University, Tai’an, 271018, hinaMar 09, 2018 · 58. gametophytic d. evelopment, interactions...

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Research area: Cell biology 1

Short title: AP-3 mediates pollen tube growth 2

For correspondence: 3

Yan Zhang, 4

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural 5

University, Tai’an, 271018, China 6

Tel: (86)538-8246365 7

Email: [email protected] 8

9

Author contributions 10

Y.Z. conceived and supervised the project; Q.F. performed the experiments with the 11

assistance of X.L.; Y.Z., S.L., and Q.F. designed the experiments and analyzed the data; 12

Y.Z. and Q.F. wrote the article. 13

14

Footnotes: 15

This work was supported by the Natural Science Foundation of China (31625003 and 16

31471304 to Y.Z., 31771558 to S.L.), by Natural Science Foundation of Shandong 17

Province (ZR2014CM027 to S.L.), and by China Postdoctoral Science Foundation 18

(2015M570605 and 2016T90643 to S.L.). Y.Z.’s laboratory is partially supported by the 19

Tai-Shan Scholar Program of the Shandong Provincial Government. 20

21

22

Plant Physiology Preview. Published on March 9, 2018, as DOI:10.1104/pp.17.01722

Copyright 2018 by the American Society of Plant Biologists

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mailto:[email protected]

https://plantphysiol.org

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The ADAPTOR PROTEIN-3 complex mediates pollen tube growth by coordinating 23

vacuolar targeting and organization 24

Qiang-Nan Feng, Xin Liang, Sha Li, Yan Zhang* 25

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural 26

University, Tai’an, 271018, China 27

28

One Sentence Summary: The dynamic organization of vacuoles and the association 29

of tonoplast cargo protein PAT10 are impaired and lead to reduced pollen tube 30

growth in adaptor protein-3 mutants. 31

32

Abstract 33

Pollen tube growth is an essential step for successful plant reproduction. Vacuolar 34

trafficking and dynamic organization are important for pollen tube growth; however, 35

the key proteins involved in these processes are not well understood. Here, we 36

report that the ADAPTOR PROTEIN-3 (AP-3) complex and its tonoplast cargo PROTEIN 37

S-ACYL TRANSFERASE10 (PAT10) are critical for pollen tube growth in Arabidopsis 38

(Arabidopsis thaliana). AP-3 is a heterotetrameric protein complex consisting of four 39

subunits, δ, β, µ, and σ. AP-3 regulates tonoplast targeting of several cargoes, such as 40

PAT10. We show that functional loss of any of the four AP-3 subunits reduces plant 41

fertility. In ap-3 mutants, pollen development was normal but pollen tube growth 42

was compromised, leading to reduced male transmission. Functional loss of PAT10 43

caused a similar reduction in pollen tube growth, suggesting that the tonoplast 44

association of PAT10 mediated by AP-3 is crucial for this process. Indeed, Ca2+ 45

gradient during pollen tube growth was significantly reduced due to AP-3 46

loss-of-function, consistent with the abnormal targeting of Calcinuerin B-like2 (CBL2) 47

and CBL3, whose tonoplast association depends on PAT10. Further, we show that the 48

pollen tubes of ap-3 mutants have vacuoles with simplified tubules and bulbous 49

structures, indicating that AP-3 affects vacuolar organization. Our results 50

demonstrate a role for AP-3 in plant reproduction and provide insights into the role 51

of vacuoles in polarized cell growth. 52

53

Keywords: pollen tube growth, vacuolar trafficking, male fertility, CBL2, calcium 54 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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55

Introduction 56

Sexual reproduction of flowering plants is a complex, multi-step process involving 57

gametophytic development, interactions between male and female gametophytes, 58

and interactions between male and female gametes (McCormick, 2004). Due to the 59

immobility of sperm cells in angiosperms, the delivery of sperm into embryo sacs 60

depends on pollen tubes, long cylindrical extensions initiated from the growing tip of 61

pollen grains. Pollen tubes penetrate female sporophytic tissues, target the micropyle 62

by sensing and responding to female guidance cues, and finally burst to release 63

sperm cells into embryo sacs (Johnson and Preuss, 2002). Tip growth of pollen tubes 64

is a specialized polar growth that involves complex intracellular activities, including a 65

tip-focused Ca2+ gradient, spatial organization of endomembrane systems, and 66

dynamic organization of the cytoskeleton (Hepler et al., 2001; Cheung and Wu, 2007; 67

Cheung et al., 2008; Cheung and Wu, 2008; Hepler et al., 2012). 68

Pollen tube growth accompanies dynamic vacuolar organization and trafficking 69

(Hicks et al., 2004). Vacuoles, as extensive tubular extensions, fill with growing pollen 70

tubes, except at the very apex (Hicks et al., 2004; Wudick et al., 2014). Functional loss 71

of VACUOLESS1 (VCL1), a gene essential for vacuolar biogenesis (Rojo et al., 2001), 72

resulted in reduced male gametophytic transmission although the vacuoles in vcl1 73

pollen tubes seemed normal (Hicks et al., 2004). Overexpression of PTEN, a 74

phosphatase that downregulates PtdIns3P, the phosphoinositide critical for vacuolar 75

fusion, resulted in defective pollen tube growth by disrupting vacuolar consumption 76

of autophagic bodies in Arabidopsis (Arabidopsis thaliana) (Zhang et al., 2011). In 77

addition, mutations at VPS41, encoding a component of the homotypic fusion and 78

vacuolar protein sorting (HOPS) complex that mediates vesicle-vacuole fusion, 79

caused defective pollen tube growth in response to female cues (Hao et al., 2016). 80

