1

RESEARCH ARTICLE 1

2 The F-box Protein SAGL1 and ECERIFERUM3 Regulate Cuticular Wax 3

Biosynthesis in Response to Changes in Humidity in Arabidopsis 4

5 Hyojin Kima,1, Si-in Yua,1, Seh Hui Jungb, Byeong-ha Leea,2, and Mi Chung Suha,2 6

7 a Department of Life Science, Sogang University, Seoul 04107, Republic of Korea 8 b Department of Bioenergy Science and Technology, Chonnam National University, 9 Gwangju 61186, Republic of Korea 10 1 These authors contributed equally to this work. 11 2 Corresponding Authors: mcsuh@sogang.ac.kr and byeongha@sogang.ac.kr. 12

13 Short title: SAGL1-CER3 function in wax biosynthesis 14

15 One-sentence summary: The Arabidopsis F-box protein SAGL1 mediates 16 proteasome-dependent degradation of ECERIFERUM3, thereby negatively regulating 17 cuticular wax biosynthesis in response to changes in humidity. 18

19 The authors responsible for distribution of materials integral to the findings presented in 20 this article in accordance with the policy described in the Instructions for Authors 21 (www.plantcell.org) are: Mi Chung Suh (mcsuh@sogang.ac.kr) and Byeong-ha Lee 22 (byeongha@sogang.ac.kr). 23

24 ABSTRACT 25

26 Cuticular waxes, which cover the aboveground parts of land plants, are essential for plant 27 survival in terrestrial environments. However, little is known about the regulatory 28 mechanisms underlying cuticular wax biosynthesis in response to changes in ambient 29 humidity. Here, we report that the Arabidopsis thaliana Kelch repeat F-box protein 30 SMALL AND GLOSSY LEAVES1 (SAGL1) mediates proteasome-dependent 31 degradation of ECERIFERUM3 (CER3), a biosynthetic enzyme involved in the production 32 of very long chain alkanes (the major components of wax), thereby negatively regulating 33 cuticular wax biosynthesis. Disruption of SAGL1 led to severe growth retardation, 34 enhanced drought tolerance, and increased wax accumulation in stems, leaves, and 35 roots. Cytoplasmic SAGL1 physically interacts with CER3 and targets it for degradation. 36 GUS expression was observed in the roots of pSAGL1:GUS plants but was barely 37 detected in aerial organs. High humidity-induced GUS activity and SAGL1 transcript 38 levels were reduced in response to abscisic acid treatment and water deficit. SAGL1 39 levels increase under high humidity, and the stability of this protein is regulated by the 40 26S proteasome. These findings indicate that the SAGL1-CER3 module negatively 41 regulates cuticular wax biosynthesis in Arabidopsis in response to changes to humidity, 42 and they highlight the importance of permeable cuticle formation in terrestrial plants 43 under high-humidity conditions. 44

Plant Cell Advance Publication. Published on July 18, 2019, doi:10.1105/tpc.19.00152

©2019 American Society of Plant Biologists. All Rights Reserved

2

INTRODUCTION 45

During evolution, land plants developed a hydrophobic cuticle that covers the surfaces of 46

their aerial organs. This adaptation is essential for plant survival in the terrestrial 47

environment, where water is scarce and plants are exposed to excess light, including 48

ultraviolet light (Riederer and Schreiber, 2001; Kunst and Samuels, 2003; Pollard et al., 49

2008; Samuels et al., 2008; Nawrath et al., 2013; Li-Beisson et al., 2013; Yeats and Rose, 50

2013). The outermost part of the cuticle is covered with epicuticular wax, which often 51

forms crystals and provides protection by reflecting incident light. The wax molecules 52

(intracuticular waxes) underneath the epicuticular wax layer are embedded in a cutin 53

polyester matrix (Pollard et al., 2008; Li-Beisson et al., 2013; Schuster et al., 2016). Both 54

epi- and intracuticular waxes are mainly composed of very long chain fatty acids (VLCFA, 55

C20 to C34), their modified derivatives (aldehydes, primary and secondary alcohols, 56

ketones), VLC alkanes, and wax esters. In addition to protecting plants from high levels of 57

irradiation and UV light, cuticular waxes minimize non-stomatal water loss. These waxes 58

also contribute to self-cleaning of the aerial surface of the plant by preventing the 59

adhesion of dust and other debris, which is important for efficient photosynthesis (Jetter 60

et al., 2006; Samuels et al., 2008; Li-Beisson et al., 2013; Lee and Suh, 2015; Wang et al., 61

2015). 62

Cuticular wax biosynthesis occurs in epidermal cells (Suh et al., 2005). C16 and 63

C18 fatty acyl groups are synthesized in plastids and activated to become acyl-CoAs (Li- 64

Beisson et al., 2013). These molecules are transported to the endoplasmic reticulum (ER) 65

where they are elongated (up to C28) by a fatty acid elongase (FAE) complex consisting 66

of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), 67

β-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR). C28 VLC 68

acyl-CoAs are further elongated (up to C34) in the ER by the combination of a FAE 69

complex and ECERIFERUM2 (CER2) or CER2-like proteins (Haslam and Kunst, 2013; 70

Lee and Suh, 2013; Li-Beisson et al., 2013). The VLCFAs are modified to form VLC 71

aldehydes, VLC alkanes, VLC ketones, and VLC secondary alcohols by an 72

alkane-forming pathway and VLC primary alcohols and wax esters by an alcohol-forming 73

pathway (Domergue et al., 2010; Li et al., 2008; Rowland et al., 2006; Greer et al., 2007). 74

The CER1/CER3/cytochrome b5 (CYTB5) complex mediates the production of VLC 75

alkanes, the major wax components in the aerial organs of Arabidopsis thaliana (Aarts et 76

al., 1995; Chen et al., 2003; Bernard et al., 2012). Cuticular wax molecules produced in 77

the ER are secreted into the extracellular space by ATP-binding cassette (ABC) 78

transporters integrated in the plasma membrane (PM) with the help of 79

3

glycosylphosphatidyl-anchored lipid transfer proteins (LTPGs) anchored in the outer 80

leaflet of the PM (Bird et al., 2007; DeBono et al., 2009; Lee et al., 2009; McFarlane et al., 81

2010; Kim et al., 2012). The wax molecules are then deposited on the outermost surfaces 82

of aboveground plant organs. Total wax loads are differentially deposited in an 83

organ-specific manner and in response to changing environments (Kosma et al., 2009; 84

Seo et al., 2011; Lee and Suh, 2015). In particular, significantly higher levels of cuticular 85

waxes are deposited in the cuticles of plants grown under arid vs. high humidity 86

conditions (Shepherd and Wynne Griffiths, 2006; Wang et al., 2016). However, limited 87

information is available about the regulatory mechanisms underlying cuticular wax 88

biosynthesis in response to changes in ambient humidity. 89

Cuticular wax biosynthesis is regulated at the transcriptional, post-transcriptional, 90

and post-translational levels (Lee and Suh, 2015). Transcriptome analysis of Arabidopsis 91

showed that genes involved in cuticular wax biosynthesis were expressed at significantly 92

higher levels in stem epidermal peels compared to stems (Suh et al., 2005), suggesting 93

that cuticular wax biosynthesis is controlled at the transcriptional level. Consistent with 94

this hypothesis, several transcription factors have been identified that are important for 95

inducing or repressing wax biosynthetic genes in response to drought, pathogen 96

infection, or darkness, including WAX INDUCER1/SHINE1, MYB30 (myeloblastosis), 97

MYB96, MYB94, and Decrease Was biosynthesis (DEWAX) (Aharoni et al., 2004; 98

Raffaele et al., 2008; Seo et al., 2011; Oshima et al., 2013; Go et al., 2014; Lee et al., 99

2016). 100

The upregulation of wax biosynthesis in stems compared to leaves is mediated by 101

the transcription factor WRINKLED4, which is abundantly expressed in the stem 102

epidermis (Park et al., 2016). Hooker et al. (2007) and Lam et al. (2012 and 2015) 103

reported that CER3 transcript levels in epidermal cells are controlled by the ribonuclease 104

CER7, a core subunit of the RNA-degrading exosome complex. In addition, 105

characterization of the cer9 mutant, with increased cuticular lipid levels and drought 106

tolerance, suggested that CER9 (a RING-type E3 ubiquitin ligase) degrades damaged, 107

misfolded, or unfolded proteins involved in cuticular lipid synthesis via the ubiquitin-26S 108

proteasome system (Lü et al., 2012). Chromatin remodeling resulting from the 109

monoubiquitination of histone H2B proteins by the RING-type E3 ligases, HISTONE 110

MONOUBIQUITINATION1 (HUB1) and HUB2, is involved in upregulating cuticular lipid 111

biosynthetic genes ATT1 (for aberrant induction of type three genes), HOTHEAD, Long 112

Chain Acyl-CoA Synthase2 (LACS2), and CER1 (Ménard et al., 2014). Two RING-type 113

E3 ligases, MYB30-INTERACTING E3 LIGASE1 (MIEL1) and DROUGHT 114

4

HYPERSENSITIVE (DHS), negatively regulate cuticular wax accumulation (Lee et al., 115

2017; Wang et al., 2018). Although MIEL1 interacts with both MYB96 and MYB30, the 116

regulation of cuticular wax biosynthesis mainly occurs via the control of MYB96 stability in 117

Arabidopsis inflorescence stems (Lee and Seo, 2016; Lee et al., 2017). Total wax loads 118

decreased in rice (Oryza sativa) plants overexpressing DHS but increased in dhs rice 119

mutants compared to the wild type. DHS shows E3 ubiquitin ligase activity and is involved 120

in the proteasome-mediated degradation of the homeodomain-leucine zipper IV protein 121

ROC4, which positively regulates wax biosynthesis (Wang et al., 2018). 122

The regulation of protein stability via the covalent attachment of ubiquitin protein is a 123

key regulatory mechanism underlying diverse biological processes during plant growth 124

and development or under abiotic stress conditions (Santner and Estelle, 2010; Shu and 125

Yang, 2017). Protein ubiquitination involves three sequential reactions catalyzed by 126

ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-ligase 127

(E3). Arabidopsis harbors 2, 37, and 1,500 genes encoding E1, E2, and E3 enzymes 128

