strigolactone and karrikin signaling pathways elicit ... · 134 . signaling, sls interact with the...
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RESEARCH ARTICLE 1
2
Strigolactone and Karrikin Signaling Pathways Elicit Ubiquitination and 3
Proteolysis of SMXL2 to Regulate Hypocotyl Elongation in Arabidopsis thaliana 4
5
Lei Wanga,b,1
, Qian Xub,c,1
, Hong Yub, Haiyan Ma
b, Xiaoqiang Li
d, Jun Yang
d, 6
Jinfang Chub,c
, Qi Xieb,c
, Yonghong Wangb,c,e
, Steven M. Smithb,f
, Jiayang Lib,c
, 7
Guosheng Xionga,g,2
, and Bing Wangb,2
8
a Agricultural Genome Institute, Chinese Academy of Agricultural Sciences, Shenzhen 9
518120, China 10 b State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research 11
(Beijing), Institute of Genetics and Developmental Biology, The Innovative Academy of 12
Seed Design, Chinese Academy of Sciences, Beijing 100101, China 13 c University of Chinese Academy of Sciences, Beijing 100039, China 14
d CAS Key Laboratory of Energy Regulation, Shanghai Institute of Organic Chemistry, 15
Chinese Academy of Sciences, Shanghai 200032, China 16 e College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, 17
China 18 f School of Natural Sciences, University of Tasmania, Hobart 7001, Australia 19
g Plant Phenomics Research Center, Nanjing Agricultural University, Nanjing 210095, 20
China 21 1These authors contributed equally to this work. 22
2Address correspondence to [email protected] or [email protected] 23
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Short title: Both SLs and KARs trigger SMXL2 degradation 25
26
One-sentence summary: Strigolactone and karrikin signaling pathways trigger 27
polyubiquitination and degradation of SMXL2 to regulate hypocotyl elongation and gene 28
expression in Arabidopsis thaliana. 29
30
The author responsible for distribution of materials integral to the findings presented in 31
this article in accordance with the policy described in the Instructions for Authors 32
(www.plantcell.org) is: Bing Wang ([email protected]). 33
34
ABSTRACT 35
Strigolactones (SLs) and karrikins (KARs) are related butenolide signaling molecules that 36
control plant development. In Arabidopsis thaliana they are recognized separately by two 37
closely related receptors but employ the same F-box protein MORE AXILLARY 38
GROWTH2 (MAX2) for signal transduction, targeting different members of the 39
SMAX1-LIKE (SMXL) family of transcriptional repressors for degradation. Both signals 40
Plant Cell Advance Publication. Published on April 30, 2020, doi:10.1105/tpc.20.00140
©2020 American Society of Plant Biologists. All Rights Reserved
2
inhibit hypocotyl elongation in seedlings, raising the question of whether signaling is 41
convergent or parallel. Here, we show that synthetic SL analog GR244DO
enhanced the 42
interaction between the SL receptor DWARF14 (D14) and SMXL2, while the KAR 43
surrogate GR24ent-5DS
induced association of the KAR receptor KARRIKIN 44
INSENSITIVE2 (KAI2) with SMAX1 and SMXL2. Both signals trigger 45
polyubiquitination and degradation of SMXL2, with GR244DO
dependent on D14 and 46
GR24ent-5DS
dependent mainly on KAI2. SMXL2 is critical for hypocotyl responses to 47
GR244DO
, and functions redundantly with SMAX1 in hypocotyl response to GR24ent-5DS
. 48
Furthermore, GR244DO
induced response of D14-LIKE2 (DLK2) and KAR-UP F-BOX1 49
(KUF1) through SMXL2, whereas GR24ent-5DS
induced expression of these genes via both 50
SMAX1 and SMXL2. These findings demonstrate that both SLs and KARs could trigger 51
polyubiquitination and degradation of SMXL2, thus uncovering an unexpected but 52
important convergent pathway in SL- and KAR-regulated gene expression and hypocotyl 53
elongation. 54
55
INTRODUCTION 56
Strigolactones (SLs) are a large diverse group of signaling compounds derived from the 57
carotenoid biosynthetic pathway and possess a characteristic butenolide ring that is 58
essential for activity. They function in many aspects of plant development, such as shoot 59
branching, internode elongation, leaf elongation and senescence, growth of primary and 60
lateral roots, anthocyanin accumulation, shoot gravitropism, and stem secondary 61
thickening (Foo et al., 2001; Stirnberg et al., 2002; Sorefan et al., 2003; Snowden et al., 62
2005; Arite et al., 2007; Simons et al., 2007; Gomez-Roldan et al., 2008; Umehara et al., 63
2008; Drummond et al., 2009; Lin et al., 2009; Agusti et al., 2011; Sang et al., 2014). 64
Mutations that block SL biosynthesis and signaling are characterized by the outgrowth of 65
axillary buds to form lateral shoots in dicotyledonous plants or tillers in grasses 66
(Al-Babili and Bouwmeester, 2015; Wang et al., 2017a; Waters et al., 2017). 67
SLs also play important roles in adaptive growth under diverse environmental 68
conditions (Saeed et al., 2017; Mostofa et al., 2018). Phosphate or water deficiency 69
significantly induces SL biosynthesis, and the accumulated SLs further mediate 70
developmental changes to reduce consumption and enhance uptake of phosphate and 71
water (Kohlen et al., 2011; Haider et al., 2018). Besides the function as an endogenous 72
3
plant hormone, SLs secreted into the rhizosphere promote symbiotic relationships with 73
arbuscular mycorrhizal fungi (AMF) to facilitate the absorption of water and nutrients 74
(Akiyama et al., 2005). In addition, such SL rhizosphere signals are exploited by parasitic 75
weeds to stimulate seed germination leading to colonization in the roots of host plants, 76
causing devastating losses to crop production (Conn et al., 2015; Toh et al., 2015; 77
Tsuchiya et al., 2015). Due to the important roles of SLs in key agronomic traits related 78
to crop yield and mechanized farming, it is important to understand the molecular 79
mechanism of SL action in plants. 80
Karrikins (KARs) are present in burned plant materials and stimulate germination of 81
dormant seeds following wildfires, facilitating seedlings establishment under favorable 82
conditions (Nelson et al., 2012). They are derived from the pyrolysis of carbohydrates, 83
and like SLs, contain a butenolide ring that is essential for activity. KARs can also 84
promote seed germination of many plant species that are not considered fire followers, 85
including weedy ephemerals such as Arabidopsis thaliana, and commercial species such 86
as Solanum lycopersicum (tomato), Zea mays (maize), Oryza sativa (rice), and Lactuca 87
sativa (lettuce) (Nelson et al., 2012; Morffy et al., 2016). Besides germination, KARs 88
influence seedling photomorphogenesis which is thought to provide an advantage to 89
seedlings in the post-fire environment (Nelson et al., 2009; Nelson et al., 2010). Recently, 90
it has been shown that exogenously applied KARs promote Arabidopsis seed germination 91
under permissive conditions but inhibit it under conditions of mild abiotic stresses, 92
suggesting a further means by which seedling establishment is safeguarded (Wang et al., 93
2018a). 94
Studies of KAR-insensitive mutants (see below) reveal pleiotropic developmental 95
abnormalities that include impaired seedling photomorphogenesis (Nelson et al., 2012; 96
Waters et al., 2012), modified leaf shape (Waters et al., 2012), impaired leaf cuticle 97
development (Li et al., 2017), abnormal root skewing and density of root hairs in 98
Arabidopsis (Swarbreck et al., 2019; Villaécija-Aguilar et al. 2019) and inability to 99
4
establish symbiosis with AMF in rice (Gutjahr et al., 2015). The defective leaf cuticle 100
results in reduced drought tolerance, indicating a possible role of KAR signaling in 101
response to water deficit (Li et al., 2017). 102
The first SLs identified are known as canonical SLs and have a tricyclic lactone, 103
comprising A, B and C rings, linked through an enol-ether bridge to a butenolide (D ring). 104
More recently non-canonical SLs have been identified that lack B and C rings 105
(Yoneyama et al., 2018). It is generally not known which SLs are responsible for each 106
biological activity, but canonical SLs are routinely used in research, either as natural 107
forms such as 5-deoxystrigol (5DS) and 4-deoxyorobanchol (4DO) or isomers of the 108
synthetic analog GR24 (Flematti et al., 2016; Yoneyama et al., 2018). 109
KARs apparently mimic an endogenous plant signaling compound, the identity of 110
which is still unknown, but structure-function assays show that a butenolide moiety is 111
essential for KAR activity (Nelson et al., 2012; Morffy et al., 2016). Early reports that 112
GR24 induced some KAR-like responses in seeds and seedlings suggested that KARs 113
could simply be SL analogues, but KARs do not stimulate germination of parasitic weeds 114
nor affect shoot branching (Nelson et al., 2009; Nelson et al., 2012). Subsequent analysis 115
recognized that commonly used preparations of GR24 comprise a racemic mixture of two 116
enantiomers with the stereochemical configurations of 5DS and its enantiomer, ent-5DS, 117
which are conveniently referred to as GR245DS
and GR24ent-5DS
. Surprisingly, while 118
GR245DS
was found to behave as a SL, GR24ent-5DS
was found to behave as a KAR 119
(Scaffidi et al., 2014). A second pair of enantiomers of GR24 is also produced during 120
chemical synthesis, and these have the stereochemical configuration of 4DO and ent-4DO. 121
These two stereoisomers are referred to as GR244DO
and GR24ent-4DO
and were similarly 122
found to act as SLs and KARs, respectively (Scaffidi et al., 2014). Furthermore, while 123
5DS and 4DO are natural SLs, their synthetic enantiomers ent-5DS and ent-4DO behave 124
as KARs. The defining feature is that analogs active as SLs have a 2'R-configured 125
butenolide D-ring while all analogs with KAR activity have a 2'S configuration (Scaffidi 126
5
et al., 2014; Flematti et al., 2016). 127
