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RESEARCH ARTICLE
The E3 Ligase DROUGHT HYPERSENSITIVE Negatively
Regulates Cuticular Wax Biosynthesis by Promoting the Degradation
of Transcription Factor ROC4 in Rice Zhenyu Wang a, Xiaojie Tian a,b , Qingzhen Zhao c, Zhiqi Liu d, Xiufeng Li a, Yuekun Ren a,b, Jiaqi Tang a,b, Jun Fang a , Qijiang Xu d, and Qingyun Bu a,1
a Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China b Graduate University of Chinese Academy of Sciences, Beijing 100049, China c School of Life Sciences, Liaocheng University, Liaocheng 252000, China d School of Life Sciences, Northeast Forestry University, Harbin 150040, China 1 Corresponding author: [email protected] Short title: DHS regulates cuticular wax and drought response One-sentence summary: The RING-type E3 ligase DHS cooperates with its putative ubiquitination substrate, the HD-ZIP transcription factor ROC4, to fine-tune wax biosynthesis and the drought stress response in rice. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Qingyun Bu ([email protected]). ABSTRACT Cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzyme-encoding genes and transcription factors involved in wax biosynthesis have been identified, but the underlying posttranslational regulatory mechanisms are poorly understood. Here, we demonstrate that DROUGHT HYPERSENSITIVE (DHS), encoding a Really Interesting New Gene (RING)-type protein, is a critical regulator of wax biosynthesis in rice (Oryza sativa). The cuticular wax contents were significantly reduced in DHS overexpression plants but increased in dhs mutants compared to the wild type, which resulted in a response opposite that of drought stress. DHS exhibited E3 ubiquitin ligase activity and interacted with the homeodomain-leucine zipper IV protein ROC4. Analysis of ROC4 overexpression plants and roc4 mutants indicated that ROC4 positively regulates cuticular wax biosynthesis and the drought stress response. ROC4 is ubiquitinated in vivo and subjected to ubiquitin/26S proteasome (UPS)-mediated degradation. ROC4 degradation was promoted by DHS but delayed in dhs mutants. ROC4 acts
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downstream of DHS, and Os-BDG is a direct downstream target of the DHS-ROC4 cascade. These results suggest a mechanism whereby DHS negatively regulates wax biosynthesis by promoting the degradation of ROC4, and they suggest that DHS and ROC4 are valuable targets for the engineering of drought-tolerant rice cultivars. INTRODUCTION 1
Rice (Oryza sativa) is a staple food for more than half of the global population. 2
However, its production is threatened by drought stress, and water resources have 3
become one of the major limiting factors for rice production due to increased 4
industrialization and water pollution (Kumar et al., 2014). Fortunately, rice has 5
evolved various strategies to cope with drought stress. Among these strategies, 6
cuticular wax provides an essential barrier for decreasing nonstomatal water loss 7
during drought stress and enhancing cuticular wax contents, thereby markedly 8
increasing drought tolerance in rice (Wang et al., 2012; Zhu and Xiong, 2013). 9
Cuticular wax is mainly composed of very-long-chain fatty acids (VLCFAs) and 10
their derivatives (including aldehydes, alcohols, alkanes, ketones, and wax esters) 11
with chain lengths ranging from C20 to C34 (Haslam and Kunst, 2013). Over the past 12
few decades, increasing numbers of genes controlling cuticular wax biosynthesis have 13
been identified via the characterization of eceriferum (cer) mutants in Arabidopsis 14
thaliana and reverse genetics approaches (McNevin et al., 1993; Greer et al., 2007), 15
and the associated biosynthetic processes have been uncovered (Yeats and Rose, 2013; 16
Borisjuk et al., 2014). Briefly, wax biosynthesis begins with a de novo C16 or C18 17
fatty acid, which is converted to C16 or C18 acyl-CoA by a long-chain 18
acyl-coenzyme A synthase (LACS) and is then used as a substrate for the fatty acid 19
elongase (FAE) complex, including β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA 20
reductase (KCR), β-hydroxy acyl-CoA dehydratase (HCD), and enoyl-CoA reductase 21
(ECR). Through a series of enzyme catalysis steps, two carbons per cycle are 22
successively added to produce VLCFAs-CoA. Finally, the resulting VLCFAs-CoAs 23
are further modified to yield various derivatives, such as primary alcohols, aldehydes, 24
and alkanes (Yeats and Rose, 2013). Several wax biosynthesis genes have been 25
identified in rice through characterizing wax crystal-sparse leaf (wsl) mutants. 26
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Mutations in these genes result in markedly reduced cuticular wax contents and 27
drought-sensitive phenotypes (Zhang et al., 2005; Yu et al., 2008; Mao et al., 2012; 28
Wang et al., 2012; Zhu and Xiong, 2013; Gan et al., 2016; Wang et al., 2017). 29
Some transcription factors have also been shown to control wax biosynthesis by 30
regulating the expression of downstream wax biosynthesis genes (Borisjuk et al., 31
2014). For example, APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/EFR) 32
family members, including WAX INDUCER1 (WIN1)/SHINE1 (SHN1) in 33
Arabidopsis, rice wax biosynthesis regulatory proteins Os-WR1 to Os-WR4, and 34
Medicago truncatula WAX PRODUCTION1 (WPX1) positively regulate wax 35
biosynthesis by directly promoting the expression of wax and cutin biosynthesis genes 36
(Broun et al., 2004; Zhang et al., 2005; Wang et al., 2012; Zhou et al., 2014). In 37
contrast, another AP2 protein in Arabidopsis, DECREASE WAX BIOSYNTHESIS 38
(DEWAX), functions as a transcriptional repressor of wax biosynthesis and 39
negatively regulates wax loads (Go et al., 2014). Several MYB family proteins are 40
also involved in cuticular wax biosynthesis and deposition via different mechanisms. 41
MYB16 and MYB106 regulate cutin biosynthesis in a similar manner to WIN1, 42
whereas MYB30 and MYB96 mediate pathogen and drought-induced wax 43
biosynthesis (Raffaele et al., 2008; Seo et al., 2011; Oshima et al., 2013). 44
Another important cuticular wax regulator is the homeodomain-leucine zipper IV 45
(HD-ZIP IV) family of transcription factors, which contain a conserved homeodomain 46
(HD) associated with a Leu zipper domain (ZIP), a steroidogenic acute 47
regulatory-related lipid transfer domain (START), and an HD-START-associated 48
domain (Nakamura et al., 2006; Ariel et al., 2007). Arabidopsis HDG1, tomato 49
(Solanum lycopersicum) CUTIN DEFICIENT2 (CD2), and maize (Zea mays) 50
OUTER CELL LAYER 1 (OCL1) are highly homologous HD-ZIP IV members. 51
These proteins regulate cutin and wax biosynthesis by directing binding to the 52
conserved L1 box cis-element in the promoters of downstream target genes, including 53
wax biosynthesis genes BODYGUARD (BDG) and FIDDLEHEAD (FDH), LIPID 54
TRANSPORTER (LTP), and ATP BINDING CASSETTE (ABC) transporters involved 55
in the transport of wax (Isaacson et al., 2009; Javelle et al., 2010; Wu et al., 2011). 56
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However,the mechanism by which HD-ZIP IV proteins are involved in cuticular wax 57
