 encodes a protein highly similar to Arabidopsis 83

(Arabidopsis thaliana (L.) Heynh.) APETALA1 (AP1) / FRUITFULL (FUL) that 84

determines meristem identity, flower organ formation, and flowering time (Yan et al., 85

2003). Vrn-H2 encodes ZCCT protein with a putative zinc finger and a CCT domain, 86

which is expected to be involved in transcriptional regulation and is expressed under 87

long photoperiods (Yan et al., 2004). Vrn-H3, which encodes an ortholog of Arabidopsis 88

flowering promoter FLOWERING LOCUS T (FT) and long photoperiods up-regulate 89

Vrn3 expression (Turner et al., 2005; Yan et al., 2006). It was proposed that they form a 90

feedback loop and interact to regulate their expression (Trevaskis et al., 2007; Distelfeld 91

et al., 2009; Shimada et al., 2009). 92



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Barley is a long-day plant in which photoperiod sensitivity delays flowering time 93

under a short photoperiod compared with that under a long photoperiod. It is well 94

known that photoperiod sensitivity greatly contributes to adaptation (Knüpffer et al., 95

2003). Two genes that influence photoperiod sensitivity are PHOTOPERIOD-H1 and 2 96

(Ppd-H1 and 2, respectively; Laurie et al., 1995). Ppd-H1 controls flowering time under 97

long photoperiods and encodes pseudo-response regulator (PRR) whose ortholog is 98

involved in circadian clock function in Arabidopsis (Turner et al., 2005). Ppd-H2 99

controls flowering time under short photoperiods and it encodes HvFT3, which belongs 100

to the gene family of FT (Kikuchi et al., 2009). 101

In addition to the above-mentioned genes, novel gene resources for early flowering 102

will be important to elucidate the genetic mechanism of the flowering time and future 103

breeding programs. Recent comparative studies in genetic pathways for flowering 104

revealed that temperate grass species share a similar gene set with dicot species 105

Arabidopsis, especially for photoperiodic pathways, although it has been disclosed 106

gradually that evolutionarily distinct genes and pathways are associated with the 107

photoperiodic pathways (Trevaskis et al., 2007; Higgins et al., 2010). These pathways 108

include photoreceptors (phytochromes, cryptochromes, and phototropin) that perceive 109

daily light/dark cycles, the circadian clock (CIRCADIAN CLOCK ASSOCIATED1, 110

TIMING OF CAB EXPRESSION 1, PRRs (like Ppd-H1), and GIGANTEA (GI; HvGI in 111

barley)), which is entrained by the signals from photoreceptors, and downstream genes 112

(CONSTANS (CO; HvCO1 in barley), FT (HvFT1 in barley), and AP1/FUL (Vrn-H1)), 113

which integrates and transmits the signals from photoreceptors and the circadian clock. 114

However, natural variation in many of such genes has not been characterized yet in 115

barley. 116



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Yasuda (1969) developed a series of near isogenic lines (NILs) carrying different 117

spring alleles Sgh2 (Vrn-H1) in a Japanese winter variety Hayakiso 2 (hereafter, HK2) 118

that had a winter allele sgh2 (vrn-H1) originally. These NILs, compared to HK2, 119

showed unexpectedly later flowering under autumn-sowing field conditions irrespective 120

of their spring growth habit. Such behavior may be ascribable to the pleiotropic effect of 121

Vrn-H1 or an unknown flowering time gene tightly linked with Vrn-H1. According to 122

previous studies (Szücs et al., 2006; Kato et al., 2008), Vrn-H1 is located closely to 123

other two candidate genes for photoperiod sensitivity, Phytochrome C (HvPhyC) and 124

Casein Kinase II alpha (HvCK2α). HvPhyC encodes the apoprotein of photoreceptor 125

PHYC, which is involved in red/far-red light perception. PhyC orthologs in other 126

species are also associated with flowering time: the rice ortholog controls photoperiod 127

sensitivity (Takano et al., 2005) and Arabidopsis alleles showed latitudinal cline, which 128

suggested the association of photoperiod sensitivity (Balasubramanian et al., 2006). 129

HvCK2α encodes the alpha subunit of CK2 protein. A rice flowering-time gene Hd6, 130

which is considered to be an ortholog of HvCK2α by cross-referencing syntheny among 131

barley, wheat (Triticum aestivum L.), and rice (Oryza sativa L.), controls photoperiod 132

sensitivity (Takahashi et al., 2001; Ogiso et al., 2010; Kato et al., 2002, 2008). 133

In this study, we first conducted genetic and linkage analyses to identify a causal 134

gene for early flowering in HK2. Secondly, the gene effect was evaluated by growing 135

the NILs under different photoperiods. Third, functional assay using a rice 136

transformation system was conducted to evaluate the functionality of the flowering-time 137

gene. Finally, based on the results of expression analysis, the role of the flowering-time 138

gene in the genetic pathways for flowering was discussed. 139

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RESULTS 142

Genetic and linkage analyses 143

HK2 was crossed with a late-flowering NIL carrying the spring allele Vrn-H1 from 144

Indo Omugi (hereafter, NIL (Vrn-H1)). Frequency distribution for flowering time in the 145

F2 population was continuous but trimodal, suggesting the segregation of early, 146

intermediate, and late types caused by a single gene (Fig. 1). For accurate genotyping, a 147

progeny test was conducted using F3 lines derived from F2 plants with flowering time 148

within the overlap regions between different types. As a result, the F2 population was 149

found to segregate 217 early, 446 intermediate, and 229 late types, which fit the ratio of 150

1:2:1 (χ2 = 0.323, P = 0.851) for single gene segregation. Hereafter, the gene was 151

designated tentatively as PS (t). 152

Linkage analysis using the same population showed that HvPhyC was linked by 153

Vrn-H1 and HvCK2α with genetic distances 1.5cM and 3.1cM, respectively, and the 154

gene order was estimated to be Vrn-H1 – HvPhyC – HvCK2α. Among these three genes, 155

HvPhyC co-segregated with PS (t) (Fig. 1A), strongly suggesting that it is the causal 156

gene for PS (t). In contrast, several critical recombinations were observed between 157

Vrn-H1 and PS (t), and between HvCK2α and PS (t) (Fig. 1B and C). From this result, 158

Vrn-H1 and HvCK2α could be ruled out as candidates. Hereafter, we designate the 159

early-flowering (HK2) and late-flowering (NIL (Vrn-H1)) alleles for the flowering-time 160

gene (HvPhyC) as HvPhyC-e and HvPhyC-l, respectively. 161

Sequence analysis of HvPhyC alleles revealed a single nucleotide polymorphism 162

(SNP) in exon 1 (at the position 1139 from the start codon) that causes non-synonymous 163

substitution at the C terminal side of the GAF (cGMP phosphodiesterase, adenylate 164



9

cyclase, FhlA) domain (at position 380) in the deduced amino acid sequence (Fig. 2A 165

and B). The deduced amino acid residue from HvPhyC-l had phenylalanine (F) at this 166

position, which was well conserved among several plant species (wheat, rice, sorghum 167