(Mazzucotelli et al., 2006; Lee and Kim, 2011), respectively. E3 ubiquitin ligases are 129

present in a single component or multicomponent complex (Cardozo and Pagano, 2004). 130

The Skp1-Cul1-F-box protein (SCF) complex belongs to a multicomponent ubiquitin 131

ligase complex. The F-box proteins in this complex are substrate recognition components 132

whose specificity is determined by C-terminal protein-protein binding domains such as 133

Leucin-Rich Repeat (LRR), Kelch, and WD-40 repeats (Cardozo and Pagano, 2004; 134

Mazzucotelli et al., 2006). Arabidopsis contains more than 100 Kelch repeat F-box 135

proteins (KFBs), which were initially identified in fruitfly (Drosophila melanogaster) (Xue 136

and Cooley, 1993; Sun et al., 2007; Schumann et al., 2011). Many plants also contain 137

proteins with Kelch motifs, including moss (Physcomitrella patens), pine (Pinus taeda), 138

poplar (Populus trichocarpa), Brassica rapa, rice, and maize (Zea mays) (Sun et al., 139

2007). KFBs contain at least one Kelch motif harboring four conserved residues: two 140

adjacent glycines (G) and a pair of tyrosine (Y) and trytophan (W) residues separated by 141

approximately six residues (Adams et al., 2000; Prag and Adams, 2003). To date, only a 142

few KFBs have been characterized in Arabidopsis. These KFBs are involved in flowering 143

time and circadian control, flavonoid biosynthesis, and the brassinosteroid signal 144

transduction pathway (Nelson et al., 2000; Yasuhara et al., 2004; Kim et al., 2007; Sawa 145

et al., 2007; Zhang et al., 2017; Zhu et al., 2017). 146

In the current study, we investigated the negative regulatory mechanisms underlying 147

posttranslational control of cuticular wax biosynthesis. Specifically, we performed 148

genetic, molecular, and biochemical analysis of the Arabidopsis sagl1-1 (small and 149

5

glossy leaves 1-1) and sagl1-2 mutants, with small, waxy leaves and severe dwarf 150

phenotypes. The SAGL1 gene encodes a Kelch-motif containing F-box protein that 151

controls the stability of CER3. SAGL1 is upregulated by relatively high humidity (>90% 152

RH) but downregulated by relatively low humidity (50–60% RH), water deficit, and 153

abscisic acid (ABA) treatment. Furthermore, increased SAGL1 protein levels were 154

observed under relatively high humidity. SAGL1 regulates CER3 protein levels in 155

response to changes in humidity. These observations indicate that the SAGL1-dependent 156

posttranslational regulatory mechanism contributes to the negative regulation of cuticular 157

wax biosynthesis in Arabidopsis. The SAGL1-CER3 module might represent a key 158

regulator that maintains the homeostasis of cuticular wax biosynthesis in terrestrial plants 159

under different moisture conditions. 160

161

162

RESULTS 163

164

Genetic Mapping of sagl1-1 and Candidate Gene Analysis Identify the SAGL1 Gene 165

166

A screen for Arabidopsis mutants showing defective growth and development resulted in 167

the identification of a mutant line with alterations in leaf morphology and development. 168

This mutant had small, thick, glossy leaves and was thus named sagl1-1 (small and 169

glossy leaves 1-1) (Figure 1A to 1D; Supplemental Data Set 1). Compared to its 170

background C24 accession, sagl1-1 plants were smaller, with shiny dark green leaves, 171

glossy stems, abnormal phyllotaxy, and altered root architecture (Figure 1E to 1J). These 172

growth phenotypes became prominent in 3-week-old plants (Figure 1K and Figure 1L). 173

To identify the gene responsible for the sagl1-1 phenotypes, we performed positional 174

cloning using previously reported or newly developed simple sequence length 175

polymorphism (SSLP) markers (Lukowitz et al., 2000). From the F2 progeny derived from 176

a cross between sagl1-1 and wild type Landsberg erecta (Ler) plants, individuals with 177

small, shiny, and dark green leaves were selected for mapping. Initial mapping using 32 178

F2 progenies suggested that the SAGL1 locus is located on the middle of chromosome 1. 179

Fine mapping further narrowed down the mutation to the F7A10 BAC clone in the middle 180

region of chromosome 1 (Figure 2A). We sequenced the genes located in this region and 181

found a putative mutation in the At1g55270 gene in sagl1-1. In sagl1-1, an 11 bp fragment 182

(392th bp to 402th bp from the start codon of the At1g55270 open reading frame [ORF]) 183

was deleted, which would result in premature termination of translation. To confirm that 184

6

we successfully cloned the SAGL1 gene, we subcloned a genomic DNA fragment 185

containing the 2001 bp region upstream of the translation start codon, the 1593 bp of the 186

ORF, and the 573 bp region downstream of the stop codon of At1g55270 into a binary 187

vector and introduced it into the sagl1-1 mutant by Agrobacterium-mediated 188

transformation. The selected transgenic plants (T1) containing the SAGL1 genomic DNA 189

fragment did not display any of the developmental defects of sagl1-1 (Figure 2B to 2E). 190

These results demonstrate that At1g55270 is SAGL1. This gene has two exons and one 191

intron with a 1305 bp coding sequence encoding a 434 amino acid protein with a 192

molecular mass of ~49.2 kD (Figure 2A and 2F). SAGL1 was annotated to encode a 193

Kelch-repeat F-box protein. Protein domain analysis predicted that SAGL1 contains a 194

F-box domain and three Kelch domains. The sagl1-1 mutation would result in the 195

absence of all of these domains (Figure 2F). 196

We also obtained a T-DNA insertion allele (SALK_097761) of sagl1 containing a 197

T-DNA insertion in the SAGL1 intron from the Arabidopsis Biological Resource Center 198

(Figure 2G). We isolated the SALK_097761 homozygote for the T-DNA insertion in 199

SAGL1 and named this allele sagl1-2. We measured SAGL1 transcript levels in sagl1-2 200

by RT-PCR. sagl1-2 failed to accumulate SAGL1 transcripts, suggesting that sagl1-2 is 201

likely a knock-out mutant (Figure 2H). Like sagl1-1, sagl1-2 displayed developmental 202

defects including small, thick, waxy, dark green leaves, a small plant stature, and altered 203

root morphology (Figure 2I to 2L). We crossed sagl1-2 to sagl1-1 and found that all F1 204

seedlings showed the sagl1 phenotypes, further confirming the notion that At1g55270 is 205

SAGL1 (Supplemental Figure 1). 206

207

sagl1 Exhibits Enhanced Drought Tolerance 208

209

One of the distinguishing features of the sagl1 mutants is their shiny but waxy leaf and 210

stem surfaces (Figure 1 and 2; Supplemental Figure 1). This observation suggests that 211

the sagl1 mutants might accumulate more cuticular waxes on their leaves than the wild 212

type (WT). Thus, we reasoned that the sagl1 mutants might exhibit enhanced drought 213

tolerance due to reduced non-stomatal conductance. To test our hypothesis, we stopped 214

supplying water to both WT and sagl1 (sagl1-1 and sagl1-2) plants for 12 days. After 12 215

days, the WT plants turned yellow and died, but the sagl1 mutants survived and still 216

appeared green (Figure 3A). We also compared water loss between WT and both sagl1-1 217

and sagl1-2 alleles after exposing detached rosette leaves of each plant to dry air. The 218

results of water loss measurements also supported our hypothesis; both sagl1 mutant 219

7

alleles showed lower water loss rates than the respective WT (C24 and Col-0) (Figure 3B; 220

Supplemental Data Set 1). To ensure that these results were indeed due to reduced 221

non-stomatal conductance, we compared the stomatal density and stomatal sensitivity to 222

ABA between WT and sagl1. The stomatal densities of the sagl1 mutants were almost 223

indistinguishable from their respective WT; in one square millimeter of leaf epidermis, 224

C24 and sagl1-1 contained 175.6 ± 10.9 and 173.4 ± 10.5 stomata, respectively, whereas 225

Col-0 and sagl1-2 contained 132.5 ± 5.9 and 136.2 ± 8.6 stomata, respectively (Figure 3C; 226

Supplemental Data Set 1). In addition, the stomatal sensitivity results (expressed as 227

relative stomatal aperture size in response to 2 PM ABA) showed that the stomata of WT 228

and sagl1 plants were closing at very similar rates (Figure 3D; Supplemental Data Set 1). 229

Taken together, these results suggest that the improved drought tolerance of the sagl1 230

mutants is likely due to reduced non-stomatal conductance and not to altered stomatal 231

regulation. 232

233

sagl1 Mutations Affect Cuticle and Cell Wall Ultrastructure and Cuticular Wax Load 234

235

The waxy phenotype and drought tolerance of the sagl1 mutants prompted us to examine 236

the ultrastructure of the sagl1 cuticle by scanning and transmission electron microscopy 237

(SEM and TEM, respectively). In the SEM images, typical epicuticular wax crystals, 238

including vertical rods, tubes, longitudinal bundles of rodlets, and horizontal and reticulate 239

platelets, were observed on both WT (C24 and Col-0) and sagl1 (sagl1-1 and sagl1-2) 240

stems (Figure 4A to 4D). The density of wax crystals was higher on sagl1 stems 241

compared to WT, particularly vertical rods (Figure 4A to 4D). TEM analysis of leaf 242

epidermal cells of WT (Col-0) and sagl1-2 revealed that the thickness of the cuticle and 243

adjacent cell walls increased by approximately 2-fold and 1.3-fold, respectively, in sagl1-2 244

compared to WT (Figure 4E to 4H; Supplemental Data Set 1). 245

We further analyzed the differences in epicuticular wax crystal abundance and cuticle 246

thickness in the sagl1 mutants by performing cuticular wax analysis using gas 247

chromatography with flame ionization detection (GC-FID). As expected, total wax loads 248

increased by approximately 2- and 1.6-fold in sagl1-1 and sagl1-2 leaves relative to C24 249

and Col-0 WT, respectively (Figure 5A; Supplemental Figure 2A; Supplemental Data Set 250

1). Similarly, the levels of alkanes (the major wax component in leaves) increased by 251

approximately 2.2-fold and 2-fold in sagl1-1 and sagl1-2 leaves, respectively vs. WT 252