biosynthesis is not clear. In addition, the posttranslational regulation of the HD-ZIP 58
IV protein remains unknown. 59
The ubiquitin/26S proteasome (UPS) pathway degrades ubiquitinated substrate 60
proteins and is extensively involved in various cellular processes. This pathway plays 61
key roles in diverse aspects of plant growth and development (Vierstra, 2009; Santner 62
and Estelle, 2010). The ubiquitination process is achieved through the sequential 63
action of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and 64
ubiquitin ligase E3. Really Interesting New Gene (RING) finger proteins, featuring 65
eight conserved Cys and His residues, are one the most important types of E3 ligases. 66
A wealth of studies have shown that RING finger proteins play key roles in plant 67
hormone signaling pathways, defense responses, and development processes (Bu et al., 68
2009; Ryu et al., 2010; Li et al., 2011; Liu and Stone, 2011; Park et al., 2012; Kim 69
and Kim, 2013; Zhang et al., 2015). Arabidopsis CER9, encoding a RING-variant 70
domain-containing protein, negatively regulates wax loads, although the E3 ligase 71
activity and mechanism have not yet been determined (Lu et al., 2012). However, the 72
roles of functional RING-type E3 ligases other than CER9 in regulating wax 73
biosynthesis are currently unclear. 74
Here, we report that DHS (DROUGHT HYPERSENSITIVE), a RING-type E3 75
ligase, regulates rice wax biosynthesis by controlling the protein stability of ROC4 76
(an HD-ZIP IV family member) via the UPS and consequently influences the drought 77
stress response. The overexpression of DHS resulted in markedly reduced wax loads 78
and strikingly drought-hypersensitive phenotypes, whereas dhs mutants showed 79
increased wax contents and enhanced drought tolerance. In addition, we found that 80
DHS possesses E3 ligase activity, interacts with ROC4, and promotes the degradation 81
of ROC4. Moreover, analysis of ROC4 overexpression plants and roc4 mutants 82
indicated that ROC4 positively regulates the wax biosynthesis and drought stress 83
response. More importantly, we discovered that ROC4 genetically acts downstream of 84
DHS. Furthermore, Os-BDG, which might control wax biosynthesis, was identified as 85
the direct target of the DHS-ROC4 cascade. Collectively, these findings demonstrate 86
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that the E3 ligase DHS cooperates with its putative ubiquitination substrate, ROC4, to 87
fine-tune wax biosynthesis and the drought stress response in rice, thereby providing 88
valuable targets for breeding drought-tolerant rice cultivars. 89
90 RESULTS 91 92
Overexpression of DHS in rice confers drought hypersensitivity 93
To identify the critical genes involved in the drought stress response in rice, we 94
screened a rice mutant library in which various transcription factor genes were 95
overexpressed. One mutant carrying LOC_Os02g45780 exhibited a strikingly 96
drought-hypersensitive phenotype and thus was selected for further analysis. 97
LOC_Os02g45780 was thus named DHS (DROUGHT HYPERSENSITIVE). 98
DHS overexpression (DHS OE) plantlets grew more slowly than WT (wild-type, 99
transformed with empty vector). After 30 d growth in regeneration culture medium, 100
DHS OE was significantly smaller than WT, exhibiting a dwarfed plant height and 101
shorter leaves (Supplemental Figure 1A–1C). The leaves of DHS OE were lighter 102
green than WT (Supplemental Figure 2A). The most obvious phenotype of DHS OE 103
was that its leaves wilted rapidly (Figure 1A, 1B). Once DHS OE seedlings were 104
removed from culture bottles and transplanted into the soil, the leaves of DHS began 105
to roll within an hour, the leaf tips turned yellow in around 3 d, and the seedlings 106
stopped growing and died gradually within 1 month (Figure 1A, 1B), which was in 107
contrast to the normal growth of WT. To confirm the strikingly 108
drought-hypersensitive phenotype of DHS OE, we analyzed the water loss rates of 109
detached leaves and discovered that DHS OE lost water much more rapidly than WT 110
(Figure 1C). As most of the independent DHS OE plants displayed similar phenotypes, 111
and the transcript level of LOC_Os02g45780 was indeed markedly increased in DHS 112
OE plants (Supplemental Figure 1D), we hypothesized that the overexpression of 113
DHS confers drought hypersensitivity in rice. 114
Plants lose water mainly via their stomata (Schroeder et al., 2001; Nilson and 115
Assmann, 2007). A preliminary examination of the stomatal density, however, 116
showed that the average stomatal density was comparable between the WT and DHS 117
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OE, which implied that stomatal water loss might be normal in DHS OE 118
(Supplemental Figure 1E). 119
Cuticular wax is the outermost membrane that protects plants from water loss, 120
and the content and structure of the waxes are closely associated with water loss rate 121
(Lu et al., 2012; Zhu and Xiong, 2013). The light green color of DHS OE is also 122
reminiscent of some rice cuticular wax-deficient mutants (Supplemental Figure 2A) 123
(Yu et al., 2008; Mao et al., 2012; Wang et al., 2017). To examine whether the 124
cuticular wax in DHS OE was defective, we performed several assays. First, we 125
soaked the rice seedlings in water and then removed them. Compared with WT, there 126
were more water drops on the leaves of DHS OE, suggesting that there may be less 127
cuticular wax in DHS OE than in WT (Supplemental Figure 2A). Second, 128
measurement of the membrane permeability demonstrated that the 129
chlorophyll-leaching rate in DHS OE was faster than that in WT (Supplemental 130
Figure 2B). Third, scanning electron microscopy (SEM) analysis revealed that the 131
platelet-like wax crystals on the leaf surface of DHS OE were sparser than those on 132
WT (Figure 1D, 1E). Fourth, ultrastructural analysis by transmission electron 133
microcopy (TEM) showed that the cuticle membranes in WT leaves were smooth and 134
contracted, in contrast to the loose, irregular, and vague membranes in DHS OE 135
leaves (Supplemental Figure 2C and 2D). Fifth, the compositions and contents of 136
cuticular waxes were analyzed by gas chromatography-mass spectrometry (GC-MS). 137
Compared with WT, the total wax loads in DHS OE were severely reduced (Figure 1F, 138
Supplemental Figure 2E). Together, these data suggest that the overexpression of 139
DHS disrupts the biosynthesis and development of cuticular wax, which results in 140
drought hypersensitivity in DHS OE. 141
To verify this notion, we generated dhs mutants via CRISPR/Cas9-mediated 142
genome editing and characterized three independent mutant alleles, dhs-1, dhs-2, and 143
dhs-3 (Supplemental Figure 3). Unlike DHS OE plants, the growth and development 144
of the dhs mutants were similar to WT, and they showed no visible phenotypes 145
(Figure 2B). Data from the GC-MS analysis, however, indicated that the cuticular 146
wax contents in dhs were increased compared with WT (Figure 2A, Supplemental 147
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Figure 4D). In addition, to some extent, the cuticular wax crystals in dhs were denser 148