(Sorghum bicolor L.), Brachypodium (Brachypodium distachyon (L.) Beauv.), and 168

Arabidopsis), while that from HvPhyC-e had serine (S), suggesting it to be a mutant 169

allele (Fig. 2C). 170

171

Effect of HvPhyC on flowering time under different photoperiods 172

Each two independent NILs carrying HvPhyC-e and HvPhyC-l (four NILs) were 173

selected out of the F4 progenies of the mapping population (Table I). All of these NILs 174

have the same genotype for the other flowering-time genes, since the alleles from the 175

NIL (Vrn-H1) were selected for Vrn-H1 and HvCK2α, which segregated in the mapping 176

population. Each two NILs carrying HvPhyC-e and HvPhyC-l were mixed together and 177

designated as NIL (HvPhyC-e) and NIL (HvPhyC-l), respectively, since the same 178

genotype NILs showed statistically the same flowering time. Under long (16h) and 179

extremely long (20h) photoperiods, NIL (HvPhyC-e) flowered much earlier than NIL 180

(HvPhyC-l) (22.5% and 24.4% reduction in days from sowing to flag leaf unfolding, 181

respectively) (Fig. 3). In contrast, no significant difference (only 4.3% reduction) was 182

observed under a short (12h) photoperiod. The result showed that HvPhyC controls 183

photoperiod sensitivity under long photoperiods. 184

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Functional assay of HvPhyC using rice transformation system 186

To evaluate the function of HvPhyC-e from HK2 and HvPhyC-l from NIL 187

(Vrn-H1), we introduced these two alleles under the control of CaMV35S promoter into 188



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a rice (Oryza sativa L.) phyA phyC double mutant line as the recipient with a genetic 189

background of the Japanese variety Nipponbare because the phyA phyC double mutant 190

line flowers significantly earlier than the original variety Nipponbare under a natural 191

(long) photoperiod (Takano et al., 2005). 192

The T1 control lines carrying the empty vector in Nipponbare and phyA phyC 193

double mutant line flowered 59.6 and 45.6 days after sowing (DAS) under a natural 194

(long) photoperiod, respectively, confirming the effect of PhyC and PhyA genes on 195

flowering time under a long photoperiod (Fig. 4). 196

One (#1-26) out of two T1 lines carrying HvPhyC-l (wild-type allele) segregated 197

late-flowering (Nip mock type) and early-flowering (phyA phyC mock-type) plants. All 198

of the late-flowering plants expressed HvPhyC-l at a high level, while early-flowering 199

plants expressed HvPhyC-l at a low level (Fig. 4). The other T1 line (#1-12) was 200

composed of only early-flowering plants that expressed HvPhyC-l at a low level. 201

Therefore, it was confirmed that high enough expression of HvPhyC-l can recover from 202

the phyA phyC double mutant phenotype. 203

One (#17-14) out of four T1 lines carrying HvPhyC-e (mutant allele) segregated 204

late-flowering plants, all of which expressed HvPhyC-e at a high level, while the others 205

(#17-1, #17-6, and #17-8) were composed of only early-flowering plants (Fig. 4). To our 206

surprise, this result strongly suggested that the HvPhyC-l and HvPhyC-e alleles are both 207

functional. However, even a lower expression level of HvPhyC-e, compared to 208

HvPhyC-l, could recover from the phyA phyC double mutant phenotype, suggesting the 209

hyperfunction of HvPhyC-e. 210

211

Expression analysis of the flowering pathway genes 212



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There were no apparent differences in HvPhyC expression between NIL 213

(HvPhyC-e) and NIL (HvPhyC-l) despite their allelic differences (Fig. 5A, 214

Supplemental Fig. S1A). In both of the NILs, HvPhyC was expressed all day and 215

seemed to show diurnal fluctuation under both long (16h) and short (8h) photoperiod 216

conditions with the trend that it was up-regulated around dusk and down-regulated 217

during the day. This result led to the hypothesis that the SNP (T1139C) in the GAF 218

domain might affect its protein function rather than its own expression pattern and also 219

the expression pattern of downstream genes interacting with HvPHYC protein. 220

Subsequently, we compared the expression of circadian clock-related genes 221

HvCK2α, HvGI, and Ppd-H1, because the circadian clock is considered to interact with 222

photoreceptors. Unexpectedly, two NILs showed similar expression patterns for all three 223

genes (Fig. 5B-D, Supplemental Fig. S1B-D). HvCK2α was expressed all day and did 224

not show apparent diurnal fluctuation under both long and short photoperiod conditions. 225

HvGI and Ppd-H1 were up-regulated during the day and down-regulated during the 226

night under both long and short photoperiod conditions, consistent with previous studies 227

(Dunford et al., 2005; Turner et al., 2005). Therefore, it was suggested that HvPhyC 228

does not affect circadian clock-related genes but affects more downstream genes of both 229

photoreceptors and the circadian clock. 230

HvCO1, which integrates signals from the circadian clock and photoreceptors, 231

showed diurnal expression patterns under long and short photoperiod conditions (Fig. 232

5E, Supplemental Fig. S1E), consistent with previous studies (Turner et al., 2005; 233

Campoli et al., 2012), although there were no clear differences between the two NILs. 234

On the other hand, clear-cut differences were detected for HvFT1 (Fig. 5F), which is 235

expected to be the direct target of HvCO1 protein and acts as the flowering promoter 236



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“florigen” (HvFT1 is also known as the vernalization requirement gene Vrn-H3). In the 237

late-flowering NIL (HvPhyC-l), HvFT1 showed a diurnal expression pattern that had a 238

small peak early in the day and a large peak around dusk under long photoperiod 239

conditions, which was consistent with Turner et al. (2005) and Campoli et al. (2012). In 240

the early-flowering NIL (HvPhyC-e), HvFT1 also showed diurnal expression, but small 241

and large peaks appeared slightly later (by 4 hours) than NIL (HvPhyC-l). Furthermore, 242

NIL (HvPhyC-e) expression of HvFT1 was considerably higher than in NIL (HvPhyC-l). 243

This result was expected from several previous studies showing that the early-flowering 244

line expressed HvFT1 at a higher level (Turner et al., 2005; Campoli et al., 2012; Faure 245

et al., 2012). Under short photoperiod conditions, HvFT1 was not expressed in either 246

NIL (Supplemental Fig. S1F), because the photoperiod was non-inductive and NILs 247

were still in the vegetative growth stage at the sampling time (14 DAS). 248

Another vernalization requirement gene Vrn-H1 showed a diurnal expression 249

pattern in both NILs under long and short photoperiod conditions (Fig. 5G, 250

Supplemental Fig. S1G), which was consistent with Campoli et al. (2012). Under long 251

photoperiod conditions, the expression level was slightly higher in NIL (HvPhyC-e) 252

than in NIL (HvPhyC-l), while no such difference was observed under short photoperiod 253

conditions. To confirm the difference under long photoperiod conditions, expression 254

analysis with real-time RT-PCR was conducted. As the result, similar trend was 255

observed as semi-quantitative RT-PCR, but the differences were not significant at most 256

of the time point (Supplemental Fig. S2). Vrn-H2 locus deploys two family genes 257