(Figure 5B; Supplemental Figure 2A; Supplemental Data Set 1). In stems, the total wax 253

loads were also greater in sagl1-1 (~1.6 fold) and sagl1-2 (~1.3 fold) relative to their 254

8

respective WT (Figure 5C; Supplemental Figure 2B; Supplemental Data Set 1) due to 255

increased levels of all wax components in the stems of both sagl1 mutants (Figure 5D; 256

Supplemental Figure 2B; Supplemental Data Set 1). In four-week-old roots, total aliphatic 257

wax loads increased by approximately 1.8-fold, but sterols and aromatic wax loads 258

decreased by approximately 1.4-fold, in sagl1-1 compared to the WT. The levels of FAs 259

(C24, C26, and C30), PAs (C22, C26, C28, and C30), and alkanes (C29 and C31) 260

increased by approximately 1.7- to 5.7-fold in sagl1-1 compared to the WT, whereas the 261

levels of C20 and C22 FAs, C22 MAGs, and alkyl coumarates significantly decreased in 262

the mutant. Interestingly, we detected alkyl ferulates and alkyl caffates in the waxes of 263

WT roots but not sagl1-1 (Figure 5E and 5F; Supplemental Figure 2C; Supplemental Data 264

Set 1). 265

We analyzed the levels and compositions of cutin monomers analyzed from the 266

leaves and stems of WT (C24 and Col-0), sagl1-1, and sagl1-2 by GC-FID and GC-mass 267

spectrometry (GC-MS). Compared to WT, the total cutin monomer loads were 268

significantly elevated in the stems and leaves of both sagl1 mutants (Figure 5G and 5H). 269

In particular, a prominent increase in C18:2 dicarboxylic acid levels was observed in the 270

leaves of sagl1-1 and stems of both sagl1 mutants, although minor increases in the levels 271

of other components were also detected (Figure 5I and J; Supplemental Figure 3; 272

Supplemental Data Set 1). 273

274

SAGL1 Decreases CER3 Stability 275

276

The complex altered wax phenotype of the sagl1 mutants, which showed elevated levels 277

of almost all wax components, did not provide clues about the potential targets of the 278

F-box protein, SAGL1, among proteins involved in cuticular wax biosynthesis and 279

deposition. Thus, we generated transgenic Arabidopsis plants overexpressing SAGL1 280

under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Supplemental 281

Figure 4A). Genomic DNA PCR and RT-PCR analyses showed that the SAGL1 gene was 282

successfully introduced and overexpressed in Arabidopsis (Col-0) (Supplemental Figure 283

4B and 4C). We analyzed cuticular wax composition and levels in the stems of 13 284

independent transgenic Arabidopsis lines overexpressing SAGL1. Compared to other 285

wax components, alkane levels were significantly reduced in SAGL1-overexpressing 286

plants (Supplemental Figure 4D), suggesting that CER1, CER3, and CYTB5D, which are 287

involved in the production of VLC alkanes from VLCFAs, are putative targets of SAGL1 288

(Bernard et al., 2012). In a subsequent analysis of cutin monomers, no remarkable 289

9

differences were observed in total cutin monomer composition and levels in stems of WT 290

vs. the SAGL1 OX lines (OX9, OX11, and OX12) (Supplemental Figure 4E; Supplemental 291

Data Set 1). 292

To examine if CER1, CER3, and/or CYTB5D proteins are substrates of SAGL1, we 293

constructed three binary vectors expressing CER1, CER3, or CYTB5D fused with the 294

MYC epitope tag under the control of the CaMV 35S promoter, introduced them into 295

Agrobacterium, and infiltrated them into Nicotiana benthamiana leaves. Total proteins 296

were extracted from N. benthamiana leaves transiently expressing MYC:CER1, 297

MYC:CER3, or MYC:CYTB5D in the presence or absence of eYFP (encoding enhanced 298

yellow fluorescent protein) or SAGL1:eYFP expression. Among the candidate target 299

proteins, the levels of MYC:CER3 were noticeably reduced in leaves co-expressing 300

MYC:CER3 and SAGL1:eYFP relative to those expressing MYC:CER3 and eYFP (Figure 301

6A; Supplemental Figure 5A and 5B). By contrast, no significant decrease in the level of 302

MYC, MYC:CER1, or MYC:CYTB5D was observed when MYC, MYC:CER1, or 303

MYC:CYTB5D was co-expressed with SAGL1:eYFP or eYFP in N. benthamiana leaves 304

(Supplemental Figure 5A and 5B). The degradation of MYC:CER3 was not observed in 305

the presence of MG132, a chemical inhibitor of the 26S proteasome (Figure 6A; 306

Supplemental Figure 5C). In addition, when MYC:CER3 was co-expressed with SAGL1 307

lacking the F-box domain (SAGL1△F), the degradation of MYC:CER3 was inhibited 308

(Figure 6B; Supplemental Figure 5D). These results suggest that SAGL1 is involved in 309

targeting CER3 for degradation by the 26S proteasome. 310

To determine if SAGL1 directly interacts with CER3, we performed 311

coimmunoprecipitation (Co-IP) assays. For this purpose, MYC:CER3 was transiently 312

expressed with or without SAGL1:eYFP in N. benthamiana leaves, and crude proteins 313

were immunoprecipitated using anti-MYC antibody-conjugated agarose beads. 314

MYC:CER1 or MYC:CYTB5D and SAGL1:eYFP were also coexpressed, 315

coimmunoprecipitated, and subjected to immunoblot analysis using anti-MYC and 316

anti-GFP antibodies as a control. When MYC:CER3 was immunoprecipitated, 317

SAGL1:eYFP was immunoprecipitated as well (Figure 6C; Supplemental Figure 5E). 318

However, SAGL1:eYFP was not visibly detected when MYC:CER1 or MYC:CYTB5D was 319

immunoprecipitated using anti-MYC antibody-conjugated agarose beads (Supplemental 320

Figure 5E). In addition, we investigated whether CER3 is ubiquitinated by SAGL1. 321

MYC:CER3 and SAGL1:eYFP were co-expressed in N. benthamiana leaves in the 322

absence or presence of MG132. MYC:CER3 was immunoprecipitated using anti-MYC 323

antibody-conjugated agarose beads and subjected to immunoblot analysis using 324

10

anti-ubiquitin antibodies. When SAGL1:eYFP was expressed, ubiquitinated MYC:CER3 325

was more clearly detected in the presence of MG132 than in its absence (Figure 6D), 326

indicating that SAGL1 is involved in the ubiquitination of CER3. In MYC:CER3-expressing 327

WT (C24) and sagl1-1 seedlings, which were generated by cross-pollination between 328

sagl1-1 and transgenic WT plants expressing MYC:CER3 under the control of the CaMV 329

35S promoter, the MYC:CER3 protein level was higher in the sagl1-1 mutant background 330

than in the WT background, but no significant differences in MYC:CER3 transcript levels 331

were observed between the two types of seedlings (Figure 6E; Supplemental Figure 5F). 332

333

SAGL1:eYFP Localizes to the Cytoplasm and Nucleus 334

335

To examine the subcellular localization of SAGL1, we translationally fused the SAGL1 336

coding sequence (CDS) to the N-terminus of eYFP in the pPZP212-eYFP vector. The 337

pPZP212-mRFP (monomeric red fluorescent protein) vector containing the mRFP gene 338

instead of eYFP was used as a cytoplasmic and nuclear protein marker (Goodin et al., 339

2002). When SAGL1:eYFP and mRFP were transiently co-expressed in N. benthamiana 340

leaves, yellow fluorescent signals were merged with blue fluorescent signals but did not 341

overlap with blue autofluorescence from chloroplasts (Figure 7A). Fluorescent signals 342

were also observed in the cytoplasm and nuclei of the cortex and endodermal cells of 343

10-day-old transgenic roots expressing SAGL1:eYFP under the control of the CaMV 35S 344

promoter (Supplemental Figure 6). These results indicate that SAGL1 is localized to the 345

cytosol and nucleus. 346

347

SAGL1 is Downregulated under Low Humidity, High ABA, or High-Salt Conditions 348

349

To measure SAGL1 transcript levels in various organs of Arabidopsis plants grown under 350

50% to 60% relative humidity (RH) conditions, we isolated total RNA from seven-day-old 351

seedlings (YS); the rosette leaves of three-week-old (R1), four-week-old (R2), and six- to 352

seven-week-old (R3) plants; and the roots (Ro), stems (St), floral buds (Bu), flowers (Fl), 353

and siliques (Si) of six- to seven-week-old plants and subjected it to qRT-PCR analysis. 354

SAGL1 transcript levels in R1, Bu, and Fl were approximately 1.5- to 1.8-fold higher than 355

those in YS and Si (Figure 7B). To investigate the temporal and spatial expression 356

patterns of SAGL1, we generated transgenic Arabidopsis plants transformed with the 357

GUS reporter gene driven by the SAGL1 5’ promoter fragment (pSAGL1:GUS). When the 358

plants were grown under 50% to 60% RH conditions, GUS expression was detected in 359

11

10-day-old seedlings and in the rosette leaves of three-week-old plants, as well as the 360

leaf hydathodes, root, nodes, anthers, and developing seeds of four to seven-week-old 361

plants. However, little or no GUS expression was detected in the aerial organs (including 362

leaves, stems, sepals, petals, and silique walls) of four to seven-week-old plants (Figure 363

7C to 7I; Supplemental Data Set 1). 364

To examine whether SAGL1 expression is regulated by changes in ambient humidity, 365

6-week-old pSAGL1:GUS plants grown in culture bottles were stained before and 32 h 366

after removing the bottle cap. GUS expression was observed in all organs including the 367

aerial parts of pSAGL1:GUS plants before removing the bottle cap but was substantially 368

reduced in the aerial parts of pSAGL1:GUS plants after removing the bottle cap (Figure 369

7J vs 7K). In 5-week-old pSAGL1:GUS plants grown in soil, GUS staining was also 370

observed in leaves wrapped with plastic sheets, but not in unwrapped leaves (Figure 7L), 371

indicating that SAGL1 is upregulated under relatively high (>90% RH) humidity conditions 372

and downregulated under low (approximately 50–60% RH) humidity conditions. In 373

addition, SAGL1 transcript levels increased by approximately 35% 15 to 30 min after 374

wrapping the leaves in plastic sheets compared to unwrapped leaves (Figure 7M; 375