than those in WT (Supplemental Figure 4A and 4B). In agreement with these results, 149
the chlorophyll-leaching rate of dhs was slower than that of WT (Supplemental Figure 150
4C). Together, these data suggest that the mutation of DHS leads to increased 151
cuticular wax biosynthesis. Compared with WT, the drought tolerance of dhs was 152
consistently significantly enhanced (Figure 2B–2D), along with a higher recovery rate 153
following dehydration treatment (Figure 2E) and a slower water loss rate (Figure 2F), 154
which further supports the notion that DHS plays negative roles in controlling wax 155
biosynthesis and consequently affects the drought stress response. 156
157 DHS has E3 ubiquitin ligase activity 158
DHS contains 165 amino acid residues as well as a predicted transmembrane 159
segment in the N-terminus and a conserved RING domain in the C-terminus of the 160
protein (Supplemental Figure 5). RING-type proteins always function as E3 ubiquitin 161
ligases (Stone et al., 2005; Bu et al., 2009; Li et al., 2011). To examine whether DHS 162
has E3 ligase activity, DHS fused with the maltose binding protein (MBP) was 163
expressed and used for an in vitro autoubiquitination assay. In the presence of E1, E2, 164
and the His-Ubiquitin protein, the MBP-DHS protein, similar to the MBP-tagged 165
ubiquitin ligase positive control MBP-CIP8 (Hardtke et al., 2002), showed clear 166
autoubiquitination, suggesting that DHS exhibits E3 ligase activity in vitro (Figure 167
3A). In contrast, when E1 or E2 were omitted, we did not detect any ubiquitination 168
(Figure 3A). In addition, an ubiquitination assay in Escherichia coli also showed that 169
DHS has E3 ligase activity (Supplemental Figure 6). Conserved Cys and His residues 170
in the RING domain are critical for E3 ligase activity (Bu et al., 2009; Li et al., 2011). 171
Thus, the mutated protein DHSC95S (Cys-95 in the RING domain was mutated to 172
Ser-95) was expressed and its activity was examined (Supplemental Figure 7A). We 173
did not detect any ubiquitination in DHSC95S (Figure 3A), suggesting that the 174
conserved RING domain is indispensable for the E3 ligase activity of DHS. 175
Furthermore, we generated DHSC95S overexpression plants (DHSC95S OE). Unlike 176
DHS-OE, DHSC95S OE was similar to WT in terms of growth speed and plant 177
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architecture (Supplemental Figure 7B). In addition, the cuticular wax contents and 178
drought stress response of DHSC95S OE were also comparable with WT (Supplemental 179
Figure 7C–7F). Taken together, these results indicate that DHS is an active E3 ligase 180
and that its E3 ligase activity is necessary for its biological function. 181
182 DHS physically interacts with ROC4 183
In general, RING-type E3 ligases function by ubiquitinating target proteins and 184
triggering their degradation via the 26S proteasome (Zhang et al., 2005; Dong et al., 185
2006; Qin et al., 2008). To reveal the role of DHS in the regulation of wax 186
biosynthesis, we attempted to identify the ubiquitination target protein of DHS. As 187
described previously, DHS functions as a negative regulator of wax biosynthesis 188
(Figure 1 and 2), and we speculated that the ubiquitinated target of DHS might be a 189
positive regulator of the cuticular wax pathway. Several transcription factors thus far 190
have been shown to play positive roles in regulating wax biosynthesis in Arabidopsis, 191
rice, tomato, and maize, including the HD-ZIP IV family (HDG1, OCL1, CD2), 192
AP2/EFR family (WIN1, Os-WR1, and Os-WR2), and MYB family (MYB16, 193
MYB106, MYB30, and MYB96; (Broun et al., 2004; Raffaele et al., 2008; Javelle et 194
al., 2010; Seo et al., 2011; Wang et al., 2012). To preliminarily screen for the possible 195
interaction partner of DHS, we used the protein sequences of the above mentioned 196
transcription factors as queries for BLAST analysis against the rice protein database, 197
and we chose the corresponding rice homologs for further analysis (Supplemental 198
Figure 8). First, we performed a yeast two-hybrid assay to examine the possible 199
interaction between DHS and the homologs in rice. We discovered that the HD-ZIP 200
IV family member ROC4 interacted with DHS (Figure 3B), whereas the homologs of 201
the AP2/EFR and MYB family members did not (Supplemental Figure 8). Second, we 202
confirmed the physical interaction between DHS and ROC4 using an in vitro 203
pull-down assay (Figure 3C). Third, an in planta luciferase complementation imaging 204
assay also showed that the co-expression of DHS with ROC4 generated strong 205
luminescence signals that were not detected in the control pairs (Figure 3D). 206
Collectively, these results suggest that DHS physically interacts with ROC4. 207
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208
ROC4 positively regulates wax development 209
ROC4, containing 813 amino acid residues, contains a typical homeobox 210
domain and a SMART domain and belongs to the HD-ZIP IV family (Supplemental 211
Figure 9). As the homologs of ROC4 in Arabidopsis, maize and tomato have been 212
shown to be involved in cuticular wax development (Isaacson et al., 2009; Javelle et 213
al., 2010; Wu et al., 2011), we investigated whether ROC4 regulates wax biosynthesis 214
in rice. For this purpose, we generated roc4 mutants (Supplemental Figure 10) and 215
ROC4 OE plants (overexpressing GFP-fused ROC4) (Supplemental Figure 11A and 216
11B). GC-MS analysis showed that there were more cuticular waxes in ROC4 OE 217
plants, but fewer in the roc4 mutant compared with WT (Figure 4A, Supplemental 218
Figure 11G). In addition, SEM analysis showed that the wax crystals were dense in 219
ROC4 OE, but they were obviously sparser in the roc4 mutant compared with WT 220
(Supplemental Figure 11C–11E). Accordingly, the chlorophyll-leaching rate was 221
slower in ROC4 OE but faster in roc4 (Supplemental Figure 11F). In agreement with 222
these finding, ROC4 OE exhibited drought tolerance, whereas roc4 was drought 223
sensitive compared with WT (Figure 4B–4E), and this result was further supported by 224
the data from the water loss assay (Figure 4F). These data thus strongly suggest that 225
ROC4 positively regulates wax biosynthesis and the corresponding drought stress 226
response. 227
228
ROC4 is subjected to UPS-dependent degradation 229
As described earlier, DHS and ROC4 physically interact with each other and 230
play opposite roles in the control of wax biosynthesis. In addition, DHS exhibits E3 231
ligase activity. These findings suggest that ROC4 might be a ubiquitination target of 232
DHS. If this is true, ROC4 protein might be unstable and modified by ubiquitination. 233
To test this hypothesis, we examined the protein stability of ROC4 in a cell-free 234
degradation assay, which indicated that ROC4 protein was unstable and degraded 235
rapidly (Figure 5A, 5B). In addition, we treated ROC4 OE callus with the protein 236
synthesis inhibitor cycloheximide (CHX) or the proteasome inhibitor MG132 for 4 h 237
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and assessed the level of ROC4 protein by immunoblot analysis. The level of ROC4 238