ZCCT-Ha and ZCCT-Hb, both of which were expressed during the day and had two 258

peaks early in the day and around dusk under long photoperiod conditions, while they 259

were not expressed under short photoperiod conditions (Fig. 5H and I, Supplemental 260



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Fig. S1H and I). Their expression patterns were consistent with Trevaskis et al. (2006). 261

There was no apparent difference in expression pattern between the NILs for both 262

ZCCT-H genes. 263

We analyzed the expression of other photoperiodic response genes, Ppd-H2 264

(HvFT3) and HvCO9, which control flowering time under short photoperiods (Kikuchi 265

et al., 2009; Kikuchi et al., 2012). There were no clear differences in either gene 266

between the NILs (Fig. 5J and K, Supplemental Fig. S1J and K)). Ppd-H2 showed a 267

diurnal expression pattern under short photoperiod conditions and much lower 268

expression under long photoperiod conditions, which were consistent with a previous 269

study (Kikuchi et al., 2009). HvCO9 also showed a diurnal expression pattern under 270

long and short photoperiod conditions and was up-regulated during the day. The 271

expression level was higher under short than long photoperiod conditions. This was 272

consistent with the observation by Kikuchi et al. (2012). 273

274

Epistatic interaction between HvPhyC and Vrn-H3 275

Flowering times under a field condition were compared among HK2 NILs 276

developed in the present study and by Yasuda (1969). NILs carrying a winter allele 277

vrn-H3 flowered on April 27.4 on average, while those carrying a spring allele Vrn-H3 278

flowered on April 16.3 on average (Table II). Within the group of NILs carrying vrn-H3, 279

the NILs with HvPhyC-e flowered earlier than those with HvPhyC-l by one week. 280

Contrary, flowering times were not different between the NILs with HvPhyC-e and 281

HvPhyC-l, all of which have Vrn-H3. The result strongly suggested that Vrn-H3 is 282

epistatic to HvPhyC. 283

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Haplotype analysis for HvPhyC and Vrn-H1 285

By using diagnostic markers (Supplemental Table S1), haplotype for HvPhyC and 286

Vrn-H1 was determined in Japanese varieties (Table III). Among twelve varieties, four 287

had the same haplotype with HK2 that deploys HvPhyC-e and vrn-H1 (identical with 288

HvVRN1 in Hemming et al. (2009)), while three had the same haplotype with NIL 289

(Vrn-H1) that deploys HvPhyC-l and Vrn-H1 (identical with HvVRN1-10 in Hemming et 290

al. (2009)). Other five varieties carried a recombinant type that was comprised of 291

HvPhyC-l and vrn-H1. 292

293

294

DISCUSSION 295

There have been several studies on phytochromes in model plant species such as 296

rice and Arabidopsis using natural variations and mutants (e.g. Takano et al., 2005; 297

Franklin and Quail, 2010). Such studies proved their importance in development and 298

physiological responses, including the control of flowering time. In contrast, in barley, 299

little is known about the effects of phytochromes, although it was suggested that one of 300

the phytochrome genes HvPhyC might be involved in photoperiod sensitivity through 301

the detection of a photoperiod-sensitivity QTL around the HvPhyC region (Szücs et al., 302

2006). In the present study, we successfully identified the natural variation for HvPhyC 303

that controlled flowering time under long photoperiods by affecting the expression of 304

flowering-time genes downstream of HvPhyC. 305

In linkage analysis, the flowering-time gene PS (t) co-segregated only with 306

HvPhyC among three candidate genes. Therefore, HvPhyC was considered to be the 307

most likely candidate gene for the difference in flowering time between HK2 and NIL 308



15

(Vrn-H1). According to Szücs et al. (2006), barley has a HvPhyC pseudo-gene in 309

addition to the intact gene. They mapped the pseudo-gene at the same position as 310

Vrn-H1, which is distinct from the intact HvPhyC by genetic mapping (Szücs et al., 311

2006). Furthermore, physical mapping located the pseudo-gene next to Vrn-H1 (Szücs 312

et al., 2006; Yan et al., 2005). Because of several recombinations between PS (t) and 313

Vrn-H1 in our linkage analysis, PS (t) was considered to be distinct from the 314

pseudo-gene. Szücs et al. (2006) also showed that the pseudo-gene was separated into 315

two segments (remnants) by a large (17kb) insertion, including retroelements and 316

MITEs. Successful PCR amplification of the pseudo-gene in both HK2 and NIL 317

(Vrn-H1) using the primers specific to the insertion and the genomic regions on opposite 318

sides of the insertion across the pseudo-gene segments (Szücs et al., 2006) implied that 319

both lines carry the pseudo-gene, which was indeed non-functional (Supplemental Fig. 320

S3). Therefore, it was concluded that the pseudo-gene is not the cause of the 321

flowering-time difference between HK2 and NIL (Vrn-H1). 322

Another flowering-related gene AGLG1 (AGAMOUS-LIKE GENE 1), encoding a 323

grass specific SEPPALLATA-like MADS-box protein, was expected to exist around 324

Vrn-H1 region because of the syntheny between barley and einkorn wheat (Triticum 325

monococcum L.) genomes (Yan et al., 2005). Although little is known about AGLG1 326

function in barley and wheat, a rice ortholog PAP2 (PANICLE PHYTOMER 2) has been 327

characterized in detail (Gao et al., 2010; Kobayashi et al., 2010; Kobayashi et al., 2012). 328

PAP2 protein is involved in inflorescence meristem identity by forming protein complex 329

with three AP1/FUL-like MADS-box proteins OsMADS14, OsMADS15, and 330

OsMADS18 (Kobayashi et al., 2012). Quadruple knockdown plants showed severely 331

affected phenotype including discordant transition from vegetative phase to 332



16

reproductive phase that resulted in delayed flowering and impaired inflorescence 333

development. In addition, PAP2 alone functions as a positive regulator of spikelet 334

meristem identity and a suppressor of the extra elongation of glumes (Gao et al., 2010; 335

Kobayashi et al., 2010). Contrary to the quadraple knockdown plants, pap2 single 336

mutant showed milder phenotype including increase in number of primary branch of 337

rachis and elongation of sterile lemma and rudimentary glume and did not affect 338

flowering time (Gao et al., 2010; Kobayashi et al., 2010). From these results, AGLG1 339

single mutation in barley was assumed to affect spike and spikelet morphology rather 340

than flowering time. However, we did not find any differences in spike and spikelet 341

morphology between HK2 and NIL (Vrn-H1). Therefore, AGLG1 was ruled out from 342

the candidate genes for PS (t). 343

By linkage analysis, the gene order was estimated to be Vrn-H1 – HvPhyC – 344

HvCK2α, which was supported by the barley EST map (CMap for Barley EST; 345

http://map.lab.nig.ac.jp:8085/cmap/; Sato et al., 2009) constructed using the DH 346

population of a Japanese variety Haruna Nijo and the Hordeum vulgare ssp. spontaneum 347

line H602. However, order Vrn-H1 – HvPhyC – HvCK2α from proximal to distal on 348

chromosome 5HL was inconsistent with previous reports. Szücs et al. (2006) mapped 349

