gain-of-function mutants of the cytokinin receptors ahk2 and … · 2017. 1. 17. · 5 102...
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Short title: Gain-of-Function Variants of Cytokinin Receptors 1
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Corresponding authors 3
Prof. Dr. Thomas Schmülling and Prof. Dr. Tomáš Werner 4
Institute of Biology/Applied Genetics 5
Dahlem Centre of Plant Sciences (DCPS) 6
Freie Universität Berlin 7
Albrecht-Thaer-Weg 6 8
D-14195 Berlin, Germany 9
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Email: [email protected], [email protected] 11
Phone: +49 30 838 55808 / 56796 12
Fax: +49 30 838 54345 13
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Gain-of-function mutants of the cytokinin receptors AHK2 and 15
AHK3 regulate plant organ size, flowering time and plant longevity 16
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Isabel Bartrina1$, Helen Jensen1$, Ondřej Novák2, Miroslav Strnad2, Tomáš Werner1,3* 18
and Thomas Schmülling1* 19 1Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie 20
Universität Berlin, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany 21 2Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany 22
ASCR, CZ-78371 Olomouc, Slechtitelu 11, Czech Republic 23 3Institute of Plant Sciences, Department of Plant Physiology, University of Graz, 24
Schubertstraße 51, 8010 Graz, Austria 25 26 $These authors have contributed equally to this work. 27
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One sentence summary: 29
Gain-of-function variants of two Arabidopsis cytokinin receptors show the impact of cytokinin 30
on regulating shoot organ size, flowering time, plant longevity and seed yield. 31
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Footnotes: 34
I.B., T.W. and T.S. designed experiments and coordinated the project; I.B. and H.J. 35
performed experiments; O.N. and M.S. carried out hormone analyses, I.B., T.W. and T.S. 36
wrote the article, all authors revised the article. 37
Plant Physiology Preview. Published on January 17, 2017, as DOI:10.1104/pp.16.01903
Copyright 2017 by the American Society of Plant Biologists
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Funding: This project was funded by grants of Deutsche Forschungsgemeinschaft in the 39
frame of CRC 429 to T.S. and T.W., in the frame of SPP 1530 to T.S. and by the Czech 40
Grant Agency (No. 15-22322S) to M.S. 41
Corresponding Author email: [email protected] and [email protected]
berlin.de 43
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ABSTRACT 44
The phytohormone cytokinin is a regulator of numerous processes in plants. In Arabidopsis 45
thaliana, the cytokinin signal is perceived by three membrane-located receptors named 46
ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3 and AHK4/CRE1. How the signal is 47
transmitted across the membrane is an entirely unknown process. The three receptors have 48
been shown to operate mostly in a redundant fashion and only very few specific roles have 49
been attributed to single receptors. Using a forward genetic approach, we isolated 50
constitutively active gain-of-function variants of the AHK2 and AHK3 genes, named repressor 51
of cytokinin deficiency2 (rock2) and rock3, respectively. It is hypothesized that the structural 52
changes caused by these mutations in the sensory and adjacent transmembrane domains 53
emulate the structural changes caused by cytokinin binding resulting in domain motion 54
propagating the signal across the membrane. Detailed analysis of lines carrying rock2 and 55
rock3 alleles revealed how plants respond to locally enhanced cytokinin signaling. Early 56
flowering time, a prolonged reproductive growth phase and, thereby, increased seed yield 57
suggest that cytokinin regulates various aspects of reproductive growth. In particular, it 58
counteracts the global proliferative arrest, a correlative inhibition of maternal growth by 59
seeds, an as yet unknown activity of the hormone. 60
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Keywords: Arabidopsis thaliana, cytokinin, cytokinin receptor, flowering time, global 63
proliferative arrest (GPA), organ size, plant development 64
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INTRODUCTION 65
The phytohormone cytokinin regulates numerous developmental processes, including cell 66
proliferation and differentiation, shoot and root growth, seed germination, and leaf 67
senescence (Werner and Schmülling, 2009; Kieber and Schaller, 2014; Zürcher and Müller, 68
2016). Cytokinin signaling is mediated by a phosphorelay system that resembles bacterial 69
two-component signaling systems (Hwang and Sheen, 2001; Müller and Sheen, 2007). In 70
Arabidopsis, there are three membrane-spanning histidine protein kinases (AHKs) which 71
serve as cytokinin receptors, AHK2, AHK3, and AHK4/CRE1 (named AHK4 in the following) 72
(Inoue et al., 2001; Ueguchi et al., 2001; Yamada et al., 2001). The hormone is recognized 73
and bound by an about 270 amino acids long extra-cytoplasmic binding domain called 74
CHASE domain (cyclin histidine kinase associated sensory). This domain is flanked by two 75
transmembrane domains and followed towards the C-terminal end on the cytoplasmic side 76
by a histidine kinase and a receiver domain (Steklov et al., 2013). The 3D structure of the 77
CHASE domain of AHK4 has been resolved by X-ray crystallography (Hothorn et al., 2011). 78
This has revealed that the receptor acts as a dimer and that the hormone is bound by a Per-79
Arnt-Sin (PAS)-like domain. A simple size exclusion mechanism allows only for the binding of 80
the free cytokinin bases, which are isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ) 81
and dihydrozeatin (DHZ). Binding of bulkier metabolites such as ribosides and nucleotides is 82
prohibited (Hothorn et al., 2011). This result has been confirmed by an in planta cytokinin 83
receptor binding assay (Lomin et al., 2015). However, it is clear that the three cytokinin 84
receptors have different affinities for the different cytokinin bases (Suzuki et al., 2001; 85
Spíchal et al., 2004; Romanov et al., 2006; Stolz et al., 2011; Lomin et al., 2015). 86
Interestingly, the ligand affinity of AHK2 resembles that of AHK4, while AHK3 differs from 87
these in its lower affinity to iP (Romanov et al., 2006; Stolz et al., 2011). 88
The bulk of cytokinin receptors is located in the endoplasmatic reticulum (Caesar et al., 89
2011; Wulfetange et al., 2011; Lomin et al., 2015). Upon ligand binding, the signal is 90
transmitted via an as yet unknown mechanism across the membrane, the cytoplasmic kinase 91
domain is activated and the protein autophosphorylates at the His residue. The high energy 92
phosphoryl group is next transferred within the same molecule to the Asp residue of the 93
receiver domain and from there to histidine phosphotransfer proteins (HPTs). These 94
translocate to the nucleus where they transfer the phosphoryl group to B-type response 95
regulators, which act as transcription factors and orchestrate downstream responses (Müller 96
and Sheen, 2007). In Arabidopsis, the transcript abundance of numerous genes is altered 97
within minutes in response to a cytokinin stimulus (Brenner et al., 2012; Bhargava et al., 98
2013; Brenner and Schmülling, 2015). 99
Loss-of-function mutations of single cytokinin receptor genes have no or only weak 100
effects on plant growth indicating strong functional redundancy (Higuchi et al., 2004; 101
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Nishimura et al., 2004; Riefler et al., 2006). Similarly, simultaneous mutation of AHK2 or 102
AHK3 together with AHK4 has only mild effects on development suggesting that both AHK2 103
and AHK3 alone are sufficient to mediate central functions of cytokinin. In contrast, ahk2 104
ahk3 mutants are dwarfed plants showing stronger root growth demonstrating the importance 105
of AHK2 and AHK3 in regulating vegetative plant growth (Riefler et al., 2006). Additional 106
comprehensive examination of the cytokinin receptor genes revealed also some 107
specialization in cytokinin receptor function (Heyl et al., 2012). For example, it was shown 108
that AHK4 alone plays a role in embryonic root development, phosphate starvation response 109
and sulfate assimilation (Mähönen et al., 2000; Maruyama-Nakashita et al., 2004; Franco-110
Zorrilla et al., 2005). Furthermore, AHK3 plays a predominant role in regulating leaf 111
senescence and cell differentiation in the transition zone of the root meristem (Kim et al., 112
2006; Riefler et al., 2006; Dello Ioio et al., 2007). No specific function has been shown for 113
AHK2 so far. 114
The functional overlap is particularly high for AHK2 and AHK3, which both contribute to 115
mediate a large number of developmental cytokinin functions from seed germination to 116
regulation of shoot growth and also responses to abiotic stresses including drought (Tran et 117
al., 2007), cold (Jeon et al., 2010) and high light (Cortleven et al., 2014). The mostly 118
redundant action of AHK2 and AHK3 is intriguing and raises the question why both genes 119
with apparently similar functions have been conserved during evolution. Consistently, AHK2 120
and AHK3 have largely overlapping expression domains, both are predominantly expressed 121
in shoots (Higuchi et al., 2004). Stolz et al. (2011) showed that both AHK2 and AHK3 122
activate cytokinin response in leaf mesophyll cells (AHK4 does not) but only AHK3 mediates 123
a response in stomata cells. Interestingly, promoter-swap and domain-swap analyses have 124
shown that AHK4 can functionally replace AHK2 but not AHK3 (Stolz et al., 2011). 125
In view of the mostly redundant action of the AHK2 and AHK3 receptors it might be 126
informative to study gain-of-function mutants to compare receptor activities. In this work, we 127
report on novel gain-of-function mutants of AHK2 and AHK3 named repressor of cytokinin 128
deficiency2 (rock2) and rock3, respectively. These mutants were identified in a screen for 129
suppressor mutants of the cytokinin deficiency syndrome displayed by 35S:CKX1 130