protein decreased markedly when treated with CHX, whereas the addition of MG132 239
efficiently blocked ROC4 degradation (Figure 5C, 5D). To confirm this result, we 240
used 2-week-old ROC4 OE seedlings to observe the green fluorescent protein (GFP) 241
fluorescence. Confocal microscopy observation showed that ROC4 was localized to 242
the nucleus, and the GFP fluorescence was enhanced by MG132 treatment but 243
reduced by CHX treatment (Figure 5E), indicating that the degradation of ROC4 244
occurs via the UPS. Additionally, ROC4 protein was immunoprecipitated from ROC4 245
OE callus using an anti-ROC4 antibody and was probed with anti-ubiquitin and 246
anti-ROC4. The higher-molecular-weight smear bands in the protein gel blot 247
indicated that ROC4 is indeed polyubiquitinated in vivo (Figure 5F). Together, these 248
data suggest that ROC4 is subjected to proteasome-mediated degradation. 249
250
DHS promotes UPS-dependent degradation of ROC4 251
We further investigated whether DHS promotes UPS-mediated degradation of 252
ROC4. First, we examined the degradation speed of ROC4 in a cell-free degradation 253
assay. For this experiment, we used calli from WT and DHS OE plants at the same 254
growth stage. Crude ROC4 protein extracted from ROC4 OE callus was divided into 255
several aliquots, and each aliquot was mixed with equal amounts of WT and DHS OE 256
protein extract. Following incubation at room temperature for the indicated time, the 257
reaction was stopped and ROC4 protein levels were examined by protein gel blot 258
analysis. This assay indicated that ROC4 degraded over time, and importantly, the 259
degradation speed of ROC4 combined with DHS OE was faster than that combined 260
with WT (Figure 6A). In contrast, a similar assay showed that the degradation speed 261
of ROC4 combined with dhs was slower than that combined with WT (Figure 6A). 262
Quantification and statistical analysis of ROC4 degradation speed from three 263
independent experiments demonstrated that the degradation of ROC4 was promoted in 264
DHS OE but was delayed in dhs (Figure 6B). Second, we transiently co-expressed 265
ROC4 with DHS or DHSC95S in a rice protoplast system. Protein gel blot analysis 266
showed that the protein level of ROC4 in the presence of DHS was much lower than 267
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that in the absence of DHS (Figure 6C, 6D). In contrast, the co-expression of DHSC95S 268
did not significantly affect ROC4 protein level (Figure 6C, 6D). Third, we examined 269
the protein level of ROC4 in different dhs allelic mutants and we found that the dhs 270
mutants accumulated more ROC4 protein than WT (Figure 6E). As a control, ROC4 271
transcript levels were similar between the dhs mutants and WT (Figure 6F). 272
Collectively, these data suggest that DHS promotes the degradation of ROC4, 273
suggesting that ROC4 is the ubiquitination target of DHS. 274
275
ROC4 genetically acts downstream of DHS 276
To examine whether ROC4 is the ubiquitinated target of DHS genetically, we 277
generated the dhs roc4 double mutant by crossing dhs-1 with roc4-1 and subjected it 278
to phenotypic analysis. SEM analysis showed that the cuticular wax in dhs roc4 was 279
sparse, as observed in roc4 (Supplemental Figure 12A–12D). In addition, GC-MS 280
analysis showed that the wax contents in dhs roc4 were lower than in dhs-1 (Figure 281
7A, Supplemental Figure 12F). Accordingly, the slower chlorophyll-leaching rate in 282
dhs-1 also was suppressed in dhs roc4 (Supplemental Figure 12E). Furthermore, the 283
results of both the water loss assay and drought stress assay demonstrated that the 284
drought tolerance of dhs roc4 was similar to that of the roc4 single mutant and was 285
significantly weaker than that of dhs-1 (Figure 7B–7F). Collectively, these data 286
clearly demonstrate that the accumulation of ROC4 is required for the dhs mutant 287
phenotype, and they support the notion that ROC4 acts downstream of DHS in 288
controlling wax biosynthesis and the corresponding drought stress response. 289
290
Os-BDG is a direct target of the DHS-ROC4 cascade 291
As described above, ROC4 acts downstream of DHS in controlling wax 292
biosynthesis. We investigated how the DHS-ROC4 cascade is involved in this 293
pathway. In Arabidopsis, BDG plays an important role in cuticular development, and 294
HDG regulates the expression of BDG by direct binding to the L1 box region in its 295
promoter (Kurdyukov et al., 2006; Wu et al., 2011). Sequence alignment 296
demonstrated that there are three homologs of Arabidopsis BDG in rice 297
12
(Supplemental Figure 13), and there are two conserved L1 boxes in the promoter 298
region of Os-BDG (LOC_Os06g04169) (Figure 8A). To investigate whether the 299
Os-BDG is direct target of ROC4, we conducted an electrophoretic mobility shift 300
assay (EMSA). ROC4 protein fused to His tag (His-ROC4) was found to bind to the 301
biotin-labeled Os-BDG promoter in an L1 box-dependent manner (Figure 8B). To 302
verify this result in vivo, we performed a chromatin immunoprecipitation (ChIP) assay 303
using ROC4 OE plants. ROC4-bound fragments enriched by immunoprecipitation 304
with anti-ROC4 antibody were used for quantitative PCR. We found that the L1 305
box-containing fragment in the Os-BDG promoter was significantly enriched in ROC4 306
OE plants (Figure 8C). Moreover, the transient expression assay in rice protoplasts 307
showed that ROC4 markedly activated the expression of Os-BDG (Figure 8D). More 308
important, the expression level of Os-BDG was significantly reduced when ROC4 309
was co-expressed with DHS, but not when co-expressed with DHSC95S (Figure 8D). 310
Furthermore, analysis of the expression of Os-BDG in ROC4 OE and roc4 showed 311
that Os-BDG expression was enhanced in ROC4 OE and reduced in roc4 (Figure 8E). 312
Together, these results indicate that Os-BDG is a direct target of ROC4. Because we 313
showed that DHS acts upstream of ROC4 genetically and negatively regulates ROC4 314
protein stability, we also analyzed the expression of Os-BDG in dhs. As shown in 315
Figure 8E, the expression of Os-BDG was higher in dhs than in WT. More important, 316
the increased expression of Os-BDG in dhs was suppressed in the dhs roc4 double 317
mutant (Figure 8E). The differential expression of Os-BDG in dhs, roc4, and the dhs 318
roc4 double mutant suggested that the DHS-ROC4 cascade regulates the wax 319
biosynthesis pathway by controlling the expression of Os-BDG. In conclusion, we 320
propose a working model in which DHS negatively regulates wax biosynthesis and 321
the drought stress response by promoting the degradation of ROC4, which directly 322
regulates the expression of the downstream target gene, Os-BDG (Figure 8F). 323
324
DISCUSSION 325
Cuticular wax plays crucial roles in protecting plants from environmental 326
stresses. In particular, increasing cuticular wax contents can reduce nonstomatal water 327
13
loss in plants, thereby improving drought tolerance. Many catalytic enzyme-encoding 328
genes and transcription factors involved in the wax biosynthesis pathway have been 329
characterized in plants (Broun et al., 2004; Raffaele et al., 2008; Yu et al., 2008; 330