Genetic studies in Arabidopsis, rice, petunia, and pea led to the discoveries of SL 128
and KAR receptors (Waters et al., 2017). The SL and KAR receptor proteins are 129
paralogous α/β-fold hydrolases known respectively as DWARF14 (D14) in rice and 130
Arabidopsis and KARRIKIN INSENSITIVE2 (KAI2) or HYPOSENSITIVE TO LIGHT 131
(HTL) in Arabidopsis. Signaling by SLs and KARs both require the F-box protein MORE 132
AXILLARY GROWTH2 (MAX2) in Arabidopsis or DWARF3 (D3) in rice. In SL 133
signaling, SLs interact with the active-site pocket of D14 which can lead to nucleophilic 134
attack by the active-site serine residue, resulting in elimination of the ABC moiety and 135
covalent attachment of a 4-carbon derivative of the D-ring to the active site serine and 136
histidine residues of D14 (Hamiaux et al., 2012; Zhao et al., 2013a; Yao et al., 2016). 137
This modification has been proposed to induce association of D14 with D3/MAX2 and 138
with repressor proteins from the rice DWARF53 (D53) or Arabidopsis SUPPRESSOR 139
OF MAX2-LIKE (SMXL) family which are subsequently ubiquitinated and degraded (de 140
Saint Germain et al., 2016; Yao et al., 2016; Yao et al., 2018b). In an alternative model, 141
SL binding to D14 triggers its association with MAX2 and SMXL proteins, resulting in 142
ubiquitination and proteolysis of the SMXL proteins, after which D14 destroys the SL by 143
hydrolysis (Seto et al., 2019). The plasticity in conformational states of D3 also plays an 144
important role in the hydrolytic activity of D14, formation of the D14-D3-D53 complex 145
and thus SL signaling (Shabek et al., 2018). 146
In Arabidopsis SMXL6, SMXL7 and SMXL8 are orthologs of D53 in rice, which 147
are repressors of axillary bud outgrowth and tiller growth, respectively. The 148
ubiquitination and degradation of SMXL6, SMXL7, SMXL8 or D53 through the 26S 149
proteasome relieves transcriptional repression of target genes controlling axillary bud 150
growth (Jiang et al., 2013; Zhou et al., 2013; Soundappan et al., 2015; Wang et al., 2015). 151
In KAR signaling, KAI2 (HTL) is expected to perceive exogenous KARs or the unknown 152
endogenous KAI2 Ligand (‘KL’) by means of a similar mechanism to SLs (Waters et al., 153
6
2012; Guo et al., 2013; Xu et al., 2016). However, whether KARs promote formation of 154
the KAI2-MAX2-SMXL complex and further trigger ubiquitination and degradation of 155
SMXL proteins is still an open question. This has greatly limited the understanding of 156
KAR signaling in plants. 157
In Arabidopsis, there are eight genes in the SMXL family, which display distinct 158
expression patterns and functions. In particular, SMXL6, SMXL7, and SMXL8 function 159
as key transcriptional repressors that regulate shoot branching and leaf development 160
(Soundappan et al., 2015; Wang et al., 2015); SUPPRESSOR OF MAX2 1 (SMAX1) 161
regulates seed germination and hypocotyl elongation through KAR signaling (Stanga et 162
al., 2013; Soundappan et al., 2015); SMXL3, SMXL4, and SMXL5 act as 163
cell-autonomous regulators of phloem formation independent of SL or KAR signaling 164
(Wallner et al., 2017). SMXL2, a close paralog of SMAX1 in Arabidopsis, mediates KAR 165
signaling and controls hypocotyl growth redundantly with SMAX1. However, SMXL2 166
does not apparently influence seed germination or leaf development (Stanga et al., 2016). 167
Furthermore, smxl2 mutant displays elongated root hairs, which is not observed in smax1 168
mutants (Villaécija-Aguilar et al. 2019). These results indicate a clear functional 169
difference between SMXL2 and SMAX1. Since exogenous SLs can also influence 170
seedling photomorphogenesis, it raises the question of whether SMXL2 might also be 171
involved in SL signaling. 172
Hypocotyl elongation of young seedlings is a key skotomorphogenic response that 173
enables seedlings to push through the soil to receive light. Upon exposure to light, 174
photomorphogenic responses are enacted including repression of hypocotyl elongation. 175
Hypocotyl elongation is controlled by numerous endogenous and environmental signals 176
(Shi et al., 2016; Wang et al., 2018b). The ratio of red to far red light, blue light, UV-B, 177
temperature, photoperiod, circadian clock, and gibberellins all regulate hypocotyl 178
elongation mainly through signaling pathways integrated by the PHYTOCHROME 179
INTERACTING FACTOR (PIF) family transcription factors (Li et al., 2016a; Zhu and 180
7
Lin, 2016). Interestingly, KARs and all stereoisomers of GR24 inhibit hypocotyl 181
elongation of Arabidopsis seedlings under continuous red light, and these responses are 182
abolished in the max2 mutant (Nelson et al., 2011; Scaffidi et al., 2014). There is 183
crosstalk between KAR signaling and ELONGATED HYPOCOTYL5 (HY5)-dependent 184
photomorphogenesis (Waters and Smith, 2013). Significantly, in low intensity white light 185
the natural SLs 5DS and 4DO inhibited hypocotyl elongation in a D14-dependent manner, 186
while the non-natural enantiomer ent-5DS inhibited hypocotyl elongation through KAI2 187
(Scaffidi et al., 2014), indicating that SLs and KARs regulate hypocotyl elongation 188
separately through both signaling pathways. Genetic analysis further showed that SMAX1 189
and SMXL2 function redundantly in KAR-induced photomorphogenesis, while SMXL6, 190
SMXL7, and SMXL8 do not regulate hypocotyl elongation (Soundappan et al., 2015; 191
Stanga et al., 2016). Thus, the molecular mechanism underlying SL-regulated 192
photomorphogenesis needs further investigation. 193
It is generally considered that SL and KAR signaling are mediated by distinct 194
members of the SMXL family (Stanga et al., 2016). In this study we show that SLs and 195
KARs specifically promote formation of the D14-MAX2-SMXL2 and 196
KAI2-MAX2-SMXL2 complexes, and subsequently trigger polyubiquitination and 197
degradation of SMXL2 in a D14- or KAI2-dependent manner, respectively. These 198
findings demonstrate a common mechanism for D14- and KAI2-mediated signaling in 199
Arabidopsis seedlings and are discussed in terms of the functions and possible 200
evolutionary relationships of these signaling pathways. 201
202
RESULTS 203
Expression of SMXL2 Is Induced by both SL and KAR Signals 204
The transcript levels of SMXL6, SMXL7, and SMXL8 in Arabidopsis and their homolog 205
D53 in rice are upregulated by a racemic mixture of GR24 (rac-GR24) consisting of 206
GR245DS
and GR24ent-5DS
, which behave preferentially as SLs and KARs respectively 207
8
(Jiang et al., 2013; Stanga et al., 2013; Zhou et al., 2013; Scaffidi et al., 2014; Lantzouni 208
et al., 2017). Therefore, expression analyses based on rac-GR24 are unable to 209
discriminate the transcript responses to SLs and KARs, except by analyses in the d14 and 210
kai2 mutants and by comparison with responses to KAR (Scaffidi et al., 2013; Waters et 211
al., 2017). To investigate the specific gene expression responding to SLs, we used 212
GR244DO
as an active SL analog because our preliminary studies showed it to be more213
active than GR245DS
. For KAR signaling we used either KAR1 or GR24ent-5DS
which has214
been shown to interact with KAI2 and to regulate gene expression, seed germination and 215
seedling development preferentially via KAI2 (Nelson, et al., 2009; Nelson et al., 2010; 216
Nelson et al., 2011; Guo et al., 2013; Scaffidi et al., 2014; Waters et al., 2015; Flematti et 217
al., 2016). 218
Phylogenetic analyses indicate that SMXL family members are probably derived 219
from duplication and functional divergence of ancient SMAX1 genes during evolution 220
(Moture et al., 2018). To investigate functions of SMXL family proteins in SL and KAR 221
signaling, we first analyzed the mRNA level of SMXL genes in response to GR244DO
222
treatment in seedlings of wild type, d14-1 and kai2-2 (Supplemental Figure 1A and 223
Figure 1). After GR244DO
treatment for 4h, transcript levels of SMXL6, SMXL7, and224
SMXL8 were upregulated in wild type. Interestingly, SMXL2 expression was also 225
upregulated upon GR244DO
treatment, whereas expression levels of SMAX1, SMXL3,226
SMXL4, and SMXL5 remained unaffected. These inductions were blocked in d14-1 but 227
were largely unaffected in kai2-2, indicating that GR244DO
could induce the expression of 228
SMXL2, SMXL6, SMXL7, and SMXL8 through D14-mediated SL signaling (Figure 1). 229
KAR1 and KAR2 have been employed to detect transcriptional changes in 230
Arabidopsis, and only a few genes including KAR-UP F-BOX1 (KUF1) and D14-LIKE2 231
(DLK2) were up-regulated, but no SMXL family member has been reported to respond, 232
even after treatment for 72 h (Nelson et al., 2009; Nelson et al., 2010; Nelson et al., 2011; 233
Waters et al., 2012). However, it was later reported that treatment of seedlings with 1 µM 234
9
KAR2 for 4 days induced a slight increase in SMXL2 expression (Stanga et al., 2013). 235
Consistent with these studies, we did not observe a significant response of SMXL family 236
members upon KAR1 treatment for 4 h (Supplemental Figure 1B), which could be 237
explained by the hypothesis that KAR1 and KAR2 are not the active ligands in vivo and 238
require activation (Waters et al., 2015; Yao et al., 2018a). To further investigate the 239
response of SMXL genes to KAR signaling, we employed GR24ent-5DS
(Supplemental 240
Figure 1A) as a KAR surrogate (Waters et al., 2015; Flematti et al., 2016) and observed 241
that GR24ent-5DS
could clearly induce the expression of SMAX1 and SMXL2 after 4 h 242
treatment in a KAI2-dependent manner, but had little effect on the expression of SMXL3, 243
SMXL4, SMXL5, SMXL7, and SMXL8 (Figure 1). GR24ent-5DS
also weakly induced 244
SMXL6 expression in wild type, but this induction was not observed in kai2-2 or d14-1, 245
suggesting that GR24ent-5DS
might regulate SMXL6 expression through both KAI2 and SL 246