These results suggested that vacuolar trafficking and organization are critical for the 81

polar and directional growth of pollen tubes. However, the key proteins targeted to 82

vacuoles and critical for pollen tube growth are yet unknown. 83

Proper targeting of proteins within the endomembrane system is crucial for cell 84

growth and responses to the environment. Adaptor proteins (AP) are key players 85

mediating protein sorting; they recognize both cargo proteins and coat proteins 86 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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during vesicle formation (Boehm and Bonifacino, 2001; Robinson, 2004; Bassham et 87

al., 2008). APs are heterotetrameric protein complexes. There are five AP complexes 88

in eukaryotes, differing in their subcellular targeting and functionalities: AP-1, AP-2, 89

AP-3, AP-4, and AP-5 (Boehm and Bonifacino, 2001; Robinson, 2004; Bassham et al., 90

2008; Hirst et al., 2011). In plants, except for the newly discovered AP-5, the other 91

types of APs have been characterized. AP-1 associates with the trans-Golgi 92

network/early endosome (TGN/EE) to sort proteins to the plasma membrane (PM), 93

to the forming cell plate, or to vacuoles (Park et al., 2013; Teh et al., 2013). 94

Functional loss of AP-1 reduced male and female fertility (Park et al., 2013; Teh et al., 95

2013; Wang et al., 2013). AP-2 participates in clathrin-mediated endocytosis and is 96

required for reproductive organ development (Fan et al., 2013; Kim et al., 2013; 97

Yamaoka et al., 2013). Another class of ancient adaptor complex termed TPLATE/TSET 98

(Gadeyne et al., 2014; Hirst et al., 2014) was reported to mediate reproduction, as 99

mutations in those components caused pollen developmental defects (Van Damme 100

et al., 2006; Gadeyne et al., 2014). AP-4 was recently reported to mediate vacuolar 101

trafficking (Fuji et al., 2016). Several studies indicated that AP-3 mediates vacuolar 102

targeting of several protein cargoes, including SUCROSE TRANSPORTER4 (SUC4) 103

(Wolfenstetter et al., 2012), the R-SNARE subfamily members VAMP713 (Ebine et al., 104

2014) and VAMP711 (Feng et al., 2017), as well as PROTEIN S-ACYL TRANSFERASE10 105

(PAT10) (Feng et al., 2017b). Functional loss of AP-3 resulted in reduced germination 106

potential of seeds (Feraru et al., 2010; Zwiewka et al., 2011) and slight defects in 107

gravitropic responses (Feraru et al., 2010). It is unclear whether AP-3 affects 108

reproductive processes. 109

In this study, we analyze mutants of all four AP-3 subunits and show that AP-3 110

and its tonoplast cargo PAT10 are important for pollen tube growth. Functional loss 111

of single AP-3 subunits reduced male transmission and caused reduced seed set. 112

Pollen development was not affected by AP-3 loss-of-function. However, the rapid 113

growth of pollen tubes both in vitro and in vivo was compromised in the ap-3 114

mutants. Functional loss of PAT10 caused a similar reduction in pollen tube growth, 115

suggesting that AP-3-mediated vacuolar targeting of PAT10 is crucial for this process. 116

We further show that AP-3 loss-of-function affected the vacuolar organization of 117

pollen tubes, as vacuoles of mutant pollen tubes were less complex tubules, with 118 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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attached bulbous structures. Our results demonstrate a role for AP-3 in plant 119

reproduction and provide insights into the role of vacuoles in polarized cell growth. 120

121

Results 122

AP-3 loss-of-function results in reduced seed set 123

To determine the potential roles of AP-3 during plant reproduction, we characterized 124

mutants of each AP-3 component, i.e., AP-3δ, AP-3β, AP-3µ, and AP-3σ (Fig. 1A). 125

Each component is encoded by a single gene in Arabidopsis (Bassham et al., 2008). 126

Mutants of AP-3δ, AP-3β, and AP-3µ used in this study were previously designated as 127

protein affected trafficking4 (pat4-2), pat2-2, and ap-3µ-2, respectively, and were 128

proven to be null mutants (Niihama et al., 2009; Feraru et al., 2010; Zwiewka et al., 129

2011). Only one T-DNA insertion mutant was available within the genomic region of 130

AP-3σ, in which the full-length transcript of AP-3σ was below RT-qPCR detection 131

(Feng et al., 2017b). To be consistent with their functional roles as AP-3 components, 132

and for simplicity, we refer to these mutants as ap-3δ, ap-3β, ap-3µ, and ap-3σ. 133

Each of the mutants was similar to wild type during the vegetative stage, as 134

reported (Feraru et al., 2010; Zwiewka et al., 2011). However, during the 135

reproductive stage, each mutant showed significantly reduced seed set (Fig. 1B), with 136

undeveloped ovules distributed predominantly at the bottom of the siliques (Fig. 1C, 137

1D). These results showed that AP-3 is important for reproduction. 138

139

Functional loss of AP-3 reduces male transmission but does not affect pollen 140

development 141

To determine the reason for the reduced seed set, we performed reciprocal crosses 142

between the wild type and each heterozygous mutant: ap-3δ, ap-3β, ap-3µ, or ap-3σ. 143

Segregation ratios of the F1 progenies showed that each mutant showed reduced 144

male but not female transmission (Table 1). These results demonstrated that AP-3 is 145

involved in male gametophytic function. 146

Several steps lead to proper male gametophytic function, specifically, pollen 147

development, pollen germination, polar and directional growth of pollen tubes, and 148

pollen tube reception (McCormick, 2004). To determine the causes of compromised 149

male gametophytic function in the ap-3 mutants, we first analyzed pollen 150 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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development using Alexander staining for cytoplasmic viability, DAPI staining for 151

nuclear structure, and scanning electron microscopy (SEM) for pollen coat structure 152

(Johnson-Brousseau and McCormick, 2004). The ap-3 mutants were no different 153

from the wild type (Supplemental Fig. 1), indicating that AP-3 is not involved in pollen 154

development. 155

156

AP-3 loss-of-function causes a reduction in pollen tube growth but not in guidance 157