Javelle et al., 2010; Seo et al., 2011; Mao et al., 2012; Nadakuduti et al., 2012; 331
Haslam and Kunst, 2013; Oshima et al., 2013; Borisjuk et al., 2014; Zhou et al., 2014; 332
Gan et al., 2016; Wang et al., 2017). In contrast, there are few reports regarding the 333
posttranslational regulation of wax biosynthesis-related transcription factors. 334
In this study, we characterized the RING-type E3 ligase DHS as a negative 335
regulator of cuticular wax biosynthesis and drought tolerance (Figure 1 and 2). We 336
also revealed that DHS interacts with the HD-ZIP IV transcription factor ROC4 337
(Figure 3, Supplemental File 1). We found that ROC4, in contrast to DHS, positively 338
regulates wax deposition and drought tolerance (Figure 4). In addition, ROC4 was 339
found to be an unstable protein, and DHS promoted the UPS-mediated degradation of 340
ROC4 (Figure 5 and 6). Moreover, we demonstrated that ROC4 acts genetically 341
downstream of DHS in controlling wax biosynthesis and the drought stress response 342
(Figure 7). Furthermore, we identified Os-BDG as a direct downstream target of 343
ROC4 and proposed that the DHS-ROC4 cascade regulates the wax biosynthesis 344
pathway by controlling the expression of Os-BDG (Figure 8). Although these data do 345
not demonstrate that DHS directly ubiquitinates ROC4, the combined physiological, 346
biochemical, and genetic data presented here strongly suggest that DHS is involved in 347
the ubiquitination and subsequent degradation of ROC4, and the data establish that 348
the DHS-ROC4 module regulates the drought stress response by controlling wax 349
biosynthesis in rice. 350
The protein structure of DHS is simple, consisting of 165 amino acid residues 351
and a transmembrane domain in the N-terminus and a RING domain in the 352
C-terminus (Supplemental Figure 5A). Using the DHS as a query for BLAST analysis 353
in the Arabidopsis protein database, we discovered a subgroup of RING-type proteins 354
(including RHA2a, RHA2b, and XERICO) that displayed homology to DHS to 355
various extents (Supplemental Figure 5B, 5C, Supplemental File 2). These RING 356
proteins play common and critical roles in the drought stress response and abscisic 357
14
acid (ABA) signaling (Ko et al., 2006; Bu et al., 2009; Li et al., 2011), although the 358
direct targets and mechanisms are not well known. Previous and current results 359
suggest that these simple-structured RING-type proteins might play major roles in the 360
abiotic stress response. It would be interesting to examine whether DHS is involved in 361
the ABA signaling response in the future. 362
In this study, we characterized DHS as a negative regulator of cuticular wax 363
biosynthesis. Similarly, the Arabidopsis RING-type protein CER9 also functions in 364
controlling wax biosynthesis (Lu et al., 2012). The underlying mechanisms of CER9 365
and DHS might differ, however. In the cer9 mutant, C22-C26 VLCFAs contents are 366
elevated, which contributes to the elevated total wax contents and enhanced drought 367
tolerance, although the contents of VLCFAs derivatives (aldehyde, alcohol, and 368
alkanes) are reduced (Lu et al., 2012). By contrast, nearly all wax composition 369
contents were reduced in DHS-OE plants, but they were increased in dhs compared to 370
WT (Figure 1F and 2A). In addition, the lower transpiration rate and improved water 371
use efficiency in the cer9 mutant also contributes to improved drought tolerance (Lu 372
et al., 2012). Moreover, the CER9 sequence is highly similar to that of Doa10, an 373
ERAD (ER-associated degradation) component. It is thought that CER9 might be 374
involved in ERAD, by which many wax biosynthesis enzymes are degraded (Lu et al., 375
2012). Future work should examine whether DHS is involved in the ERAD process. 376
Compared with dhs, roc4, and ROC4-OE, DHS-OE showed more severe changes 377
in wax loads and more strikingly drought-hypersensitive phenotypes. Because DHS 378
exhibits E3 ligase activity, we speculated that DHS might have multiple 379
ubiquitination targets in addition to ROC4 and that these targets might play redundant 380
or distinct roles, consequently contributing to the severe phenotypes of DHS-OE. 381
Supporting this notion, we discovered that DHS also interacts with ROC5, which 382
shares the highest sequence similarity with ROC4 (Supplemental Figure 8, 9, 383
Supplemental File 3). Further investigation is required to examine whether DHS 384
promotes the ubiquitination and degradation of ROC5 and whether ROC5 is involved 385
in wax biosynthesis and the relative drought stress response. In addition, we found 386
that ROC4 was still degraded far more slowly in the dhs mutant than in the WT 387
15
(Figure 6A and 6B), which implies that ROC4 might be ubiquitinated by other E3 388
ligases as well. 389
ROC4 belongs to rice HD-ZIP IV gene family, which contains nine members 390
(ROC1 to ROC9), with five members specifically expressed in the epidermis (Ito et 391
al., 2003). ROC5 controls leaf rolling by regulating bulliform cell number and size 392
(Zou et al., 2011), whereas the functions of other ROC members remain unknown. In 393
this study, we discovered that ROC4 positively regulates cuticular wax biosynthesis, 394
thereby influencing the relative drought stress response. These data point to divergent 395
functions among ROC members, which is not unexpected. For example, the 396
expression patterns of the majority of maize OCL genes encoding HD-ZIP IV proteins 397
are restricted to the epidermal and subepidermal layers of various organs (Ingram et 398
al., 2000). OCL4, however, controls anther and trichome development (Vernoud et al., 399
2009), whereas OCL1 is involved in root and kernel development (Khaled et al., 400
2005). Subsequently, through the identification and analysis of OCL1 targets, it was 401
shown that OCL1 also positively regulates cuticular wax biosynthesis by directly 402
modulating the expression of wax and lipid transporter genes (WBC11a and LtpII.12) 403
and the wax biosynthesis gene FAR1 (Javelle et al., 2010). Another possible role of 404
ROC4 in wax biosynthesis was obtained through the characterization of CFL1 405
(CURLY FLAG LEAF1), which controls both leaf rolling and cuticular wax 406
development (Wu et al., 2011). In CFL1 overexpression plants, cuticular wax contents 407
and cutin compositions are severely affected. In addition, HDG1, a homolog of ROC4, 408
was identified as the interaction partner of CFL1 and was found to be required for the 409
functioning of CFL1 (Wu et al., 2011). These findings suggest that HD-ZIP IV 410
proteins play multiple roles in various aspects of plant development and that different 411
members function in different developmental processes. 412
Rice plants have high water requirements, and drought has become a major 413
limiting factor for rice production due to water shortages. There is an urgent demand 414
for the breeding of drought-tolerant rice cultivars (Kumar et al., 2014). The 415
overexpression of Mt-WXP1, At-CER1, Os-WR1 and Os-WR2 results in elevated total 416
wax loads, reduced water loss, and less chlorophyll leaching, and consequently, 417
16