overexpressing (CKX1ox) plants (Niemann et al., 2015). Reversion of the cytokinin 131
deficiency phenotype was due to a constitutive activity of the mutant receptors. The receptor 132
variants do have differential impact on individual phenotypic traits and are thus informative 133
about cytokinin signaling during plant development. The results yield new information on the 134
functions of these evolutionary closely related receptors and highlight their roles in regulating 135
flowering time and plant longevity. Given the promoting effect of the rock2 and rock3 alleles 136
on shoot organ growth and, in particular, seed yield, we propose their potential value for 137
biotechnological approaches. 138
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RESULTS 139
rock2 and rock3 Suppress the Cytokinin-Deficiency Phenotype 140
To identify molecular factors required for establishing the CK deficiency syndrome displayed 141
by CKX1ox plants (Werner et al., 2003), we searched for suppressor mutants reverting the 142
dwarf shoot phenotype of CKX1ox plants (Niemann et al., 2015). Amongst others, two 143
mutants named rock2 and rock3 which grew larger than CKX1ox (Fig. 1A, C) were identified 144
and selected for further study. Genetic analysis showed that rock2 and rock3 are two 145
dominant second site mutations (Supplemental Table S1). The reversion of the cytokinin-146
deficient phenotype was already obvious early after germination. In the CKX1ox background, 147
rock2 and rock3 developed strongly enlarged cotyledons with longer petioles, which even 148
exceeded the size of wild-type cotyledons (Fig. 1B). rock2 CKX1ox and rock3 CKX1ox plants 149
developed larger rosette leaves, grew taller inflorescence stems with more flowers (Fig. 1A, 150
C) and the flowers of both suppressor lines were enlarged (Fig. 1D). In addition, both 151
mutations suppressed the late flowering phenotype of CKX1ox plants under long-day 152
conditions (Werner et al., 2003). The rescue was partial in the case of rock3 CKX1ox 153
whereas rock2 CKX1ox flowered even earlier than wild-type plants (Fig. 1E). Under short-154
day conditions, the flowering transition defect of CKX1ox plants were more severe as these 155
plants remained in the vegetative stage. Interestingly, only the rock2 mutation was able to 156
suppress the non-flowering phenotype of CKX1ox under short-day conditions (Fig. 1F). In 157
contrast, rock3 CKX1ox still failed to flower under short-day conditions. This indicates that 158
rock2 particularly regulates processes associated with the change from vegetative to 159
reproductive development under different light periods. 160
Cytokinin is known to delay leaf senescence (Richmond and Lang, 1957; Gan and 161
Amasino, 1995; Riefler et al., 2006) and, consistently, leaves of CKX1ox plants showed a 162
reduced chlorophyll retention in a detached leaf assay (Fig. 1G). Both, rock2 and rock3, 163
retarded the breakdown of chlorophyll of CKX1ox plants during dark-induced senescence 164
compared to wild-type and CKX1ox plants. After seven days in the dark, the leaf chlorophyll 165
content was reduced by almost 80% in wild-type and by 90% in CKX1ox plants while it was 166
decreased only by 65 and 35% in rock2 CKX1ox and rock3 CKX1ox mutants, respectively. 167
This indicates that rock3 plays a more prevalent role than rock2 in delaying dark-induced leaf 168
senescence. 169
To study whether the rock2 and rock3 eventually exert a differential influence also on 170
root development we compared primary root elongation in the mutants and wild type. Figure 171
1H shows that the roots of rock2 CKX1ox seedlings displayed a complete and rock3 CKX1ox 172
seedlings a partial reversion of the enhanced primary root elongation caused by the reduced 173
cytokinin content of CKX1ox seedlings (Werner et al., 2003). 174
175
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rock2 and rock3 Increase Sensitivity Towards Exogenous Cytokinin 176
What could be the cause for the suppression of the cytokinin deficiency phenotype of 177
CKX1ox plants? Analysis of the expression of the CKX1 transgene in both the rock2 and 178
rock3 mutant showed that it was not affected by the mutation (Fig. 2A). In addition, 179
outcrossing the 35S:CKX1 transgene from the rock2 CKX1ox or rock3 CKX1ox background 180
revealed that the transgene was functionally intact (data not shown). Comparison of the 181
cytokinin content of wild type, CKX1ox, rock2 and rock3 seedlings showed that the mutations 182
did not increase the endogenous cytokinin content compared to CKX1ox (Fig. 2B and 183
Supplemental Table S2). This suggested that a mechanism different from interference with 184
35S:CKX1 gene expression or metabolic compensation must be the cause for the phenotypic 185
reversion. 186
Next, we analyzed whether rock2 and rock3 change the plants´ sensitivity to 187
exogenously applied cytokinin. Figure 2C shows that wild-type seedlings grew smaller on 188
media containing 25 nM BA, however, with the exception of few yellowing leaves, most 189
leaves stayed green. CKX1ox seedlings were less sensitive and grew on BA-containing 190
media similar as on medium without BA. In contrast, rock2 CKX1ox and rock3 CKX1ox 191
seedlings showed strong hypersensitive reaction to cytokinin. They stayed smaller than 192
control plants grown on standard media and formed yellow leaves, which is a typical reaction 193
to high exogenous cytokinin concentrations (Ainley et al., 1993). The altered growth 194
responses indicate that rock2 and rock3 enhance the plants´ cytokinin sensitivity and 195
suggest that the altered sensitivity may be causal for the suppression of the cytokinin-196
deficient phenotype. 197
198
rock2 and rock3 Encode Novel Dominant Gain-of-Function Alleles of AHK2 and AHK3 199
Intriguingly, genetic mapping of the rock2 and rock3 mutations revealed that both were 200
located in genes coding for different cytokinin receptors. The rock2 mutation turned out to be 201
a C to T transition in the 7th exon of the AHK2 (At5g35750) gene leading to a semi-202
conservative substitution of aliphatic leucine by aromatic phenylalanine at amino acid 203
position 552 (L552F) in the fourth predicted transmembrane domain (Fig. 3A, B). The rock3 204
mutation was identified as a single base change from C to T in the second exon of AHK3 205
(At1g27320). This results in a non-conservative amino acid change from polar threonine to 206
aliphatic isoleucine at position 179 (T179I) located closely to the predicted cytokinin-binding 207
domain of the AHK3 receptor (Fig. 3A, B). A second independent allele, named rock3-2, was 208
identified during the course of this work causing exchange of negatively charged glutamic 209
acid to positively charged lysine at position 182 (E182K) in close proximity to the T179I 210
mutation (Fig. 3A). The first identified allele rock3-1 was used for all analyses described in 211
this article and is called rock3 throughout. 212
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To prove that the identified mutant alleles of AHK2 and AHK3 are causal for the 213
suppression of the cytokinin-deficient phenotype, the mutant gene variants were cloned 214
under the control of their native promoters and transformed in the CKX1ox background. 215
Transforming CKX1ox was inherently difficult, nevertheless, two independent transgenic 216
AHK2:rock2 CKX1ox lines and three independent AHK3:rock3 CKX1ox lines were identified. 217
All five transgenic lines showed a suppression of the cytokinin deficiency phenotype similar 218
or even stronger than the rock2 CKX1ox and rock3 CKX1ox suppressor lines (Fig. 3, C-J). 219
This unequivocally confirmed that the phenotypes were caused by the mutations in the AHK2 220
and AHK3 genes. Thus, rock2 and rock3 encode two novel dominant gain-of-function 221
variants of the cytokinin receptors AHK2 and AHK3. 222
223
rock2 and rock3 Enhance Cytokinin Signaling 224
The revertant morphology and the enhanced cytokinin sensitivity indicated that the rock2 and 225
rock3 alleles might code for cytokinin receptors with increased signaling activity. To test 226
whether the mutant receptors act in a cytokinin-independent manner or could be activated by 227
lower cytokinin concentrations we used the yeast complementation assay described by Inoue 228
et al. (2001). In this assay, AHK4 rescues, in a cytokinin-dependent manner, the survival of a 229
yeast mutant that lacks the endogenous histidine kinase SLN1 (Inoue et al., 2001). It is 230
known that the yeast Δsln1 strain carrying one of the other two cytokinin receptors (AHK2 or 231
AHK3) can grow in the absence of cytokinin, but growth may be accelerated in the presence 232
of cytokinin (Mähönen et al., 2006; Tran et al., 2007). However, under our laboratory 233
conditions the cytokinin-dependent faster growth rate of strains containing AHK2 or AHK3 234
receptor genes was not observed and, therefore, we were not able to test the original rock2 235
or rock3 mutation in this system. Instead, we constructed rock2 and rock3 variants of AHK4 236
(named AHK4rock2 and AHK4rock3) as the positions affected by the rock2 and rock3 mutations 237
are conserved in all three cytokinin receptors of Arabidopsis (Fig. 3A). Both rock variants of 238
AHK4 suppressed the lethality of the Δsln1 mutation even without the addition of cytokinin to 239
the medium while the mutant carrying the wild-type AHK4 allele showed strictly cytokinin-240
dependent growth rescue (Fig. 4A). This shows that the His kinase activity of AHK4rock2 and 241
AHK4rock3 is independent of cytokinin and that rock2 and rock3 may be constitutively active 242
receptor proteins. 243
To test further the effects of rock2 and rock3 on cytokinin signaling output in planta we 244
compared the expression level of the well-established cytokinin reporter ARR5 in wild type 245
and both rock mutants. Fig. 4B shows increased steady state mRNA levels of ARR5 in rock2 246
and rock3 indicating enhanced cytokinin signaling in planta. Next we introgressed the mutant 247
alleles (without the 35S:CKX1 transgene) into a line harboring the ARR5:GUS reporter 248
(D'Agostino et al., 2000). Figures 4B-J show strongly increased GUS activity in all analyzed 249