signaling. 247
248
SL Treatment Stimulates Interaction of SMXL2 with D14 but KAR Triggers its 249
Association with KAI2 250
To study the role of SMXL2 in SL signaling we investigated the physical association of 251
the SL receptor D14 with SMXL2, SMAX1, SMXL5 and SMXL6. In a 252
co-immunoprecipitation (Co-IP) assay using Arabidopsis protoplasts, hemagglutinin 253
(HA)-tagged D14 associated with Green Fluorescent Protein (GFP)-tagged SMXL2 and 254
GFP-SMXL6 proteins, and these interactions were significantly enhanced by GR244DO
. 255
However, neither SMAX1 nor SMXL5 could interact with D14, and GR244DO
treatment 256
had no effect (Figure 2A). This result is consistent with stimulation of interaction 257
between D14 and SMXL6 by rac-GR24 (Soundappan et al., 2015; Wang et al., 2015) and 258
indicates that GR244DO
enhances the interaction between D14 and SMXL2 in vivo. We 259
further examined interaction between HA-D14 and GFP-SMXL2 in the absence or 260
presence of KAR1, GR24ent-5DS
, GR245DS
, or GR244DO
, and found that the D14-SMXL2 261
10
interaction was significantly enhanced by GR244DO
and GR245DS
, but showed little 262
change upon KAR1 or GR24ent-5DS
treatment (Figure 2B). Furthermore, the interaction 263
between HA-D14 and GFP-SMXL2 was enhanced by GR244DO
with dose dependency 264
(Figure 2C). These results collectively demonstrate that SLs could specifically enhance 265
the interaction between D14 and SMXL2 in vivo. 266
Interaction between D14 and MAX2 is triggered by SLs in numerous plant species 267
(Hamiaux et al., 2012; Jiang et al., 2013; Zhou et al., 2013; Wang et al., 2015; Liang et al., 268
2016). However, biochemical evidence for interaction between KAI2 and MAX2 induced 269
by KARs has been difficult to obtain. In yeast two-hybrid assays, KAR1 promoted a weak 270
interaction between KAI2 (AtHTL) and MAX2 (Toh et al., 2014). Recently however, 271
strong autoactivation in the yeast two hybrid system by KAI2 was reported, but no 272
interaction between KAI2 and MAX2 could be observed (Yao et al., 2018a). To detect the 273
interaction between KAI2 and MAX2 in vivo, we co-expressed HA-KAI2 and 274
GFP-MAX2 in Arabidopsis protoplasts and conducted Co-IP assays. In the absence of 275
GR24ent-5DS
, HA-KAI2 was found to interact with GFP-MAX2 with low efficiency, while 276
addition of GR24ent-5DS
greatly enhanced the association, demonstrating that KAI2 forms 277
a complex with MAX2 in Arabidopsis (Supplemental Figure 2). 278
Although genetic evidence showed that SMAX1 and SMXL2 function downstream of 279
KAI2 and MAX2 in Arabidopsis (Stanga et al., 2013; Soundappan et al., 2015; Stanga et 280
al., 2016), there has been no evidence for physical interaction of SMXL proteins with 281
KAI2. We thus conducted Co-IP assays to test the association of HA-KAI2 with 282
GFP-SMAX1, GFP-SMXL2, GFP-SMXL5 and GFP-SMXL6 (Figure 3A). In the 283
absence of chemical treatment association between KAI2 and SMXLs was hardly 284
detected, but the KAI2-SMAX1 and KAI2-SMXL2 interactions were strongly induced by 285
GR24ent-5DS
treatment. SMXL6 could weakly interact with KAI2 under GR24ent-5DS
286
treatment, but SMXL5 showed no interaction, suggesting that SMXL6 is able to form a 287
complex with KAI2 with low efficiency in the protoplast system employed. We further 288
11
compared the effects of KAR1, GR24ent-5DS
, GR245DS
, and GR244DO
on KAI2-SMXL2 289
interaction and found that GR245DS
and GR244DO
could barely stimulate protein 290
association, indicating that GR24ent-5DS
preferentially and effectively triggers the 291
interaction between SMXL2 and KAI2 in vivo (Figure 3B). KAR1 did not trigger protein 292
association in this experiment, which might be due to insufficient time or capability for 293
the activation of KAR1 in this system. Together, these data clearly show that interaction 294
of SMXL2 with D14 and KAI2 is promoted by ligands that selectively activate SL and 295
KAR signaling, respectively. 296
297
SMXL2 Undergoes Ubiquitination upon SL or KAR Treatment 298
Polyubiquitination and degradation of rice D53 and Arabidopsis SMXL6, SMXL7 and 299
SMXL8 proteins in response to SL treatment are core events in SL signaling (Jiang et al., 300
2013; Zhou et al., 2013; Soundappan et al., 2015; Wang et al., 2015; Liang et al., 2016). 301
However, the biochemical mechanism underlying KAR signaling remains to be 302
determined. We therefore investigated the polyubiquitination of SMXL2 upon activation 303
of SL and KAR signaling in Arabidopsis leaf protoplasts. Clear polyubiquitination of 304
GFP-SMXL2 was observed upon treatment with rac-GR24 for 1.5 h but not by treatment 305
with KAR1 (Supplemental Figure 3), which is consistent with its inability to induce 306
interaction with SMXL2 in the protoplast system. We then investigated the 307
polyubiquitination of SMXL2 using a more responsive system. In transgenic plants 308
overexpressing SMXL2-GFP under the control of the cauliflower mosaic virus (CaMV) 309
35S promoter, GR244DO
or GR24ent-5DS
could trigger the ubiquitination of SMXL2-GFP 310
recombinant protein within 20 min, although KAR1 showed no effect (Figure 4A). 311
However, when the KAR1 treatment was prolonged to 2 h, a weak signal for the 312
ubiquitination of SMXL2-GFP could be detected, indicating a slow SMXL2 313
polyubiquitination induced by KAR1 (Figure 4B). 314
We next evaluated the contribution of SL or KAR receptor to the ubiquitination of 315
12
SMXL2 induced by GR244DO
and GR24ent-5DS
. Seedlings of transgenic plants expressing 316
35S:SMXL2-GFP in wild-type, d14-1 or kai2-2 backgrounds were treated for 20 min with 317
GR244DO
. The ubiquitination of SMXL2-GFP was observed in the wild type and kai2-2 318
but was very weak in the d14-1 background (Figure 4C). By contrast, the 319
polyubiquitination of SMXL2-GFP triggered by GR24ent-5DS
was dramatically impaired in 320
the kai2-2 background compared with the wild type, but only slightly weakened in the 321
d14-1 background (Figures 4C), indicating that the SMXL2 ubiquitination induced by 322
GR24ent-5DS
depends largely on KAI2. We then examined the ubiquitination level of 323
GFP-SMXL2 in protoplasts from wild type and the d14-1 kai2-2 double mutant (Figure 324
4D). The SMXL2 ubiquitination induced by GR244DO
and GR24ent-5DS
was not detected in 325
d14-1 kai2-2, indicating that ubiquitination of SMXL2 in the d14-1 and kai2-2 single 326
mutants was probably due to incomplete specificity of GR24 stereoisomers for D14 and 327
KAI2. 328
We further examined the effects of GR244DO
and GR24ent-5DS
on SMXL6 329
ubiquitination in protoplasts and found that SMXL6 underwent ubiquitination after 330
GR244DO
or GR24ent-5DS
treatment, but the GR24ent-5DS
-induced ubiquitination of SMXL6 331
was weaker than that of SMXL2 (Figure 4E). More importantly, the GR244DO
-induced 332
SMXL6 ubiquitination was not detected in d14-1 but largely unaffected in kai2-2. The 333
GR24ent-5DS
-induced SMXL6 ubiquitination was obviously impaired in both d14-1 and 334
kai2-2 single mutants (Figure 4E), suggesting that GR244DO
induced SMXL6 335
ubiquitination through D14, but the GR24ent-5DS
-triggered SMXL6 ubiquitination could 336
function through both KAI2 and D14. This phenomenon is consistent with previous 337
reports that ubiquitination and degradation of SMXL6-GFP upon rac-GR24 treatment is 338
completely blocked in d14-1 (Wang et al. 2015). Taken together, these data demonstrate 339
that SL and KAR signaling trigger the ubiquitination of SMXL2 in a manner that 340
predominantly depends on D14 and KAI2, respectively. 341
342
13
SMXL2 Is Degraded upon SL or KAR Treatment 343
Since polyubiquitination can regulate the stability and function of target proteins 344
(Mukhopadhyay and Riezman, 2007; Wang et al., 2017b), we examined the stability of 345
SMXL2 in response to SL and KAR treatments of seedlings harboring the stably 346
transformed SMXL2-GFP transgene. We observed that GR244DO
and GR24ent-5DS
347
triggered significant degradation of the SMXL2-GFP recombinant protein within 20 min, 348
whereas KAR1 induced a slower rate of degradation, which was apparent after 60 min but 349
took 120-240 min to achieve comparable degradation (Figure 5A). We further observed 350
the GFP fluorescence in root tips of 35S:SMXL2-GFP transgenic plants using Z-stack 351
screening of confocal images and found that it was distributed throughout in root tip 352
(Supplemental Figure 4). To visualize the endogenous degradation of SMXL2-GFP in 353
response to GR244DO
and GR24ent-5DS
treatments, we quantified the GFP fluorescence 354
signal in single plane of fixed focus and examined its changes during the time course of 355
chemical treatment. These results established that the GFP fluorescence was significantly 356
reduced after GR244DO
and GR24ent-5DS
treatment for 10 min, 20 min and 30 min, but 357
remained stable under mock treatment (Figure 5B and 5C). These results collectively 358
demonstrate that either SL or KAR signaling is able to induce ubiquitination and 359
degradation of SMXL2 in Arabidopsis thaliana. 360
To evaluate the contributions of SL and KAR receptors to GR244DO
- and 361
GR24ent-5DS
-induced SMXL2 degradation, seedlings of transgenic plants expressing 362
35S:SMXL2-GFP in wild-type, d14-1 or kai2-2 backgrounds were treated with GR244DO
363
and GR24ent-5DS
. The degradation of SMXL2-GFP induced by GR244DO
was blocked in 364
d14-1 but was weakly attenuated in kai2-2 (Figure 5D), indicating that SMXL2 365
undergoes a D14-dependent ubiquitination and degradation in response to GR244DO
. By 366
contrast, the GR24ent-5DS
-triggered SMXL2-GFP degradation was clearly impaired in 367
kai2-2 compared with the wild type, but only slightly delayed in d14-1 (Figures 5D), 368