HvPhyC on the proximal side of Vrn-H1 using the Dicktoo x Morex double haploid 350

(DH) mapping population. HvCK2α was mapped to a more distal region on 5HL using 351

the Steptoe x Morex DH population (Kato et al., 2008). Taken together, their results 352

suggested that the order is HvPhyC – Vrn-H1 – HvCK2α from proximal to distal on 5HL. 353

This order is consistent with that in the corresponding regions of wheat (Yan et al., 354

2003; Kato et al, 2002), Brachypodium (Brachypodium distachyon Gbrowse; 355

http://gbrowse.brachypodium.org/cgi-bin/gbrowse/brachypodium-gbrowse/), rice 356



17

(RAP-DB; http://rapdb.dna.affrc.go.jp/), and sorghum (Sorghum bicolor GDB; 357

http://www.plantgdb.org/XGDB/phplib/resource.php?GDB=Sb), showing that this gene 358

order is prevalent in grass species. Therefore, it was considered that the chromosomal 359

rearrangement in this region might be shared in some barley varieties, including the 360

Japanese varieties Hayakiso 2 and Haruna Nijo, Taiwanese variety Indo Omugi, and H. 361

vulgare ssp. spontaneum line H602. 362

A “supergene” is formed by a group of co-segregating genes whose allelic 363

combinations (haplotypes) facilitate integrated control of adaptive phenotypes. 364

“Supergenes” have been described in various species: e.g. S locus controlling 365

heterostyly and self-incompatibility in Primula species (Primula sinensis L.) (Mather, 366

1950) and P locus controlling wing color pattern in mimetic butterflies (Clarke et al., 367

1968; Brown et al., 1974, Nijhout, 2003). In a mimetic butterfly Heliconius numata, 368

chromosomal rearrangements (inversions) around the P locus prevented recombination 369

within this supergene locus, and inversions that formed specific haplotypes were 370

completely associated with corresponding wing color patterns (Joron et al., 2011). In the 371

present study, formation of a “supergene” was expected in chromosome 5HL where four 372

flowering-related genes (Vrn-H1, HvPhyC, HvCK2α, and HvAGLG1) are clustered and 373

closely linked to each other. Especially for the inversion including the Vrn-H1 – 374

HvPhyC region might played evolutionary and adaptive roles by establishing specific 375

haplotypes (vrn-H1 / HvPhyC-e (HK2 type) and Vrn-H1 / HvPhyC-l (NIL (Vrn-H1) 376

type). Contrary to expectation, our preliminary analysis using Janapese varieties showed 377

that there was a recombinant type (vrn-H1 / HvPhyC-l) in addition to above two types 378

(Table III). To clarify the evolutionary and adaptive roles of Vrn-H1 and HvPhyC as 379

well as other flowering-related genes, worldwide landrace collection needs to be 380



18

analyzed comprehensively. 381

To conduct functional assay of HvPhyC, we adopted the Agrobacterium-mediated 382

transformation system of rice. The result showed that even the mutant allele 383

(HvPhyC-e) is functional and may have a hyperfunction compared to the wild-type 384

allele (HvPhyC-l). If this is the case, functional PhyC has a promoting effect in long-day 385

plant barley under long photoperiod conditions and, conversely, it has a delaying effect 386

in short-day plant rice under the same conditions. There is an another example in which 387

orthologous genes affect flowering time conversely between long-day and short-day 388

plants: in the long-day plant Arabidopsis, the functional CO promotes flowering under 389

long photoperiod conditions, whereas the functional rice ortholog Hd1 delays flowering 390

under the same condition (Putterill et al., 1995; Yano et al., 2000). Rice is a reasonable 391

model plant for functional assay of HvPhyC gene, because phyC null mutant lines are 392

available in a Nipponbare genetic background (Takano et al., 2005) which facilitates 393

transformation. On the other hand, transformation of barley is possible only in a 394

“Golden Promise” genetic background and no HvPhyC null mutant genes are available 395

so far. However, the best way to evaluate precise function of HvPhyC is complemention 396

analysis introducing HvPhyC-e and HvPhyC-l under the control of native promoter into 397

a HvPhyC deficient mutant of barley (native genetic background), because it is unclear 398

how the ectopic expression from viral promoter and how the differences of 399

photoperiodic pathways between barley and rice affect HvPhyC gene function in rice 400

(heterogenous) genetic background. 401

Phytochrome protein deploys several domains: a GAF, a PHY (phytochrome 402

specific GAF-related), three PAS, and two histidine kinase-related domains (Fig. 1B). 403

The first half of this protein, including N-terminal PAS (PER, ARNT, SIM), GAF, and 404



19

PHY domains, is considered to be crucial for light perception and signal transduction. 405

And several missense mutations in this region caused a deficiency in signal transduction, 406

chromophore incorporation and spectral integrity, and Pfr stabilization (reviewed by 407

Nagatani, 2010). In the present study, an exon 1 SNP (T1139C) was found near the 3’ 408

end of GAF domain containing a chromophore binding site. This mutation resulted in 409

non-synonymous substitution at position 380 from nonpolar and hydrophobic 410

phenylalanine to polar and hydrophilic serine. Because mutation at this position is 411

unknown in plants including Arabidopsis, it remains unknown how the mutation affects 412

the biochemical function of the protein. However, this amino acid residue is well 413

conserved among many plant species (wheat, Brachypodium, rice, sorghum, and 414

Arabidopsis), suggesting its importance for protein function (Fig. 1C). On the 415

C-terminal side of the protein, both HK2 and NIL (Vrn-H1) had a 8 amino acid insertion, 416

which was previously reported in Morex by Szücs et al. (2006) (Fig. 1B). No such 417

insertion was found in wheat, Brachypodium, rice, and sorghum PHYC proteins. 418

However, this insertion is located close to the C-terminal end and outside the histidine 419

kinase-like ATPase (HKL) domain (Fig. 1B). In addition, the N-terminal side is rather 420

important for light perception and signal transduction as compared with the C-terminal 421

side, although it is associated with dimerization (Nagatani et al., 2010). Therefore, the 422

effect of the insertion on flowering time was considered to be small. Consistent with 423

this idea, constitutive expression of both HvPhyC alleles in rice phyA phyC mutant 424

could recover the wild-type phenotype although the recovering levels were different 425

(Fig. 4). 426

Expression analysis showed that SNP (T1139C) in HvPhyC did not affect the 427

expression pattern of HvPhyC itself, circadian clock genes, and most of the downstream 428



20

genes (Fig. 5A-E, H-K, Supplemental Fig. S1A-K). The only exception was HvFT1 429

(Vrn-H3) which was up-regulated by the mutant allele HvPhyC-e under a long 430

photoperiod (Fig. 5F). This was consistent with physiological analysis in which 431

