2 3 corresponding author - plant physiology83 sgh1, and sgh3, respectively). vrn-h1 encodes a...
TRANSCRIPT
1
Running head: Barley Phytochrome C controls long-day flowering 1
2
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
8
Research area: Genes, Development and Evolution 9
10
11
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.
2
Title of article: Phytochrome C Is A Key Factor Controlling Long-day Flowering in Barley 12 (Hordeum vulgare L.) 13
14
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
18
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
26
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
31
32
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
3
33
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
38
Corresponding autor (e-mail): Kenji Kato ([email protected]) 39
40
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
45
46
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
4
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
67
68
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
5
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
6
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
7
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
140
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
8
141
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
185
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
10
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
11
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
12
“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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
13
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
284
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
14
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
LITERATURE CITED 585
Balasubramanian S, Sureshkumar S, Agrawal M, Michael TP, Wessinger C, 586
Maloof JN, Clark R, Warthmann N, Chory J, Weigel D (2006) The 587
PHYTOCHROME C photoreceptor gene mediates natural variation in flowering 588
and growth responses of Arabidopsis thaliana. Nat Genet 38: 711-715 589
Brown KS, Benson WW (1974) Adaptive polymorphism associated with multiple 590
Müllerian mimicry in Heliconius numata. Biotropica 6: 205–228 591
Campoli C, Drosse B, Searle B, Coupland G, von Korff M (2012) Functional 592
characterisation of HvCO1, the barley (Hordeum vulgare) flowering time ortholog 593
of CONSTANS. Plant J 69: 868–880 594
Clack T, Shokry A, Moffet M, Liu P, Faul M, Sharrock RA (2009) Obligate 595
heterodimerization of Arabidopsis phytochromes C and E and interaction with the PIF3 596
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
27
basic helix-loop-helix transcription factor. Plant Cell 21: 786-799 597
Clarke CA, Sheppard PM, Thornton IWB (1968) The genetics of the mimetic 598
butterfly Papilio memnon. Philos Trans R Soc Lond B 254: 37–89 599
Distelfeld A, Li C, Dubcovsky J (2009) Regulation of flowering in temperate cereals. 600
Curr. Opin. Plant Biol 12: 178-184 601
Distelfeld A, Dubcovsky J (2010) Characterization of the maintained vegetative phase 602
deletions from diploid wheat and their effect on VRN2 and FT transcript levels. 603
Mol Genet Genomics 283: 223-232 604
Dunford RP, Griffiths S, Christodoulou V, Laurie DA (2005) Characterisation of a 605
barley (Hordeum vulgare L.) homologue of the Arabidopsis flowering time 606
regulator GIGANTEA. Theor Appl Genet 110: 925–931 607
Faure S, Turner AS, Gruszka D, Christodoulou V, Davis SJ, von Korff M, Laurie 608
DA (2012) Mutation at the circadian clock gene EARLY MATURITY 8 adapts 609
domesticated barley (Hordeum vulgare) to short growing seasons. Proc Natl Acad 610
Sci USA 109: 8328–8333 611
Franklin KA, Quail PH (2010) Phytochrome functions in Arabidopsis development. J 612
Exp Bot 61: 11-24. 613
Hemming MN, Fieg S, Peacock WJ, Dennis ES, Trevaskis B (2009) Regions 614
associated with repression of the barley (Hordeum vulgare) VERNALIZATION1 615
gene are not required for cold induction. Mol Genet Genomics 282: 107-117 616
Hemming MN, Peacock WJ, Dennis ES, Trevaskis B (2008) Low-temperature and 617
daylength cues are integrated to regulate FLOWERING LOCUS T in barley. Plant 618
Physiol 147: 355-366 619
Higgins JA, Bailey PC, Laurie DA (2010) Comparative genomics of flowering time 620
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