improved drought adaptability (Zhang et al., 2005; Bourdenx et al., 2011; Wang et al., 418
2012; Zhou et al., 2014). DWA1 (DROUGHT-INDUCED WAX 419
ACCUMULATION1) is a critical enzyme that positively controls drought-induced 420
wax production, and plants overexpressing DWA1 and dwa1 mutants exhibit clear 421
changes in wax contents and opposite drought responses (Zhu and Xiong, 2013). 422
These studies clearly indicate that drought tolerance could be improved by enhancing 423
cuticular wax deposition. In this study, we demonstrated that DHS and its putative 424
ubiquitination target, ROC4, are critical regulators of wax biosynthesis. More 425
important, dhs and ROC4 OE plants showed significantly enhanced drought tolerance, 426
suggesting that these genes are valuable targets for engineering drought-tolerant rice 427
cultivars. 428
429
METHODS 430
Plant materials and growth conditions 431
Rice cultivar Longjing 11 (O. sativa ssp. japonica) was used to generate the DHS 432
and ROC4 overexpression plants and knockout mutants. The seedlings were grown in 433
a growth chamber (white fluorescent tubes, 200-300 µmol m-2s-1) at 30°C for 14 h 434
(day) and 24°C for 10 h (night) at 70% humidity or in the field (natural long-day 435
conditions). 436
437
Generation of transgenic plants and mutants 438
The coding sequences of DHS and ROC4 were cloned from Nipponbare (Oryza 439
sativa ssp. japonica) cDNA using a standard reverse transcription (RT)-PCR protocol. 440
The full-length coding region of DHS was cloned into the binary vector pCAMBIA 441
1300-221-HA to generate a DHS overexpression vector in which DHS was driven by 442
the CaMV35S promoter. To produce the ROC4 overexpression construct, the 443
full-length coding region of ROC4 was cloned into pENTR/D-TOPO (Invitrogen, 444
Carlsbad, CA, USA) and subcloned into the binary vector pH7WGF2 by LR reaction 445
to generate 35Spro:GFP-ROC4. To generate the dhs and roc4 mutants, two and one 446
sgRNAs were designed to target DHS and ROC4, respectively. The sgRNA cassettes 447
17
were sequentially ligated into the CRISPR/Cas9 binary vectors 448
pYLCRISPR/Cas9Pubi-H (Ma et al., 2015). All primers used for these constructs are 449
listed in Supplemental Table 1. The constructs were introduced into Agrobacterium 450
tumefaciens strain EHA105, and rice cultivar Longjing11 was used as the recipient for 451
Agrobacterium-mediated transformation as described previously (Tian et al., 2015). 452
Homozygous T2 transgenic rice seedlings were used for phenotype analysis. 453
454
Total RNA isolation and RT-qPCR analysis 455
Total RNA was extracted using TRIzol (Invitrogen) and treated with DNaseI. 456
cDNA was synthesized from 2 µg of total RNA using Superscript II Reverse 457
Transcriptase (Invitrogen). RT-qPCR was performed with SYBR Green PCR master 458
mix (Takara, Okinawa, Japan). Data were collected using a Bio-Rad Chromo 4 459
Real-time PCR Detector. All expression levels were normalized against the ACTIN 460
gene (Os03g0718100). The primers used are listed at Supplemental Table 1. 461
462
Water loss assay 463
The water loss assay was performed as previously described with some 464
modifications (Tian et al., 2015) using 4-week-old rice seedlings grown in climate 465
chambers. Leaves at the same growth stages were detached from the plants, left on a 466
laboratory bench, and weighed at the indicated time points. Time-course analysis of 467
water loss was performed and represented as the percentage of initial fresh weight at 468
each time point. 469
470
Chlorophyll leaching assays 471
Chlorophyll leaching assays were used to measure the epidermal permeability of 472
rice leaves as described previously (Mao et al., 2012). The third leaf from the top was 473
sampled from 4-week-old seedlings. The leaf was cut into segments (~2 cm) and 474
immersed in 30 mL 80% ethanol at room temperature. Aliquots of 0.5 mL were 475
removed for chlorophyll quantification and returned to the same tube at the indicated 476
time point. The chlorophyll concentration was quantified using a Thermo 477
18
BIOMATE3 spectrophotometer at wavelengths of 664 and 647 nm. Chlorophyll 478
efflux at each time point was expressed as a percentage of total chlorophyll extracted 479
after 24 h of immersion. 480
481
Scanning and transmission electron microscopy 482
SEM was performed as previously described (Mao et al., 2012). Leaf blades 483
excised from 4-week-old plants were used for SEM analysis. Samples were pre-fixed 484
with 2.5% glutaraldehyde-sodium phosphate buffer (0.1 M) at room temperature and 485
post-fixed in 1% OsO4 at 4°C. The samples were dehydrated through an ethanol series 486
and dried with a critical point dryer. The dried samples were coated with platinum 487
using sputtering equipment and examined by scanning electron microscopy (SEM, 488
S-4800, Hitachi, Tokyo, Japan) at an accelerating voltage of 10 kV. For TEM, the 489
samples were processed as described previously (Mao et al., 2012). Mature expanded 490
leaves were cut into 3 × 1 mm segments between the midvein and the leaf margin. 491
Ultrathin sections (80 nm) were cut using an Ultracut E Ultramicrotome (Leica, 492
Wetzlar, Germany) and mounted on copper grids. The sections were stained with 493
uranyl acetate and lead citrate solution and observed by TEM (TEM; H-7650, 494
Hitachi). 495
496
Cuticular wax analysis 497
Cuticular wax was extracted and measured as described previously (Mao et al., 498
2012). Briefly, leaves of 8-week-old seedlings were immersed in 30 mL n-hexane at 499
67°C for 30 s, with 50 µg n-tetracosane as an internal standard. The n-hexane was 500
then evaporated under gaseous N2 and the residue was derivatized with 100 µL of 501
bis-N, N-(trimethylsilyl) trifluoroacetamide (BSTFA, Sigma, St. Louis, MO, USA) 502
and 100 µL of pyridine for 60 min at 70°C. All wax samples were analyzed with an 503
Agilent (Santa Clara, CA, USA) 7000C GC-MS/MS device on a 30 m HP-1MS 504
column. The column was operated with helium as the carrier gas and splitless 505
injection at 250°C. The oven temperature was increased from 50°C to 200°C at 20°C 506
min-1, held for 2 min at 200°C, increased at 2°C min-1 to 320°C, and held at 320°C for 507
19
14 min. The total amount of cuticular wax was expressed per unit area of the leaf 508
surface. Leaf area was measured using an LI-3000C Portable Area Meter (LI-COR 509
Biosciences, San Jose, CA, USA). 510
511
Multiple sequence alignments and phylogenetic analysis 512
Multiple sequence alignments were constructed using the ClustalX2 software. A 513
phylogenetic analysis was conducted by MEGA version 4.0 using the 514
neighbor-joining method with 1000 bootstrap replications. See alignments in 515
Supplemental Files 2–4. 516
Yeast two-hybrid assay 517
The coding sequence of DHS was cloned into the EcoR I and Pst I sites of the 518
pGBKT7 vector to generate the BD-DHS construct. The coding sequence of ROC4 519
was cloned into the EcoR I and Xho I sites of the pGADT7 vector to generate the 520
AD-ROC4 construct. The resulting constructs were transformed into yeast strain Y2H 521
Gold. The presence of the transgenes was confirmed by growth on an SD/-Leu/-Trp 522