To determine the cause of reduced male transmission in the ap-3 mutants, we next 158

performed in vitro pollen germination (Fig. 2A-E). All ap-3 mutants were comparable 159

to wild type in germination potential (Fig. 2F), indicating that AP-3 is not essential for 160

pollen germination. In the first two hours of pollen germination, there was no 161

difference in pollen tube length between the ap-3 mutants and the wild type 162

(Supplemental Fig. 2). However, at later stages (4 hr and 6 hrs), pollen tube growth in 163

the ap-3 mutants was significantly reduced (Fig. 2B-E) compared to the wild type (Fig. 164

2A) at the same time points (Fig. 2G, Supplemental Fig. 2). In addition, pollen tube 165

width of the ap-3 mutants was slightly but significantly increased (Fig. 2H), 166

suggesting compromised tube polarity. 167

To test whether pollen tube growth in the ap-3 mutants was also affected in vivo, 168

we emasculated and hand-pollinated wild-type pistils with pollen from the ap-3 169

mutants or wild type. Using aniline blue staining of pistils at 9 hours after pollination 170

(HAP), we determined that wild-type pollen tubes could grow to the bottom of the 171

pistils (Fig. 3A), whereas pollen tubes of the ap-3 mutants could not (Fig. 3B-E). 172

Among the ap-3 mutants, ap-3σ showed the least severe phenotypic defect in pollen 173

tube growth in vivo (Fig. 3E), consistent with its seed set reduction (Fig. 1D, 1E) and 174

pollen tube growth in vitro (Fig. 2E, 2G). Even at 24 HAP, pollen tubes of the ap-3 175

mutants could hardly reach the bottom of the pistils (Supplemental Fig. 3), which 176

explains the incidence of unfertilized ovules at the bottom of the pistils (Fig. 1). 177

Despite the reduced growth, pollen tubes of ap-3 mutants were able to reach 178

and properly target ovules that were accessible (Fig. 3G-J), similarly to the wild type 179

(Fig. 3F). SEM of wild-type pistils emasculated and hand-pollinated with ap-3 pollen 180

also confirmed normal pollen tube guidance (Fig. 3K-O). Because ap-3δ contained 181

ProLAT52:GUS in the T-DNA, we could perform histochemical GUS staining of wild-type 182 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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pistils pollinated either with ProLAT52:GUS pollen or ap-3δ pollen. All ovules accessible 183

to mutant pollen tubes were targeted (Supplemental Fig. 4), confirming intact pollen 184

tube guidance in the ap-3 mutants. 185

186

AP-3 loss-of-function compromises the vacuolar organization of pollen tubes 187

Pollen tube growth requires dynamic vacuolar organization (Hicks et al., 2004; 188

Wudick et al., 2014), which is likely crucial for turgor regulation and ion homeostasis. 189

Functional loss of AP-3 affected vacuolar organization in seeds and root cells (Feraru 190

et al., 2010; Zwiewka et al., 2011). Therefore, we were interested in determining the 191

vacuolar organization of ap-3 mutant pollen tubes. To this purpose, we used a 192

fluorescent-fusion of the V-ATPase a3 subunit, VHA-a3-YFP, to label the tonoplast. 193

VHA-a3 is targeted to the tonoplast via an AP-3-independent route (Viotti et al., 2013; 194

Feng et al., 2017). In wild-type pollen tubes, VHA-a3-labeled tubular vacuoles were 195

extensive and penetrated to the subapical area right beneath the clear zone (Fig. 4A, 196

Supplemental Movie 1). In contrast, VHA-a3-labeled vacuoles in ap-3σ pollen tubes 197

were less complex and far behind the subapical region (Fig. 4B, Supplemental Movie 198

2). In addition to tubular structures resembling those in wild type (Fig. 4A, 199

Supplemental Movie 3), there were often VHA-a3-labeled ring-shaped or bulbous 200

structures associated with tubular vacuoles in ap-3σ pollen tubes (Fig. 4B, 201

Supplemental Movie 4). These results showed that functional loss of AP-3 202

compromises vacuolar organization in pollen tubes. 203

204

PAT10, whose tonoplast association depends on AP-3, mediates pollen tube growth 205

Because AP-3 functions through sorting vacuolar cargoes at the Golgi (Bassham et al., 206

2008; Uemura and Ueda, 2014), we questioned whether mis-targeting of some AP-3 207

cargoes resulted in reduced pollen tube growth. In Arabidopsis, AP-3 was reported to 208

mediate the tonoplast localization of SUC4 (Wolfenstetter et al., 2012), VAMP713 209

(Ebine et al., 2014), VAMP711 (Feng et al., 2017), and PAT10 (Feng et al., 2017b). To 210

determine whether these AP-3 cargoes were involved in pollen tube growth, we first 211

examined their expression in mature pollen grains by RT-qPCR. We found that SUC4 212

was barely expressed in pollen, while PAT10 and the genes encoding R-SNAREs were 213

expressed (Supplemental Fig. 5). 214 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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Functional loss of PAT10 showed reproductive defects, specifically reduced male 215

transmission (Zhou et al., 2013). Although the role of PAT10 in 216

sporophytically-controlled pollen development has been characterized (Zhou et al., 217

2013), its roles during pollen tube growth are not known. First, we introduced 218

PAT10g-GFP (Zhou et al., 2013) into ap-3δ and examined PAT10 targeting in ap-3δ 219

pollen tubes. VHA-a3-RFP was co-expressed to label the tonoplast both in wild-type 220

and ap-3δ pollen tubes. In wild type, PAT10-GFP labeled dynamic tubular structures 221

overlapping those of VHA-a3-RFP (Fig. 5A), indicative of the tonoplast (Hicks et al., 222

