1  

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: buqingyun@iga.ac.cn 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 (buqingyun@iga.ac.cn). 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

  2  

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  

  4  

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  

  5  

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  

  6  

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  

  7  

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  

  8  

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  

  10  

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  

  11  

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

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