Supplemental Figure 7A). 376

We then investigated whether SAGL1 expression is regulated by exogenous ABA 377

treatment, osmotic stress, or salt stress. The detached leaves of 4-week-old 378

pSAGL1:GUS plants grown in soil were incubated with 1/2 MS liquid medium or the same 379

medium supplemented with 100 μM ABA, 200 mM mannitol, or 200 mM NaCl for 24 h. 380

GUS staining and activity increased markedly in leaves floated on 1/2 MS liquid medium 381

(MOCK) compared to non-treated leaves taken directly from soil-grown plants (Non). The 382

intense GUS staining and high activity decreased to a great extent in leaves of 383

pSAGL1:GUS plants in response to ABA treatment, salt, and osmotic stress relative to 384

MOCK (Figure 7N and 7O) but were still higher than in the non-treated leaves (Non). 385

qRT-PCR analysis of SAGL1 steady-state transcript accumulation confirmed these 386

trends (Supplemental Figure 7B; Supplemental Data Set 1). These results indicate that 387

the expression of SAGL1 is negatively regulated by ABA treatment, salt, and osmotic 388

stress. 389

390

SAGL1 Stability Increases under Relatively High Humidity Conditions, and SAGL1 391

Regulates MYC:CER3 Protein Levels in Response to Changes in Humidity 392

393

12

To investigate SAGL1 protein levels in response to different ambient humidity levels, we 394

generated transgenic Arabidopsis plants expressing SAGL1:eYFP driven by the CaMV 395

35S promoter. Seven-day-old WT and transgenic Arabidopsis seedlings overexpressing 396

SAGL1:eYFP were grown in Petri dishes and transferred to soil under 50–60% humidity 397

conditions. The seedlings were then covered with an opaque plastic lid for 1 day, 398

uncovered for 0, 12, 24, and 48 h, and subjected to immunoblot analysis. SAGL1:eYFP 399

protein levels significantly decreased in transgenic seedlings 24 and 48 h after being 400

uncovered (50–60% RH) relative to covered (>90% RH) transgenic seedlings (Figure 401

8A). 402

To examine if the stability of SAGL1 is regulated by the 26S proteasome complex, we 403

incubated 4-day-old WT (Col-0) and transgenic seedlings expressing SAGL1:eYFP in 1/2 404

Murashige and Skoog (MS) liquid medium supplemented with the inhibitor MG132 or 405

dimethyl sulfoxide (DMSO) for 4 h, followed by incubation in 1/2 MS liquid medium 406

supplemented with cycloheximide (CHX) after removing the MG132. The intensity of the 407

SAGL1:eYFP bands was stronger in the presence of MG132 than in the presence of 408

DMSO but gradually decreased in response to CHX treatment (Figure 8B), suggesting 409

that the stability of SAGL1:eYFP is controlled by the 26S proteasome complex. 410

Next, we examined the relationship between CER3 and SAGL1 in Arabidopsis under 411

different humidity conditions. Seven-day-old MYC:CER3-expressing WT (C24) and 412

sagl1-1 seedlings grown in Petri dishes (>90% RH) were exposed to 50–60% humidity 413

conditions for 0 and 12 h, followed by immunoblot analysis. MYC:CER3 protein levels 414

significantly increased in WT seedlings exposed to 50–60% humidity conditions 415

compared to WT seedlings grown under >90% humidity conditions, but no noticeable 416

differences were observed in MYC:CER3 protein levels between sagl1-1 seedlings 417

exposed to relatively high and low humidity conditions (Figure 8C). These results indicate 418

that SAGL1 regulates MYC:CER3 protein levels in response to changes in humidity. 419

Finally, we grew WT (Col-0) and sagl1-2 plants under 50%–60% RH and >90% RH 420

conditions and stained the leaves of 4-week-old plants with 0.05% toluidine blue (TB) 421

solution. Under relatively low humidity conditions, no TB staining (indicative of cuticle 422

permeability) was observed in WT or sagl1-2 leaves (Figure 8D). By contrast, strong TB 423

staining was clearly visible in WT leaves, but not in sagl1-2 leaves, in plants grown under 424

relatively high humidity conditions (Figure 8D), revealing that WT leaves possess a more 425

permeable cuticle than sagl1-2 leaves. These findings demonstrate that the presence of 426

SAGL1 results in the formation of a permeable cuticle under high humidity conditions. In 427

13

addition, the phenotype of the sagl1-1 mutant was more severe under 50%–60% humidity 428

than under <30% humidity conditions (Supplemental Figure 8). 429

430

431

DISCUSSION 432

The cuticle covers the primary aerial surfaces of land plants and serves as a defensive 433

barrier against various abiotic and biotic stresses (Kunst and Samuels, 2003; Pollard et 434

al., 2008; Samuels et al., 2008; Yeats and Rose, 2013; Serrano et al., 2014). Thus, the 435

regulation of cuticle formation is essential for the optimal growth and development of 436

plants under various environmental conditions (Kosma et al., 2009; Go et al., 2014; Kim 437

et al., 2017). Recent advances in our understanding of the molecular mechanisms 438

underlying cuticle biosynthesis underscore the importance and complexity of its 439

regulatory networks. One long-standing question in cuticle biology is how land plants 440

differentially control cuticular wax biosynthesis in response to changes in ambient 441

humidity. In this study, we showed that the Kelch repeat F-box protein, SAGL1, negatively 442

regulates cuticular wax biosynthesis by modulating the stability of CER3, a biosynthetic 443

enzyme involved in the production of VLC alkanes; these compounds account for 444

approximately 60 to 75% of total wax loads in the cuticles Arabidopsis leaves and stems 445

(Bernard et al., 2012; Li-Beisson et al., 2013). The presence of E3 ubiquitin ligases, which 446

are evolutionarily conserved and function as highly diverse key regulators (Chen and 447

Hellmann, 2013) in plants, suggests that regulatory mechanisms similar to that employing 448

the SAGL1-CER3 module might also be involved in regulating cuticular wax biosynthesis 449

in other plants including rapeseed (Brassica napus) and tomato (Solanum lycopersicum), 450

whose major wax components are VLC alkanes (Supplemental Figure 9, Supplemental 451

Data Set 2; Lee and Suh, 2015). 452

Even though cuticular wax biosynthesis is known to be regulated by transcriptional, 453

post-transcriptional, and post-translational regulatory mechanisms, to date, the 454

post-translational mechanism of wax deposition has not been studied in much detail 455

(Hooker et al., 2007; Lam et al., 2012, 2015; Lü et al., 2012; Lee and Suh, 2013, 2015; 456

Ménard et al., 2014; Marino et al., 2013; Lee and Seo, 2016; Wang et al., 2018). The 457

discovery of the E3 ubiquitin ligase, CER9, whose defects result in increased levels of 458

cutin monomers, pointed to the possible post-translational regulation of cuticular wax 459

biosynthesis (Lü et al., 2012). More recently, four RING-containing E3 ubiquitin ligases, 460

HUB1, HUB2, MIEL1, and DHS, were shown to be involved in cuticular wax biosynthesis; 461

HUB1 and HUB2 alter the stability of histone proteins involved in the formation of the 462

14

chromatin structure of cuticular lipid biosynthesis genes (Ménard et al., 2014), MIEL1 463

targets two cuticular wax biosynthesis regulators (the transcription factors MYB96 and 464

MYB30) (Marino et al., 2013; Lee and Seo, 2016), and DHS promotes the degradation of 465

the transcription factor ROC4, which positively regulates wax biosynthesis (Wang et al., 466

2018). Our characterization of the role of the F-box protein SAGL1 in the degradation of 467

CER3 by the 26S proteasome further contributes to our understanding of 468

post-translational regulation of cuticular wax biosynthesis. 469

GUS expression was detected in the roots and rosette leaves of 3-week-old 470

pSAGL1:GUS plants but was barely detected in the aerial organs, whose surfaces are 471

covered with cuticular waxes. In addition, we observed the humidity-dependent 472

expression of GUS driven by the SAGL1 5’ promoter fragment; high humidity (i.e., 473

belowground or wrapped conditions, >90% RH) induced GUS expression, whereas low 474

humidity (i.e., aboveground or unwrapped conditions, 50–60% RH) diminished the 475

increase in GUS expression, in pSAGL1:GUS plants. Furthermore, SAGL1 was more 476

stable under relatively high humidity conditions (>90% RH) and was degraded by the 26 477

proteasome under relatively low humidity conditions (Figure 8A and 8B). Co-expression 478

and Co-IP analyses of SAGL1 and CER3 clearly showed that SAGL1 mediates the 479

breakdown of CER3 by the 26S proteasome complex. Therefore, we speculate that the 480

SAGL1-CER3 module is a key determinant of total wax loads on the surfaces of above- 481

and belowground organs of Arabidopsis in response to changes in ambient humidity. 482

Interestingly, no differences in growth or development were observed between WT and 483

the SAGL1 overexpressing lines, indicating that alkane biosynthesis is not severely 484

impaired in the stems of the SAGL1 overexpressing plants. Indeed, VLC alkane levels in 485

the stems of SAGL1 overexpressing plants were reduced by only approximately 15% 486

compared to WT under relatively low humidity conditions (50–60% RH; Supplemental 487

Figure 4D), suggesting that CER3 is only partially degraded in these organs due to the 488

instability of SAGL1. Computational analysis (Supplemental Figure 10) using an 489

open-source tool for visualization of proteoforms (http://topcons.net/; 490

http://wlab.ethz.ch/protter/start/) predicted that cytoplasm-localized SAGL1 might interact 491

with two cytoplasmic loop domains or the cytoplasmic C-terminal region of CER3, an ER 492

membrane-localized protein (Kamigaki et al., 2009). Forty-two Lys residues in the 493

cytoplasmic regions of CER3 are putative target sites for ubiquitination (Bernard et al., 494

2012), further supporting the idea that CER3 is the target of the E3 ubiquitin ligase 495

SAGL1. This hypothesis is also supported by the finding that two cytoplasm-localized 496