2004). However, PAT10-GFP was distributed into punctate vesicles in ap-3δ pollen 223

tubes, whereas VHA-a3-RFP still showed tubular tonoplast localization (Fig. 5B). The 224

tonoplast association of PAT10-GFP in ap-3δ pollen tubes, as well as reduced pollen 225

tube length, were restored by introducing AP-3δg-RFP (Fig. 5C-D), confirming 226

AP-3δ-dependent tonoplast targeting of PAT10 and tube growth. 227

Next, we used PAT10g-GFP/-;pat10-2 plants for pollen tube growth studies in 228

vitro, in which fluorescent pollen tubes were equivalent to wild type and 229

non-fluorescent ones were pat10-2. Indeed, non-fluorescent pollen tubes from the 230

PAT10g-GFP/-;pat10-2 plants were significantly shorter than GFP-expressing pollen 231

tubes (Supplemental Fig. 6), demonstrating the role of PAT10 in pollen tube growth. 232

PAT10 mediates the S-acylation-dependent tonoplast association of 233

CALCINEURIN B-LIKE2 (CBL2) and CBL3 (Zhou et al., 2013; Zhang et al., 2015). To 234

determine whether CBL2 and CBL3 were mis-targeted in the ap-3 pollen tubes due to 235

mis-targeting of PAT10, we analyzed the subcellular localization of CBL2-RFP in each 236

ap-3 mutant. In contrast to the tubular distribution of CBL2-RFP in wild-type pollen 237

tubes (Fig. 5E), RFP signals were distributed into the PM and cytoplasmic vesicles in 238

the ap-3 pollen tubes (Fig. 5F-I). Because mutations at CBL2 and CBL3 cause defects 239

in pollen tube growth (Steinhorst et al., 2015), these results further supported the 240

idea that the mis-targeting of PAT10 and its downstream components contributes to 241

the reduced pollen tube growth of the ap-3 mutants. 242

243

AP-3 loss-of-function compromises the tip-focused Ca2+ gradient in pollen tubes 244

Pollen tube growth relies on a tip-focused Ca2+ gradient (Konrad et al., 2011; 245

Steinhorst and Kudla, 2013), while CBL2 and CBL3 are calcium sensors functioning in 246 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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pollen tube growth (Steinhorst et al., 2015). Thus, we hypothesized that ap-3 pollen 247

tubes might be compromised in their Ca2+ gradient or signaling, leading to reduced 248

pollen tube growth. To test this hypothesis, we introduced a ProUBQ10:YC3.6 transgene 249

(Monshausen et al., 2008; Behera et al., 2015), which expresses the Förster 250

(fluorescence) resonance energy transfer (FRET)-based genetically modified Ca2+ 251

indicator Yellow Cameleon YC3.6, into ap-3δ. FRET analysis showed that compared to 252

wild-type pollen tubes (Fig. 6A, 6C), growing ap-3δ pollen tubes exhibited a 253

significantly reduced Ca2+ gradient at the tip (Fig. 6B, 6C), consistent with the reduced 254

growth of mutant pollen tubes. 255

To provide further evidence that a reduced Ca2+ gradient resulted in 256

compromised pollen tube growth in the ap-3 mutants, we tested the effect of 257

enhanced Ca2+ concentration by supplementing the growth medium with a higher 258

concentration of extracellular Ca2+, using 5 mM instead of 2 mM Ca2+ in the regular 259

germination medium. Although both wild-type and ap-3δ pollen tubes responded to 260

the increased Ca2+ by enhanced tube growth (Fig. 6D), ap-3δ pollen tubes were more 261

sensitive than those of wild type. The ap-3δ pollen tubes doubled their length after 4 262

hr germination, whereas those of wild type only increased around 40% (Fig. 6D). This 263

result supports the idea that there is reduced Ca2+ signaling in ap-3δ pollen tubes. 264

265

Discussion 266

The roles of AP-1 and AP-2 in plants have been well characterized, especially during 267

reproductive processes (Fan et al., 2013; Park et al., 2013; Wang et al., 2016). Here 268

we demonstrated that AP-3, the AP complex that sorts proteins at the Golgi to 269

vacuoles, mediates pollen tube growth and male fertility (Fig. 1, Fig. 2). Mutants in 270

each AP-3 component showed a reduction of pollen tube growth both in vitro and in 271

vivo (Fig. 3), suggesting AP-3 functions as a complex. The phenotypic defects of ap-3σ 272

were the weakest (Fig. 2, Fig. 3). Because the σ subunit stabilizes AP complexes 273

(Boehm and Bonifacino, 2001; Robinson, 2004), we speculate that this subunit is not 274

essential for AP-3. Alternatively, the σ subunits of other APs may perform redundant 275

roles as a compensation mechanism. 276

Among the established tonoplast cargoes that are sorted by AP-3 during their 277

vacuolar trafficking, PAT10 is an important player in pollen tube growth (Fig. 5, 278 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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Supplemental Fig. 6); functional loss of PAT10 affected male fertility (Zhou et al., 279

2013). By using sporophytically-complemented transgenic materials, we 280

demonstrated that PAT10 functions in male gametophytes to promote pollen tube 281

growth (Supplemental Fig. 6). CBL2 and CBL3, two calcium sensors, rely on PAT10 for 282

their tonoplast association and functionality (Zhou et al., 2013; Zhang et al., 2015). 283

Indeed, functional loss of both CBL2 and CBL3 impaired male fertility (Steinhorst et 284

al., 2015). The CBL2/CBL3 study was performed using a homozygous cbl2 cbl3 mutant, 285

in which sporophytic defects would have enhanced the defects of pollen tube growth 286

(Steinhorst et al., 2015). Nevertheless, these results suggest a key role for the 287

tonoplast calcium sensors in pollen tubes. Indeed, by FRET analysis of the YC3.6 288

probe, we demonstrated that growing pollen tubes with a functional loss of AP-3 289

contained a reduced Ca2+ gradient (Fig. 6), which is consistent with the reduced 290

pollen tube growth (Hepler et al., 2012). 291

Functional loss of AP-3 caused not only mis-targeting of PAT10, which is 292

important for pollen tube growth, but also defects in dynamic vacuolar organization 293