indicating that the degradation of SMXL2 induced by GR24ent-5DS
depends largely on 369
14
KAI2. 370
371
The RGKT Motif of SMXL2 Is Essential for its Ubiquitination and Degradation 372
Induced by SL and KAR Signaling 373
The ubiquitination and degradation of SMXL6 and SMXL7 in Arabidopsis and D53 in 374
rice depend on the conserved RGKT motif within the C-terminal portion of these proteins 375
(Jiang et al., 2013; Zhou et al., 2013; Wang et al., 2015; Liang et al., 2016). Comparison 376
of amino acid sequences of the Arabidopsis SMXL family showed that the RGKT motif 377
is conserved in SMXL proteins in SL and KAR signaling pathways including SMAX1, 378
SMXL2, SMXL6, SMXL7, and SMXL8, but is absent from SMXL3, SMXL4, and 379
SMXL5, which are not implicated in SL or KAR signaling (Supplemental Figure 5). This 380
analysis prompted us to investigate roles of the RGKT motif in ubiquitination and 381
degradation of SMXL2 induced by SLs and KARs to determine if it is important in both 382
signaling pathways. 383
We generated 35S:SMXL2D-GFP transgenic plants that over-express a mutated 384
SMXL2 protein bearing a deletion specifically of the RGKT motif (Supplemental Figure 385
6). We found that the ubiquitination of SMXL2-GFP was detected after GR244DO
386
treatment for 20 min, but the ubiquitination of SMXL2D-GFP was abolished (Figure 6A). 387
Similarly, deletion of the RGKT motif prevented the ubiquitination of SMXL2 induced 388
by GR24ent-5DS
treatment for 20 min or KAR1 for 60 min (Figures 6B and 6C). 389
Furthermore, GR244DO
, GR24ent-5DS
and KAR1 could trigger the degradation of 390
SMXL2-GFP but showed no effect on the amount of SMXL2D-GFP protein (Figures 6D 391
and 6E). The GFP fluorescence signals in root tips of 35S:SMXL2D-GFP transgenic 392
plants were stable throughout the 30 min treatment with GR244DO
and GR24ent-5DS
393
(Figure 6F and 6G, Supplemental Figure 4). Therefore, the ubiquitination and 394
degradation of SMXL2 in response to SL and KAR treatments require the RGKT motif. 395
396
15
SMXL2 Is Important for GR244DO
- and GR24ent-5DS
-Regulated Hypocotyl 397
Elongation 398
To explore the function of SMXL2 in seedling development, we examined the hypocotyl 399
length of Col-0, smax1, smxl2, smax1 smxl2 (double mutant) and smxl6 smxl7 smxl8 400
(triple mutant, hereafter referred to as smxl6,7,8) after treatment with GR244DO
, 401
GR24ent-5DS
, and GR244DO
plus GR24ent-5DS
. Hypocotyls of the smax1 and smxl2 single 402
mutants were somewhat shorter than those of wild type, whereas the hypocotyl length of 403
the smax1 smxl2 double mutant was only 22% of the wild type (Figure 7A). These results 404
are largely consistence with previously reported observations (Stanga et al., 2016). More 405
importantly, GR244DO
and GR24ent-5DS
repressed hypocotyl elongation and showed an 406
additive effect in Col-0, and the repression by GR244DO
and GR24ent-5DS
in smax1 was 407
stronger than that in Col-0, indicating that SMXL2 plays an important role in hypocotyl 408
responses to both GR244DO
and GR24ent-5DS
. In the smxl2 mutant, although the effect of 409
GR24ent-5DS
was similar with that in Col-0, the effect of GR244DO
was rather weak. This 410
weak effect might be due to the possible ‘leakiness’ of the smxl2 mutant or the possibility 411
that other proteins also participate in these hypocotyl responses, which is supported by 412
the further suppression of hypocotyl length in the smax1 smxl2 double mutant in response 413
to GR244DO
and GR24ent-5DS
treatments (Figure 7A). The hypocotyl response of the 414
smxl6,7,8 triple mutant to chemical treatment was similar to the wild type, indicating that 415
SMXL6, SMXL7 and SMXL8 are not involved in the regulation of hypocotyl elongation 416
by SLs and KARs (Figure 7A). These results indicate that SMAX1 and SMXL2 display 417
functional redundancy in the hypocotyl response to GR24ent-5DS
, and SMXL2 plays 418
important roles in hypocotyl responses to both GR244DO
and GR24ent-5DS
. 419
We next compared the influence of SMXL2D and SMXL6D in the hypocotyl 420
response to GR244DO
and GR24ent-5DS
. Seedlings of 35S:SMXL2D-GFP/Col-0 transgenic 421
plants grew longer hypocotyls than the wild type and were insensitive to either GR244DO
422
or GR24ent-5DS
, but the 35S:SMXL6D-GFP/Col-0 transgenic plants displayed similar 423
16
phenotypes to wild type (Figure 7B), further supporting the importance of SMXL2 in 424
hypocotyl responses to GR244DO
and GR24ent-5DS
. In addition, the abundance of 425
SMXL2-GFP in the hypocotyl of 35S:SMXL2-GFP seedlings grown in the presence of 426
GR244DO
plus GR24ent-5DS
was only about 6% of that in 35S:SMXL2-GFP seedlings 427
without chemical treatment (Supplemental Figure 7), indicating that GR244DO
and 428
GR24ent-5DS
induced degradation of SMXL2-GFP in the hypocotyl. Importantly, seedlings 429
of 35S:SMXL2-GFP/Col-0 transgenic plants were sensitive to GR244DO
and GR24ent-5DS
430
treatments, but the 35S:SMXL2-GFP/d14-1 transgenic plants were insensitive to 431
GR244DO
and sensitive to GR24ent-5DS
treatment. By contrast, the 432
35S:SMXL2-GFP/kai2-2 transgenic plants were insensitive to GR24ent-5DS
and sensitive 433
to GR244DO
treatment (Figure 7C). These results demonstrated that SMXL2 mediates 434
both SL- and KAR-regulated hypocotyl elongation in Arabidopsis. 435
In Arabidopsis, SMXL6, SMXL7 and SMXL8 promote shoot branching mainly 436
through repressing BRANCHED 1 (BRC1) expression and enhancing polar auxin 437
transport (Soundappan et al., 2015; Wang et al., 2015). They are degraded in response to 438
D14-dependent SL signaling, leading to repression of shoot branching. Since SMXL2 is 439
also degraded in response to SL signaling, we investigated whether SMXL2 regulates 440
shoot branching. We found that numbers of primary rosette branches of smax1, smxl2, 441
and smax1 smxl2 were similar to Col-0 whereas d14-1 had many more branches 442
(Supplemental Figure 8A). We further found that although the 35S:SMXL2D-GFP and 443
35S:SMXL6D-GFP transgenic plants contained comparable levels of the corresponding 444
SMXL2D-GFP and SMXL6D-GFP proteins in their rosette lateral buds, the primary 445
branch number of 35S:SMXL6D-GFP transgenic plants was significantly increased while 446
that of 35S:SMXL2D-GFP transgenic plants was indistinguishable from the wild type 447
(Supplemental Figure 8B, 8C). These data suggest that SMXL2 does not apparently 448
regulate shoot branching even when ectopically expressed in lateral shoot buds, at least in 449
Col-0 background. This further emphasizes the importance of SMXL2 in seedling 450
17
development. 451
452
Regulation of DLK2 and KUF1 by SLs Depends on SMXL2 Whereas KAR Signaling 453
Employs either SMAX1 or SMXL2 454
To investigate the function of SMXL2 in the regulation of gene expression, we analyzed 455
the transcript levels of DLK2 and KUF1, which have been reported to respond to KARs 456
and SLs (Scaffidi et al., 2013; Scaffidi et al., 2014; Waters et al., 2015), in seedlings of 457
wild type, smax1, smxl2, smax1 smxl2 double mutant and smxl6,7,8 triple mutant. 458
Expression levels of DLK2 and KUF1 were comparable in the wild type, smax1, smxl2 459
and smxl6,7,8 triple mutant, but were remarkably elevated in the smax1 smxl2 double 460
mutant, indicating that both SMAX1 and SMXL2 repress the expression of DLK2 and 461
KUF1 (Figure 8A). These results are consistent with previously reported observations 462
(Stanga et al., 2016). However, MAX4 expression was downregulated in the smxl6,7,8 463
triple mutant compared with wild type, but remained unchanged in smax1, smxl2, and 464
smax1 smxl2 double mutant (Figure 8A). Thus, SMAX1 and SMXL2 mainly mediate the 465
transcriptional response of DLK2 and KUF1, whereas the SMXL6, SMXL7, SMXL8 466
group mainly mediates expression of the SL-responsive gene MAX4. More importantly, 467
expression of DLK2 and KUF1 were decreased in the SL-biosynthetic mutants max1 and 468
max4, suggesting that endogenous SLs could activate expression of DLK2 and KUF1 in 469
seedlings (Figure 8B). 470
Furthermore, exogenously applied GR244DO
could significantly induce the471
expression of DLK2 and KUF1 in wild type, smxl6,7,8 triple mutant, and smax1 single 472
mutant, indicating that GR244DO
promotes expression of DLK2 and KUF1 independently473
of SMXL6, SMXL7, SMXL8 and SMAX1 (Figure 8C). Interestingly, GR244DO
showed474
no effect in smxl2 or smax1 smxl2, indicating that SMXL2 is essential for stimulation of 475
DLK2 and KUF1 expression by SL signaling (Figure 8C). We further evaluated the 476
contribution of SMXL family members in mediating the expression of DLK2 and KUF1 477
18
induced by KAR signaling and found that GR24ent-5DS
could trigger an increase in478
transcript abundance of DLK2 and KUF1 in wild-type, smxl6,7,8 triple mutant, and 479
smax1 and smxl2 single mutant plants, but could not induce DLK2 and KUF1 expression 480
in the smax1 smxl2 double mutant (Figure 8C). This result indicates that SMAX1 and 481
SMXL2 are both required for activation of DLK2 and KUF1 expression in KAR 482
signaling. Moreover, both GR244DO
and GR24ent-5DS
could induce the expression of DLK2483
and KUF1 in seedlings of Col-0 and 35S:SMXL2-GFP transgenic plants, but had no 484
effect on seedlings of 35S:SMXL2D-GFP, indicating that SMXL2 induces the expression 485
of DLK2 and KUF1 in response to GR244DO
and GR24ent-5DS
in an “RGKT”-dependent486
manner (Figure 8D). Together, these results establish that SL stimulates DLK2 and KUF1 487
expression through SMXL2, whereas KAR induces DLK2 and KUF1 expression through 488
SMAX1 and SMXL2 (Figure 8E). 489
490
DISCUSSION 491
SMXL2 Is Involved in Both SL and KAR Signaling Pathways 492
Based on findings in this and earlier studies, we propose an overview model of SL and 493
KAR signaling in Arabidopsis (Figure 9). Previous research has shown that in the 494