F-box proteins, EIN2-TARGETING PROTEIN1 (ETP1) and ETP2, interact with the 497

15

C-terminus of ER-localized EIN2 and target it for proteasome-mediated degradation in 498

the absence of ethylene (Qiao et al., 2009). 499

Changes in endogenous ABA levels are regulated by the balance of ABA 500

biosynthesis and catabolism (Dong et al., 2015). Under drought conditions, ABA 501

accumulation is mediated by the enhanced expression of the 9-cis-epoxycarotenoid 502

dioxygenase 3 gene and an increase in its protein stability (Luchi et al., 2001; Endo et al., 503

2008). Conversely, high humidity activates the expression of CYP707A, encoding ABA 504

8c-hydroxylase, a key enzyme in ABA catabolism (Okamoto et al., 2009). ABA treatment 505

and water deficit result in an increase in total wax loads in Arabidopsis leaves, including a 506

significant increase in alkane levels (Kosma et al., 2009; Seo et al., 2011). By contrast, 507

the density of epicuticular wax crystals, total wax loads, and alkane and secondary 508

alcohol levels significantly decrease in Brassica oleracea leaves exposed to high air 509

humidity (Koch et al., 2006). Arabidopsis mutants with defects in the core ABA signaling 510

pathway, including pyrabacin resistance1 (pyr1) pyr1-like1 (pyl1) pyl2 pyl4 pyl5 pyl8 and 511

snf1-related protein kinase2.2 snrk2,3 snrk2,6, with permeable cuticles, are better 512

adapted to water-saturated conditions than the WT, suggesting that the core ABA 513

signaling pathway plays a negative role in regulating plant responses to increased 514

humidity (Cui et al., 2016). In the current study, GUS activity in pSAGL1:GUS plants and 515

SAGL1 transcript levels increased under high humidity conditions (>90% RH) but 516

decreased under low humidity conditions (50–60% RH) and ABA treatment, indicating 517

that the regulation of SAGL1 expression is dependent on humidity levels, which is 518

inversely correlated with endogenous ABA levels (Waadt et al., 2014). In addition, SAGL1 519

protein levels increased under high humidity conditions (>90% RH). Therefore, the 520

SAGL1-dependent posttranslational regulatory mechanism plays a key role in regulating 521

cuticular wax biosynthesis in terrestrial plants in response to different humidity levels. 522

The leaf plasticity of terrestrial plants is critical for increasing oxygen uptake and 523

preventing the depletion of carbohydrates, which function as substrates for respiration 524

under flooding conditions (Mommer and Visser, 2005). Amphibious plants, which are able 525

to survive in both aquatic and terrestrial environments, show a change in the thickness of 526

their cuticular membranes when exposed to aquatic or aerial conditions. Indeed, aquatic 527

cuticular membranes have lower resistance to O2 or CO2 diffusion than aerial cuticular 528

membranes (Frost-Christensen et al., 2003; Frost-Christensen and Floto, 2007), 529

suggesting that this change facilitates gas exchange under submergence or flooding 530

conditions. Therefore, the SAGL1-mediated formation of a permeable cuticle may be 531

important for plant acclimation to humid environments. 532

16

The sagl1-1 and sagl1-2 mutants exhibit the overaccumulation of wax and 533

dwarfism. Because the balanced distribution of VLCFAs (common metabolic precursors 534

used for cuticular wax and sphingolipid biosynthesis) is essential for optimal plant growth, 535

the dwarf phenotype of the sagl1-1 and sagl1-2 mutants may be a consequence of 536

excessive wax production (Zheng et al., 2005; Bach et al., 2008; Beaudoin et al., 2009; 537

Bourdenx et al., 2011; Seo et al., 2011). However, the phenotypes of sagl1-2 cer3-6 538

double mutants were similar to the dwarf phenotype of sagl1-2 and the wax-deficient 539

phenotype of cer3-6 (Rowland et al., 2007; Supplemental Figure 11), indicating that 540

SAGL1 has additional targets. Yu et al. (2019) recently reported that both sagl1-1 and 541

sagl1-2 plants show enhanced accumulation of anthocyanins and lignin derived from the 542

phenylpropanoid pathway. Phenylalanine ammonia lyase activity is increased in both 543

sagl1-1 and sagl1-2 leaves but reduced in SAGL1 OX leaves compared to the WT. When 544

SAGL1 and PHENYLALANINE AMMONIA LYASE1 (PAL1):GFP were co-expressed in 545

N. benthamiana leaves, PAL1:GFP levels decreased, suggesting that PAL1 is another 546

target of SAGL1. However, it is still unclear if the growth retardation of the sagl1 mutants 547

is related to the enhanced activity of PAL1. 548

In conclusion, we demonstrated that the Kelch-domain containing F-box protein, 549

SAGL1, negatively regulates cuticular wax biosynthesis by promoting the degradation of 550

CER3. SAGL1 transcript and protein levels increased under high ambient humidity (>90% 551

RH), whereas the high humidity-induced expression of SAGL1 was repressed by 552

exposure to ABA or low humidity, suggesting that the SAGL1-CER3 module plays a key 553

role in regulating cuticular wax biosynthesis in Arabidopsis in response to different 554

humidity conditions, e.g., drought and flooding. 555

556

557

METHODS 558

Plant Materials and Growth Conditions 559

Arabidopsis thaliana seeds were surface sterilized in bleach solution and sown directly in 560

soil (SunGro Mix #5) or half-strength MS agar medium [MS salts (Caisson Laboratories) 561

containing 0.3% Gelrite (Duchefa), 1% sucrose, pH 5.8]. After 2-3 day stratification at 562

4qC, the plants were grown at 22–23qC under long-day conditions (16-h light/8-h dark 563

cycles) in white fluorescent light (100 µmol photons/m2/s). Seeds of the sagl1-2 T-DNA 564

mutant (SALK_097761) were obtained from the Arabidopsis Biological Resource Center 565

(http://www.arabidopsis.org). Wild tobacco plants (N. benthamiana) were grown at 22–566

23qC under the above-mentioned long-day conditions. 567

17

568

Positional Cloning 569

For genetic mapping of the sagl1-1 mutation, the mapping population was derived from F1 570

plants obtained from a cross between sagl1-1 (in the C24 accession background) and WT 571

Landsberg erecta. F2 seedlings with the sagl1-1 phenotypes (small, thick, waxy, dark 572

green leaves) were selected and their genomic DNA was extracted. SSLP markers were 573

used for mapping. When necessary, new SSLP markers were developed using the 1001 574

Genomes project sequence database (http://1001genomes.org). The newly developed 575

SSLP markers are listed in Supplemental Table 1. 576

577

Gene Expression and gDNA Analysis 578

For RNA analysis, total RNA was extracted from 2-week-old plants using the RNAiso Plus 579

reagent (Takara) and an RNeasy Plant Mini Kit (Qiagen). Genomic DNA contamination 580

was removed by DNase I treatment (NEB). Reverse transcription was performed using 581

PrimeScript® RT Master Mix (Takara), and the resulting cDNA was used for RT-PCR 582

analysis. qRT-PCR was carried out using KAPA SYBR FAST qPCR Kit Master Mix (2X) 583

Universal (KAPA Biosystems) on a CFX96 Real-Time PCR system (Bio-Rad). The 584

reaction mixture (total volume of 20 µL) consisted of 1 µL cDNA, 10 PL KAPA SYBR 585

master mix, 7 µL of H2O, and 2 µL of 10 pmol gene-specific primer set listed in 586

Supplemental Table 1. PP2A (At1g13320) was used as a reference gene (Czechowski et 587

al., 2005). The qRT-PCR cycle was as follows: preincubation at 95qC for 10 min, 45 588

cycles of 2 step amplification at 95qC for 20 sec and 60qC for 20 sec. For genomic DNA 589

(gDNA) analysis, gDNA was isolated using extraction buffer (200 mM Tris-HCl, pH 7.5, 590

250 mM NaCl, 25 mM EDTA and 0.5% SDS) from mutants or 2-week-old leaves of T1 591

generation transgenic plants expressing SAGL1. Insertion of T-DNA was confirmed by 592

PCR using gene- and vector-sequence specific primer sets listed in Supplemental Table 593

1. 594

595

Plasmid Construction and Plant Transformation 596

To complement the sagl1-1 mutant, a 4,167 bp genomic DNA fragment of SAGL1 597

including 2,001 bp upstream of the start codon was amplified using ExTaq polymerase 598

(Takara). C24 genomic DNA was used as a template with the primer pair 599

F7A10_58.5K_SacIFg and F7A10_60K_KpnIRg. The amplified DNA fragment was 600

cloned into pCAMBIA1200 between the SacI and KpnI sites, resulting in the 601

pCAM1200-SAGL1g construct. To generate the SAGL1:eYFP fusion construct, the 602

18

SAGL1 CDS without the stop codon was amplified by PCR with the following primers: 603

SAGL1_SacF and SAGL1_SmaR. The SAGL1 CDS fragment was cloned into the 604

pPZP212 binary vector harboring a CaMV 35S promoter, enhanced yellow fluorescent 605

protein (eYFP), and Rubisco small subunit terminator (Hajdukiewicz et al., 1994; Go et al. 606

2014) between the SacI and SmaI sites. To generate the F-box-deleted SAGL1, the two 607

parts of the SAGL1 CDS were amplified by PCR with the primer pairs 608

At1g55270_cXbaIF/SAGL1(-Fbox)-R and SAGL1(-Fbox)-F/At1g55270_cKpnIR. The two 609

PCR products were ligated by overlap PCR with the primer pair At1g55270_cXbaIF and 610

At1g55270_cKpnIR. The resulting F-box-deleted SAGL1 CDS fragment was cloned into 611

the pBIB superbinary vector. The F-box-deleted SAGL1 CDS without the stop codon was 612

then amplified by PCR using the SAGL1_SacF and SAGL1_SmaR primers. The DNA 613

fragments were inserted into the pPZP212 binary vector between the SacI and SmaI sites, 614

resulting in SAGL1'F:eYFP. To generate the MYC:CER3 (At5g57800) fusion construct, 615

the CER3 CDS was amplified by PCR with the CER3_smF and CER3_scR primers. The 616

CDS of CER3 was cloned into the pBA002 binary vector harboring the CaMV 35S 617

promoter, 6xMYC, and nopaline synthase (Nos) terminator between the SmaI and SacI 618

sites. To generate the MYC:CER1 and MYC:CYTB5D fusion constructs, the CER1 and 619