plate. To assess protein interactions, the transformed yeast cells were suspended in 523
liquid SD/-Leu/-Trp to OD600 = 1.0. The suspended cells were spread on plates 524
containing SD/-His/-Leu/-Trp medium. Interactions were observed after 4 d of 525
incubation at 30°C. 526
527
Pull-down assay 528
The full-length coding region of ROC4 in pENTR/D-TOPO was subcloned into 529
the expression vector pDEST15 to generate the glutathione S-transferase 530
(GST)-ROC4 fusion vector. The coding region of DHS was ligated into the pMAL-c2x 531
vector (New England Biolabs, Ipswich, MA, USA) to generate the MBP-DHS 532
construct. The resulting vectors were transformed into E. coli strain BL21 (DE3) to 533
express the protein. The recombinant proteins MBP-DHS and GST-ROC4 were 534
affinity purified using amylose resin (BioLabs, E8021S) and glutathione Sepharose 535
4B beads (GE Healthcare), respectively. 536
For the in vitro pull-down assay, bacterial lysates containing ~2 µg of MBP-DHS, 537
20
~2 µg of GST-ROC4 fusion proteins, and amylose resin were added to pull-down 538
buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM 539
DTT) with continuous rocking at 4°C for 1 h. The beads were washed five times with 540
wash buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100), 541
and the pull-downed protein was separated by 10% SDS-PAGE and detected by 542
immunoblot analysis with anti-GST (1:5000; Abmart, M20007) and anti-MBP 543
antibodies (1:3000; CWBIO, CW0288), respectively. 544
545
Protein gel blot analysis 546
For the secondary antibody in the protein gel blot assay, peroxidase-labeled goat 547
anti-rabbit antibody (1:4000; Abcam, ab6789 XXX) or goat anti-mouse (1:4000; 548
Abcam, ab6721) was utilized. Membranes were developed with the SuperSignal West 549
Pico Chemiluminescent Substrate Kit (Pierce Biotechnology) and the signal was 550
detected by chemiluminescence imaging (Tanon 5200). 551
552
Luciferase complementation imaging assays 553
For the luciferase (LUC) complementation imaging assays, the coding regions of 554
DHS and ROC4 were ligated into pCAMBIA-nLUC and pCAMBIA-cLUC, 555
respectively, and the nLUC-DHS and cLUC-ROC4 constructs were generated. The 556
nLUC-/cLUC-derivative constructs were transformed into A. tumefaciens strain 557
GV3101. After overnight culture, the Agrobacteria were suspended in infiltration 558
buffer (0.2% MgCl2, 100 µM acetosyringone, and 10 mM MES) at OD600 = 1.0. 559
Equal volumes of Agrobacteria resuspension carrying the nLUC and cLUC derivative 560
constructs were mixed and co-infiltrated into N. benthamiana leaves. LUC activity in 561
infiltrated leaves was analyzed at 48 h after infiltration using chemiluminescence 562
imaging (Tanon 5200). 563
564
In vitro ubiquitination assay 565
Purified MBP-DHS and MBP-DHSC95S were used for the ubiquitination assay. 566
To generate MBP-DHSC95S, the Cys-95 of DHS was mutated to Ser-95 using a point 567
21
mutation kit. The ubiquitination assay was performed as described previously (Zhao 568
et al., 2012). Briefly, purified wheat E1 (GI: 136632, approximately 40 ng), 569
Arabidopsis Ubc10 (E2, approximately 100 ng), Arabidopsis UBQ14 (At4g02890) 570
fused with His tag (approximately 1 µg), and recombinant MBP-DHS (approximately 571
500 ng) were prepared for the E3 ubiquitin ligase activity assay. The reaction was 572
stopped by adding 5× SDS sample buffer and boiled before SDS-PAGE separation. 573
Ubiquitinated proteins were analyzed using the anti-His antibody (1:4000, , Santa 574
Cruz Biotechnology, sc8036). The ubiquitination assay in the E. coli system was 575
performed following a recently published protocol (Han et al., 2017). DHS and ROC4 576
were ligated into the appropriate Duet expression vectors. The auto-ubiquitination of 577
DHS was analyzed using anti-Myc (1:5000, Abmart, M20002) and anti-FLAG 578
antibodies (1:5000, Abmart, M20008). 579
580
Detection of ROC4 ubiquitination in vivo 581
In vivo ubiquitination of ROC4 proteins was assayed as described previously 582
with some modifications (Shen et al., 2008). Briefly, approximately 1 g ROC4 OE 583
callus was treated with 20 µM MG132 for 4 h and ground into a powder in liquid 584
nitrogen to extract protein using extraction buffer (100 mM sodium phosphate, pH 7.8, 585
100 mM NaCl, 0.1% NP-40, 2 mM PMSF, complete protease inhibitor cocktail, and 586
50 µM MG132). Crude extracts containing 500 µg proteins were co-incubated with 587
anti-ROC4 polyclonal antibodies and protein A MagBeads (GenScript, China) to 588
immunoprecipitate the protein complex. After 3 h incubation, the immunoprecipitated 589
complexes were washed three times with wash buffer (100 mM sodium phosphate, 590
pH 7.8, 100 mM NaCl, 0.5% NP-40, 2 mM PMSF, complete protease inhibitor 591
cocktail, and 50 µM MG132), followed by the addition of 5× SDS buffer and boiling 592
for 5 min. The samples were separated on a 10% SDS-polyacrylamide gel and 593
detected by immunoblot analysis with anti-ROC4 (1:200; made by Abmart) and 594
anti-ubiquitin (1:500;Santa Cruz Biotechnology, sc8017) antibodies. 595
596
In vitro degradation assay 597
22
Total protein was isolated from ROC4 OE callus using degradation buffer (25 598
mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT, and 599
10 mM ATP). At the indicated time, an aliquot of the extract was removed and 5× 600
SDS buffer was added to stop the degradation, followed by boiling for 5 min. The 601
samples were loaded onto a 10% SDS-PAGE gel for immunoblotting with anti-ROC4 602
antibody (1:200; made by Abmart). 603
To compare the degradation speeds of ROC4 in WT, DHS OE, and dhs, ROC4 604
was extracted from ROC4 OE callus and divided into equal parts. Each aliquot was 605
incubated with an equal amount of crude protein extract from WT, DHS OE, and dhs 606
calli. The degradation of ROC4 was stopped at the indicated time point and examined 607
by immunoblotting with anti-ROC4(1:200; made by Abmart) and anti-HSP antibody 608
(1:5000; BGI Tech, AbM51099). 609
610
In vivo degradation assay of ROC4 611
ROC4 OE callus were treated with 50 mM CHX or 40 mM MG132 for 4 h. 612
ROC4 protein was extracted in buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.2% 613
NP-40, 0.1% Triton X-100, 5 mM EDTA, complete protease inhibitor cocktail, and 614
50 µM MG132), followed by the addition of 5× SDS buffer and boiling for 5 min. 615
The samples were loaded on a 10% SDS-PAGE gel for immunoblotting with 616
anti-ROC4 and anti-HSP antibodies. Simultaneously, to assess GFP fluorescence, 617
2-week-old ROC4 OE seedlings were treated with 50 mM CHX or 50 µM MG132 for 618
4 h, and GFP fluorescence in ROC4 OE roots was observed and imaged under a Zeiss 619
LSM 510 Meta UV confocal microscope. 620
621
Transient expression assay in rice protoplasts 622
The coding regions of ROC4, DHS, and DHSC95S were ligated into the pRT107 623
vector to generate the 35Spro:ROC4, 35Spro:DHS and 35Spro:DHSC95S constructs. Rice 624
protoplasts were isolated from stem and sheath tissues of young WT seedlings as 625
described previously (Chen et al., 2006). Different combinations of plasmid DNA 626
(approximately 10 µg DNA of each construct) were transiently expressed in 627
23