presence of SLs, D14 forms a complex with MAX2 and one or more of SMXL6, SMXL7 495
and SMXL8. The complex triggers ubiquitination and degradation of SMXLs, dependent 496
on the RGKT motif, followed by de-repression of key genes that suppress shoot 497
branching, such as BRC1 (Soundappan et al., 2015; Wang et al., 2015). We now show that 498
in seedlings, SLs stimulate the association of D14 with SMXL2 (Figure 2), which is 499
ubiquitinated and degraded (Figures 4 and 5), also dependent on the RGKT motif (Figure 500
6). Degradation of SMXL2 presumably relieves the repression of genes that function to 501
inhibit hypocotyl elongation (Figure 7) and also relieves repression of DLK2 and KUF1 502
(Figure 8). Similarly, for KAR signaling, KAR1 or the KAR surrogate GR24ent-5DS
, induce503
KAI2 to form a complex with MAX2 (Supplemental Figure 2) and with SMXL2 (Figure 504
19
3), triggering the ubiquitination and degradation of SMXL2 in a manner dependent on the 505
RGKT motif (Figures 4 to 6), and further regulating gene expression (Figure 8) and 506
hypocotyl elongation (Figure 7). GR24ent-5DS
also induces interaction between KAI2 and 507
SMAX1 (Figure 3) and is speculated to trigger ubiquitination and degradation of SMAX1, 508
although we have not yet demonstrated this. Importantly, these results collectively show 509
that SMXL2 is a common target for proteolysis in both SL and KAR signaling and it 510
functions as a repressor protein regulating hypocotyl elongation and gene expression in 511
Arabidopsis (Figure 9). 512
Members of the SMXL protein family in Arabidopsis have been considered to play 513
specific roles in response to different signaling molecules (Mach, 2015; Soundappan et 514
al., 2015; Wang et al., 2015; Stanga et al., 2016; Wallner et al., 2017). SMXL6, SMXL7 515
and SMXL8 function as key transcriptional repressor proteins in SL signaling, while 516
SMAX1 and SMXL2 are important for KAR-regulated changes in transcripts 517
(Soundappan et al., 2015; Wang et al., 2015; Stanga et al., 2016). However, SLs and 518
KARs also regulate the expression of several common downstream genes, for instance 519
SALT TOLERANCE H-BOX7 (STH7), DLK2, KUF1, INDOLE-3-ACETIC ACID 520
INDUCIBLE5 (IAA5), and IAA6 (Scaffidi et al., 2013; Scaffidi et al., 2014; Waters et al., 521
2015). Because F-box protein MAX2 is required for targeting and proteolysis of SMXL 522
proteins, we speculated that SMXL2 could be responsible for this convergence in 523
regulation of gene expression by SLs and KARs. Interestingly, disruption of SMXL6, 524
SMXL7, and SMXL8 collectively, or SMAX1 or SMXL2 has no effect on the induction of 525
DLK2 and KUF1, but disruption of both SMAX1 and SMXL2 greatly enhanced the 526
expression of DLK2 and KUF1 (Figure 8A) (Stanga et al., 2016) and completely 527
abolished the upregulation in response to SLs and KARs (Figure 8C). Thus, biochemical 528
and genetic data in this study clearly demonstrate that SMXL2 is common to both SL and 529
KAR signaling, and plays a key role in integrating responses to SLs and KARs at the 530
transcription level. 531
20
532
Specificity of GR244DO
and GR24ent-5DS
in SL and KAR signaling in Arabidopsis 533
A strong connection between KARs and SLs is seen in their chemical structures, 534
with common butenolide moieties (Smith and Li, 2014). The natural SL isomers 5DS and 535
4DO regulate shoot branching, hypocotyl elongation and gene expression in a 536
D14-dependent manner, while the non-natural enantiomer ent-5DS selectively regulates 537
seed germination and hypocotyl elongation through KAI2 (Scaffidi et al., 2014). The 538
structure-activity relationship and receptor recognition studies have demonstrated that the 539
configuration at C-2' plays a critical role in signal perception of SLs and KARs in 540
Arabidopsis and rice (Scaffidi et al., 2014; Umehara et al., 2015), but the chemical 541
specificity is not absolute and might vary at different growth stages or in different 542
experimental systems (Li et al. 2016b; Villaécija-Aguilar et al. 2019). We found that 543
GR244DO
could induce downstream gene expression, the ubiquitination and degradation 544
of SMXL2 and SMXL6, and repress hypocotyl elongation of the 35S:SMXL2-GFP 545
transgenic plants in a D14-dependent manner, indicating that GR244DO
specifically 546
displayed SL activity in Arabidopsis (Figures 1, 4C, 5D, and 7C). GR24ent-5DS
induced a 547
strong interaction between KAI2 and SMXL2 but also a very weak interaction between 548
D14 and SMXL2 in Co-IP assays (Figures 2B, 3A and 3B). GR24ent-5DS
could also 549
stimulate a relatively strong ubiquitination of SMXL2 that was weaker in d14-1 but 550
largely blocked in kai2-2 (Figure 4C). These data indicate that GR24ent-5DS
displays 551
strong KAR-like activity and low SL activity in Arabidopsis. 552
Although KAR1 and GR24ent-5DS
induced transcripts of DLK2 after treatment for 24 553
h to 72 h in a KAI2-dependent manner (Sun et al., 2016), we found that compared with 554
GR24ent-5DS
, KAR1 was less effective in the experimental systems detecting protein 555
ubiquitination, degradation, protein-protein interactions and regulation of transcript levels, 556
especially within short-term treatments in the region of an hour. These observations are 557
consistent with the view that KAR1 might require metabolism in vivo to produce a ligand 558
21
that can be recognized by the receptor KAI2 (Waters et al., 2015; Waters et al., 2017). 559
However, until the endogenous ligand for KAI2 is identified, it is difficult to fully assess 560
the factors that result in KAI2-mediated regulation of seed germination and seedling 561
development. Therefore, discovery of the endogenous KAI2 ligand, KL, is becoming 562
increasingly important (Morffy et al., 2016; Sun et al., 2016; Waters et al., 2017; Yao et 563
al., 2018a; Smith, 2019). 564
565
Co-Evolution of the SMXL and KAI2/D14 Families 566
Evolutionary analysis shows that KAI2 orthologs exist in basal land plants, and the 567
endogenous KL is predicted to exist throughout land plants from basal to seed plants 568
(Waters et al., 2012; Conn and Nelson, 2015; Gutjahr et al 2015; Kameoka and Kyozuka, 569
2015; Waters et al., 2017; Carbonnel et al 2019;). Interestingly, SMXL family members 570
are not apparent in the charophytes, but have been identified in non-vascular plants 571
including liverworts and mosses, and display 60-65% sequence identity with Arabidopsis 572
SMAX1. These ancient forms of SMAX1 subsequently underwent duplication in land 573
plants and evolved into four major clades consisting of homologs of SMAX1, and 574
SMXL2 as clade I, SMXL6, SMXL7 and SMXL8 as clade II, SMXL3 as clade III, and 575
SMXL4 and SMXL5 as clade IV (Moturu et al., 2018). Our current studies indicate that 576
SMAX1 and SMXL2 mainly control seedling development especially hypocotyl 577
elongation, which is important throughout land plants. By contrast, shoot branching and 578
vascular development are notable features of vascular plants, for which SMXL clades II, 579
III and IV have been shown to be required. The correlation between evolutionary history 580
and functional differentiation of SMXL proteins provides new evidence for the 581
hypothesis of co-evolution between the SMXL family and KAI2/D14 family (Moturu et 582
al., 2018). More importantly, we now show that SMXL2 interacts with both the KAR 583
receptor KAI2 and the SL receptor D14 (Figures 2 and 3), and undergoes 584
polyubiquitination and degradation to regulate gene expression and seedling development 585
22
(Figures 3 to 8). GR24ent-5DS
induced a weak interaction between SMXL6 and KAI2 586
(Figure 3A) and a weak ubiquitination that was impaired in kai2-2 (Figure 4E), 587
suggesting that SMXL6 could form a complex with KAI2 and undergo ubiquitination 588
with low efficiency in response to GR24ent-5DS
treatment. This may reflect the 589
evolutionary origin of interactions between the KAR receptor and SMXL-type repressor 590
proteins, although KAI2 and SMXL6 genes are not thought to be co-expressed normally. 591
The demonstration that KAR and SL signaling employ the same mechanism of 592
MAX2-dependent SMXL polyubiquitination and proteolysis provides strong support for 593
the proposed co-evolution of SL signaling and SMXL functions from an ancestral 594
KAI2-SMAX1 system (Moturu et al., 2018; Machin et al., 2020). 595
596
SL- and KAR-Regulated Seedling Development 597
Hypocotyl elongation is important to push the apex of young seedlings out of the 598
soil or deep water to receive light and continue vegetative and reproductive development. 599
Hypocotyl elongation is regulated by various phytohormones, including auxin, 600
gibberellin, ethylene, brassinosteroid and jasmonate (Yang et al., 2011; Li et al., 2016a; 601
Shi et al., 2016; Reed et al., 2018; Yi et al., 2020). It has been reported that hypocotyls of 602
wild-type seedlings under red light are sensitive to both SL and KAR treatment; and 603
SMAX1 and SMXL2 collectively mediate the KAR-repressed hypocotyl development 604
(Stanga et al., 2016). However, the molecular mechanism underlying SL-regulated 605
hypocotyl development has been unclear. Here we show that transgenic plants 606
overexpressing degradation-resistant SMXL2 possessed longer hypocotyls that were 607
insensitive to exogenously applied SL and KAR, but overexpressing degradation-resistant 608
SMXL6 showed no effect on hypocotyl response, and the hypocotyl response to 609
exogenously applied SL was blocked in the d14 background (Figure 7B and 7C). Based 610
on the roles of SMXL2 in SL signaling, these results clearly show that it is SMXL2 rather 611
than SMXL6, SMXL7, or SMXL8 that functions as a repressor protein in SL-regulated 612
23
hypocotyl development in Arabidopsis. 613