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tissues of rock2 ARR5:GUS and rock3 ARR5:GUS seedlings. Seven days after germination 250
increased GUS activity was detected in the vascular tissue of cotyledons and leaves, in the 251
shoot apex, the root vasculature and the vascular procambium and root cap of the primary 252
roots. rock2 caused an overall stronger increase in GUS activity than rock3 in all analyzed 253
tissues. For example, rock2 mutants showed high GUS activity along the whole root, 254
whereas in rock3 roots the GUS signal was increased only weakly in the apical part of the 255
root. This correlates well with the observed stronger capacity of rock2 to suppress the 256
cytokinin deficiency phenotypes in shoot and root. The enhanced expression of the cytokinin 257
response gene ARR5:GUS as an output of the cytokinin signaling system, together with the 258
results of the yeast complementation assay, shows that the constitutively active rock2 and 259
rock3 activate more strongly the cytokinin signal transduction pathway. 260
Previous work has provided indications that cytokinin signaling is intimately linked to 261
metabolic homeostasis of the hormone (Riefler et al., 2006). The identified more active 262
receptors provide an opportunity to test this link further. We, therefore, analyzed whether the 263
endogenous cytokinin concentration responds to the enhanced cytokinin signaling and 264
determined the cytokinin levels of wild type, rock2, and rock3 seedlings and seedlings 265
expressing AHK2:rock2. Both rock mutants and the transgenic line showed a similar 266
reduction of 45-56% of the total cytokinin content (Table 1). Stronger differences between 267
rock2 and rock3 were found for the biologically active free bases. Both the rock2 mutation 268
and transgenic expression of AHK2:rock2 caused a decrease of about 40% for iP and cZ and 269
of about 55% for tZ in comparison to wild type (Table 1). rock3 seedlings contained only 24% 270
iP, 11% tZ and 14% cZ in comparison to wild-type seedlings. Taken together, the gain-of-271
function alleles of AHK2 and AHK3 have an impact on cytokinin homeostasis and lower the 272
cytokinin content supporting a feedback regulation of cytokinin metabolism by the cytokinin 273
signaling pathway. Moreover, the results corroborate the hypothesis that these receptor 274
variants display a high signaling activity independent of the steady state cytokinin 275
concentration. 276
277
rock2 and rock3 Increase Shoot Growth and Leaf Size 278
The functional redundancy within the cytokinin receptor family makes it difficult to dissect the 279
biological function of individual receptor proteins. In this situation, gain-of-function receptor 280
variants can be useful to obtain information on their individual functions. We have, therefore, 281
analyzed the consequences of the rock2 and rock3 mutations on growth and development in 282
more detail using lines containing the original rock mutations in the wild-type background as 283
well as transgenic lines expressing the rock2 and the rock3 coding sequence under their 284
native promoters. Results are shown for homozygous progeny of lines AHK2:rock2-10 and 285
AHK3:rock3-1. Two other independent lines, AHK2:rock2-7 and AHK3:rock3-5, showed 286
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similar results. qRT-PCR analysis showed that the levels of AHK2 transcripts were similar in 287
wild type, rock2 and the transgenic AHK2:rock2 line (Supplemental Fig. S1A). Likewise, wild 288
type, rock3 and the transgenic AHK3:rock3 line had comparable AHK3 transcript levels 289
(Supplemental Fig. S1B). 290
Figure 5 shows different aspects of the shoot development in these plants. rock2 291
mutants and transgenic plants grew significantly taller than wild type (Fig. 5A, B). The 292
inflorescence stems of 50-d-old wild-type and rock3 mutant plants measured 33.2 ± 1.3 cm 293
and 35.4 ± 2.3 cm in height (7% increase). rock2 mutant plants grew 42%, transgenic 294
AHK3:rock3 plants 60% and transgenic AHK2:rock2 plants even 85% taller than wild type. All 295
the mutant and transgenic lines had also an increased stem diameter (Figs. 5C to E). Also in 296
this case the transgenic lines showed the strongest increase, the stem diameter was 297
increased up to about 36% in comparison to wild type. Transverse sections of the primary 298
inflorescence stem showed that stem morphology was normal in all genotypes, but the 299
number of cells in stems of transgenic lines was higher than in wild-type stems. Figure 5E 300
shows as an example transverse sections of wild-type and AHK3:rock3 inflorescence stems. 301
The increased cell number could be due to a higher cambial activity, which has been 302
previously shown to be regulated by cytokinin (Matsumoto-Kitano et al., 2008; Nieminen et 303
al., 2008; Bartrina et al., 2011). This will, however, require further clarification. 304
Further inspection of the shoot phenotype revealed that rock2 and rock3 also positively 305
regulate shoot lateral organ size. rock2 and rock3 seedlings developed strongly enlarged 306
cotyledons (Fig. 6A), a phenotype which was more prominent in transgenic rock2 and rock3 307
lines. Later during development, rock2 and rock3 mutants formed larger rosette leaves (Fig. 308
6B) with the AHK2:rock2 and AHK3:rock3 transgenic lines forming the largest leaves. The 309
biomass of rosette leaves was increased in rock2 and rock3 mutants (Fig. 6C). As organ size 310
is influenced by cell number and cell expansion, we analyzed cell size of epidermal cells in 311
the sixth fully developed rosette leaf of wild-type and rock2 plants. The epidermal cell size of 312
rock2 plants was slightly but not significantly reduced (Fig. 6D). This together with the 313
increased leaf surface (Fig. 6E) revealed that rock2 rosette leaves formed about 40-50% 314
more epidermal cells compared to wild type. In conclusion, the rock2-dependent changes in 315
organ size are due to prolonged mitotic activity and/or faster cell proliferation during leaf 316
growth. 317
318
rock3 Prolongs the Life Span of Leaves 319
It is known that cytokinin delays leaf senescence (Gan and Amasino, 1995; Kim et al., 2006). 320
We compared the natural senescence as well as dark-induced senescence of detached 321
leaves of rock2 and rock3 with that of wild type. Visual examination of the sixth rosette 322
leaves throughout their life spans showed that the onset of natural leaf senescence was 323
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delayed particularly in rock3 mutant plants (Fig. 7A). rock3 leaves had an about seven days 324
longer life span compared to wild-type leaves, whereas rock2 leaves showed only a slightly – 325
up to two days – delayed leaf senescence (Fig. 7A). These results were confirmed by 326
measuring the photosynthetic efficiency of photosystem II (Fv/Fm), which was maintained 327
about four days longer at a high level in leaves of rock2 and about 8 days longer in rock3 328
plants (Fig. 7B). Figure /C shows that also dark-induced leaf senescence was strongly 329
retarded in rock3 mutant plants. After seven days in the dark, the chlorophyll content was 330
reduced to 10% in wild-type and rock2 plants, whereas rock3 leaves were still green with a 331
remaining chlorophyll content of almost 60%. These results show that a gain-of-function 332
mutation in the AHK3 cytokinin receptor significantly delays different senescence-associated 333
symptoms in Arabidopsis. Enhanced activity of AHK2 on the other hand has only a minor but 334
still significant impact on delaying leaf senescence. 335
The strong delay of leaf senescence in rock3 mutants prompted us to compare rock3 336
with another AHK3 allele, ore12, reported to delay leaf senescence (Kim et al., 2006). Both 337
the visual inspection and the Fv/Fm values showed a significantly later onset of senescence 338
in rock3 than in ore12 mutants (Supplemental Figure S2), indicating quantitatively different 339
effects of these mutations on AHK3 receptor activity. 340
341
rock2 and rock3 Alter Flowering Time, and Increase Flower Size and Seed Yield 342
Under long-day conditions, rock2 mutants and AHK2:rock2 transgenic lines flowered 343
significantly earlier than wild type, as indicated by their lower number of rosette leaves at 344
flowering time (Fig. 8A). Likewise, the AHK3:rock3 transgenic plants, but not the rock3 345
mutant, flowered earlier. Interestingly, all mutant and transgenic lines showed also a 346
markedly increased duration of flowering and increased total life span. Seven-week-old wild-347
type plants ceased to form new flowers, while rock genotypes continued to flower for at least 348
one more week and up to two weeks in case of AHK2:rock2 transgenic plants (Fig. 8B). 349
The size of flowers was significantly enhanced in rock2 and rock3 mutants (Fig. 8C). To 350
determine whether the increase in petal size was a result of an increased cell proliferation, 351
cell expansion or both, abaxial petal epidermal cell size was analyzed exemplarily in 352
AHK2:rock2 flowers. Fig. 8D shows that the cell size was not significantly altered, indicating 353
that an enhanced cell proliferation was the cause for the larger petals, similar as was found 354
for rosette leaf size regulation (Fig. 6D and E). 355
rock2, AHK2:rock2, and AHK3:rock3 plants formed more siliques than wild-type plants 356
(Fig. 8E). The transgenic AHK2:rock2 line showed the largest increase, producing almost 357
twice as many siliques as wild type. Interestingly, despite activity of cytokinin in regulating 358
shoot meristem activity and size (Bartrina et al., 2011), microscopic analysis did not reveal 359
an increased size of rock2 or rock3 inflorescence meristems (Supplemental Fig. S3). 360
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Consistently, analysis of cytokinin signaling output in the inflorescence meristem of rock2 361
plants using the TCSn:GFP marker gene (Zürcher et al., 2013) showed no significant change 362
in comparison to wild type (Supplemental Fig. S3) suggesting dampening of the increased 363