CYTB5D CDS regions were amplified by PCR with the CER1_XmaF and CER1_SacR 620

primers and CYTB5_smF and CYTB5_scR primers, respectively. Each CDS region of 621

CER1 and CYTB5D was cloned into the pBA002 binary vector harboring the CaMV 35S 622

promoter, 6xMYC, and nopaline synthase (Nos) terminator between the SmaI or XmaI 623

and SacI sites. For the SAGL1 promoter-driven GUS construct, a 1,493 bp fragment 624

upstream of the SAGL1 start codon was amplified by PCR using C24 genomic DNA and 625

the primer pair F7A10(SAGL1g)pBamHI-F and F7A10(SAGL1g)pNcoI-R. The SAGL1 626

promoter fragment was subcloned into pCAMBIA1381 between the BamHI and NcoI 627

sites, resulting in pCAM1381-SAGL1p-GUS. The binary constructs were transformed into 628

Agrobacterium tumefaciens GV3101:pMP90 and the transformed Agrobacterium was 629

used for plant transformation by the floral dip method for Arabidopsis (Col-0, C24, or 630

sagl1-1) or leaf infiltration for N. benthamiana (Bent, 2006; Li, 2011). 631

632

Drought Treatment and Water Loss Measurements 633

Three-week-old C24 and sagl1-1 plants and 2-week-old Col-0 and sagl1-2 634

(Salk_097761) plants grown under the above-mentioned conditions were subjected to 635

drought stress by withholding water for 12 days. To ensure that both WT and sagl1 plants 636

experienced equal drought stress, the same amount of soil per pot was used and the pots 637

19

were soaked fully with water just before withholding water. Rosette leaves of 25-day-old 638

WT and sagl1 mutants were detached and placed under room conditions. The fresh 639

weights of the leaves were measured at the indicated time points (0, 1, 2, 3 and 4 h).640

641

Stomatal Sensitivity Assay 642

Stomatal sensitivity was measured as previously described (Guo et al., 2002). To 643

examine the stomatal response to ABA, seedlings grown under long-day conditions were 644

placed under high humidity in the light for 12 to16 h immediately before the assay. The 645

epidermal layers from leaves were incubated in stomatal opening solution [10 mM 646

MES-Tris (pH 6.15), 50 mM KCl] for 2 h. Using the epidermal layer from the leaves, 647

stomatal apertures were measured under a light microscope (Leica ICC50 HD) after 648

treatment with 2 μM ABA. For a proper comparison, relative values were used, with 649

stomatal apertures of the respective wild type at 0 μM ABA serving as a reference value. 650

651

SEM and TEM Analysis 652

WT and sagl1 plants grown in soil under the above-mentioned conditions for 6 weeks 653

were used for SEM and 4-week-old plants were used for TEM analysis. For SEM analysis, 654

live WT and mutant stems and leaves were coated with platinum particles using a sputter 655

coater for 10 min on a round grid. The samples were loaded and directly observed by 656

SEM (Hitachi S2400 FE-SEM) (Kim et al., 2017). Wax crystals on stem and leaf surfaces 657

were observed under 2,000x and 3,000x magnification. For TEM analysis, leaves were 658

fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate 659

buffer (pH 7.4). The samples were dehydrated in an ethanol series (10 to 100%) and 660

embedded in Spurr’s epoxy resin (ERL 4206; TedPella Inc.) for 2 d. Thin sections were 661

prepared using an ultramicrotome (RMC MT X) after staining with uranyl acetate and lead 662

citrate. 663

664

Cuticular Wax Analysis 665

Ten-day-old WT, sagl1, and SAGL1 overexpressing plants grown on half-strength MS 666

plates containing 1% sucrose and 0.7% agar were transplanted to soil. After 6 weeks, 667

inflorescence stems, rosette leaves, and primary roots were used to measure wax 668

content and composition. Waxes from stems and rosette leaves were extracted by 669

dipping in chloroform for 30 s, and three internal standards (20 µg n-octacosane, 10 µg 670

docosanoic acid, and 10 µg 1-tricosanol per 0.1 g fresh weight for stem waxes; 2 µg 671

n-octacosane, 1 µg docosanoic acid, and 1 µg 1-tricosanol per 0.1 g fresh weight for672

20

rosette leaf wax) were added to the wax extracts. After evaporating the wax extracts 673

under nitrogen gas, 100 µL of pyridine and N,O-Bis(trimethylsilyl)trifluoroacetamide were 674

added to the samples, followed by incubation at 95°C for 30 min. The reactants were 675

evaporated again and dissolved in heptane:toluene (1:1, v/v) solvent. Wax load and 676

composition analyses were performed using GC-FID and GC-MS as described previously 677

(Seo et al., 2011). 678

679

Cutin Analysis 680

For cutin polymer analysis, NaOMe depolymerization and acetylation of monomers were 681

performed as described previously (Li-Beisson et al. 2013), with slight modifications. 682

Six-week-old inflorescence stems and rosette leaves were used to measure cutin content 683

and composition. Arabidopsis tissues were immersed in boiling (85°C) isopropanol for 10 684

min, and soluble lipids were extracted using chloroform/methanol and methanol. The 685

residues were dried under nitrogen gas, depolymerized using NaOMe, and acetylated. 686

Methyl heptadecanoate and a ω-pentadecalactone were used as internal standards (100 687

µg per 0.1 g dry residues). Polyester monomers were identified and quantified by GC-FID 688

and GC-MS as described previously (Seo et al., 2011). 689

690

In vitro Immunoblot Assay 691

Agrobacterium cells harboring the pPZP212 construct (for the control), the MYC:CER3, 692

MYC:CER1 or MYC:CYTB5D construct, and the SAGL1 or SAGL1ΔF construct were 693

co-infiltrated into the abaxial sides of N. benthamiana leaves with expression buffer [10 694

mM MES (pH 5.7), 10 mM MgCl2, 0.5 mM acetosyringone]. Plant tissues were harvested 695

1.5 d after Agrobacterium infiltration. Plant tissues pretreated with 100 µM MG132 were 696

treated with infiltration medium 12 h before sampling. Harvested plant materials were 697

ground in liquid nitrogen and dissolved in extraction buffer [125 mM Tris (pH 6.8), 4% 698

SDS, 2% DTT, 20% glycerol, and proteinase inhibitors including PMSF, pepstain A, 699

aprotinin, leupeptin, and MG132]. The total cellular extracts were analysed by 700

SDS-PAGE (10% gels) and blotted onto polyvinylidene difluoride membranes. 701

Epitope-tagged proteins were immunologically hybridized using anti-MYC (1:2,000 702

dilution; Millipore) and anti-GFP (1:8,000 dilution; Clontech) antibodies. All images were 703

developed using Thermo Scientific Pierce ECL Western Blotting Substrate and the AI600 704

chemidoc imaging system (GE Healthcare). 705

For the Co-IP and ubiquitination assays, Agrobacterium cells carrying the 706

SAGL1:eYFP and MYC:CER3, MYC:CER1 or MYC:CYTB5D construct were 707

21

co-infiltrated into N. benthamiana leaves. At 24 h after Agrobacterium infiltration, the N.708

benthamiana leaves were treated with 100 µM MG132 or DMSO using a syringe. After an 709

additional 12 h, the leaves were harvested and ground using a mortar and pestle under 710

liquid nitrogen. Total proteins were extracted with extraction buffer [RIPA buffer: 50 mM 711

Tris-HCl (pH 7.5); 150 mM NaCl; 0.1% sodium dodecyl sulfate (SDS); 0.5% 712

deoxycholate; 1% Triton X-100; and proteinase inhibitors including phenylmethane 713

sulfonyl fluoride (PMSF), pepstain A, aprotinin, leupeptin, and MG132]. Anti-c-MYC 714

conjugated agarose beads (A7470, Sigma) were added to the protein extracts (total 715

amount 1.5 mg) and incubated on an orbital shaker for 2 h at 4 °C. After centrifugation, 716

the anti-c-MYC conjugated agarose beads were thoroughly washed with RIPA buffer. 717

The washed beads were resuspended in sample buffer [125 mM Tris-HCl (pH 6.8), 4% 718

SDS, 2% dithiothreitol, 20% glycerol, and 0.025% bromophenol blue]. The protein 719

samples were subjected to an immunoblot assay as described above. eYFP-tagged 720

proteins and ubiquitinated proteins were immunologically detected using anti-GFP 721

(1:8,000 dilution; Clontech) and anti-Ub (1:2,000 dilution; SantaCruz) antibodies, 722

respectively. 723

For the SAGL1 protein stability assay, four-day-old transgenic Arabidopsis seedlings 724

overexpressing SAGL1:eYFP driven by the CaMV 35S promoter were collected and 725

incubated in 1/2 MS liquid medium containing 100 PM MG132 or DMSO on a rocker for 4 726

h. The seedlings were washed with 1/2 MS liquid medium to remove the MG132 or727

DMSO and incubated in 1/2 MS liquid medium containing 100 PM CHX on a rocker for 728

0.5, 1, and 2 h. The protein stability of SAGL1:eYFP under different humidity conditions 729

was examined in the rosette leaves of three- to four-week-old transgenic Arabidopsis 730

overexpressing SAGL1:eYFP that were unwrapped or wrapped with plastic sheets for 12, 731

24, or 36 h. To measure CER3 proteins levels in WT (C24) and sagl1-1 Arabidopsis 732

overexpressing MYC:CER3, the rosette leaves of three- to four-week-old plants were 733

unwrapped or wrapped with plastic sheets for 24 h. Immunoblot analysis for 734

SAGL1:eYFP and MYC:CER3 was carried out as described above. 735

736

Subcellular Localization 737

Agrobacterium cells harboring SAGL1:eYFP or mRFP in the pPZP212 vector were 738

inoculated into N. benthamiana leaf epidermal cells with expression buffer [50 mM MES 739

(pH 5.7), 10 mM MgSO4, 5% D-glucose and 0.1 mM acetosyringone] to a final 740

concentration of OD600=0.5. After 24 h of incubation, N. benthamiana leaf protoplasts 741

were isolated as previously described (Abel and Theologis, 1998). The protoplasts were 742

22

visualized under a TCS SP5 AOBS/Tandem confocal laser-scanning microscope (Leica). 743