Recent evolutionary analyses have shown that SMXL2 appears due to a recent 614
duplication of SMAX1 in the Brassicaceae and has not been observed in the genome of 615
plants belonging to other families (Walker et al., 2019). Interestingly, the rice SL mutants 616
d10, d14, and d17 form elongated mesocotyl in comparison to wild type (Hu et al., 2010; 617
Hu et al., 2013), but the Arabidopsis SL mutants max1, max3, max4 and d14 form 618
hypocotyls indistinguishable from wild type (Nelson et al., 2011; Scaffidi et al., 2013; 619
Scaffidi et al., 2014). This may be due to the possible role of D53 and D53L in 620
SL-regulated rice mesocotyl development. Meanwhile, the rice d3 mutant (equivalent to 621
Arabidopsis max2) displays a more elongated mesocotyl phenotype than d14 and SL 622
biosynthesis-deficient mutants (Hu et al., 2010), suggesting a potential role of the KAR 623
signaling pathway in mesocotyl development (Kameoka and Kyozuka, 2015). Further 624
investigation of SL and KAR signaling and the roles of D53-family proteins in mesocotyl 625
elongation could provide a breakthrough in understanding rice seedling growth and 626
development. This is important for the future agricultural practice of direct seeding to 627
avoid seedling transplantation in rice crop production. In other crops, increased 628
elongation of hypocotyl or mesocotyl could permit deeper sowing of seeds in soils in 629
which the surface layers are at risk from drying. Thus, our research enables further 630
understanding of the control of seed germination, seedling skotomorphogenesis and 631
photomorphogenesis, which provides major opportunities for future improvements in 632
agriculture. 633
634
METHODS 635
Plant Materials and Growth Conditions 636
Arabidopsis thaliana ecotype Columbia (Col-0) was used as wild type. The smxl6,7,8 637
triple mutant (Wang et al., 2015), d14-1 (Waters et al., 2012), smax1-2 (Stanga et al., 638
2013), smxl2-1 (Stanga et al., 2016) and smax1-2 smxl2-1 double mutant (Stanga et al., 639
24
2016), max1-1 (Stirnberg et al., 2002), and max4-5 (Bennett et al., 2006) mutants have 640
been described previously. The kai2-2 (Waters et al., 2012) mutant in Ler background 641
was backcrossed to Col-0 six times by Dr. Mark Waters (University of Western Australia) 642
and was provided by Dr. Ruifeng Yao (Tsinghua University). Seeds were surface 643
sterilized, vernalized at 4 ºC, and germinated on 0.5× Murashige and Skoog (MS) 644
medium containing 1.0% (w/v) sucrose and 0.7% (w/v) agar. For morphological 645
observations, 10-day-old seedlings were transferred to pots containing a 1:1 646
vermiculite:soil mixture saturated with 0.3× MS medium. Plants were grown under 647
controlled conditions at about 21 ºC, 16-h light, 8-h dark cycle, and fluorescent lamps 648
with a light intensity of 60-80 μE m−2
s−1
as described previously (Dai et al., 2006). 649
650
Chemicals and Reagents 651
Synthetic rac-GR24 and KAR1 were obtained from Chiralix (Nijmegen, The 652
Netherlands). The SL analogue GR244DO
, GR245DS
and GR24ent-5DS
were synthesized 653
through the following method. GR24 stereoisomers were prepared as previously 654
described (Mangnus et al., 1992). Then flash chromatography was performed to give two 655
kinds of enantiomers. The enantiomers moving faster were further separated into 656
GR24ent-5DS
and GR245DS
, and the enantiomers moving slower were separated into 657
GR24ent-4DO
and GR244DO
using HPLC fitted with a Chiral HPLC Column (150 mm long, 658
4.6-mminternal diameter). Separation was achieved using a flow rate of 1.0 mL min-1
of a 659
1:1 mixture of hexane:ethyl alcohol. The corresponding components were collected and 660
obtained pure optical isomers. MG132 was obtained from Calbiochem (California, USA). 661
The stock solution of rac-GR24, KAR1, GR244DO
, GR245DS
, or GR24ent-5DS
was prepared 662
in acetone, and stock solution of MG132 was prepared in Dimethyl sulfoxide (DMSO). 663
664
Vector Construction and Plant Transformation 665
To construct the 35S:SMXL2-GFP plasmid, the coding sequence of SMXL2 was 666
25
amplified with primer pairs of SMXL2-OE-F and SMXL2-OE-R (Supplemental Table 1), 667
and then cloned into KpnI- and SalI-digested pWM101. To construct the 668
35S:SMXL2D-GFP plasmid, SMXL2D was amplified through site-directed mutagenesis 669
using primer pairs of SMXL2-DEL-F and SMXL2-DEL-R, and then cloned into 670
pWM101. The 35S:SMXL2-GFP and 35S:SMXL2D-GFP recombinant plasmids were 671
introduced into Agrobacterium tumefaciens strain EHA105 and used to transform 672
wild-type, d14-1 and kai2-2 plants through the Agrobacterium-mediated floral dip 673
method. To construct the plasmids of 35S:GFP-SMXL2, 35S:GFP-SMXL5 and 674
35S:3×HA-KAI2, the coding sequence of SMXL2, SMXL5 and KAI2 were amplified with 675
primer pairs of SMXL2-PD-F, SMXL2-PD-R, SMXL5-PD-F, SMXL5-PD-R and 676
KAI2-PD-F, KAI2-PD-R (Supplemental Table 1), then cloned into the Gateway Entry 677
vector by BP reaction and recombined to pBeacon-eGFP and pBeacon-3×HA (Wang et 678
al., 2015) through LR reaction. To construct the plasmids of 35S:GFP-SMAX1, the 679
codon-optimized SMAX1 coding sequence was amplified from the Flag-SMAX1 plasmid 680
(Yao et al., 2017) with primer pairs of SMAX1-PD-F, SMAX1-PD-R, then cloned into 681
the pBeacon-eGFP vector through BP and LR reaction. 682
683
Gene Expression Analysis 684
The wild type, mutants of smax1, smxl2, smax1 smxl2, smxl6,7,8, max1, max4, d14-1, 685
kai2-2, 35S:SMXL2-GFP/Col-0 and 35S:SMXL2D-GFP/Col-0 were grown on agar with 686
0.5× MS medium for ten days, collected and equilibrated in 0.5× MS liquid medium for 2 687
h, then treated with GR244DO
, GR24ent-5DS
, KAR1 or acetone in the light at about 21 ºC 688
for 4 h. The whole seedlings were frozen and ground into power in liquid nitrogen. Total 689
RNA was prepared using a TRIzol kit (Invitrogen, USA) according to the user manual. 690
RNA samples (each containing 12.5 μg RNA) were treated with TUBRO DNase 691
(Ambion, USA). Then first-strand cDNA was synthesized using oligo (dT) and random 692
primers with the SSIII first-strand synthesis system (Invitrogen, USA). Real-time PCR 693
26
was performed using gene-specific primers (see Supplemental Table 1) on a CFX 96 694
real-time PCR detection system (BioRad, USA) in a total volume of 10 μL with 2 μL 695
diluted cDNA, 0.3 μM gene-specific primers, and 5 μL SsoFast EvaGreen supermix 696
(BioRad, Singapore). The real deltaCt values are determined relative to the Arabidopsis 697
ACTIN2 (ACT2) gene (Weissgerber et al. 2015). 698
699
Co-Immunoprecipitation (Co-IP) Assay 700
Protoplasts were generated from mesophyll cells of Arabidopsis leaves (Yoo et al., 2007) 701
and transformed with transient expression plasmids including 35S:3×HA-D14 (Wang et 702
al., 2015), 35S:GFP-SMXL2, 35S:GFP (Wang et al., 2015), 35S:3×HA-KAI2, and 703
35S:GFP-MAX2 (Wang et al., 2015). After incubation at 21 ºC for 11 h followed by 704
another 1 h with 100 µM GR244DO
, GR24ent-5DS
, KAR1, or GR245DS
, protoplasts were705
collected in protein extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% 706
(v/v) glycerol, 1% (v/v) Nonidet P-40) and 1× ‘Complete Protease Inhibitor Cocktail’ 707
(Cat#04693132001, lot no. 41353700; Roche, Switzerland). The lysate was centrifuged at 708
18,000 g for 15 min at 4 ºC, and the supernatant was collected for Co-IP assay. In brief, 709
30 μl of the agarose-conjugated anti-GFP monoclonal antibody (D153-8, lot no. 048; 710
MBL, Japan) was added into 1 ml total extracted protein solution and incubated at 4 ºC 711
for 3 h in the presence or absence of GR244DO
, GR24ent-5DS
, KAR1, and GR245DS
. The712
beads were washed three times with extraction buffer containing 0.1% (v/v) NP-40, then 713
eluted with 25 μl of SDS–PAGE sample buffer for immunoblot analysis. The levels of 714
GFP-SMXL2, GFP-MAX2, and GFP protein were detected using mouse anti-GFP 715
monoclonal anti-body (Cat#11814460001, lot no.11751700; Roche, Switzerland) at a 716
1:2,000 dilution and ‘One Step Western Kit HRP (Mouse)’ (CW2030M, lot no. 717
03803/40449; CoWin Biosciences, China) at 1:200 dilution. The levels of HA-D14 and 718
HA-KAI2 protein were detected using rabbit anti-HA polyclonal antibody (H6908, lot No. 719
106M4792; Sigma, Germany) at a 1:2,000 dilution and anti-rabbit IgG HRP linked whole 720
27
Ab (NA934V, lot no. 9761194; GE Healthcare, USA) at 1:10,000 dilution. Signals were 721
then detected using ‘Amersham ECL Select Western Blotting Detection Reagent’ 722
(RPN2235, lot no. 16926118; GE Healthcare, USA) and X-ray film. 723
724
In Vivo Ubiquitination Assay 725
The transgenic plants of 35S:SMXL2-GFP/Col-0, 35S:SMXL2-GFP/d14-1, 726
35S:SMXL2-GFP/kai2-2, and 35S:SMXL2D-GFP/Col-0 were grown for 7 d, collected 727
and pretreated with 50 µM MG132 for 1 h, then treated with KAR1, rac-GR24, GR244DO
, 728
GR24ent-5DS
or acetone for the indicated time in 0.5× MS liquid medium at 21 °C. Equal 729
weights of plant materials were collected for protein extraction using protein extraction 730
buffer containing 1× ‘Complete Protease Inhibitor Cocktail’ (Cat# 04693132001, lot No. 731
41353700; Roche, Switzerland). Then 30 μl of anti-GFP monoclonal antibody-conjugated 732
agarose (D153-8, lot No. 048; MBL, Japan) was added into 1 ml total proteins extract and 733
incubated at 4 ºC for 3 h with gentle rotation. The beads were washed three times with 734
the protein extraction buffer containing 0.1% (v/v) NP-40, and then eluted with 25 μl of 735
the SDS–PAGE sample buffer for protein blotting. The polyubiquitination level was 736
detected using mouse anti-ubiquitin monoclonal antibody (Zhao et al., 2013b) at a 737
1:3,000 dilution and anti-mouse IgG HRP linked whole Ab (NA931V, lot No. 9761190; 738