HvPhyC affected flowering time only under long photoperiods. Therefore, HvPhyC was 432

considered to bypass most of the genes that function in relatively upstream parts of the 433

genetic pathways for flowering (e.g. circadian clock genes and HvCO1) and affect the 434

gene (HvFT1) that functions in the most downstream part of the genetic pathways. 435

Consistent with this, complete loss of three phytochromes in rice did not affect the 436

expression of circadian clock genes and CO while it affected the expression of FT 437

(Izawa et al., 2002), although the sole effect of PhyC remains unknown. Furthermore, 438

field experiment in the present study also supported our idea (Table II). HvPhyC-e, 439

compared to HvPhyC-l, had a promoting effect when co-existing with vrn-H3, while it 440

did not have such effect when co-existing with Vrn-H3 which was derived from a 441

Finnish variety Tammi. Tammi allele for Vrn-H3 was shown to include four copies of 442

HvFT1 which is associated with earlier transcriptional up-regulation of themselves 443

(Nitcher et al., 2013). Therefore, it was considered that the spring allele Vrn-H3 (high 444

expression of HvFT1) is epistatic to that of HvPhyC-e and HvPhyC functions in the 445

same pathway as Vrn-H3. 446

Based on the feedback loop models (Trevaskis et al., 2007; Distelfeld et al., 2009; 447

Shimada et al., 2009), up-regulation of HvFT1 was expected to give rise to concordant 448

up-regulation of Vrn-H1 and down-regulation of Vrn-H2. Our result showed that Vrn-H1 449

expression tended to be slightly higher in NIL (HvPhyC-e) than NIL (HvPhyC-l) (Fig. 450

5G, Supplemental Fig. S2). However, their differences were confirmed to be 451

non-significant (Supplemental Fig. S2). This might be attributable to that both of the 452



21

NILs carry the spring allele Vrn-H1. Since the spring allele Vrn-H1 expresses at a high 453

level even in young seedlings (Trevaskis et al., 2003; von Zitzewitz et al., 2005), further 454

up-regulation of Vrn-H1 by higher expression of HvFT1 will be marginal even if it 455

occurs. Consistent with this idea, the expression levels of Vrn-H2 were not different 456

between both NILs (Fig. 5H and I). This might also be attributable to that expression of 457

Vrn-H2 was repressed by the spring allele Vrn-H1 (Hemming et al., 2008) to a 458

considerably low level where the differences were not detectable. Distelfeld and 459

Dubcovsky (2010) suggested that lack of the entire TaPhyC gene down-regulated the 460

ZCCT genes in the mvp (maintained vegetative phase) mutant of diploid wheat (T. 461

monococcum L.). To disclose the effect of HvPhyC on Vrn-H2 (perhaps through 462

affecting Vrn-H1), a F2 population segregating for HvPhyC and Vrn-H2 and carrying the 463

winter allele vrn-H1 need to be developed. 464

Molecular genetic studies of Arabidopsis have disclosed the essential roles of 465

phytochromes in transcriptional and post-transcriptional control of CO. PHYB is 466

associated with post-transcriptional regulation of CO by degrading CO protein early in 467

the day under a long photoperiod (Valverde et al., 2004). PHYB protein is known to 468

form not only a homodimer but also heterodimers with PHYC and PHYD proteins, 469

while PHYC and PHYD proteins do not form a homodimer (Clack et al., 2009). If 470

PHYB/PHYC heterodimer participates in CO protein stability, PhyC may affect 471

flowering time through a CO-mediated photoperiodic pathway. Similarly, PHYC protein 472

can participate in transcriptional regulation of CO by forming a heterodimer with PHYB. 473

Clack et al. (2009) showed that PHYC protein, probably by forming a heterodimer with 474

PHYB protein, can interact with PIF3 (Phytochrome Interacting Factor 3), which is 475

involved in signal transduction from phytochromes. Oda et al. (2004) showed that 476



22

suppression of PIF3 resulted in up-regulation of CO and FT but did not affect the 477

expression of circadian clock genes, including LHY and CCA1, irrespective of 478

possessing a possible binding sequence in their promoter. Our expression analysis 479

suggests that the mutation (T1139C) in HvPhyC affects the post-transcriptional control 480

of CO rather than the transcriptional control of CO, because HvFT1 was up-regulated 481

but HvCO1 was not in NIL (HvPhyC-e). Taken together, our results indicate that 482

HvPhyC is a key factor to control long-day flowering directly. 483

484

MATERIALS AND METHODS 485

Plant Materials and Growth Conditions 486

NIL (Vrn-H1) was developed by backcrossing more than ten times using a 487

Japanese barley (Hordeum vulgare ssp. vulgare L.) variety Hayakiso 2 (HK2) as the 488

recurrent parent and a Taiwanese variety Indo Omugi as the non-recurrent parent 489

(Vrn-H1 donor) (this NIL was originally developed by Dr. Shozo Yasuda who conducted 490

backcrossings six times (Yasuda, 1969). After publication, he conducted further 491

backcrosses more than four times.). 492

The F2 population (918 individuals) and F3 lines (87 lines, 10–15 plants each) of 493

HK2 x NIL (Vrn-H1) were subjected to genetic and linkage analyses for flowering-time 494

QTL. They were grown under natural conditions in the experimental field of the Faculty 495

of Agriculture, Okayama University (34° 41’N, 133° 55’E, 4m above sea level). The 496

sowing date of the F2 population was 25th November, 2005, and those of F3 lines were 497

20th December, 2006, 21st December, 2007, and 21st November, 2008. Flowering time 498

was scored for each plant as the date when the first ear appeared from the sheath. 499

For evaluation of the QTL effect on photoperiod sensitivity and the expression 500



23

pattern of flowering-time genes, the NILs with different alleles for HvPhyC (Table I) 501

were selected from F4 families using allele-specific DNA markers, as summarized in 502

Supplemental Table S1. For the evaluation of photoperiod sensitivity, seeds of NIL 503

(HvPhyC-e) and NIL (HvPhyC-l) were soaked in water at 4ºC for 24h, and subsequently 504

kept at 20ºC for 24h for germination. Germinated seeds were sown on the soil (1:1 505

mixture of field soil and bark compost). Each three plants per line (six plants for each 506

genotype) were grown under short photoperiod (12h light/12h dark), long photoperiod 507

(16h light/8h dark), and extremely long (20h light/4h dark) photoperiod conditions in 508

growth chambers (LH-350SP; Nippon Medical & Chemical Instruments Co. Ltd., 509

Japan) where temperature was kept at 20ºC. The light source was fluorescent lamps and 510

photon flux density was ca. 160 mol/m2/s. For expression analysis, the same lines were 511

grown in the same way under short (8h light/16h dark) and long (16h light/8h dark) 512

photoperiods. From 15 days after sowing, second and third leaves (three biological 513

replicates for each genotype at every time point) were collected at four-hour intervals 514

for two days. 515

For validation of the interaction between HvPhyC and Vrn-H3, flowering time of 516

additional two HK2 NILs carrying Vrn-H3 (Yasuda, 1969) as well as the NILs described 517

above were evaluated under a natural condition. Five plants per each line were grown in 518

the same field as described above. Sowing date was 17th December, 2010. 519

Young seedlings of twelve Japanese varieties were grown for the HvPhyC / Vrn-H1 520

haplotype analysis (Table III). Leaves of these varieties were collected for DNA 521

extraction. 522

523

Functional assay of HvPhyC using rice transformation system 524



24

We adopted the rice (Oryza sativa L.) phyA phyC double mutant line as the 525

recipient, instead of the phyC single mutant line, with a genetic background of Japanese 526

variety Nipponbare (Takano et al., 2005). This is because the phenotypic effect of the 527