28
pathways using Brachypodium distachyon as a model for the temperate grasses. 621
PLoS ONE 5: e10065 622
Izawa T, Oikawa T, Sugiyama N, Tanisaka T, Yano M, Shimamoto K (2002) 623
Phytochrome mediates the external light signal to repress FT orthologs in 624
photoperiodic flowering of rice. Genes Dev 16: 2006-2020 625
Joron M, Frezal L, Jones RT, Chamberlain NL, Lee SF, Haag CR, Whibley A, 626
Becuwe M, Baxter SW, Ferguson L, Wilkinson PA, Salazar C, Davidson C, 627
Clark R, Quail MA, Beasley H, Glithero R, Lloyd C, Sims S, Jones MC, 628
Rogers J, Jiggins CD, ffrench-Constant RH (2011) Chromosomal 629
rearrangements maintain a polymorphic supergene controlling butterfly mimicry. 630
Nature 477: 203-208 631
Kato K, Kidou S, Miura H, Sawada S (2002) Molecular cloning of the wheat CK2α 632
gene and detection of its linkage with Vrn-A1 on chromosome 5A. Theor Appl 633
Genet 104: 1071-1077 634
Kato K, Kidou S, Miura H (2008) Molecular cloning and mapping of casein kinase 2 635
alpha and beta subunit genes in barley. Genome 51: 208-215 636
Kikuchi R, Kawahigashi H, Ando T, Tonooka T, Handa H (2009) Molecular and 637
functional characterization of PEBP genes in barley reveal the diversification of 638
their roles in flowering. Plant Physiol 149: 1341–1353 639
Kikuchi R, Kawahigashi H, Oshima M, Ando T, Handa H (2012) The differential 640
expression of HvCO9, a member of the CONSTANS-like gene family, contributes 641
to the control of flowering under short-day conditions in barley. J Exp Bot 63: 642
773-784 643
Knüpffer H, Terentyeva I, Hammer K, Kovaleva O, Sato K (2003) Ecogeographical 644
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
29
diversity – a Vavilovian approach. In R von Bothmer, T van Hintum, H Knüpffer, 645
K Sato, eds, Diversity in Barley (Hordeum vulgare). Elsevier, Amsterdam, pp. 646
54–76 647
Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newberg LA 648
(1987) MAPMAKER: an interactive computer package for constructing primary 649
genetic linkage maps of experimental and natural populations. Genomics 1: 650
174-181 651
Laurie DA, Pratchett N, Snape JW, Bezant JH (1995) RFLP mapping of five major 652
genes and eight quantitative trait loci controlling flowering time in a winter x 653
spring barley (Hordeum vulgare L.) cross. Genome 38: 575-585 654
Mather K (1950) The genetical architecture of heterostyly in Primula sinensis. 655
Evolution 4: 340–352 656
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant 657
DNA. Nucleic Acids Res 8: 4321-4325 658
Nagatani A (2010) Phytochrome: structural basis for its functions. Curr Opin Plant Biol 659
13: 565-570 660
Nijhout HF (2003) Polymorphic mimicry in Papilio dardanus: mosaic dominance, big effects, 661
and origins. Evol Dev 5: 579–592 662
Nitcher R, Distelfeld A, Tan C, Yan L, Dubcovsky J (2013) Increased copy number at the 663
HvFT1 locus is associated with accelerated flowering time in barley. Mol Genet Genomics 664
288: 261-275 665
Oda A, Fujiwara S, Kamada H, Coupland G, Mizoguchi T (2004) Antisense suppression of 666
the Arabidopsis PIF3 gene does not affect circadian rhythms but causes early flowering 667
and increases FT expression. FEBS lett 557: 259-264 668
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
30
Ogiso E, Takahashi Y, Sasaki T, Yano M, Izawa T (2010) The role of casein kinase II 669
in flowering time regulation has diversified during evolution. Plant Physiol 152: 670
808-820 671
Putterill J, Robson F, Lee K, Simon R, Coupland G (1995) The CONSTANS gene of 672
arabidopsis promotes flowering and encodes a protein showing similarities to zinc 673
finger transcription factors. Cell 80: 847-857 674
Sato K, Nankaku N, Takeda K (2009) A high-density transcript linkage map of barley 675
derived from a single population. Heredity 103: 110-117 676
Shimada S, Ogawa T, Kitagawa S, Suzuki T, Ikari C, Shitsukawa N, Abe T, 677
Kawahigashi H, Kikuchi R, Handa H, Murai K (2009) A genetic network of 678
flowering time genes in wheat leaves, in which an APETALA1/FRUITFULL-like 679
gene, VRN1, is upstream of FLOWERING LOCUS T. Plant J 58: 668-681 680
Szücs P, Karsai I, von Zitzewitz J, Mészáros K, Cooper LLD, Gu YQ, Chen THH, 681
Hayes PM, Skinner JS (2006) Positional relationships between photoperiod 682
response QTL and photoreceptor and vernalization genes in barley. Theor Appl 683