receptor signaling in the inflorescence meristem. This indicates that the formation of more 364
flowers and, consequently, of more siliques was due to a longer flowering phase. Analysis of 365
seed yield did not show an increase in plants harboring a rock2 allele, which was probably 366
accountable to heterostylous flowers displaying disproportionally elongated gynoecia in 367
comparison to the stamen, which caused reduced self-fertilization (Supplemental Fig. S4). 368
However, AHK3:rock3 plants lacking this morphological defect produced about 40% more 369
seeds than wild-type plants. A similar result was found for a second transgenic rock3 line 370
(AHK3:rock3-5), confirming that seed yield is positively influenced by the rock3 mutation. 371
372
rock2 and rock3 Reduce Root Growth 373
In contrast to its promotional role in shoot organs, cytokinin is a negative regulator of root 374
development (Werner and Schmülling, 2009). As expected from the observed suppressor 375
activity (Fig. 1H), root growth was inhibited in all tested rock seedlings grown under in vitro 376
conditions (Fig. 9A). Primary root elongation was reduced by 37 and 28% in rock2 and rock3 377
seedlings, respectively. The transgenic rock lines AHK2:rock2-10 and AHK3:rock3-1 showed 378
slightly milder root phenotypes, with a reduction in root elongation by 23% and 18%, 379
respectively (Fig. 9A). Consistently, root meristem size was decreased in both the rock2 and 380
rock3 mutants (Fig. 9B, C). The formation of lateral roots was strongly inhibited as well. On 381
average, the number of lateral roots in wild-type plants was around 1.8 to 2.4 times greater 382
than that of the rock mutants, with rock2 showing the strongest reduction of lateral root 383
formation (Fig. 9D). The similar or even weaker consequences of transgenic rock gene 384
expression as compared to their original alleles contrasts with their generally stronger effects 385
on the shoot phenotype. 386
387
388
DISCUSSION 389
From the analysis of the rock2 and rock3 mutant alleles and transgenic lines carrying these 390
alleles we obtained valuable information about the cytokinin signaling mechanism and the 391
roles of these redundantly acting receptors in regulating various facets of the plant´s 392
phenotype. Both aspects are discussed in the following sections. 393
394
rock2 and rock3 Mutations Provide Insight into Transmembrane Signaling by 395
Cytokinin Receptors 396
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13
How the cytokinin signal is transmitted across the membrane is an entirely unknown process. 397
The rock2 and rock3 mutations identified several amino acid residues that are relevant for 398
transmembrane signaling. Presumably, they induce changes in the receptor structure 399
resembling those induced by cytokinin binding to the CHASE domain and thus cause 400
constitutive cytokinin signaling. The fact that the AHK4 receptors harboring rock2 and rock3 401
mutations conferred growth rescue of Δsln1 yeast to a great extent similar to that caused by 402
cytokinin-induced signaling of wild-type AHK4 suggests that these mutations cause strong, if 403
not maximal, activation of the receptors. 404
We isolated two independent rock3 mutations which are located in close proximity to 405
each other in a region of the CHASE domain linking the long α-helical stalk domain with the 406
membrane-distal (ligand-binding) PAS domain (Hothorn et al., 2011). More precisely, they 407
are located in a slightly bent region comprising a 310-helix which connects helix α2 with the 408
first β-strand of the membrane-distal PAS domain (Hothorn et al., 2011)(see Figure 9). This 409
region, which is not involved in ligand binding, could participate in the receptor domain 410
movement expected to be triggered by cytokinin binding. The rock3 mutations, which alter 411
evolutionary highly conserved residues (Heyl et al., 2007; Steklov et al., 2013), may provoke 412
a similar intramolecular movement. 413
There is an intriguing structural similarity between the CHASE domain and the sensing 414
domain of a bacterial methyl-accepting chemotaxis protein (MCP), which is a membrane-415
bound His kinase recognizing isoleucine. The sensory domains of both receptor types have a 416
long α-helical stalk important to keep the receptor dimers together and to hold two PAS 417
domains of which the membrane-distal one binds the ligand (Hothorn et al., 2011; Liu et al., 418
2015). Analysis of the ligand-induced conformational changes of the PAS sensing domain of 419
MCP revealed that it likely signals by a piston displacement mechanism (Liu et al., 2015). 420
The signal-binding PAS domain fluctuates between a closed (ligand bound) and open form 421
resulting in a conformational change of the proximal PAS domain and movement towards 422
and away from the membrane. This movement is propagated causing a displacement of the 423
TM helix towards the cytoplasm thus generating a transmembrane signal (Liu et al., 2015). 424
Considering the close structural similarity between cytokinin receptors and MCP as well as 425
the fact that piston-type signaling is a common theme among other type of His kinases 426
(Chervitz and Falke, 1996; Cheung and Hendrickson, 2009; Moore and Hendrickson, 2009; 427
Bhate et al., 2015), we hypothesize that also the CHASE domain undergoes upon ligand 428
binding structural changes emulating a piston-like domain motion resulting in transmembrane 429
signaling (Fig. 10). Interestingly, the last β-strand of the membrane-proximal PAS domain is 430
linked to the stalk helix by a disulfide bridge (Hothorn et al., 2011), which might limit the 431
degree of structural rearrangements. Hence, the domain displacement will probably be of a 432
subtle nature, as known for other His kinases (Chervitz and Falke, 1996). 433
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14
The rock2 mutation is located in the TM domain connecting the CHASE domain and the 434
cytosolic His-kinase domain. In the proposed signaling mechanism (Figure 9), this TM 435
domain is essential for transmitting the signal from the lumen of the endoplasmic reticulum to 436
the cytoplasm. Noteworthy, this TM domain shows a high degree of sequence conservation 437
among different cytokinin receptors, in contrast to the TM domain delimiting the CHASE 438
domain N-terminally (Steklov et al., 2013). The L552F substitution in the rock2 receptor 439
variant affects the highly conserved Leu residue of the helix core motif 440
Axxx(S/A)x(G/L)x(L/F)VIx(L/F)LxG(Y/H)I (Leu552 underlined). Furthermore, this residue is 441
located in immediate vicinity of two gain-of-function mutations of the AHK4 receptor that have 442
been reported to cause constitutive cytokinin signaling in a bacterial assay (Miwa et al., 443
2007) and that are partially conserved in AHK2. Presumably, the described mutations cause 444
constitutively conformational alterations of the TM domain which normally occur during 445
signaling. Although no crystal structure for any TM domain of any His kinase has been 446
resolved to date, there is a growing body of knowledge (Bhate et al., 2015) allowing to 447
predict that the structural changes may involve a lateral or vertical displacement of the 448
neighboring TM helices, and that such a displaced conformation is being locked by the rock2 449
and related mutations. 450
451
Constitutive Active Receptors Yield Novel Information About Cytokinin-Regulated 452
Processes 453
The analysis of mutants and transgenic lines expressing constitutive active variants of two 454
cytokinin receptors has revealed how plants with locally strongly enhanced cytokinin 455
signaling look like. Only a single gain-of-function cytokinin receptor mutant, ore12, has been 456
described previously in Arabidopsis (Kim et al., 2006) but the description has been limited to 457
the impact on leaf senescence. In addition, the ore12 mutation achieved a lower activation of 458
the AHK3 receptor as compared to the rock3 mutations described here, suggesting that this 459
receptor signals in a gradual rather than in an all-or-nothing fashion. Others reported that the 460
production of transgenic plants expressing constitutively active receptors was difficult if not 461
impossible (Miwa et al., 2007). Studying the influence of enhanced cytokinin signaling on the 462
plant phenotype is relevant as, up to now, our knowledge on the consequences of an 463
enhanced cytokinin status has been derived largely from plants ectopically expressing a 464
cytokinin-synthesizing IPT (Rupp et al., 1999; Sun et al., 2003) or LOG gene (Kuroha et al., 465
2009) or carrying mutations in cytokinin-degrading CKX genes (Bartrina et al., 2011). 466
However, the impact of an enhanced production of the hormone may differ from the 467
consequences of increased signaling. The former involves a mobile signal that can induce 468
local but also systemic effects through all cytokinin receptors that are present in a given cell 469
or tissue, while the latter acts in a cell-autonomous fashion involving only a single activated 470
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15
receptor. In accord with this notion, the expression of the ARR5:GUS gene confirmed 471
enhanced cytokinin signaling by rock2 and rock3 and showed that signaling stays generally 472
limited to those tissues previously known to activate the cytokinin reporter by the 473
corresponding wild-type receptor (D'Agostino et al., 2000; Stolz et al., 2011). Interestingly, 474
rock2 and rock3 mutants have reduced levels of all analyzed cytokinin metabolites. This is in 475
agreement with the result that ahk loss-of-function mutants have an increased cytokinin 476
content (Riefler et al., 2006) and thus underpins the existence of homeostatic control 477
mechanisms. Part of these control mechanisms is an influence of cytokinin signaling on the 478
transcript level of cytokinin metabolism genes (Miyawaki et al., 2004; Werner et al., 2006; 479
Brenner et al., 2012). 480
Both rock2 and rock3 mutations affect most morphological aspects of the cytokinin 481
deficiency syndrome but revert them to a different degree. The similar although not identical 482