(Fig. 4). By using an AP-3-independent tonoplast protein VHA-a3, we demonstrated 294

that pollen tubes of the ap-3 mutants contained a simplified structure of vacuoles 295

(Fig. 4). Unlike the extensive tubular vacuoles in wild-type pollen tubes, which 296

extended to the subapical region right behind the apex, those in ap-3 pollen tubes 297

were excluded from the subapical zone (Fig. 4). Indeed, mutations in other AP-3 298

subunits also resulted in defects in morphology and function of lytic vacuoles in root 299

cells (Feraru et al., 2010; Zwiewka et al., 2011). Because of the key role of vacuoles in 300

providing turgor pressure and facilitating ion homeostasis, such defective vacuolar 301

organization might have contributed to the reduction in pollen tube growth. 302

Despite the role of AP-3 in pollen tube growth, defects of ap-3 mutants have low 303

penetrance (Table 1), whereas mutations at the HOPS complex, involved in 304

vesicle-vacuole fusion, resulted in male gametophytic lethality (Hao et al., 2016). A 305

likely explanation is that compensating pathways of vacuolar trafficking, such as 306

Rab5-mediated (Cui et al., 2014; Ebine et al., 2014; Singh et al., 2014) and 307

AP-4-mediated (Fuji et al., 2016), might help to maintain the dynamic organization of 308

vacuoles and provide ion homeostasis. 309

310 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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Materials and Methods 311

Plant materials, growth, and transformation 312

The T-DNA insertion lines ap-3δ/pat4-2 (SALK_069881) (Niihama et al., 2009; 313

Zwiewka et al., 2011), ap-3β/pat2-2 (SAIL_1258_G03) (Niihama et al., 2009; Feraru et 314

al., 2010), ap-3µ (SALK_064486) (Niihama et al., 2009), and ap-3σ (SAIL_269_F04) 315

were obtained from the Arabidopsis Biological Resource Center (ABRC). Other 316

materials, including ProLAT52:GUS (Li et al., 2013), ProUBQ10:YC3.6 (Behera et al., 2015), 317

PAT10g-GFP (Zhou et al., 2013), ProUBQ10:VHA-a3-YFP (Feng et al., 2017), and 318

ProUBQ10:CBL2-RFP (Zhang et al., 2015) were described previously. The Arabidopsis 319

(Arabidopsis thaliana) Columbia-0 ecotype was used as the wild type. Transgenic 320

plants were selected on half-strength MS medium supplemented with 30 µg/mL 321

Basta salts (Sigma-Aldrich) or 50 µg/mL Hygromycin (Roche). 322

323

RT-qPCR 324

The extraction of total RNA, reverse transcription, and RT-qPCR were performed as 325

described (Zhou et al., 2013). Primers for all AP-3 mutants were described (Feng et al., 326

2017b). Primers used for RT-qPCR were ZP5314/ZP5315 for VAMP711, 327

ZP5316/ZP5317 for VAMP712, ZP5318/ZP5319 for VAMP713, ZP5363/ZP5364 for 328

SUC4, and ZP691/ZP692 for PAT10. Primers for GAPDH and TUBULIN2 were as 329

described (Zhou et al., 2013). All primers are listed in Supplemental Table 1. 330

331

DNA constructs 332

Primers used for cloning of AP-3δ are ZP4427/ZP4428. The entry vector for AP-3δ 333

was generated in the pENTR/D/TOPO backbone (Invitrogen). The entry vectors for 334

VHA-a3 (Feng et al., 2017) and CBL2 (Zhang et al., 2015) were described previously. 335

Expression vectors were generated by combining entry vectors and the destination 336

vector ProUBQ10:GW-RFP (Zhang et al., 2015) in LR reactions using LR Clonase II 337

(Invitrogen). PCR amplifications used Phusion hot-start high-fidelity DNA polymerase 338

with the annealing temperature and extension times recommended by the 339

manufacturer (Thermofisher). Entry vectors were sequenced, and sequences were 340

analyzed using Vector NTI. All primers are listed in Supplemental Table 1. 341

342 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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Phenotypic analysis of pollen development and tube growth 343

Methods to analyze pollen development, including Alexander staining, DAPI staining, 344

and SEM, were performed as described (Johnson-Brousseau and McCormick, 2004; Li 345

et al., 2013). Methods to analyze pollen tube growth in vitro were performed as 346

described (Boavida and McCormick, 2007). Methods to analyze pollen tube growth in 347

vivo by aniline blue staining and by histochemical GUS staining were performed as 348

described (Li et al., 2013). 349

350

Fluorescence microscopy 351

Fluorescent images were captured using a Zeiss LSM 880 confocal laser scanning 352

microscope (CLSM) with a 40/1.3 oil objective. GFP and YFP fusions were excited at 353

488 nm with a VIS-argon laser; RFP fusions were excited at 561 nm with a 354

VIS-DPSS561 laser diode. Pollen tubes double-labeled with GFP and RFP fusions were 355

captured using alternate line switching mode with a multi-track function. 356

Fluorescence was detected using a 505- to 550-nm band-pass filter for GFP/YFP/OG 357

or a 575- to 650-nm band-pass filter for RFP. Z-stack images were recorded with a 358

step size of 0.3 μm, image dimension of 512 512, pinhole at 1 airy unit. Time-lapse 359

imaging for generating movie clips was performed as an interval of 0.5 sec. Each 360

movie clip was generated from 100 slides of still images. Image processing was 361

performed with the Zeiss LSM image processing software (Zeiss). 3-D surface 362

renderings were performed with Imaris7.0 software. 363

364

Accession Numbers. Arabidopsis Genome Initiative locus identifiers for the genes 365

mentioned in this article are: AP-3δ, At1g48760; AP-3β, At3g55480; AP-3µ, 366

At1g56590; AP-3σ, At3g50860; CBL2, At5g55990; PAT10, At3g51390; SUC4, 367

At1g09960; VAMP711, At4g32150; VAMP712, At2g25340; VAMP713, At5g11150; 368

VHA-a3, At4g39080. 369

370

Supplemental Data 371

The following materials are available in the online version of this article. 372

Supplemental Fig. 1. Functional loss of AP-3 does not compromise pollen 373

development. 374 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.