The excitation wavelength was 500 nm and the emission wavelength was 535 nm for YFP 744

signals. The excitation range was 494 to 540 nm and the emission range was 570 to 620 745

nm for RFP signals. Auto-fluorescence from chloroplasts was detected at 514 nm 746

excitation and 561 nm emission wavelengths. 747

748

Histochemical and Toluidine Blue O Staining and Histological Analysis 749

Transgenic plants were selected via the application of 30 µg/mL hygromycin B. The T2 750

generation plants were used to observe GUS expression. For the high humidity test, 751

seeds were germinated on half-strength MS agar medium in a covered container for 3 to 752

6 weeks. The container cover was removed for 32 h and the samples were stained. Some 753

of the seedlings were transplanted to soil and grown for 2 weeks. All tissues were stained 754

with GUS staining solution with 100 mM X-Gluc at 37qC overnight as previously described 755

(Jefferson et al., 1987). After overnight staining, chlorophyll was removed with 75% 756

ethanol and the samples were examined under a microscope. 757

Rosette leaves from 6-week-old pSAGL1:GUS transgenic plants were detached 758

and floated on half-strength MS (pH 5.7) solution with or without ABA (100 μM), mannitol 759

(200 mM), or NaCl (200 mM) for 24 h. The leaves were washed with distilled water and 760

subjected to GUS assays as described above. To quantify GUS activity, total protein was 761

extracted from ground rosette leaves using extraction buffer [50 mM sodium phosphate 762

buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% SDS, and 10 mM 763

β-mercaptoethanol]. Ten μg of protein was used to catalyze the 764

4-methylumbelliferyl-β-D-glucuronide (MUG) reaction. After incubation at 37qC for 1 h,765

the samples were analyzed using a luminescence reader (GLOMAX-20/20). 766

For Toluidine blue O staining, 4-week-old Arabidopsis plants were immersed in 767

Toluidine blue solution (0.05% with 0.01% Tween-20) for 10 min, washed with distilled 768

water, and photographed.769

770

Phylogenetic Analysis of SAGL1 771

The deduced amino acid sequences of Arabidopsis thaliana SAGL1 (At1g55270; 772

GenBank, NP_001320632.1) and its homologues from Brachypodium distachyon773

(Bradi1G73860.2; GenBank, XP_003562030.2), Brassica rapa (Brara.H00068.1; 774

GenBank: RID49257.1), Glycine max (Glyma.08G112900.1; GenBank, KRH42810.1), 775

Oryza sativa (LOC Os10G26990.1; GenBank, XP_015614781.1), Medicago truncatula776

(Medtr8G085600.1; GenBank, AET04178.1), Populus trichocarpa (Potri.003G217700.1; 777

23

GenBank, XP_002304010.2), Ricinus communis (29835.m000632; GenBank, 778

XP_002527061.1), Sorghum bicolor (Sobic.001G251800.1; GenBank, XP_002464634.1),779

Vitis vinifera (GSVIVT01025417001; GenBank, CBI16496.3), and Zea mays780

(GRMZM2G147402T01; GenBank, ACN32130.1) were obtained from Phytozome (v12.1; 781

https://phytozome.jgi.doe.gov/) and National Center for Biotechnology Information (NCBI; 782

https://www.ncbi.nlm.nih.gov/). Phylogenetic analysis was generated using MEGA6 by 783

ClustalW alignment and the neighbor-joining method based on the JTT matrix-based 784

model with bootstrap value percentages of 500 replicates (Tamura et al., 2013). The 785

scanning of F-box domain was performed using SMART (Simple Modular Architecture 786

Research Tool, http://smart.embl-heidelberg.de) (Letunic and Bork, 2018). 787

788

Accession Numbers 789

Sequence data in this article can be found in the TAIR (http://www.arabidopsis.org/) or 790

Phytozome 12 database (https://phytozome.jgi.doe.gov/) under the following accession 791

numbers: SAGL1 (AT1G55270), CER3 (AT5G57800), CER1 (At1g02205), CYTB5D 792

(At5g48810), PP2A (AT1G13320). 793

794

Supplemental Data 795

Supplemental Figure 1. Allelsm test between sagl1 mutants. 796

Supplemental Figure 2. Cuticular wax compositions and loads on the leaves, stems and 797

roots of WT and sagl1 plants. 798

Supplemental Figure 3. Cutin monomer compositions and levels in the leaves and 799

stems of WT and sagl1 plants. 800

Supplemental Figure 4. Cuticular wax and cutin compositions and loads on the stems of 801

WT (Col-0) and transgenic plants overexpressing SAGL1.802

Supplemental Figure 5. Immunoblot and Co-IP analysis of CER1 or CYTB5D 803

associated with SAGL1:eYFP protein. 804

Supplemental Figure 6. Subcellular localization of SAGL1:eYFP in 10-day-old 805

transgenic Arabidopsis roots overexpressing SAGL1:eYFP under the control of the 806

CaMV 35S promoter. 807

Supplemental Figure 7. Quantitative RT-PCR analysis of SAGL1 expression. 808

Supplemental Figure 8. Growth phenotypes of C24 and sagl1-1 under different humidity 809

conditions. 810

Supplemental Figure 9. Phylogenetic analysis and amino acid sequence alignment of 811

SAGL1 from various plant species. 812

24

Supplemental Figure 10. Topology of the CER3 protein. 813

Supplemental Figure 11. Phenotypes of the sagl1-2 cer3-6 double mutant. 814

Supplemental Table 1. List of DNA primers used in this study. 815

Supplemental Data Set 1. ANOVA tables. 816

Supplemental Data Set 2. Alignment of sequences used for neighbor-joining method. 817

818

ACKNOWLEDGEMENTS 819

820

We thank Su Bin Lee, Juyoung Kim, Reo Jin Kim, and Saet Buyl Lee for experimental 821

assistance, Young Woo Seo (Korea Basic Science Institute, Gwangju) for confocal 822

imaging, and Ljerka Kunst (University of British Columbia, Canada) for critical reading of 823

the manuscript. 824

825

AUTHOR CONTRIBUTIONS 826

827

B.-h.L. and M.C.S. conceived and designed the experiments. H.K., S.-i.Y., and S.H.J. 828

conducted experiments. B.-h.L. and M.C.S. wrote the paper with the help of H.K., S.-i.Y., 829

and S.H.J. 830

831

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(2005). Cuticular lipid composition, surface structure, and gene expression in 1064

Arabidopsis stem epidermis. Plant Physiol. 139: 1649-1665. 1065

Sun, Y., Zhou, X., and Ma, H. (2007). Genome-wide analysis of kelch repeat containing 1066

F-box family. J. Integr. Plant Biol. 49: 940-952.1067

Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013) MEGA6: 1068

Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30:2725-2729. 1069

Waadt, R., Hitomi, K., Nishimura, N., Hitomi, C., Adams, S.R., Getzoff, E.D., and 1070

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acid concentration changes and distribution in Arabidopsis. eLife 3: e01739. 1072

Wang, Y., Wang, J., Chai, G., Li, C., Hu, Y., Chen, X., and Wang, Z. (2015). 1073

Developmental changes in composition and morphology of cuticular waxes on leaves 1074

and spikes of glossy and glaucous wheat (Triticum aestivum L.). PLoS One 10: 1075

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Wang, M., Wang, Y., Wu, H., Xu, J., Li, T., Hegebarth, D., Jetter, R., Chen, L., and Wang, 1077

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F-box protein KIB1 mediates Brassinosteroid-induced inactivation and degradation of1102

GSK3-like kinases in Arabidopsis. Mol. Cell 66: 648-657.1103

1104

1105

Figure 1. Morphological phenotypes of the sagl1-1mutant.

(A) Comparison of leaf size between C24 (left) and sagl1-1 (right). The 6th rosette leaves from 5-week-oldplants are shown. Bar = 0.5 cm.(B) Cross sections of C24 (top) and sagl1-1 (bottom) leaves in the middle of the lamina. The 6th rosetteleaves from 5-week-old plants are shown. Bar = 20 μm.(C) Comparison of leaf thickness between C24 and sagl1-1. The 5th to 6th rosette leaves from 5-week-oldplants were measured. Averages from 60 rosette leaves from 30 plants and their SD are shown. Thevalues were statistically tested using ANOVA (*** indicates significant difference at P < 0.001).(D) Comparison of leaf reflection between C24 (left) and sagl1-1 (right) leaves. The arrow points to aglossy reflection in the sagl1-1 leaf. The 6th rosette leaves from 5-week-old plants are shown. Bar = 0.1cm.(E) to (J) Comparison of leaf (E, F), stem (G, H), and root morphology (I, J) of 4 to 6-week-old C24 (E, G,I) and sagl1-1 (F, H, J) plants.(K) Comparison of the growth habits of 6-week-old C24 (left) and sagl1-1 (right) plants.(L) Comparison of fresh weight between 4-week-old C24 and sagl1-1. Averages from 5 biologicalreplicates and their SD are shown. The values were statistically tested using ANOVA (** indicatessignificant difference at P < 0.01).

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Figure 2. Molecular cloning and complementation of sgal1-1.

(A) Map-based cloning of SAGL1. 791 F2 seedlings derived from a Ler x sagl1-1 cross were used for mapping.SSLP markers for mapping were F14C21, T7N22, F7A10, T18I3, and T5A14 from left to right. The number ofrecombinations is shown below each marker locus.(B) to (E) Molecular complementation of sagl1-1 with the SAGL1 gene. (B) C24, (C) sagl1-1 with the SAGL1gene, and (D) sagl1-1 (E) Overall phenotypes of each genotype.(F) Predicted domains in SAGL1. Asterisk represents the sagl1-1 mutation site.(G) Position of the T-DNA insert in the sagl1-2 allele.(H) SAGL1 expression in WT (Col-0) and sagl1-2, as determined by RT-PCR.(I) Comparison of the leaf phenotypes of each allele and the respective WT control. Three-week-old plants areshown.(J) Growth and development of Col-0 (left) and sagl1-2 (right and inset). Six-week-old plants are shown.(K) and (L) Root morphology of Col-0 (K) and sagl1-2 (L).

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34th SGLKTVVEARKFVPGSKLCIQPDINPNSGCRSKEVCSWFKALYST

52th aa Stop

Wild Typesagl1-1

Wild Typesagl1-1

Figure 3. sagl1mutants displayed enhanced drought tolerance compared with WT.