Genet 112: 1277-1285 684
Takahashi Y, Shomura A, Sasaki T, Yano M (2001) Hd6, a rice quantitative trait locus 685
involved in photoperiod sensitivity, encodes the alpha subunit of protein kinase 686
CK2. Proc Natl Acad Sci USA 98: 7922-7927 687
Takahashi R, Yasuda S (1970) Genetics of earliness and growth habit in barley. In RA 688
Nilan, ed, Barley genetics II. Proc 2nd Int Balrey Genet Symp. Washington State 689
University Press, Pullman, pp. 388-408 690
Takano M, Inagaki N, Xie X, Yuzurihara N, Hihara F, Ishizuka T, Yano M, 691
Nishimura M, Miyao A, Hirochika H, Shinomura T (2005) Distinct and 692
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
31
cooperative functions of phytochromes A, B, and C in the control of deetiolation 693
and flowering in rice. Plant Cell 17: 3311–3325 694
Trevaskis B, Bagnall DJ, Ellis MH, Peacock WJ, Dennis ES (2003) MADS box 695
genes control vernalization-induced flowering in cereals. Proc Natl Acad Sci U S 696
A. 100: 13099-13104 697
Trevaskis B, Hemming MN, Dennis ES, Peacock WJ (2007) The molecular basis of 698
vernalization-induced flowering in cereals. Trends Plant Sci 12: 352-357 699
Trevaskis B, Hemming MN, Peacock WJ, Dennis ES (2006) HvVRN2 responds to 700
daylength, whereas HvVRN1 is regulated by vernalization and developmental 701
status. Plant Physiol 140: 1397-1405 702
Turner A, Beales J, Faure S, Dunford RP, Laurie DA (2005) The pseudo-response 703
regulator Ppd-H1 provides adaptation to photoperiod in barley. Science 310: 704
1031-1034 705
Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland G (2004) 706
Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. 707
Science 303: 1003-1006 708
von Bothmer R, Sato K, Komatsuda T, Yasuda S, Fischbeck G (2003) The 709
domestication of cultivated barley. In R von Bothmer, T van Hintum, H Knüpffer, 710
K Sato, eds, Diversity in Barley (Hordeum vulgare). Elsevier, Amsterdam, pp. 711
9–27 712
von Zitzewitz J, Szucs P, Dubcovsky J, Yan L, Francia E, Pecchioni N, Casas A, 713
Chen TH, Hayes PM, Skinner JS (2005) Molecular and structural 714
characterization of barley vernalization genes. Plant Mol Biol 59: 449-467 715
Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M, Sanchez A, Valarik M, 716
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
32
Yasuda S, Dubcovsky J (2006) The wheat and barley vernalization gene VRN3 is 717
an orthologue of FT. Proc Natl Acad Sci USA 103: 19581-19586 718
Yan L, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, SanMiguel P, 719
Bennetzen JL, Echenique V, Dubcovsky J (2004) The wheat VRN2 gene is a 720
flowering repressor down-regulated by vernalization. Science 303: 1640-1644 721
Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J (2003) 722
Positional cloning of the wheat vernalization gene VRN1. Proc Natl Acad Sci 723
USA 100: 6263-6268 724
Yan L, von Zitzewitz J, Skinner JS, Hayes PM, Dubcovsky J (2005) Molecular 725
characterization of the duplicated meristem identity genes HvAP1a and HvAP1b 726
in barley. Genome 48: 905-912 727
Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, 728
Yamamoto K, Umehara Y, Nagamura Y, Sasaki T (2000) Hd1, a major 729
photoperiod sensitivity quantitative trait locus in rice, is closely related to the 730
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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
w
ww
.plantphysiol.orgon A
ugust 29, 2020 - Published by
Dow
nloaded from
Copyright ©
2013 Am
erican Society of P
lant Biologists. A
ll rights reserved.
38
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
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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.
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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.
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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).
w
ww
.plantphysiol.orgon A
ugust 29, 2020 - Published by
Dow
nloaded from
Copyright ©
2013 Am
erican Society of P
lant Biologists. A
ll rights reserved.
#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.
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.
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.
www.plantphysiol.orgon August 29, 2020 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.