GE Healthcare, USA) at 1:10,000 dilution. The levels of SMXL2-GFP and 739
SMXL2D-GFP proteins were detected using the mouse anti-GFP monoclonal antibody 740
(Cat# 11814460001, lot No. 11751700; Roche, Switzerland) at a 1:2,000 dilution and 741
‘One Step Western Kit HRP (Mouse)’ (CW2030M, lot No. 03803/40449; CoWin 742
Biosciences, China) at 1:200 dilution. Signals were then detected using ‘Amersham ECL 743
Select Western Blotting Detection Reagent’ (RPN2235, lot No. 16926118; GE 744
Healthcare, USA) and X-ray film. 745
In the ubiquitination system using protoplasts, the GFP-SMXL2 and GFP-SMXL6 746
recombinant proteins were expressed in Arabidopsis protoplasts made from mesophyll 747
28
cells (Yoo et al., 2007). Equal volumes of protoplasts were pre-treated with MG132 for 1 748
h and treated with indicated chemicals for 1.5 h, then underwent protein extraction using 749
lysis buffer. Equal volumes of GFP monoclonal antibody-conjugated agarose beads 750
(D153-8, lot No. 048; MBL, Japan) were added to each sample, incubated for 3 h, 751
washed for 3 times, and eluted with 30 µl 2× SDS loading buffer. Then 10 µl eluted 752
sample was used to detect polyubiquitination and GFP levels in immunoblots. The 753
polyubiquitination level was detected using anti-ubiquitin monoclonal antibody (Zhao et 754
al., 2013b) at a 1:3,000 dilution and anti-mouse IgG HRP linked whole Ab (NA931V, lot 755
No. 9761190; GE Healthcare, USA) at 1:10,000 dilution. The levels of GFP-SMXL2 and 756
GFP-SMXL6 proteins were detected using the mouse anti-GFP monoclonal antibody 757
(Cat#11814460001, lot No. 11751700; Roche, Switzerland) at a 1:2,000 dilution and 758
‘One Step Western Kit HRP (Mouse)’ (CW2030M, lot No. 03803/40449; CoWin 759
Biosciences, China) at 1:200 dilution. Signals were then detected using ‘Amersham ECL 760
Select Western Blotting Detection Reagent’ (RPN2235, lot No. 16926118; GE 761
Healthcare, USA) and X-ray film. 762
763
SMXL2 Degradation 764
The transgenic plants of 35S:SMXL2-GFP/Col-0, 35S:SMXL2-GFP/d14-1, 765
35S:SMXL2-GFP/kai2-2, and 35S:SMXL2D-GFP/Col-0 were grown for 7 d, collected 766
and treated with GR244DO
, GR24ent-5DS
, or KAR1 for the indicated time in 0.5× MS liquid767
medium at 21 °C. Equal weight of plant materials were collected for protein extraction 768
using lysis buffer containing 1× ‘Complete Protease Inhibitor Cocktail’ (Cat# 769
04693132001, lot No. 41353700; Roche, Switzerland). Protein levels of SMXL2-GFP 770
and SMXL2D-GFP were detected by immunoblotting with mouse anti-GFP monoclonal 771
antibody (Cat#11814460001, lot No.11751700; Roche, Switzerland) at a 1:2,000 dilution 772
and ‘One Step Western Kit HRP (Mouse)’ (CW2030M, lot No. 03803/40449; CoWin 773
Biosciences, China) at 1:200 dilution. In addition, abundances of SMXL2-GFP in the root, 774
29
hypocotyl and cotyledons of 35S:SMXL2-GFP/Col-0 seedlings were determined by 775
immunoblotting with mouse anti-GFP monoclonal antibody (Cat#11814460001, lot 776
No.11751700; Roche, Switzerland) at a 1:2,000 dilution and ‘One Step Western Kit HRP 777
(Mouse)’ (CW2030M, lot No. 03803/40449; CoWin Biosciences, China) at 1:200 778
dilution, and endogenous ACTIN levels were determined using mouse anti-Actin 779
monoclonal antibody (M20009L, lot No. 313860; Abmart, China) at a 1:10000 dilution 780
and anti-mouse IgG HRP linked whole Ab (NA931V, lot No. 9761190; GE Healthcare, 781
USA) at 1:10,000 dilution. Signals were then detected using ‘Amersham ECL Select 782
Western Blotting Detection Reagent’ (RPN2235, lot No. 16926118; GE Healthcare, USA) 783
and X-ray film. 784
785
GFP Fluorescence 786
To detect endogenous degradation of SMXL2-GFP, roots of 7-d-old 787
35S:SMXL2-GFP/Col-0 and 35S:SMXL2D-GFP/Col-0 seedlings were treated with 2 µM 788
GR244DO
or GR24ent-5DS
in 0.5× MS liquid medium for the time indicated on glass slides, 789
and imaged by confocal laser scanning microscopy at excitation wavelength of 488 nm 790
(FluoView 1000; Olympus, Japan). The same offset and gain settings for GFP detection 791
were used within experiments for each transgenic line. The signal intensities of GFP were 792
quantitatively determined with the FV10-ASW 4.0 viewer 1000 software (Olympus, 793
Japan). To observe the SMXL2-GFP expression profiles in root tips of 794
35S:SMXL2-GFP/Col-0 and 35S:SMXL2D-GFP/Col-0 seedlings grown for 5 days under 795
long-day conditions, Z stacks containing 5 focal planes were imaged and collected by 796
confocal laser scanning microscopy at excitation wavelength of 488 nm (FluoView 1000; 797
Olympus, Japan). 798
799
Hypocotyl Measurements 800
For hypocotyl elongation assays, surface-sterilized seeds were sown on 0.5× MS media 801
30
containing 1.0% (w/v) sucrose, 0.7% (w/v) agar, 100 nM GR244DO
, GR24ent-5DS
, or 802
GR244DO
plus GR24ent-5DS
(100 nM of each chemical). The seeds were stratified in the 803
dark at 4 ºC for 72 h, exposed to white light from fluorescence tube (approximately 60-80 804
μE m−2
s−1
) for 3 h at 21 ºC, transferred to dark for 21 h, and then exposed to continuous 805
red light (approximately 20-30 μE m−2
s−1
) for 4 d at 21 ºC as previously described 806
(Stanga et al., 2013). The lengths of the hypocotyls were measured using ImageJ 807
(http://rsbweb.nih.gov/ij/). 808
809
Statistical analysis 810
For gene expression analysis, phenotype observation, and GFP fluorescence 811
quantification, statistical analysis was assessed as described in the figure legends. P 812
values were calculated by two-tailed Student’s t-test using Excel 2016 or by two-way 813
ANOVA multiple comparison with a Tukey test using GraphPad Prism 7.00. 814
815
Accession Numbers 816
Sequence data were obtained from the GenBank/EMBL libraries under the following 817
accession numbers: AT5G57710 (SMAX1), AT4G30350 (SMXL2), AT3G52490 (SMXL3), 818
AT4G29920 (SMXL4), AT5G57130 (SMXL5), AT1G07200 (SMXL6), AT2G29970 819
(SMXL7), AT2G40130 (SMXL8), AT3G03990 (AtD14), AT4G37170 (KAI2), AT2G26170 820
(MAX1), AT2G42620 (MAX2), AT4G32810 (MAX4), AT3G24420 (DLK2), AT1G31350 821
(KUF1), and AT3G18780 (ACT2). 822
823
Supplemental Data 824
Supplemental Figure 1. Expression of SMXL gene family members shows no response 825
to KAR1 treatment. 826
Supplemental Figure 2. GR24ent-5DS
induces interaction between KAI2 and MAX2 in 827
vivo. 828
31
Supplemental Figure 3. Ubiquitination of SMXL2 after KAR1 or rac-GR24 treatment of 829
protoplasts. 830
Supplemental Figure 4. GFP fluorescence signals in 35S:SMXL2-GFP and 831
35S:SMXL2D-GFP transgenic lines. 832
Supplemental Figure 5. Alignment of Arabidopsis SMXL family proteins. 833
Supplemental Figure 6. Diagram showing domain structure of SMXL2 and the amino 834
acid changes in SMXL2D protein. 835
Supplemental Figure 7. SMXL2-GFP abundance in the hypocotyl of 35S:SMXL2-GFP 836
seedlings. 837
Supplemental Figure 8. SMXL2 has little effect on shoot branching. 838
Supplemental Table 1. Primers used in this study. 839
Supplemental Data Set 1. Statistical analysis data. 840
841
ACKNOWLEDGEMENTS 842
We thank Dr. Mark Waters at University of Western Australia and Dr. Daoxin Xie at 843
Tsinghua University for providing the kai2-2 mutant, Dr. Ruifeng Yao at Hunan 844
University for providing the d14-1 kai2-2 mutant, Dr. David Nelson at University of 845
California, Riverside and Dr. Mark Waters at University of Western Australia for 846
providing smax1-2, smxl2-1 and smax1-2 smxl2-1 mutants, and Dr. Ottoline Leyser at 847
Cambridge University for providing max1-1 and max4-5 mutants. This work was 848
supported by National Natural Science Foundation of China (31788103, 31600221, 849
31661143025), the Chinese Academy of Sciences President’s International Fellowship 850
Initiative (2018VBA0025), and Youth Innovation Promotion Association of the Chinese 851
Academy of Sciences (2019099). 852
853
32
AUTHOR CONTRIBUTIONS 854
L.W., B.W. and Q.X. designed and performed experiments; H.Y., H.M., X.L., J.Y., J.C. 855
Q.X., and Y.W. contributed to some of the experiments. B.W., G.X., and J.L. designed 856
research, conceived and supervised the project. B.W., L.W., S.M.S. and J.L. analyzed the 857
data and wrote the paper. All authors commented on the manuscript. 858
859
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1149
FIGURE LEGENDS 1150
43
Figure 1. Expression of SMXL family genes upon GR244DO
or GR24ent-5DS
treatment. 1151
Expression of SMXL gene family members in 10-day-old seedlings of wild type (Col-0), 1152
d14-1 and kai2-2 treated with 5 μM GR244DO
or GR24ent-5DS
for 4 h. Expression values 1153
are determined relative to ACTIN2. Values are means ± SD. *P < 0.05; **P < 0.01; ns, no 1154
significance (n = 3, two-tailed Student’s t-test, three independent experiments; 1155
Supplemental Data set 1). 1156
1157
Figure 2. GR244DO
induces interaction between D14 and SMXL2 in vivo. 1158
(A) Association of HA-D14 with GFP-SMAX1, GFP-SMXL2, GFP-SMXL5, and 1159
GFP-SMXL6 revealed by Co-IP assay in wild-type (Col-0) protoplasts in the absence or 1160
presence of 100 µM GR244DO
. 1161
(B) Interaction between HA-D14 and GFP-SMXL2 revealed by Co-IP assay in wild-type 1162
(Col-0) protoplasts in the absence or presence of 100 µM KAR1, GR24ent-5DS
, GR245DS
, 1163
or GR244DO
. 1164
(C) Interaction between HA-D14 and GFP-SMXL2 revealed by Co-IP assay in wild-type 1165
(Col-0) protoplasts in the presence of 0, 50, 100, and 150 µM GR244DO
. 1166
The HA-D14 recombinant protein was detected with anti-HA monoclonal antibody; the 1167
GFP-SMAX1, GFP-SMXL2, GFP-SMXL5, GFP-SMXL6 fusion proteins and GFP were 1168
detected with anti-GFP monoclonal antibody. All experiments were repeated at least three 1169
times with similar results. 1170
1171
Figure 3. GR24ent-5DS
induces interaction between KAI2 and SMXL2 in vivo. 1172
(A) Association of HA-KAI2 with GFP-SMAX1, GFP-SMXL2, GFP-SMXL5, and 1173