effects of these mutations on the plant phenotype reflect their high degree of functional 483
redundancy revealed by loss-of-function mutants (Higuchi et al., 2004; Nishimura et al., 484
2004; Riefler et al., 2006). There are a number of notable quantitative differences between 485
the rock2 and rock3 effects, which are also seen in the differential activation of the 486
ARR5:GUS reporter in different tissues. For example, the rock2 mutation has a stronger 487
impact on primary root elongation and root meristem size, which is surprising in view of the 488
proposed central role that AHK3 (but not AHK2) plays in the root apical meristem (Dello Ioio 489
et al., 2007). Similarly, the late flowering phenotype of CKX1ox plants was only partially 490
reverted by rock3 whereas rock2 CKX1ox flowered even earlier than wild type. In contrast, 491
rock3 had, for example, a stronger effect in retarding leaf senescence. These mostly gradual 492
differences may partly be due to differences in signaling strength caused by the mutations 493
but may also reflect differences in coupling to downstream signaling processes. Indeed, 494
ectopic expression of the rock2 and rock3 alleles under the transcriptional control of the 495
same promoters causes in Arabidopsis partly different responses (A. Stolz and T.S., 496
unpublished results). This indicates that, although the cytoplasmic domains of both receptors 497
interact with the same AHP proteins in a yeast two-hybrid assay (Dortay et al., 2006), the 498
affinities between signaling proteins may differ in planta. Their interaction can also be 499
modulated by accessory proteins in a similar fashion as known from bacterial two component 500
systems (Jung et al., 2012). 501
One important result derived from the phenotypic analysis is that cytokinin signaling is 502
limiting for the growth of different shoot organs and that rock2/rock3-dependent changes in 503
organ size are due to prolonged cell proliferation. This supports the hypothesis that cytokinin 504
primarily controls the duration of the cell proliferation phase in shoot organ primordia by 505
delaying the onset of cell differentiation (Holst et al., 2011). Consistently, a lower cytokinin 506
activity causes a reduced leaf size, and genetic analysis has revealed that this is redundantly 507
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16
controlled by AHK2 and AHK3 (Werner et al., 2003; Higuchi et al., 2004; Nishimura et al., 508
2004; Riefler et al., 2006). It has been proposed that plant organ growth displays bell‐shaped 509
dose-responses to cytokinin (Ferreira and Kieber, 2005) and that cytokinin limits leaf size in 510
wild type (Efroni et al., 2013). The enhanced growth of different shoot organs in rock2 and 511
rock3 mutants underpins this view. However, the range of cytokinin activity promoting growth 512
appears to be limited since cytokinin overproduction may result in the formation of smaller 513
leaves (Hewelt et al., 1994; van der Graaff et al., 2001; Sun et al., 2003). This is likely due to 514
strongly increased cell proliferation and the concomitant inability of proper cell differentiation 515
and expansion. Cell number is apparently sensed in plant leaves (Hisanaga et al., 2015) and 516
an increase in cell number above a certain threshold may interfere with cell expansion 517
resulting in smaller leaves (Dewitte et al., 2003). The increased cell proliferation in rock2 and 518
rock3 mutants was linked to normal cell expansion suggesting that this threshold was not 519
reached. The present work further revealed that flower organ size is very sensitive to 520
enhanced cytokinin signaling too. The particularly strongly enlarged petals and gynoecia 521
suggest that cytokinin is especially involved in regulating development of these organs. 522
Since long time it is known that exogenously applied cytokinin can promote flowering in 523
Arabidopsis (Michniewicz and Kamienska, 1967; Besnard-Wibaut, 1981; Dennis et al., 1996; 524
D'Aloia et al., 2011). However, it has remained unclear whether also endogenous cytokinin 525
would have the same promotive activity. rock2 suppresses the late-flowering phenotype of 526
CKX1ox plants stronger than rock3. This effect is most obvious under short-day conditions 527
where CKX1ox remain in the vegetative state and rock2, but not rock3, reverts this non-528
flowering phenotype. It seems clear that in Arabidopsis a certain cytokinin threshold signal is 529
indispensable for flower induction in short days and that cytokinin has a promotive effect on 530
flowering also in long days. The fact that rock2 CKX1ox mutants start to flower even earlier 531
than wild type underpins the importance of cytokinin signaling for flowering time control. The 532
mechanistic basis of flowering time control by cytokinin is poorly understood. It has been 533
shown that cytokinin promotes flowering independently of FLOWERING LOCUS T, but 534
through transcriptional activation of its paralogue TWIN SISTER OF FT (TSF; D'Aloia et al., 535
2011). However, the question whether the flowering response to cytokinin is mediated 536
entirely through transcriptional activation of TSF in leaves or whether cytokinin might also 537
promote flowering by direct action in the shoot apical meristem (Corbesier et al., 2003; 538
D'Aloia et al., 2011) needs to be further scrutinized. 539
An unexpected phenotype was the increased plant longevity and prolonged reproductive 540
growth phase, which was particularly marked in AHK3:rock3 transgenic plants. The 541
prolonged reproductive growth resulted in a strongly increased number of flowers and 542
siliques. This is a very interesting observation, because the monocarpic plant Arabidopsis 543
generates dependent on the growth conditions a specific number of flowers which is followed 544
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17
by cessation of reproductive meristematic activity. This correlative inhibition of maternal 545
growth is caused by the offspring (seeds) and is referred to as global proliferative arrest 546
(GPA; Hensel et al., 1994). The molecular mechanism underlying this phenomenon is largely 547
unknown. A recent study has suggested that the low mitotic activity in meristems linked to 548
GPA represents a form of bud dormancy (Wuest et al., 2016). Given that cytokinin is a major 549
factor determining the proliferative activity in axillary buds (Shimizu-Sato et al., 2009), it is 550
tempting to hypothesize that cytokinin counteracts GPA by maintaining cell division activity. 551
This scenario would predict that a drop in cytokinin activity is required for meristematic arrest 552
during GPA and that the constitutive signaling in rock3 transgenic plants delays it. How 553
cytokinin could affect GPA is currently unclear. One possibility is that rock3, and thus 554
cytokinin, acts locally in the reproductive meristem by sustaining its activity. This idea is 555
consistent with the previous hypothesis that meristematic sink activity, rather than the leaf 556
source capacity, is decisive for the activity of the meristem, and that sink strength is 557
particularly triggered by cytokinin (Werner et al., 2008). A second possibility is that the strong 558
capacity of rock3 to delay leaf senescence prolongs the activity of the reproductive 559
meristems and in addition provides sufficient source capacity to support the development of 560
supernumerary seeds. However, it has been also discussed that the activity of the 561
reproductive meristem is uncoupled from rosette leaf senescence in Arabidopsis (Hensel et 562
al., 1993; Noodén and Penney, 2001; Wuest et al., 2016). The two possibilities are not 563
mutually exclusive and further research is needed to understand the mechanism underlying 564
the action of cytokinin during GPA. 565
In the above context, it is noteworthy that the size of the inflorescence meristems of 566
rock2 and rock3 mutants was not increased although these receptors are expressed and 567
active in the meristem (Riefler et al., 2006; Stolz et al., 2011; Gruel et al., 2016). This is 568
surprising considering that plants producing more or less cytokinin develop a larger or 569
smaller inflorescence meristem, respectively (Bartrina et al., 2011). One plausible 570
explanation for these apparently incongruous observations might be that in tissues where 571
AHK2 and AHK3 expression overlaps with that of AHK4, as it is the case in the inflorescence 572
meristem (Gruel et al., 2016), the enhanced signaling activity might be dampened by the 573
phosphatase activity of AHK4 (Mähönen et al., 2006). In the presence of low cytokinin (as in 574
the rock2 and rock3 plants), the phosphatase activity of the AHK4 receptor may prevail and 575
alleviate downstream signaling thus counteracting the enhanced kinase activity of rock2 and 576
rock3 receptors. Consistent with this idea the TCSn:GFP cytokinin reporter indicated similar 577
signaling output activity in wild-type and rock2 inflorescence meristems. It this respect, it 578
would be interesting to analyze the rock2 and rock3 mutations in the ahk4 knockout 579
background (Inoue et al., 2001). In contrast to rock2/rock3 plants, ckx3 ckx5 mutation 580
(Bartrina et al., 2011) presumably increases the signaling output of all three cytokinin 581
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18
receptors and lowers the phosphatase activity of AHK4; together this might lead to a 582
quantitatively and qualitatively different cytokinin output signal. 583
Last but not least, transgenic expression of the rock3-1 allele caused an about 50% 584
higher seed yield, which is similar to that brought about by ckx3 ckx5 mutation (Bartrina et 585
al., 2011). However, the mechanisms leading to increased seed yield appear to be at least 586
partly different. ckx3 ckx5 mutants have a larger inflorescence meristem forming an 587
increased number of flowers and siliques. In addition, siliques contain more seeds due to an 588
increased placenta activity producing more ovules (Bartrina et al., 2011). In the case of rock3 589
transgenic plants enhanced yield was mainly due to delayed GPA (see above) leading to 590