protoplasts via polyethylene glycol-mediated transfection. Following overnight 628
incubation in the dark at 28°C, total proteins were isolated from the protoplasts with 629
extraction buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 0.1% Triton 630
X-100, 5 mM EDTA, complete protease inhibitor cocktail, and 50 µM MG132), 631
followed by the addition of 5× SDS buffer and boiling for 5 min. The samples were 632
loaded onto a 10% SDS-PAGE gel for immunoblotting with anti-ROC4 and anti-HSP 633
antibodies. For the transactivation assay in rice protoplasts, total RNA was extracted 634
from protoplasts after overnight incubation and used for RT-qPCR analysis 635
636
Electrophoretic Mobility Shift Assay (EMSA) 637
The full-length coding region of ROC4 in pENTR/D-TOPO was subcloned into 638
the expression vector pDEST17 to generate the histidine (His)-ROC4 fusion vector. 639
Purified His-ROC4 was used for the EMSA. Oligonucleotide probes 48 bp long 640
containing a wild-type L1-box (TAAATGYA) or mutated L1-box (TACGCGAA) 641
motifs were synthesized and labeled with biotin using an 642
EMSA Probe Biotin Labeling Kit (Beyotime, Cat. No. GS008). For competition with 643
unlabeled probe, unlabeled probe was added to the reactions. EMSA was performed 644
using a Chemiluminescent EMSA kit (Beyotime, Cat. No. GS009). Probe sequences 645
are shown in Supplemental Table 1. 646
647
Chromatin Immunoprecipitation (ChIP) Assay 648
ROC4-OE was used for the ChIP assay as previously described (Tian et al., 649
2017). Briefly, approximately 2 g of rice seedling tissue was cross-linked in 1% 650
formaldehyde under a vacuum. The cross-linking was stopped by the addition of 651
0.125 M glycine. The sample was ground to a powder in liquid nitrogen and used to 652
isolate nuclei. Anti-ROC4 (1:200 dilution) was used to immunoprecipitate the 653
protein-DNA complex, and the precipitated DNA was used for quantitative PCR. 654
Chromatin precipitated without antibody was used as a control. The data are presented 655
as means ± SE of three independent experiments. Primers used for ChIP-qPCR are 656
listed in Supplemental Table 1. 657
24
658
Accession Numbers 659
Sequence data from this article can be found in the GenBank Database or Rice 660
Genome Annotation Project under the following accession numbers: DHS: 661
LOC_Os02g45780; Os-ROC4: LOC_Os04g48070; Os-BDG: LOC_Os06g04169; 662
At-RHA2a, AEE29264.1; At-RHA2b, ABF58928.1; At-XERICO, AEC05812.1; 663
Os-ROC5, BAC77158; At-HDG1, NP_191674; Zm-OCL1, CAG38614; At-BDG: 664
AAO63446.1 665
666
Supplemental Data 667
Supplemental Figure 1. Phenotypic analysis of DHS OE plants. 668
Supplemental Figure 2. Cuticular wax structure and composition analysis of DHS 669
OE plants. 670
Supplemental Figure 3. Identification of dhs mutants generated by 671
CRISPR/Cas9-mediated genome editing. 672
Supplemental Figure 4. Wax content is increased in dhs vs. wild type. 673
Supplemental Figure 5. Protein structure and bioinformatics analysis of DHS. 674
Supplemental Figure 6. Ubiquitination assay in an Escherichia coli system showing 675
that DHS has E3 ligase activity. 676
Supplemental Figure 7. Intact RING domain in DHS is essential for its biological 677
function. 678
Supplemental Figure 8. Interaction between DHS and various transcription factors in 679
a yeast two-hybrid assay. 680
Supplemental Figure 9. Protein structure and bioinformatics analysis of ROC4. 681
Supplemental Figure 10. Identification of roc4 mutants generated by 682
CRISPR/Cas9-mediated genome editing. 683
Supplemental Figure 11. ROC4 positively regulates wax loads. 684
Supplemental Figure 12. Characterization of the wax crystal structure and 685
composition in the dhs roc4 double mutant. 686
Supplemental Figure 13. Protein structure and bioinformatics analysis of Os-BDG. 687
25
Supplemental Table 1. Primers used in this study. 688
Supplemental File 1. Uncut pictures of protein gel blot in this study. 689
Supplemental File 2. Alignment used to produce the phylogenetic tree shown in 690
Supplemental Figure 5. 691
Supplemental File 3. Alignment used to produce the phylogenetic tree shown in 692
Supplemental Figure 9. 693
Supplemental File 4. Alignment used to produce the phylogenetic tree shown in 694
Supplemental Figure 13. 695
ACKNOWLEDGEMENTS 696
We thank our laboratory members for their helpful comments and discussions 697
during the article preparation. We thank Prof. Jianmin Wan, Prof. Xiaoquan Qi, Dr. 698
Lu Gan, and Dr. Lixin Duan for their assistance in measuring wax contents. We thank 699
Prof. Yaoguang Liu for sharing the plasmid used for gene editing. We also thank Prof. 700
Dongping Lv for helping with the ubiquitination assay. This study was supported by 701
the National Natural Science Foundation of China (Grant No. 31701058 and 702
31671653), the Strategic Priority Research Program of Chinese Academy of Sciences 703
(Grant No. XDA08040101), the Natural Science Foundation of Heilongjiang (Grant 704
No. ZD2015005, C2017071), and the Hundred Talents Program of the Chinese 705
Academy of Sciences to Q.Y. Bu 706
707
AUTHOR CONTRIBUTIONS 708
Q.B. conceived and supervised the entire project, analyzed the data, and wrote the 709
article. Z.W. performed most of the experiments, analyzed the data, and drafted the 710
article. Q.Z performed the autoubiquitination assay. Z.L., X.T., X. L., W.Z., Y.R., and 711
J.T. performed some of the experiments and provided technical assistance. J.F. and 712
Q.X. helped with the discussion of the work. All authors discussed the results and 713
contributed to the final article. 714
715
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Figure 1. Overexpression of DHS leads to drought hypersensitive phenotypes and disrupts cuticular wax structure and composition. (A, B) Phenotypes of three independent DHS OE lines and WT at 5 min (A) and 60 min (B) after the seedlings were transplanted into soil from culture bottles. (C) DHS OE plants lose water more rapidly than the WT. Leaves of DHS OE and WT at the same developmental stages were excised and weighed at various time points after detachment. Water loss is represented as the percentage of initial fresh weight at each time point. Values are means ± SE of three individual plants per genotype. (D, E) SEM images of cuticular wax crystal patterns on the surfaces of leaf blades in WT (D) and DHS OE (E). Scale bar is 1 µm. (F) Cuticular wax composition on the leaf surfaces of WT and DHS OE plants analyzed by GC-MS. Wax constituents are grouped by carbon chain length and chemical class. Data are means ± SE of three biological replicates using independent seedling samples grown at the same condition. Asterisks denote significant differences from WT (*P<0.05, **P<0.01) determined by Student's t test.
DOI 10.1105/tpc.17.00823; originally published online December 13, 2017;Plant Cell
Fang, Qijiang Xu and Qingyun BuZhenyu Wang, Xiaojie Tian, Qingzhen Zhao, Zhiqi Liu, Xiufeng Li, Yuekun Ren, Jiaqi Tang, Jun
Biosynthesis by Promoting the Degradation of Transcription Factor ROC4 in RiceThe E3 Ligase DROUGHT HYPERSENSITIVE Negatively Regulates Cuticular Wax
This information is current as of October 6, 2020
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