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Supplemental Fig. 2. Functional loss of AP-3 reduces pollen tube growth in vitro. 375

Supplemental Fig. 3. Functional loss of AP-3 reduces pollen tube growth in vivo. 376

Supplemental Fig. 4. Functional loss of AP-3δ does not impair pollen tube 377

guidance. 378

Supplemental Fig. 5. Expression of genes encoding cellular cargos of AP-3 in 379

pollen. 380

Supplemental Fig. 6. AP-3-mediated tonoplast protein PAT10 is important for 381

pollen tube growth. 382

Supplemental Movie 1. Vacuolar dynamics in a wild-type pollen tube. 383

Supplemental Movie 2. Vacuolar dynamics in an ap-3δ pollen tube. 384

Supplemental Movie 3. 3D surface rendering of VHA-a3-YFP-labeled vacuoles in 385

a wild-type pollen tube. 386

Supplemental Movie 4. 3D surface rendering of VHA-a3-YFP-labeled vacuoles in 387

an ap-3δ pollen tube. 388

Supplemental Table 1. Oligos used in this study. 389

390

Acknowledgements 391

We thank ABRC for plant materials. We are grateful for language editing by Prof. 392

Sheila McCormick. The authors declare that there is no conflict of interest. 393

394

Table 1. AP-3 loss-of-function resulted in defective male transmission.

Parents F1 progenies

Female X Male Genotype Expected Ratio Observed Ratio

ap-3δ +/- X wild type ap-3δ +/+:+/- 1:1 74:68

wild type X ap-3δ +/- ap-3δ +/+:+/- 1:1 87:7 a

ap-3δ +/- X ap-3δ +/- ap-3δ +/+:+/-:-/- 1:2:1 13:20:2 b

ap-3β +/- X wild type R:S* 1:1 94:87

wild type X ap-3β +/- R:S* 1:1 11:159 a

ap-3β +/- X ap-3β +/- R:S* 3:1 369:211 c

ap-3μ +/- X wild type ap-3μ +/+:+/- 1:1 99:107



- 14 -

wild type X ap-3μ +/- ap-3μ +/+:+/- 1:1 170:8 a

ap-3μ +/- X ap-3μ +/- ap-3μ +/+:+/-:-/- 1:2:1 42:38:16 b

ap-3σ +/- X wild type R:S* 1:1 125:110

wild type X ap-3σ +/- R:S* 1:1 39:56 a

ap-3σ +/- X ap-3σ +/- R:S* 3:1 409:150 c

a Significantly different from the segregation ratio 1:1 (2, P<0.01 ).

b Significantly different from the segregation ratio 1:2:1 (2, P<0.01).

c Significantly different from the segregation ratio 3:1 (22, P<0.01).

*: R for Basta-resistant and S for Basta-sensitive. Basta sensitivity was used to

distinguish the mutant copy of ap-3β or ap-3σ, resistant to Basta salt, from its

wild-type copy.

395

Figure Legends 396

Figure 1. Mutations at AP‐3 subunits result in reduced seed set. 397

(A) Schematic illustration of T-DNA insertions within the genomic regions of AP-3 398

subunit-encoding genes, AP-3δ, AP-3β, AP-3µ, and AP-3σ. Arrowheads point at the 399

T-DNA insertion sites. Arrows at the genomic region of AP-3σ indicate the binding 400

sites of RT-qPCR primers. (B) Quantitative analysis of seed set in wild type (WT) and 401

the ap-3 mutants. Results are means ± standard deviation (SD, N=20). Each mutant is 402

significantly different from wild type in seed set as indicated by asterisks (One-way 403

ANOVA, Dunnett’s multiple comparison test, P<0.05). (C-D) From left to right: a 404

representative silique from WT, ap-3δ, ap-3β, ap-3µ, or ap-3σ. Close-up of the 405

bottom part of (C) is shown in (D). Arrowheads point at undeveloped ovules. Bars = 2 406

mm for (C); 500 µm for (D). 407

408

Figure 2. Functional loss of AP‐3 reduces pollen tube growth in vitro. 409

(A-E) In vitro pollen tubes from WT (A), ap-3δ (B), ap-3β (C), ap-3µ (D), or ap-3σ (E) at 410

4 hr after germination. Two representative pollen tubes from each genotype are 411

highlighted in pink. (F-H) The germination ratio (F), length (G), or apical width (H) of 412

pollen tubes after 4 hours incubation in germination medium. Results shown are 413

means ± SD (n=300 for F, n= 100 for G, n=60 for H). All mutants are not significantly 414

different from wild type in the germination ratio (One-way ANOVA, Dunnett’s 415

multiple comparison test, P>0.05). Each mutant is significantly different from wild 416

type in the length or width of pollen tubes as indicated by asterisks (One-way ANOVA, 417

Dunnett’s multiple comparison test, P<0.05). Bars = 50 µm. 418

419

Figure 3. Functional loss of AP‐3 affects the growth but not the guidance of pollen 420

tubes in vivo. 421

(A-J) Representative aniline blue staining of wild-type pistils emasculated and 422

hand-pollinated with pollen from wild type (A, F), ap-3δ (B, G), ap-3β (C, H), ap-3µ (D, 423

I), or ap-3σ (E, J) at 9 hours after pollination (HAP). Arrows point at the front of in 424 https://plantphysiol.orgDownloaded on May 29, 2021. - Published by