PhyC gene is prominent when PhyA is non-functional: the phyA phyC double mutant 528

line flowers much earlier than the original variety Nipponbare and even earlier than the 529

phyC single mutant line under a natural (long) photoperiod, while the phyA single 530

mutant line flowers at the same time as Nipponbare under the same conditions (Takano 531

et al., 2005). 532

HvPhyC-e and HvPhyC-l cDNAs driven by CaMV35S promoter were introduced 533

into the phyA phyC double mutant line via Agrobacterium-mediated transformation, as 534

described by Kikuchi et al. (2009). Four T1 lines expressing HvPhyC-e and two T1 lines 535

expressing HvPhyC-l developed from independent T0 plants were subjected to the 536

analysis. T1 lines carrying the empty vector (mock) with the phyA phyC and Nipponbare 537

background (phyA phyC mock and Nip mock, respectively) were used as the control. 538

Their seeds were sown on 26th July, 2012 in plastic pots and plants were grown until 539

flowering under a natural photoperiod in a greenhouse, as described by Kikuchi et al. 540

(2009). Flag leaves were collected at 9:00 a.m. at the end of September for HvPhyC 541

expression analysis. 542

543

DNA Extraction and Genotyping 544

Genomic DNA was extracted from each plant of the F2 population, F3 lines, their 545

parents, NILs, and varieties following the CTAB method (Murray and Thompson, 546

1980). 547

For linkage analysis, PCR amplification was conducted using allele-specific DNA 548



25

markers for Vrn-H1, HvPhyC, and HvCK2α (Supplemental Table S1). PCR products or 549

digested PCR products were separated in agarose gels by electrophoresis. PCR products 550

were visualized with ethidium bromide. A genetic map was constructed using 551

MAPMAKER/EXP3.0 (Lander et al., 1987). 552

In addition to Vrn-H1, HvPhyC, and HvCK2α, other flowering-time genes, Ppd-H1, 553

Ppd-H2, Vrn-H2, and Vrn-H3, were also genotyped for HK2 and its NILs using 554

diagnostic markers (Supplemental Table S1). 555

556

Sequence Analysis 557

The HvPhyC gene region was amplified by long PCR using appropriate primers 558

(Supplemental Table S2) and Phusion High-Fidelity DNA polymerase (Thermo 559

Scientific, USA), and were cloned using the TOPO TA Cloning Kit (Invitrogen, USA) 560

following the manufacturers’ instructions. Three clones per single PCR product were 561

sequenced using a PRISM 3730 DNA Analyzer (Applied Biosystems, USA). 562

563

Gene Expression Analysis 564

Young leaves (100mg) of barley NILs and transgenic rice were frozen in liquid 565

nitrogen and ground using a Multi-beads shocker (Yasui apparatus, Japan). Total RNA 566

was extracted from the ground leaf using Sepasol RNAI Super (Nacalai Tesque, Japan) 567

and first-strand cDNA was synthesized using ReverTra Ace (TOYOBO, Japan) 568

following manufacturers’ instructions. Semi-quantitative RT-PCR was conducted using 569

specific primers for flowering-time genes and internal controls (Supplemental Table S3). 570

PCR products were electrophoresed in agarose gels and stained by ethidium bromide. 571

Band intensity was analyzed by Scion Image (Scion Corporation, USA). 572



26

573

Accession Numbers 574

Sequence data from this article can be found in the GenBank database under the 575

following accession numbers: HvPhyC-e (AB827939) and HvPhyC-l (AB827940). 576

577

ACKNOWLEDGMENTS 578

We are grateful to Dr. Shozo Yasuda at Institute of Plant Science and Resources, 579

Okayama University and Dr. Makoto Takano at National Institute of Agrobiological 580

Sciences for providing HK2 NILs and the rice phyA phyC double mutant line, 581

respectively. We are also grateful to Dr. Koichiro Ushijima at Okayama University for 582

technical advice on the real-time PCR analysis. 583

584

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Arabidopsis flowering time gene CONSTANS. Plant Cell 12: 2473-2483 731

Yasuda S (1969) Physiology and genetics of ear emergence in barley and wheat. VIII. 732

Effects of four genes for spring habit on earliness in barley. Nogaku Kenkyu 53: 733

99-113. (In Japanese) 734

735



33

Figure legends 736

737

Figure 1. Frequency distribution of flowering time in the F2 population derived from 738

a cross between Hayakiso 2 (HK2) and its near isogenic line carrying the spring allele 739

for Vrn-H1 (NIL (Vrn-H1)) under field conditions. The 892 F2 plants could be classified 740

into three types by the genotype of (A) a causal gene for flowering time (PS (t)) as well 741

as its candidate genes (A) HvPhyC, (B) Vrn-H1, and (C) HvCK2α. White, gray, and 742

black rectangles indicate homozygote for HK2 allele, heterozygote, and homozygote for 743

NIL (Vrn-H1) allele. 744

745

Figure 2. Structure of HvPhyC gene and its protein. (A) HvPhyC gene sequences 746

from HK2 and NIL (Vrn-H1) were compared with those from Morex (DQ238106), 747

Dicktoo (DQ201145), and Kompolti Korai (DQ201146). Filled boxes and horizontal 748

lines between them indicate exons and introns, respectively. Vertical line indicates 749

single nucleotide polymorphism (SNP) when NIL (Vrn-H1) was compared with other 750

varieties. Open triangle indicates a 24 bp insertion. HK2, NIL (Vrn-H1), and Morex 751

have this insertion, which results in 8 amino acid insertion. Numbers and letters at 752

polymorphic sites indicate the position and polymorphic varieties, respectively. H, M, D, 753

and K indicate HK2, Morex, Dicktoo, and Kompolti Korai, respectively. (B) Deduced 754

amino acid sequences were compared based on the HvPhyC gene sequences. Filled 755

boxes indicate domains. PAS: PER, ARNT, SIM domain, GAF: cGMP 756

phosphodiesterase, adenylate cyclase, FhlA domain, PHY: Phytochrome-specific 757

GAF-related domain, HK: histidine kinase domain, HKL: histidine kinase-like ATPase 758

domain. Polymorphisms are shown in the same way as HvPhyC gene. (C) Deduced 759



34

amino acid sequences around the C-terminal end of GAF domain were compared based 760

on the genomic sequences of barley, wheat (GenBank: AY672995.1, AY673002.1, 761

AY672998.1, AY672999.1, AY673000.1), Brachypodium distachyon (Brachypodium 762

distachyon Gbrowse: Bradi1g08400.1), Oryza sativa (RAP-DB: Os03g0752100-01), 763

Sorghum bicolor (Sorghum bicolor GDB: Sb01g007850.1). The same and similar 764

sequences are highlighted in black and gray, respectively. 765

766

Figure 3. Photoperiodic response of the NILs carrying different HvPhyC alleles. Days 767

from sowing to flag leaf unfolding (parallel with flowering time) of NIL (HvPhyC-e) 768

and NIL (HvPhyC-l) were compared under three different (20h, 16h, and 8h) 769

photoperiod conditions. ***indicates that the difference between the NILs is statistically 770

significant (P <0.001). 771

772

Figure 4. Functional assay of HvPhyC in rice by introducing different HvPhyC alleles 773