taller plants with more siliques (rock2 transgenic plants formed even more flowers but 591
suffered from reduced fertilization). In sum, our results corroborate the role of cytokinin as a 592
regulator of seed yield (Ashikari et al., 2005; Bartrina et al., 2011) and demonstrated that 593
different developmental mechanisms can be involved. We propose the rock2 and rock3 594
genes as a novel biotechnological tool to achieve yield enhancement. Because coupling of 595
the receptors to downstream signaling components is promiscuous, they could be used 596
directly for a gain-of-function approach in crop plants to increase cytokinin signaling in a 597
targeted and cell-autonomous fashion. 598
599
MATERIALS AND METHODS 600
Plant Material and Growth Conditions 601
The Columbia (Col-0) ecotype of Arabidopsis thaliana was used as the wild type. The 602
following lines were described previously: 35S:CKX1-11 (Werner et al., 2003), ahk2-5 603
(Riefler et al., 2006) ARR5:GUS (D'Agostino et al., 2000) and ore12-1 (Kim et al., 2006). All 604
plants were grown on soil or in vitro on half-strength Murashige and Skoog (MS) medium 605
under long-day (16 h of light/8 h of darkness) or short-day conditions (8 h of light/16 h of 606
darkness) at 22°C. For root growth assays, seedlings were grown on vertical plates, and the 607
length of the primary root was measured between day 4 and 12 after germination from digital 608
images using Scion Image software (http://www.scioncorp.com). For the cytokinin sensitivity 609
assay, benzyladenine (BA) dissolved in DMSO or DMSO as a solvent control were added to 610
the medium. 611
612
Mutagenesis and Gene Mapping 613
The rock2 and rock3 mutants were identified in a screen of an M2 population of 35S:CKX1 614
plants mutagenized with ethyl methanesulfonate (Niemann et al., 2015). Mapping 615
populations for rock2 and rock3 were generated by crossing the rock2 35S:CKX1 and rock3 616
35S:CKX1 plants with the Landsberg erecta ecotype. F2 progenies resistant to hygromycin 617
(co-segregating with 35S:CKX1) and showing the cytokinin deficiency syndrome were used 618
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19
to map the recessive (wild-type) alleles of rock2 and rock3. By analyzing 535 F2 619
recombinants, rock2 was mapped to a 1.45 Mbp region (~21.1 cM). To map the rock3 locus 620
927 F2 recombinants were analyzed and a 350 kb interval (~0.4 cM) was identified. The 621
rock2 and the rock3 mutation were identified by sequencing candidate genes in these 622
intervals and subsequent complementation of 35S:CKX1 transgenic plants by the mutant 623
alleles of the respective gene. 624
625
DNA Cloning 626
The rock2 and rock3 point mutations were introduced into the AHK2:AHK2 and AHK3:AHK3 627
genes (Stolz et al., 2011) using the QuickChange site-directed mutagenesis kit (Stratagene, 628
La Jolla, USA). Both constructs were introduced into Agrobacterium tumefaciens strain 629
GV3101, and 35S:CKX1 and wild-type plants were transformed using the floral-dip method 630
(Clough and Bent, 1998). Transgenic lines were selected on medium containing 50 mg/L 631
kanamycin. 632
633
Analysis of Transcript Levels by Quantitative Reverse Transcription-PCR 634
Total RNA was extracted from seedlings with the TRIzol method (Thermo Fisher Scientific). 635
Equal amounts of starting material (1 µg of RNA) were used in a 10 µL SuperScript III 636
Reverse Transcriptase reaction (Thermo Fisher Scientific). First strand complementary DNA 637
synthesis was primed with a combination of oligo(dT) primers and random hexamers. Real 638
time PCR using FAST SYBR Green I technology was performed on a CFX96 Touch Real-639
Time PCR Detection System (Bio Rad). The qPCR temperature program consisted of the 640
following steps: 95°C for 15 min; 40 cycles of 95°C 15 s, 55°C 15 s, 72°C 15 s; followed by 641
melting curve analysis. Relative transcript abundance of each gene was calculated based on 642
the ΔΔCt method (Livak and Schmittgen, 2001). PP2AA2 (At3g25800) was used for 643
normalization. Primers used for reference genes and genes of interest are listed in 644
Supplemental Table S3. 645
646
Yeast Complementation Assay 647
The yeast complementation assay was performed as described before (Inoue et al., 2001; 648
Mähönen et al., 2006). The rock2 and rock3 mutations were introduced into the AHK4 gene 649
(named AHK4rock2 and AHK4rock3) of plasmid p423TEF-CRE1 (Mähönen et al., 2006) using 650
the QuickChange site-directed mutagenesis kit. After sequence confirmation, plasmids were 651
introduced into yeast strain (TM182) ∆sln1 (Inoue et al., 2001) and the yeast 652
complementation assay was performed with either 2% galactose or 2% glucose with 0.1 or 653
10 µM tZ added to the medium. OD600 was measured after 20 h. 654
655
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20
GUS Staining, Microscopy and Scanning Electron Microscopy 656
GUS staining was performed as described in Köllmer et al. (2014). For microscopic analysis, 657
tissues were cleared according to Malamy and Benfey (1997). Hand-cut cross sections of 658
stems from 5-week-old plants were stained for 5 min in 0.02% aqueous toluidine blue O, 659
rinsed, and mounted in water. All samples were viewed with an Axioskop 2 plus microscope 660
(Zeiss, Jena, Germany). The inflorescence meristem of the main stem from 4-week-old soil 661
grown plants was dissected and analyzed by scanning electron microscopy as described 662
before (Bartrina et al., 2011). TCSn:GFP fluorescence was analyzed according to Zürcher et 663
al. (2013) using a Leica SP5 confocal microscope. Root meristem size was determined as 664
described by Dello Ioio et al. (2007). 665
666
Determination of the Cytokinin Content 667
Plants were grown in vitro for 14 days. For each sample, 100 mg of seedlings were pooled, 668
and five independent samples were analyzed for each genotype. The cytokinin content was 669
determined by ultra-performance liquid chromatography-electrospray tandem mass 670
spectrometry (Novak et al., 2008). 671
672
Analysis of Leaf Senescence and Photosynthetic Parameters 673
For the analysis of dark-induced leaf senescence, seedlings were grown in vitro for 18 days. 674
The sixth rosette leave was detached and floated on distilled water. After 7 days in the dark 675
at room temperature, chlorophyll was extracted as described before (Köllmer et al., 2011). 676
The maximum efficiency of PSII photochemistry (Fv/Fm ratio) of dark adapted plants was 677
measured with FluorCam (Photon Systems Instruments, Brno, Czech Republic). 678
679
Determination of Flowering Time, Stem Diameter, Plant Height and Yield Parameters 680
For flowering time analysis, seeds were stratified for three days at 4°C and sown on soil. 681
Onset of flowering was defined as the plant age when the first flower was visible. Termination 682
of flowering was defined as the time point when no new flowers were formed at the main 683
inflorescence. The number of rosette leaves was scored at the onset of flowering. The 684
diameter of the main inflorescence stem was determined when individual stems reached the 685
height of 15 cm. The hand-made transverse sections were taken 1 cm above the rosette. 686
The final plant height and the number of siliques were determined after termination of 687
flowering. For analysis of seed yield, the weight of all fully ripened and desiccated seeds was 688
determined. 689
690
Petal Surface Area and Cell Size Measurement 691
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21
The petal surface area was measured from digital images of fully expanded organs with the 692
Scion Image. Petals were cleared (Malamy and Benfey, 1997), and average cell sizes were 693
calculated from the number of cells per unit area of digital micrographs. 694
695
696
ACKNOWLEDGEMENTS 697
We thank Ildoo Hwang for seeds of the ore12 mutant and Bruno Müller for the TCSn:GFP 698
line. We acknowledge funding by Deutsche Forschungsgemeinschaft (CRC 429, SPP 1530) 699
and the Czech Grant Agency (grant no. 15-22322S). I.B. and H.J. were supported by Elsa-700
Neumann-Scholarships of the state of Berlin. 701
702
SUPPLEMENTAL DATA 703
Supplemental Figure 1. Expression of AHK2 and AHK3 in different genotypes. 704
Supplemental Figure 2. Comparison of leaf senescence in wild type (WT), rock2, rock3 705
and ore12. 706
Supplemental Figure 3. Size and cytokinin activity in inflorescence meristems. 707
Supplemental Figure 4. Flower size and morphology of rock2 and rock3 mutants 708
compared to wild type (WT). 709
Supplemental Table 1. Genetic analysis of the rock2 and rock3 mutation. 710
Supplemental Table 2. Cytokinin content of rock2 CKX1ox and rock3 CKX1ox. 711
Supplemental Table 3. Oligonucleosis used in this study. 712
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22
TABLES 713 714
Table 1: Cytokinin content of rock2 and rock3 715
716 Arabidopsis seedlings (14 DAG) were analyzed. iP, N6-(Δ2-isopentenyl)adenine; iPR, N6-(Δ2-717
isopentenyl)adenosine; iPRMP, N6-(Δ2-isopentenyl)adenosine 5’-monophospate; iP9G, N6-718
(Δ2-isopentenyl)adenine 9-glucoside; tZ, trans-zeatin; tZR, trans-zeatin riboside; tZRMP, 719
trans-zeatin riboside 5’-monophosphate; tZ9G, trans-zeatin 9-glucoside; tZOG, trans-zeatin 720
O-glucoside; tZROG, trans-zeatin riboside O-glucoside; cZ, cis-zeatin; cZR, cis-zeatin 721
riboside; cZRMP, cis-zeatin riboside 5’-monophosphate; cZOG, cis-zeatin O-glucoside; 722
cZROG, cis-zeatin riboside O-glucoside. Data shown are pmol/g fresh weight ± SD; n = 3. 723
724
725
genotype iP iPR iPRMP iP9G tZ tZR tZRMP Z9G
WT 1.91 ± 0.33 1.28 ± 0.18 2.18 ± 0.55 1.38 ± 0.09 0.71 ± 0.17 0.67 ± 0.19 1.29 ± 0.38 5.74 ± 0.90
rock2 1.13 ± 0.26 1.01 ± 0.18 2.23 ± 0.03 0.72 ± 0.06 0.32 ± 0.04 0.24 ± 0.05 0.40 ± 0.12 1.56 ± 0.38
AHK2:rock2 1.06 ± 0.29 1.25 ± 0.21 1.99 ± 0.31 0.93 ± 0.11 0.31 ± 0.01 0.23 ± 0.02 0.44 ± 0.09 1.88 ± 0.48
rock3 1.45 ± 0.41 1.08 ± 0.21 1.31 ± 0.07 0.84 ± 0.10 0.63 ± 0.11 0.20 ± 0.02 0.23 ± 0.06 1.30 ± 0.10
genotype tZOG tZROG cZ cZR cZRMP cZOG cZROG
WT 5.66 ± 1.00 0.46 ± 0.10 0.23 ± 0.04 0.89 ± 0.20 4.40 ± 0.76 12.26 ± 1.82 3.95 ± 1.27
rock2 1.83 ± 0.36 0.20 ± 0.03 0.14 ± 0.02 0.75 ± 0.23 3.15 ± 0.12 3.65 ± 0.18 4.46 ± 0.13