GFP-SMXL6 revealed by Co-IP assay in wild-type (Col-0) protoplasts in the absence or 1174
presence of 100 µM GR24ent-5DS
. 1175
(B) Interaction between HA-KAI2 and GFP-SMXL2 revealed by Co-IP assay in 1176
wild-type (Col-0) protoplasts in the absence or presence of 100 µM KAR1, GR24ent-5DS
, 1177
44
GR245DS
, or GR244DO
. 1178
The HA-KAI2 recombinant protein was detected with anti-HA monoclonal antibody; the 1179
GFP-SMAX1, GFP-SMXL2, GFP-SMXL5, GFP-SMXL6 fusion proteins and GFP were 1180
detected with anti-GFP monoclonal antibody. All experiments were repeated at least three 1181
times with similar results. 1182
1183
Figure 4. SMXL2 undergoes ubiquitination after GR244DO
, KAR1 or GR24ent-5DS
1184
treatment. 1185
(A) Ubiquitination of SMXL2-GFP proteins in 7-day-old 35S:SMXL2-GFP transgenic 1186
plants after GR244DO
, GR24ent-5DS
or KAR1 treatment. Seedlings were treated with 50 µM 1187
MG132 for 1 h, then with 2 µM GR244DO
, GR24ent-5DS
or KAR1 for 20 min in 0.5× MS 1188
liquid medium. 1189
(B) Ubiquitination of SMXL2-GFP proteins in 7-day-old 35S:SMXL2-GFP transgenic 1190
plants with 50 µM MG132 for 1 h, then with 2 µM KAR1 for 2 h or 2 µM GR244DO
for 1191
20 min in 0.5× MS liquid medium. Mock treatments were 2 h. 1192
(C) Ubiquitination of SMXL2-GFP in wild-type, d14-1 and kai2-2 Arabidopsis seedlings 1193
containing 35S:SMXL2-GFP transgene. Seedlings were treated after 7 days growth with 1194
50 µM MG132 for 1 h, then with 2 µM GR244DO
or GR24ent-5DS
for 20 min in 0.5× MS 1195
liquid medium. 1196
(D) Ubiquitination of GFP-SMXL2 in protoplasts made from wild-type and d14-1 kai2-2 1197
seedlings. 1198
(E) Ubiquitination of GFP-SMXL6 in protoplasts made from wild-type, d14-1 and kai2-2 1199
seedlings. 1200
In (D) and (E), protoplasts were transformed with 35S:GFP-SMXL2 or 35S:GFP-SMXL6 1201
plasmid, incubated overnight for protein synthesis, then pretreated with 50 µM MG132 1202
for 1 h and treated with 100 µM GR244DO
and GR24ent-5DS
for 1.5 h. 1203
Proteins were detected by immunoblotting with anti-ubiquitin (Ubi) polyclonal antibody 1204
45
or anti-GFP monoclonal antibody. All experiments were repeated at least three times with 1205
similar results. 1206
1207
Figure 5. SMXL2 undergoes degradation after GR244DO
, GR24ent-5DS
or KAR1 treatment. 1208
(A) Degradation of SMXL2-GFP proteins in 7-day-old 35S:SMXL2-GFP transgenic 1209
plants after 2 µM GR244DO
, GR24ent-5DS
or KAR1 treatment for the time indicated. 1210
(B) GFP fluorescence in single plane of fixed focus in root tips of 7-day-old 1211
35S:SMXL2-GFP transgenic plants treated with 2 µM GR244DO
or GR24ent-5DS
in 0.5× 1212
MS liquid medium for the time indicated. Bar, 30 μm. 1213
(C) Relative abundance of SMXL2-GFP in root tips was determined by fluorescence 1214
measurement at the time indicated after 2 µM GR244DO
or GR24ent-5DS
treatment, with the 1215
zero-time signal set as 1.00. Values are means ± SD. Fluorescence signal after GR244DO
1216
or GR24ent-5DS
treatment was compared with that of mock treatment at indicated time. 1217
**P < 0.01 (n = 12 nuclei, two-tailed Student’s t-test; Supplemental Data set 1). 1218
(D) Degradation of SMXL2-GFP proteins in wild-type (Col-0), d14-1 and kai2-2 1219
Arabidopsis seedlings containing 35S:SMXL2-GFP transgene. Seedlings were treated 1220
after 7 days growth with 2 µM GR244DO
or GR24ent-5DS
in 0.5× MS liquid medium for the 1221
time indicated. 1222
In (A and D), proteins were detected by immunoblotting with anti-GFP monoclonal 1223
antibody. Relative abundances of SMXL2-GFP were determined by densitometry and 1224
normalized to loadings determined by Ponceau staining (red), with the zero-time signal 1225
set as 1.00. All experiments were repeated at least three times with similar results. 1226
1227
Figure 6. Ubiquitination and degradation of SMXL2-GFP depends on the RGKT motif. 1228
(A to C) Ubiquitination of SMXL2-GFP and SMXL2D-GFP in wild-type seedlings 1229
containing 35S:SMXL2-GFP or 35S:SMXL2D-GFP transgene. Seedlings were treated 1230
after 7 d growth with 50 µM MG132 for 1 h, then with 2 µM GR244DO
for 20 min (A), 2 1231
46
µM GR24ent-5DS
for 20 min (B), or 10 µM KAR1 for 1 h (C) in 0.5× MS liquid medium. 1232
(D and E) Levels of SMXL2-GFP and SMXL2D-GFP proteins in wild-type seedlings 1233
containing 35S:SMXL2-GFP or 35S:SMXL2D-GFP transgene. Seedlings were treated 1234
after 7 days growth with 2 µM GR244DO
, GR24ent-5DS
(D), or 10 µM KAR1 (E) in 0.5× 1235
MS liquid medium for the time indicated. 1236
(F) GFP fluorescence in single plane of fixed focus in root tip of 7-day-old 1237
35S:SMXL2D-GFP transgenic plants treated with 2 µM GR244DO
or GR24ent-5DS
in 0.5× 1238
MS liquid medium for the time indicated. Bar, 30 μm. 1239
(G) Relative abundance of SMXL2D-GFP in root tips was determined by fluorescence 1240
measurements at the time indicated after 2 µM GR244DO
or GR24ent-5DS
treatment, with 1241
the zero-time signal set as 1.00. Values are means ± SD. Fluorescence signal after 1242
GR244DO
or GR24ent-5DS
treatment was compared with that of mock treatment at indicated 1243
time. ns, no significance (n = 12 nuclei, two-tailed Student’s t-test; Supplemental Data set 1244
1). 1245
In (A to C), proteins were detected by immunoblotting with anti-ubiquitin (Ubi) 1246
polyclonal antibody or anti-GFP monoclonal antibody. In (D and E), proteins were 1247
detected by immunoblotting with anti-GFP monoclonal antibody. Relative abundances of 1248
SMXL2-GFP were determined by densitometry and normalized to loadings determined 1249
by Ponceau staining (red), with the zero-time signal set as 1.00. All experiments were 1250
repeated at least three times with similar results. 1251
1252
Figure 7. Hypocotyl response to GR244DO
, GR24ent-5DS
, or GR244DO
plus GR24ent-5DS
. 1253
(A) Seedlings of Col-0 (wild type), smax1, smxl2, smax1 smxl2, and smxl6,7,8 triple 1254
mutant were grown on 0.5× MS media containing 100 nM of each compound for 4 d 1255
under continuous red light at 21 ºC. Values are means ± SD. Different letters indicate 1256
significant difference P < 0.05 (n = 20 different plants, Two-way ANOVA multiple 1257
comparison, Tukey test, three independent experiments; Supplemental Data set 1). 1258
47
(B) Seedlings of Col-0 (wild type), 35S:SMXL2D-GFP/Col-0, and 1259
35S:SMXL6D-GFP/Col-0 were grown on 0.5× MS media containing 100 nM of each 1260
compound for 4 d under continuous red light at 21 ºC. Values are means ± SD. Different 1261
letters indicate significant difference P < 0.05 (n = 15 different plants, Two-way ANOVA 1262
multiple comparison, Tukey test, three independent experiments; Supplemental Data set 1263
1). 1264
(C) Seedlings of 35S:SMXL2-GFP/Col-0, 35S:SMXL2-GFP/d14-1, and 1265
35S:SMXL2-GFP/kai2-2 were grown on 0.5× MS media containing 100 nM of each 1266
compound for 4 d under continuous red light at 21 ºC. Values are means ± SD. Different 1267
letters indicate significant difference P < 0.05 (n = 15 different plants, Two-way ANOVA 1268
multiple comparison, Tukey test, three independent experiments; Supplemental Data set 1269
1). 1270
1271
Figure 8. SMXL2 is required for induction of DLK2 and KUF1 by signaling of SL and 1272
KAR in seedlings. 1273
(A) Relative expression of DLK2, KUF1 and MAX4 in 10-day-old seedlings of Col-0 1274
(wild type), smxl6,7,8 triple mutant, smax1, smxl2, and smax1 smxl2 double mutant. 1275
(B) Relative expression of DLK2 and KUF1 in 10-day-old seedlings of Col-0 (wild type), 1276
max1, and max4. 1277
(C) Relative expression of DLK2 and KUF1 in 10-day-old seedlings of Col-0 (wild type), 1278
smxl6,7,8 triple mutant, smax1, smxl2, and smax1 smxl2 double mutant treated with 1 μM 1279
GR244DO
or GR24ent-5DS
for 4 h. 1280
(D) Relative expression of DLK2 and KUF1 in 10-day-old seedlings of Col-0 (wild type), 1281
35S:SMXL2-GFP and 35S:SMXL2D-GFP treated with 1 μM GR244DO
or GR24ent-5DS
for 1282
4 h. 1283
(E) Schematic representation of SL and KAR-regulated gene expression mediated by 1284
SMAX1 and SMXL2. 1285
48
In (A to D), expression values are determined relative to ACTIN2. Values are means ± SD. 1286
*P < 0.05; **P < 0.01; ns, no significance (n = 3, two-tailed Student’s t-test, three 1287
independent experiments; Supplemental Data set 1). 1288
1289
Figure 9. Roles of SMXLs in the control of Arabidopsis development by KAR and SL 1290
signaling. 1291
KARs trigger association of KAI2 with SMXL2 and MAX2, leading to ubiquitination 1292
and degradation of SMXL2. KARs also trigger interaction between KAI2 and SMAX1 1293
and are proposed to trigger ubiquitination and degradation of SMAX1. SLs induce 1294
formation of the D14-MAX2-SMXL2, D14-MAX2-SMXL6, D14-MAX2-SMXL7, and 1295
D14-MAX2-SMXL8 complexes, and subsequent ubiquitination and degradation of 1296
SMXL2, SMXL6, SMXL7, and SMXL8. Degradation of SMXL2 represses hypocotyl 1297
elongation and induces the expression of DLK2 and KUF1, and the same functions are 1298
proposed for SMAX1. By contrast, degradation of SMXL6, SMXL7, and SMXL8 1299
represses shoot branching and MAX4 expression. SMXL2 is therefore a common target of 1300
proteolysis in SL and KAR signaling and functions to regulate hypocotyl elongation and 1301
gene expression in Arabidopsis thaliana. U, ubiquitin. 1302
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DOI 10.1105/tpc.20.00140; originally published online April 30, 2020;Plant Cell
Wang, Steven M Smith, Jiayang Li, Guosheng Xiong and Bing WangLei Wang, Qian Xu, Hong Yu, Haiyan Ma, Xiaoqiang Li, Jun Yang, Jinfang Chu, Qi Xie, Yonghong
Regulate Hypocotyl Elongation in Arabidopsis thalianaStrigolactone and Karrikin Signaling Pathways Elicit Ubiquitination and Proteolysis of SMXL2 to
This information is current as of August 18, 2020
Supplemental Data /content/suppl/2020/04/30/tpc.20.00140.DC1.html
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