(T1 generation). Mutant allele (HvPhyC-e; red filled marks) and wild-type allele 774

(HvPhyC-l; blue filled marks) were introduced into rice phyA phyC double mutant lines 775

with a Nipponbare genetic background via the Agrobacterium-mediated transformation 776

method. As the control, empty vector was introduced into phyA phyC double mutant 777

(phyA phyC mock; red open circle) and Nipponbare (Nip mock; blue open triangle). All 778

of the T1 lines were grown under natural (long) photoperiod conditions in a greenhouse. 779

Expression level of HvPhyC was analyzed by semi-quantitative RT-PCR using OsActin 780

as the internal control. 781

782

Figure 5. Expression pattern of flowering time genes under a long (16h) photoperiod. 783



35

Red circles and blue diamonds indicate the near isogenic lines NIL (HvPhyC-e) and NIL 784

(HvPhyC-l), respectively. Expression level is the average of band intensity obtained by 785

semi-quantitative RT-PCR from three biological replications. White and black 786

horizontal bars below each graph indicate light and dark conditions, respectively. 787

Vertical bar indicates standard deviation. Asterisks above plots indicate that the 788

expression level was significantly different between two lines when they were 789

compared at the same time points. *, **, and *** indicate significance at P = 0.05, 0.01, 790

and 0.001, respectively. 791

792

793



36

794

Table I Genotype for flowering time genes in Hayakiso 2 and its near isogenic lines determined by diagnostic markers

Photoperiod sensitivity Vernalization requirement

Variety / Line Flowering time HvPhyC HvCK2αa Ppd-H1b Ppd-H2c Vrn-H1d Vrn-H2d Vrn-H3d Growth habit

Hayakiso 2 (HK2) Early HvPhyC-e H Ppd-H1 Ppd-H2 vrn-H1 Vrn-H2 vrn-H3 Winter

NIL (Vrn-H1) Late HvPhyC-l N Ppd-H1 Ppd-H2 Vrn-H1 Vrn-H2 vrn-H3 Spring

NIL (HvPhyC-l) Late HvPhyC-l N Ppd-H1 Ppd-H2 Vrn-H1 Vrn-H2 vrn-H3 Spring

NIL (HvphyC-e) Early HvphyC-e N Ppd-H1 Ppd-H2 Vrn-H1 Vrn-H2 vrn-H3 Spring

a: H and N indicate the alleles from HK2 and NIL (Vrn-H1), respectively.

b: Recessive allele for Ppd-H1 confers late-flowering under long photoperiod.

c: Dominant allele for Ppd-H2 confers early-flowering under short photoperiod.

d: Dominant allele for Vrn-H1 (identical with HvVRN1-10 in Hemming et al. (2009)), recessive allele for Vrn-H2, dominant allele for Vrn-H3 confer spring growth habit.

795



37

796

Table II Flowering time of Hayakiso 2 (HK2) NILs carrying different alleles for HvPhyC, Vrn-H1, and Vrn-H3

Genotype1

Line HvPhyC Vrn-H12 Vrn-H3 HvCK2α3 Flowering time ± standard deviation4

HK2 HvPhyC-e vrn-H1 vrn-H3 H 24.6 ± 1.1 a

NIL (HvPhyC-e) HvPhyC-e Vrn-H1 vrn-H3 N 21.1 ± 1.3 b

NIL (HvPhyC-l) HvPhyC-l Vrn-H1 vrn-H3 N 30.0 ± 0.9 c

NIL (Vrn-H1, Vrn-H3) HvPhyC-l Vrn-H1 Vrn-H3 N 16.2 ± 1.3 d

NIL (Vrn-H3) HvPhyC-e vrn-H1 Vrn-H3 H 16.4 ± 1.8 d

NIL (Vrn-H1) HvPhyC-l Vrn-H1 vrn-H3 N 33.8 ± 1.1 e

1: All lines carry Vrn-H2 (winter allele), Ppd-H1(early-flowering allele), and Ppd-H2 (early-flowering allele) in common.

2: All the dominant alleles for Vrn-H1 are identical with HvVRN1-10 (Hemming et al. (2009)). They were derived from Indo Omugi except for the allele

in NIL (Vrn-H1, Vrn-H3) that was derived from a Japanese variety Marumi 16.

3: H and N indicate the alleles from HK2 and NIL (Vrn-H1), respectively.

4: 1st of April = 1. Values with different letters indicate significant difference (P<0.01) by the Tukey test.

797

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798

Table III Haplogypes for HvPhyC and Vrn-H1 in twelve Japanese varieties

Haplotype Growth

Variety HvPhyC Vrn-H1a Habit

Ishuku shirazu HvPhyC-e vrn-H1 Spring

Ichibanboshi HvPhyC-e vrn-H1 Winter

Kawasaigoku HvPhyC-e vrn-H1 Spring

Haruna Nijo HvPhyC-e vrn-H1 Spring

Silky Snow HvPhyC-l vrn-H1 Winter

Kirarimochi HvPhyC-l vrn-H1 Spring

Sayakaze HvPhyC-l vrn-H1 Spring

Suzukaze HvPhyC-l vrn-H1 Spring

Shunrai HvPhyC-l vrn-H1 Spring

Hozoroi HvPhyC-l Vrn-H1 Spring

Marumi 16 HvPhyC-l Vrn-H1 Spring

Kinai 5 HvPhyC-l Vrn-H1 Spring

a: vrn-H1 and Vrn-H1 are identical with HvVRN1 and HvVRN1-10 (Hemming

et al., 2009), respectively.

799



Num

ber o

f pla

nts

40

60

80

100

120

140PS (t) / HvPhyCA

N

0

20

40

13 15 17 19 21 23 25 27 29 31 33 35

100

120

140

f pla

nts

Vrn-H1B

0

20

40

60

80

13 15 17 19 21 23 25 27 29 31 33 35140

Num

ber o

f

H CK2C

20

40

60

80

100

120

Num

ber o

f pla

nts

HvCK2aC

Flowering time (1st of April = 1)

013 15 17 19 21 23 25 27 29 31 33 35

Figure 1. Frequency distribution of flowering time in the F2population derived from a cross between Hayakiso 2 (HK2) andits near isogenic line carrying the spring allele for Vrn-H1 (NILg y g p g ((Vrn-H1)) under field conditions. The 892 F2 plants could beclassified into three types by the genotype of (A) a causal genefor flowering time (PS (t)) as well as its candidate genes (A)HvPhyC, (B) Vrn-H1, and (C) HvCK2α. White, gray, and blackrectangles indicate homozygote for HK2 allele, heterozygote,and homozygote for NIL (Vrn-H1) allele.