- 15 -

vivo growing pollen tubes; arrowheads point at the micropyle where pollen tubes 425

arrive. The relative distance pollen tubes travel within the transmitting tract is 426

quantified with 15 pistils. Results are shown at the bottom (means ± SD). Each 427

mutant is significantly different from wild type in the length of in vivo growing pollen 428

tubes (One-way ANOVA, Dunnett’s multiple comparison test, P<0.05). (K-O) Scanning 429

electron micrographs (SEMs) of wild-type ovules hand-pollinated with pollen from 430

wild type (K), ap-3δ (L), ap-3β (M), ap-3µ (N), or ap-3σ (O) at 9 HAP. Pollen tubes 431

growing toward the micropyle are false-colored pink. Bars=200 μm for (A-E); 100 μm 432

for (F-J); 20 μm for (K-O). 433

434

Figure 4. Vacuolar organization is compromised in ap‐3 pollen tubes. 435

(A-B) a growing wild-type (A) or ap-3δ pollen tube (B) expressing VHA-a3-YFP. From 436

top to bottom: the YFP channel image at the mid-optical plane; merge of the YFP and 437

transmission channels; 3D-surface rendering of CLSM images. Dotted lines illustrate 438

the silhouettes of the pollen tubes. Over 30 pollen tubes for each genotype were 439

examined with similar results. Bars = 10 μm. 440

441

Figure 5. Tonoplast association of PAT10 and CBL2 relies on AP‐3 in pollen tubes. 442

(A-B) Confocal laser scanning fluorescence micrographs (CLSMs) of a pollen tube 443

expressing PAT10g-GFP;VHA-a3-RFP in wild type (A) or in ap-3δ (B). (C) CLSM of a 444

pollen tube expressing PAT10g-GFP in the complemented AP-3δ-RFP;ap-3δ. (D) 445

Pollen tube length. Results shown are means ± SD (n=100). Means with different 446

letters indicate significant difference of pollen tube length (One-way ANOVA, 447

Dunnett’s multiple comparison test, P<0.05). (E-F) A pollen tube stably transformed 448

with ProUBQ10:CBL2-RFP in wild type (E) or in ap-3δ (F). (G-I) A pollen tube stably 449

transformed with ProUBQ10:CBL2-RFP;PAT10g-GFP in ap-3β (G), ap-3µ (H), or ap-3σ (I). 450

G/T or R/T indicates merge of the GFP or RFP channel and the transmission channel; 451

G/R/T indicates merge of the GFP channel, the RFP channel, and the transmission 452

channel. Over 30 pollen tubes for each genotype were examined with similar results 453

for data shown in (A-C) and (E-I). Bars = 10 μm. 454

455

Figure 6. AP‐3 loss‐of‐function compromises the tip‐focused Ca2+ gradient. 456

(A-B) CLSM of a pollen tube expressing the Ca2+ sensor YC3.6 in wild type (A) or in 457

ap-3δ (B). Images are representative of 20 pollen tubes for each genotype. Cytosolic 458

Ca2+ levels were calibrated as described in “Materials and Methods” and 459

pseudocolored according to the scale at the left. (C) Quantification of fluorescence 460

intensity as the ratio between YFP signals and CFP signals. Results are means SD 461

(n=20). Asterisk indicates significant difference (t-test, P<0.01). (D) Pollen tube length 462

after 4 hr germination in a medium containing 2 mM Ca2+ or 5 mM Ca2+. Results are 463

means ± SD (n=100). Asterisk indicates significantly different responses of pollen tube 464

growth upon increased exogenous Ca2+ levels (t-test, P<0.01). Bars = 10 μm. 465

466

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Figure 1. Mutations at AP‐3 subunits resulted in reduced seed set. (A) Schematic illustration of T‐DNA insertions within the genomic regions of AP‐3 subunit‐encoding genes, AP‐3δ, AP‐3β, AP‐3µ, and AP‐3σ. Arrowheads point at the T‐DNA insertion sites. Arrows at the genomic region of AP‐3σ indicate the binding sites of RT‐PCR primers. (B) Quantitative analysis of seed set in wild type (WT) and the ap‐3 mutants. Results are means ± standard deviation (SD, N=20). Each mutant is significantly different from wild type in seed set as indicated by asterisks (One‐way ANOVA, Dunnett’s multiple comparison test, P<0.05). (C‐D) From left to right: a representative silique from WT, ap‐3δ, ap‐3β, ap‐3µ, or ap‐3σ. Close‐up of the bottom part of (C) is shown in (D). Arrowheads point at undeveloped ovules. Bars = 2 mm for (C); 500 µm for (D).



Figure 2. Functional loss of AP‐3 reduced pollen tube growth in vitro. (A‐E) In vitro pollen tubes from WT (A), ap‐3δ (B), ap‐3β (C), ap‐3µ (D), or ap‐3σ (E) at 4 hr after germination. Two representative pollen tubes from each genotype are highlighted in pink. (F‐H) The germination ratio (F), length (G), or apical width (H) of pollen tubes after 4 hours incubation in germination medium. Results shown are means ± SD (n=300 for F, n= 100 for G, n=60 for H). All mutants are not significantly different from wild type in the germination ratio (One‐way ANOVA, Dunnett’s multiple comparison test, P>0.05). Each mutant is significantly different from wild type in the length or width of pollen tubes as indicated by asterisks (One‐way ANOVA, Dunnett’s multiple comparison test, P<0.05). Bars = 50 µm.