AHK2:rock2 1.67 ± 0.35 0.26 ± 0.03 0.13 ± 0.01 0.93 ± 0.18 3.84 ± 0.73 6.11 ± 1.78 4.33 ± 0.81
rock3 0.98 ± 0.04 0.21 ± 0.01 0.20 ± 0.05 1.02 ± 0.20 3.63 ± 1.03 3.92 ± 0.82 3.13 ± 0.45
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23
FIGURE LEGENDS 726
Figure 1. The rock2 and rock3 mutations suppress the CKX1ox phenotype. A, Morphology of 727
wild-type (WT), CKX1ox, rock2 CKX1ox and rock3 CKX1ox plants at the rosette stage. 728
Plants were grown for 25 days under long-day conditions. B, rock2 CKX1ox and rock3 729
CKX1ox seedlings have larger cotyledons than WT and CKX1ox seedlings. The photo was 730
taken 10 days after germination. C, Adult phenotype of 46-d-old WT, CKX1ox, rock2 CKX1ox 731
and rock3 CKX1ox plants. D, Flowers of WT, CKX1ox, rock2 CKX1ox and rock3 CKX1ox 732
plants (from left to right). E and F, Flowering time of WT and mutant plants grown under long-733
day (E) or short-day (F) conditions. Crosses indicate that no transition to flowering occurred 734
(n = 20). G, Relative leaf chlorophyll content of the 4th and 5th leaf of 3-week-old soil-grown 735
plants after 7 days in the dark. Chlorophyll content before the start of dark incubation was set 736
100% (n = 3). H, Root elongation of seedlings between day 3 and 9 after germination (n ≥ 737
25). Error bars represent SD. Statistical significance of differences was calculated by two-738
way ANOVA. *, P < 0.01 compared to WT; °, P < 0.05 compared to CKX1ox plants. DAG, 739
days after germination. 740
741
Figure 2. rock2 and rock3 mutations alter cytokinin sensitivity. A, qRT-PCR analysis of CKX1 742
transcript levels in seedlings grown in vitro for 10 days. Expression values were normalized 743
to PP2AA2 and expression in WT was set to 1. Values are averages of three biological 744
replicates ± SE. B, Total cytokinin (CK) content of two-week-old seedlings. Contents of 745
individual cytokinin metabolites are shown in Supplemental Table S1. Error bars represent 746
SD. *, P < 0.05 compared to wild type as calculated by two-way ANOVA. C, Phenotype of 747
14-d-old seedlings grown on medium without (-CK) or with 25 nM BA (+CK). rock2 CKX1ox 748
and rock3 CKX1ox develop pale yellow leaves and show reduced shoot growth. 749
750
Figure 3. rock2 and rock3 are novel gain-of-function alleles of the AHK2 and AHK3 gene. A, 751
Two segments of the sequence alignment between the cytokinin receptor proteins AHK2, 752
AHK3 and AHK4. The amino acid residues which are mutated (shown in orange) in rock2 753
and rock3 are conserved in all three receptors. Full-length AHK protein sequences were 754
aligned using ClustalW. B, Schematic representation of the AHK2 and AHK3 protein domains 755
and the position of amino acid substitutions (red arrows) corresponding to the rock2 and 756
rock3 mutations. C-J, Genetic complementation of the CKX1ox phenotype by AHK2:rock2 757
and AHK3:rock3. Two independent transgenic AHK2:rock2 (G and H) and AHK3:rock3 (I and 758
J) lines in CKX1ox background in comparison to wild type (C), CKX1ox (D), rock2 CKX1ox 759
(E) and rock3 CKX1ox (F) at 18 DAG. 760
761
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24
Figure 4. The rock2 and rock3 genes code for constitutively active receptors. A, Suppression 762
of the lethal sln1∆ mutation by histidine kinase activity of AHK4rock2 and AHK4rock3. Growth of 763
sln1∆ yeast strain expressing AHK4rock2 or AHK4rock3 was independent of the presence of 764
cytokinin. Error bars represent SD (n = 3). B, Relative expression levels of ARR5 in the rock 765
mutants compared with wild type. Quantitative RT-PCR analysis was performed using 5-d-766
old seedlings grown in vitro. Values are averages of three biological replicates ± SE. C-K, 767
Histochemical analysis of ARR5:GUS activity in wild type (C-E), rock2 (F-H) and rock3 (I-K) 768
background. Pictures show whole seedlings and shoot apices of 5-d-old plants stained 769
overnight and primary root tips of 7-d-old plants after 30 min of staining (from left to right). 770
Statistical significance of differences compared to wild type was calculated by ANOVA. *, P < 771
0.01. 772
773
Figure 5. Enhanced shoot growth of rock2 and rock3 mutants and transgenic plants. A and 774
B, Height of main inflorescence stem after termination of flowering (50 days after 775
germination). C and D, Primary inflorescence stems 3 cm above the rosette (C) and their 776
diameter (D). E, Stem sections of wild-type (WT) and AHK3:rock3 plants at the base of 777
primary inflorescence stems. Sections were stained with toluidine blue. Scale bar = 2 mm 778
(C). Error bars represent SD. Statistical significance of differences compared to wild type 779
was calculated by ANOVA. *, P < 0.001. 780
781
Figure 6. Leaf phenotype of rock2 and rock3 mutants and transgenic plants. A, Cotyledon 782
size of 5-d-old seedlings. Scale bar = 1 mm. B, Cotyledons and rosette leaves in the order of 783
appearance (from left to right) at 24 DAG. Scale bar = 1 cm. C, Fresh weight of rosette 784
leaves 32 DAG. D, Average size of abaxial epidermal cells of the sixth rosette leaf of wild-785
type (WT) and rock2 plants (n = 14). E, Leaf area of the sixth fully grown rosette leaf. 786
Statistical significance of differences compared to wild type was calculated by ANOVA. *, P < 787
0.001. 788
789
Figure 7. Natural and dark-induced leaf senescence. A, Age-dependent senescence 790
phenotype of the sixth leaf of WT and rock2/rock3 mutants grown under long-day conditions 791
starting at 16 days after leaf emergence (DAE). B, The photochemical efficiency of PSII 792
(Fv/Fm) of the sixth leaf at time points shown in A. C, Dark-induced senescence in a 793
detached leaf assay. The chlorophyll content of the sixth leaf was examined after 7 days in 794
the dark. The leaf chlorophyll content before the start of dark incubation was set at 100% for 795
each genotype tested. (n = 10). Error bars represent SD. Statistical significance of 796
differences compared to WT was calculated by ANOVA. *, P < 0.001. 797
798
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25
Figure 8. rock2 and rock3 positively regulate flowering time and flower size. A, Number of 799
rosette leaves at the start of flowering of plants grown und long-day conditions. B, rock2 and 800
rock3 mutants and transgenic plants flower longer than wild type (WT). Shown are days until 801
termination of flowering. (n = 10). C, Flowers of rock2 and rock3 mutants and transgenic lines 802
compared to WT. D, Average size of abaxial epidermal cells of petals at stage 13 (Smyth et 803
al., 1990) (n = 5). E, Number of siliques on the main stem. Siliques, including unfilled and 804
partially filled siliques, were counted after the end of flowering (n = 15). F, Seed yield of rock2 805
and rock3 mutants and transgenic lines compared to WT. Seed yield of WT was set to 100%. 806
Error bars represent SD. Statistical significance of differences compared to WT was 807
calculated by Student’s t test. *, P < 0.005. 808
809
Figure 9. The rock2 and rock3 mutants have a reduced root system. A, Elongation of 810
primary roots between day 4 and 12 after germination (n = 20). B, Root meristems of wild 811
type, rock2 and rock3. Roots were analyzed 5 days after germination. White and black 812
arrowheads indicate, respectively, the QC and the start of the transition zone. Scale bar 50 813
µm. C, Number of cortex cells between the QC and the start of the transition zone (n = 10). 814
D, Number of lateral roots at 12 DAG (n = 20). Error bars represent SD. Statistical 815
significance of differences compared to WT was calculated by Student’s t test *, P < 0.005. 816
817
Figure 10. Model of conformational changes associated with transmembrane signaling 818
following cytokinin perception. Schematic topology of the sensory module of cytokinin 819
receptors based on the crystal structure of AHK4 (Hothorn et al., 2011). The N-terminal 820
helices (α1, α2 and its neighboring 310-helix) of the CHASE domain are shown in orange, the 821
two PAS domains are depicted schematically. The model predicts that cytokinin binding 822
causes reversible conformational changes (double-headed arrows) causing a piston-type 823
displacement of the different subdomains and ultimately resulting in transmission of the 824
signal across the membrane. rock2 and rock3 mutations (arrows) are predicted to mimic 825
those changes locking the receptor in a constitutively active conformation. 826
827
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26
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Werner T, Schmülling T (2009) Cytokinin action in plant development. Curr Opin Plant Biol 1039
12: 527-538 1040
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Physiol 156: 1808-1818 1045
Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T, Mizuno T 1046
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cytokinin signals across the membrane. Plant Cell Physiol 42: 1017-1023 1048
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Figure 1. The rock2 and rock3mutations suppress the CKX1ox phenotype. A, Morphology of wild‐type(WT), CKX1ox, rock2 CKX1ox and rock3 CKX1ox plants at the rosette stage. Plants were grown for 25days under long‐day conditions. B, rock2 CKX1ox and rock3 CKX1ox seedlings have larger cotyledonsthan WT and CKX1ox seedlings. The photo was taken 10 days after germination. C, Adult phenotypeof 46‐d‐old WT, CKX1ox, rock2 CKX1ox and rock3 CKX1ox plants. D, Flowers of WT, CKX1ox, rock2CKX1ox and rock3 CKX1ox plants (from left to right). E and F, Flowering time of WT and mutant plantsgrown under long‐day (E) or short‐day (F) conditions. Crosses indicate that no transition to floweringoccurred (n = 20). G, Relative leaf chlorophyll content of the 4th and 5th leaf of 3‐week‐old soil‐grownplants after 7 days in the dark. Chlorophyll content before the start of dark incubation was set 100%(n = 3). H, Root elongation of seedlings between day 3 and 9 after germination (n ≥ 25). Error barsrepresent SD. Statistical significance of differences was calculated by two‐way ANOVA. *, P < 0.01compared to WT; °, P < 0.05 compared to CKX1ox plants. DAG, days after germination.