GTC CCT TCC CCG CTCHK21139

Val Pro Ser Pro Leu

HvPhyC

906D

1491K, D

2100M

3612D

1139H

4115M

A

4294K, D

24bp

NIL (Vrn-H1) GTC CCT TTC CCG CTCVal Pro Ser Pro Leu

Val Pro Phe Pro Leu

1 kb

GAF PHY PAS PAS HK HKL

8aa

BHvPHYCprotein

PAS

Chromophore

8aa

Phe SerGln His100 aa

protein302D

380H

Gln Arg

1052D

1131K, D

C GAF domain

HK2 365: 409NIL (Vrn-H1) 365: 409i i 36 09

W G L V V C H H T S P R F V P S P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EChinese Spring (5A) 365: 409

Soleil (5A) 365: 409Soleil (5B) 365: 409Chinese Spring (5D) 365: 409Soleil (5D) 365: 409Brachypodium distachyon 365: 409Oryza sativa 364: 408Sorghum bicolor 363:: 407Arabidopsis thaliana 357: 401

W G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K E

W G L V V C H H A S P R F V P F P L R Y A C E F L T Q V F G V Q I N K E A E S A V L L K EW G L V V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A A Q A K EW G L M V C H H T S P R F V P F P L R Y A C E F L L Q V F G I Q I N K E V E L A A Q A K EW G L V V C H H S S P R F V P F P L R Y A C E F L L Q V F G I Q L N K E V E L A S Q A K E

Figure 2. Structure of HvPhyC gene and its protein. (A) HvPhyC gene sequences from HK2 and NIL (Vrn-H1) werecompared with those from Morex (DQ238106), Dicktoo (DQ201145), and Kompolti Korai (DQ201146). Filled boxes andhorizontal lines between them indicate exons and introns, respectively. Vertical line indicates single nucleotidepolymorphism (SNP) when NIL (Vrn-H1) was compared with other varieties. Open triangle indicates a 24 bp insertion.HK2, NIL (Vrn-H1), and Morex have this insertion, which results in 8 amino acid insertion. Numbers and letters atpolymorphic sites indicate the position and polymorphic varieties, respectively. H, M, D, and K indicate HK2, Morex,Dicktoo, and Kompolti Korai, respectively. (B) Deduced amino acid sequences were compared based on the HvPhyC genesequences. Filled boxes indicate domains. PAS: PER, ARNT, SIM domain, GAF: cGMP phosphodiesterase, adenylatecyclase, FhlA domain, PHY: Phytochrome-specific GAF-related domain, HK: histidine kinase domain, HKL: histidineki lik ATP d i P l hi h i th H Ph C (C) D d d i idkinase-like ATPase domain. Polymorphisms are shown in the same way as HvPhyC gene. (C) Deduced amino acidsequences around the C-terminal end of GAF domain were compared based on the genomic sequences of barley, wheat(GenBank: AY672995.1, AY673002.1, AY672998.1, AY672999.1, AY673000.1), Brachypodium distachyon(Brachypodium distachyon Gbrowse: Bradi1g08400.1), Oryza sativa (RAP-DB: Os03g0752100-01), Sorghum bicolor(Sorghum bicolor GDB: Sb01g007850.1). The same and similar sequences are highlighted in black and gray, respectively.



6080

100120140

flag

leaf

unf

oldi

ng

*** *** NIL (HvPhyC-e)NIL (HvPhyC-l)

02040

20h 16h 12h

Day

s to

fl

Photoperiod

Figure 3. Photoperiodic response of the NILs carrying differentFigure 3. Photoperiodic response of the NILs carrying differentHvPhyC alleles. Days from sowing to flag leaf unfolding (parallelwith flowering time) of NIL (HvPhyC-e) and NIL (HvPhyC-l)were compared under three different (20h, 16h, and 8h)photoperiod conditions. *** indicates that the differencebetween the NILs is statistically significant (P <0.001).

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#1-26#1-12

40

50

60

70

#17-1#17-6#17-8#17-14

T1 line with HvPhyC-l

T1 line with HvPhyC-e

T1 line with empty vector

Figure 4. Functional assay of HvPhyC in rice by introducingdifferent HvPhyC alleles (T1 generation). Mutant allele(HvPhyC-e; red filled marks) and wild-type allele (HvPhyC-l;blue filled marks) were introduced into rice phyA phyC double

HvPhyC / OsActin

300.0 1.0 2.0 3.0 4.0 5.0

phyA phyC mockNip mock

1 e w e p y vec o

mutant lines with a Nipponbare genetic background via theAgrobacterium-mediated transformation method. As the control,empty vector was introduced into phyA phyC double mutant(phyA phyC mock; red open circle) and Nipponbare (Nip mock;blue open triangle). All of the T1 lines were grown under natural(long) photoperiod conditions in a greenhouse. Expression levelof HvPhyC was analyzed by semi-quantitative RT-PCR usingOsActin as the internal control.



0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.5

1.0

1.5

2.0

0.5

1.0

1.5

2.0

2.5

*

*

***

**

A E I

0.0

0 5

1.0

1.5

2.0

2.5

* **

0.0

0.5

1.0

1.5

***

***

** **

**

0.0

1 0

2.0

3.0

4.0

5.0B F J

0.0

0.5

1.0

1.5

2.0

*

0.0

1.0

1.5**

**

**

**** * *

*

0.0

1.0

1.5

2.0

2.5

3.0

3.5C G K

0.0

0.5

2.0

2.5

3.0

0.0

0.5

1.5

2.0*** ** Time (h)

8 16 24 32 40 480

0.0

0.5

1.0

1.5

D H

0.0

0.5

1.0

1.5

*

0.0

0.5

1.0

** *

Figure 5 Expression pattern of flowering time genes under a long (16h) photoperiod Red circles and blue

Time (h)8 16 24 32 40 480

Time (h)8 16 24 32 40 480

Figure 5. Expression pattern of flowering time genes under a long (16h) photoperiod. Red circles and bluediamonds indicate the near isogenic lines NIL (HvPhyC-e) and NIL (HvPhyC-l), respectively. Expression level is theaverage of band intensity obtained by semi-quantitative RT-PCR from three biological replications. White and blackhorizontal bars below each graph indicate light and dark conditions, respectively. Vertical bar indicates standarddeviation. Asterisks above plots indicate that the expression level was significantly different between two lines whenthey were compared at the same time points. *, **, and *** indicate significance at P = 0.05, 0.01, and 0.001,respectively.



2 3 corresponding author - plant physiology83 sgh1, and sgh3, respectively). vrn-h1 encodes a...

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