(A) Differences between WT and sagl1 phenotypes under drought stress.(B) Water loss from rosette leaves of WT (squre symbols) and sagl1 (circle symbols) at the indicated timepoints. Averages from 6 biological replicates and their SD are shown. The values were statistically tested usingANOVA with post-hoc Tukey HSD test (P < 0.05). The lowercase letters indicate statistical differenciation.(C) Comparison of stomatal density on the abaxial surfaces between WT and sagl1 leaves. Averages from 15biological replicates and their SD are shown. The values were statistically tested using ANOVA with post-hocTukey HSD test (P < 0.05). The lowercase letters indicate statistical differenciation.(D) Relative stomatal aperture in response to 2 μM ABA treatment. Averages from 50 biological replicates andtheir SD are shown. The values were statistically tested using ANOVA with post-hoc Tukey HSD test (P < 0.05).The lowercase letters indicate statistical differenciation.

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Figure 4. Scanning electron microscopy (SEM) of stems and transmission electron microscopy (TEM)of leaves of WT and sagl1 plants.

(A) to (D) SEM images of cuticular wax crystals on the stems of C24 (A), sagl1-1 (B), Col-0 (C) and sagl1-2(D). Bars = 5 μm. The typical shapes of epicuticular wax crystals, rod (blue arrow), tube (yellow arrow) andhorizontal plate (red arrow), are shown on the surfaces of the Arabidopsis stems.(E) and (F) TEM images of the cuticle in the leaves of Col-0 (E) and sagl1-2 (F). The large black squaresshow magnified views of the imiages in the small black squares. Bars = 1 μm.(G) and (H) Cuticle thickness (G) and cell wall thickness (H) measurements in Col-0 and sagl1-2. Averagesfrom 5 biological replicates and their SD are shown. The values were statistically tested using ANOVA (*indicates significant difference at P < 0.01).

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Figure 5. Cuticular wax and cutin composition and levels in the leaves, stems and roots of WT andsagl1 plants.

(A) to (F) Cuticular waxes were extracted from rosette leaves (A, B), stems (C, D) and roots (E, F) of 4 to5-week-old Arabidopsis plants. (A, C and E) total wax loads. (B, D and F) compositions of cuticular waxes.(G) to (J) Cutin monomers were extracted from rosette leaves (G, H) and stems (I, J) of 4 to 5-week-oldArabidopsis plants. (G and I) total cutin loads. (H and J) compositions of cutin monomers.Averages from three biological replicates from two individual experiments and their SD are shown. Thevalues were statistically tested using ANOVA with post-hoc Tukey HSD test (P < 0.05) or ANOVA (*,P<0.05. **, P<0.01). Ak, alkanes; Ke, ketone; PA, primary alcohols; Al, aldehydes; FA and FFA, fatty acids;SA, secondary alcohols; MAG, monoacylglycerol; HFA, hydroxy fatty acids; DCA, dicarboxylic acids; Un,unidentified. FW, fresh weight.

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Figure 6. Immunoblot analysis and Coimmunoprecipitation assay of CER3 associated with SAGL1.

(A) and (B) MYC:CER3 and SAGL1:eYFP (A) or mutated SAGL1F:eYFP with a deleted F-box domain region (B)under the control of the CaMV 35S promoter were transiently co-expressed in N. benthamiana leaves. MG132 (100μM) was used to inhibit the degradation of proteins by the 26S proteasome system. Anti-MYC or anti-GFPantibodies were used for immunoblot analysis to detect MYC:CER3 or SAGL1/SAGL1F:eYFP. Equal proteinloading was assured using Ponceau-S staining.(C) Coimmunoprecipitation (Co-IP) assays to detect MYC:CER3 and SAGL1:eYFP interactions. Total proteinextracts were obtained from N. benthamiana leaves infiltrated with Agrobacterium suspensions harboring theMYC:CER3 or SAGL1:eYFP construct. Input, Total protein extracts were subjected to immunoblot analysis usinganti-GFP or anti-MYC antibodies. IP: α-MYC, Total protein extracts were immunoprecipitated with anti-MYC agaroseand subjected to immunoblot analysis using anti-GFP or anti-MYC antibodies.(D) Ubiquitination assay of MYC:CER3. SAGL1:eYFP and MYC:CER3 were transiently co-expressed in N.benthamiana leaves. MG132 (100 μM) was infiltrated into the leaves to inhibit 26S proteasome activity. Total proteinextracts obtained from N. benthamiana leaves were immunoprecipitated using anti-MYC conjugated to agarosebeads and subjected to immunoblot analysis using anti-MYC or anti-Ub antibodies. WB, western blotting(immunoblot analysis).(E) MYC:CER3 protein and transcripts levels in 14-day-old WT (C24) and sagl1-1 seedlings overexpressingMYC:CER3 under the control of the CaMV 35S promoter. MYC:CER3 protein levels were determined in total proteinextracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-Sstaining. MYC:CER3 transcript levels were determined by RT-PCR. PP2A was used as an endogenous referencegene.

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Figure 7. Subcellular localization of SAGL1:eYFPin N. benthamiana protoplasts and GUSexpression driven by the SAGL1 5’ promoterfragment in Arabidopsis.

(A) SAGL1:eYFP driven by the CaMV 35S promoterwas transiently co-expressed in N. benthamianaleaves with mRFP in pPZP212 (left panel), which wasused as a cytoplasmic and nuclear protein marker(Goodin et al. 2002). Protoplasts isolated from N.benthamiana leaves were used to visualizefluorescent signals under a confocal laser-scanningmicroscope 48 h after infiltration. Autofluorescentsignals from chloroplasts are shown in blue (middlepanel) and merged images are shown on the right.Scale bars = 10 µm.(B) SAGL1 expression in various Arabidopsis organs.Total RNA was isolated from 7-day-old seedlings (YS),rosette leaves of 3-week-old (R1), 4-week-old (R2),and 6- to 7-week-old (R3) plants, and roots (Ro),stems (St), buds, (Bu), flowers (Fl) and siliques (Si; 1–10 days after flowering) of 6- to 7-week-old plants andsubjected to qRT-PCR analysis. PP2A was used asan endogenous reference gene for normalizationacross samples, and data are presented as 2−ΔCtvalues (Livak and Schmittgen, 2001). Averages fromfour biological replicates from two individualexperiments and their SD are shown.(C) to (L) and (N) Histochemical analysis oftransgenic Arabidopsis expressing GUS under thecontrol of the SAGL1 5’ promoter fragment. (C), Ten-day-old transgenic Arabidopsis seedling grown in aPetri dish; (D) and (E), Three-week-old and four-week-old transgenic Arabidopsis in a soil. (F) to (I),Root (F), stems and cauline leaves (G), floral organs(H), and silique (I) of 5- to 7-week-old transgenicArabidopsis. (J) and (K), Six-week-old transgenicArabidopsis grown in a culture bottle before (J) and 32h after (K) removing the bottle cap. (L), Some leavesof 5-week-old transgenic Arabidopsis grown in soilwere not covered (left), but others were covered withplastic sheets for 3 d (right).(M) qRT-PCR analysis of SAGL1. Total RNA wasisolated from rosette leaves unwrapped or wrapped inplastic sheets for the indicated times and subjected toqRT-PCR analysis. The PP2A gene was used tomeasure the quality and quantity of cDNA. Averagesfrom two biological replicates from six individualexperiments and their SD are shown. Data werestatistically analyzed using an ANOVA with post-hocTukey HSD test (P < 0.05).(N) and (O) GUS staining (N) and activity (O) inrosette leaves of 4-week-old transgenic Arabidopsisbefore (Non) and 24 h after incubation in half-strengthMS liquid medium without (MOCK) and supplementedwith 100 μM ABA, 200 mM mannitol or 200 mM NaCl.(O), Averages from two biological replicates fromthree individual experiments and their SD are shown.RLU, Relative Light Units.

Figure 8. Immunoblot analysis of SAGL1:eYFP and MYC:CER3 in transgenic Arabidopsis plants,and cuticle permeability of WT and sagl1-2 under relatively low and high humidity conditions.

(A) Assay of SAGL1:eYFP protein levels under different humidity conditions. Seven-day-old WT (Col-0)and transgenic Arabidopsis seedlings overexpressing SAGL1:eYFP grown in Petri dishes weretransferred to soil. The seedlings were covered with an opaque plastic lid for 1 day (>90% RH),uncovered (50–60% RH) for the indicated time points, and subjected to immunoblot analysis.SAGL1:eYFP protein levels were determined in total protein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equal protein loading was assured using Ponceau-Sstaining.(B) SAGL1:eYFP protein stability assay. Four-day-old WT (Col-0) and transgenic Arabidopsis seedlingsoveresxpressing SAGL1:eYFP were treated with the MG132 (100 μM) or DMSO before treatment with100 μM CHX for the indicated amounts of time. SAGL1:eYFP protein levels were determined in totalprotein extracts by immunoblot analysis using anti-GFP antibodies at the indicated time points. Equalprotein loading was assured using Ponceau-S staining.(C) Seven-day-old transgenic WT (C24) and sagl1-1 seedlings overexpressing MYC:CER3 grown inPetri dishes were exposed to 50–60% RH for 0 and 12 h and subjected to immunoblot analysis.MYC:CER3 protein levels were determined in total protein extracts by immunoblot analysis using anti-MYC antibodies. Equal protein loading was assured using Ponceau-S staining.(D) Arabidopsis WT (Col-0) and sagl1-2 plants were transferred to soil after germination on half-strengthMS medium and uncovered or covered with a lid to maintain high humidity (>90% RH). Four-week-oldArabidopsis WT and sagl1-2 leaves were immersed in Toluidine blue O solution (0.05% with 0.01%Tween 20) for 10 min, washed with distilled water, and photographed.

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DOI 10.1105/tpc.19.00152; originally published online July 18, 2019;Plant Cell

Hyojin Kim, Si-in Yu, Seh Hui Jung, Byeong-ha Lee and Mi Chung Suhto Changes in Humidity in Arabidopsis

The F-box Protein SAGL1 and ECERIFERUM3 Regulate Cuticular Wax Biosynthesis in Response

 This information is current as of January 6, 2020

 

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