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BA
C CKX1ox rock2 CKX1ox rock3 CKX1oxWT
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CK content (pmol/g FW
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Figure 2. rock2 and rock3 mutations alter cytokinin sensitivity. A, qRT‐PCR analysis of CKX1 transcriptlevels in seedlings grown in vitro for 10 days. Expression values were normalized to PP2AA2 andexpression in WT was set to 1. Values are averages of three biological replicates ± SE. B, Totalcytokinin (CK) content of two‐week‐old seedlings. Contents of individual cytokinin metabolites areshown in Supplemental Table S1. Error bars represent SD. *, P < 0.05 compared to wild type ascalculated by two‐way ANOVA. C, Phenotype of 14‐d‐old seedlings grown on medium without (‐CK)or with 25 nM BA (+CK). rock2 CKX1ox and rock3 CKX1ox develop pale yellow leaves and showreduced shoot growth.
1
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100Relative
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Figure 3. rock2 and rock3 are novel gain‐of‐function alleles of the AHK2 and AHK3 gene. A, Twosegments of the sequence alignment between the cytokinin receptor proteins AHK2, AHK3 and AHK4.The amino acid residues which are mutated (shown in orange) in rock2 and rock3 are conserved in allthree receptors. Full‐length AHK protein sequences were aligned using ClustalW. B, Schematicrepresentation of the AHK2 and AHK3 protein domains and the position of amino acid substitutions(red arrows) corresponding to the rock2 and rock3 mutations. C‐J, Genetic complementation of theCKX1ox phenotype by AHK2:rock2 and AHK3:rock3. Two independent transgenic AHK2:rock2 (G andH) and AHK3:rock3 (I and J) lines in CKX1ox background in comparison to wild type (C), CKX1ox (D),rock2 CKX1ox (E) and rock3 CKX1ox (F) at 18 DAG.
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301 KIPSAIDQRTFEEYTERTNFERPLTSGVAYAL162 KIPSAIDQRTFSEYTDRTSFERPLTSGVAYAM174 KNPSAIDQETFAEYTARTAFERPLLSGVAYAE
rock3-1 (T179I)
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rock3-2 (E182K)
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Figure 4. The rock2 and rock3 genes code for constitutively active receptors. A, Suppression of thelethal sln1∆ mutation by histidine kinase activity of AHK4rock2 and AHK4rock3. Growth of sln1∆ yeaststrain expressing AHK4rock2 or AHK4rock3 was independent of the presence of cytokinin. Error barsrepresent SD (n = 3). B, Relative expression levels of ARR5 in the rock mutants compared with wildtype. Quantitative RT‐PCR analysis was performed using 5‐d‐old seedlings grown in vitro. Values areaverages of three biological replicates ± SE. C‐K, Histochemical analysis of ARR5:GUS activity in wildtype (C‐E), rock2 (F‐H) and rock3 (I‐K) background. Pictures show whole seedlings and shoot apices of5‐d‐old plants stained overnight and primary root tips of 7‐d‐old plants after 30 min of staining (fromleft to right). Statistical significance of differences compared to wild type was calculated by ANOVA. *,P < 0.01.
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Figure 5. Enhanced shoot growth of rock2 and rock3 mutants and transgenic plants. A and B, Heightof main inflorescence stem after termination of flowering (50 days after germination). C and D,Primary inflorescence stems 3 cm above the rosette (C) and their diameter (D). E, Stem sections ofwild‐type (WT) and AHK3:rock3 plants at the base of primary inflorescence stems. Sections werestained with toluidine blue. Scale bar = 2 mm (C). Error bars represent SD. Statistical significance ofdifferences compared to wild type was calculated by ANOVA. *, P < 0.001.
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Figure 6. Leaf phenotype of rock2 and rock3 mutants and transgenic plants. A, Cotyledon size of 5‐d‐old seedlings. Scale bar = 1 mm. B, Cotyledons and rosette leaves in the order of appearance (fromleft to right) at 24 DAG. Scale bar = 1 cm. C, Fresh weight of rosette leaves 32 DAG. D, Average size ofabaxial epidermal cells of the sixth rosette leaf of wild‐type (WT) and rock2 plants (n = 14). E, Leafarea of the sixth fully grown rosette leaf. Statistical significance of differences compared to wild typewas calculated by ANOVA. *, P < 0.001.
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Figure 7. Natural and dark‐induced leaf senescence. A, Age‐dependent senescence phenotype of the sixth leaf of WT and rock2/rock3 mutants grown under long‐day conditions starting at 16 days after leaf emergence (DAE). B, The photochemical efficiency of PSII (Fv/Fm) of the sixth leaf at time points shown in A. C, Dark‐induced senescence in a detached leaf assay. The chlorophyll content of the sixth leaf was examined after 7 days in the dark. The leaf chlorophyll content before the start of dark incubation was set at 100% for each genotype tested. (n = 10). Error bars represent SD. Statistical significance of differences compared to WT was calculated by ANOVA. *, P < 0.001.
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Figure 7:
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Figure 8. rock2 and rock3 positively regulate flowering time and flower size. A, Number of rosetteleaves at the start of flowering of plants grown und long‐day conditions. B, rock2 and rock3 mutantsand transgenic plants flower longer than wild type (WT). Shown are days until termination offlowering. (n = 10). C, Flowers of rock2 and rock3 mutants and transgenic lines compared to WT. D,Average size of abaxial epidermal cells of petals at stage 13 (Smyth et al., 1990) (n = 5). E, Number ofsiliques on the main stem. Siliques, including unfilled and partially filled siliques, were counted afterthe end of flowering (n = 15). F, Seed yield of rock2 and rock3 mutants and transgenic lines comparedto WT. Seed yield of WT was set to 100%. Error bars represent SD. Statistical significance ofdifferences compared to WT was calculated by Student’s t test. *, P < 0.005.
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0
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Meristem size(cellnumber)
Figure 9. The rock2 and rock3 mutants have a reduced root system. A, Elongation of primary roots between day 4 and 12 after germination (n = 20). B, Root meristems of wild type, rock2 and rock3. Roots were analyzed 5 days after germination. White and black arrowheads indicate, respectively, the QC and the start of the transition zone. Scale bar 50 µm. C, Number of cortex cells between the QC and the start of the transition zone (n = 10). D, Number of lateral roots at 12 DAG (n = 20). Error bars represent SD. Statistical significance of differences compared to WT was calculated by Student’s t test *, P < 0.005.
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CK
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E182K
N C
PASproximal
PASdistal
Figure 10. Model of conformational changes associated with transmembrane signaling followingcytokinin perception. Schematic topology of the sensory module of cytokinin receptors based on thecrystal structure of AHK4 (Hothorn et al., 2011). The N‐terminal helices (α1, α2 and its neighboring310‐helix) of the CHASE domain are shown in orange, the two PAS domains are depictedschematically. The model predicts that cytokinin binding causes reversible conformational changes(double‐headed arrows) causing a piston‐type displacement of the different subdomains andultimately resulting in transmission of the signal across the membrane. rock2 and rock3 mutations(arrows) are predicted to mimic those changes locking the receptor in a constitutively activeconformation.
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