Figure 3. Functional loss of AP‐3 affected the growth but not guidance of pollen tubes in vivo. (A‐J) Representative aniline blue staining of wild‐type pistils emasculated and hand‐pollinated with pollen from wild type (A, F), ap‐3δ (B, G), ap‐3β (C, H), ap‐3µ (D, I), or ap‐3σ (E, J) at 9 hours after pollination (HAP). Arrows point at the front of in vivo growing pollen tubes; arrowheads point at the micropyle where pollen tubes arrive. The relative distance pollen tubes travel within the transmitting tract is quantified with 15 pistils. Results are shown at the bottom (means ± SD). Each mutant is significantly different from wild type in the length of in vivo growing pollen tubes (One‐way ANOVA, Dunnett’s multiple comparison test, P<0.05). (K‐O) Scanning electron micrographs (SEMs) of wild‐type ovules hand‐pollinated with pollen from wild type (K), ap‐3δ (L), ap‐3β (M), ap‐3µ (N), or ap‐3σ (O) at 9 HAP. Pollen tubes growing toward the micropyle are false‐colored pink. Bars=200 μm for (A‐E); 100 μm for (F‐J); 20 μm for (K‐O).



Figure 4. Vacuolar organization was compromised in ap‐3 pollen tubes. (A‐B) a growing wild‐type (A) or ap‐3δ pollen tube (B) expressing VHA‐a3‐YFP. From top to bottom: the YFP channel image at the mid‐optical plane; merge of the YFP and transmission channels; 3D‐surface rendering of CLSM images. Dotted lines illustrate the silhouettes of the pollen tubes. Bars = 10 μm.



Figure 6. AP‐3 loss‐of‐function compromised the tip‐focused Ca2+ gradient. (A‐B) CLSM of a pollen tube expressing the Ca2+ sensor YC3.6 in wild type (A) or in ap‐3δ (B). Cytosolic Ca2+ levels were calibrated as described in “Materials and Methods” and pseudocolored according to the scale at the left. (C) Quantification of fluorescence intensity as the ratio between YFP signals and CFP signals. Results are means SD (n=20). Asterisk indicates significant difference (t‐test, P<0.01). (D) Pollen tube length after 4 hr germination in a medium containing 2 mM Ca2+ or 5 mM Ca2+. Results are means ± SD (n=100). Asterisk indicates significantly different responses of pollen tube growth upon increased exogenous Ca2+ levels (t‐test, P<0.01). Bars = 10 μm.



Parsed CitationsBassham DC, Brandizzi F, Otegui MS, Sanderfoot AA (2008) The secretory system of Arabidopsis. Arabidopsis Book 6: e0116

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Behera S, Wang N, Zhang C, Schmitz-Thom I, Strohkamp S, Schultke S, Hashimoto K, Xiong L, Kudla J (2015) Analyses of Ca2+dynamics using a ubiquitin-10 promoter-driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and differences incytoplasmic Ca2+ responses in Arabidopsis and rice (Oryza sativa) roots. New Phytol 206: 751-760


Boavida LC, McCormick S (2007) Temperature as a determinant factor for increased and reproducible in vitro pollen germination inArabidopsis thaliana. Plant J 52: 570-582


Boehm M, Bonifacino JS (2001) Adaptins: the final recount. Mol Biol Cell 12: 2907-2920Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cheung AY, Duan QH, Costa SS, de Graaf BH, Di Stilio VS, Feijo J, Wu HM (2008) The dynamic pollen tube cytoskeleton: live cellstudies using actin-binding and microtubule-binding reporter proteins. Mol Plant 1: 686-702


Cheung AY, Wu HM (2007) Structural and functional compartmentalization in pollen tubes. J Exp Bot 58: 75-82Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cheung AY, Wu HM (2008) Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu Rev Plant Biol59: 547-572


Cui Y, Zhao Q, Gao C, Ding Y, Zeng Y, Ueda T, Nakano A, Jiang L (2014) Activation of the Rab7 GTPase by the MON1-CCZ1 complex isessential for PVC-to-vacuole trafficking and plant growth in Arabidopsis. Plant Cell 26: 2080-2097


Ebine K, Inoue T, Ito J, Ito E, Uemura T, Goh T, Abe H, Sato K, Nakano A, Ueda T (2014) Plant vacuolar trafficking occurs throughdistinctly regulated pathways. Curr Biol 24: 1375-1382


Fan L, Hao H, Xue Y, Zhang L, Song K, Ding Z, Botella MA, Wang H, Lin J (2013) Dynamic analysis of Arabidopsis AP2σ subunit reveals akey role in clathrin-mediated endocytosis and plant development. Development 140: 3826-3837


Feng QN, Song SJ, Yu SX, Wang JG, Li S, Zhang Y (2017) Adaptor Protein-3-dependent vacuolar trafficking involves a subpopulation ofCOPII and HOPS tethering proteins. Plant Physiol 174: 1609-1620


Feng QN, Zhang Y, Li S (2017) Tonoplast targeting of VHA-a3 relies on a Rab5-mediated but Rab7-independent vacuolar traffickingroute. J Integr Plant Biol 59: 230-233


Feraru E, Paciorek T, Feraru MI, Zwiewka M, De Groodt R, De Rycke R, Kleine-Vehn J, Friml J (2010) The AP-3 β adaptin mediates thebiogenesis and function of lytic vacuoles in Arabidopsis. Plant Cell 22: 2812-2824


http://www.ncbi.nlm.nih.gov/pubmed?term=Bassham DC%2C Brandizzi F%2C Otegui MS%2C Sanderfoot AA %282008%29 The secretory system of Arabidopsis%2E Arabidopsis Book 6%3A e0116&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bassham DC, Brandizzi F, Otegui MS, Sanderfoot AA (2008) The secretory system of Arabidopsis. Arabidopsis Book 6: e0116

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bassham&hl=en&c2coff=1

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Niihama M, Takemoto N, Hashiguchi Y, Tasaka M, Morita MT (2009) ZIP genes encode proteins involved in membrane trafficking of theTGN-PVC/vacuoles. Plant Cell Physiol 50: 2057-2068


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Teh OK, Shimono Y, Shirakawa M, Fukao Y, Tamura K, Shimada T, Hara-Nishimura I (2013) The AP-1 µ adaptin is required for KNOLLElocalization at the cell plate to mediate cytokinesis in Arabidopsis. Plant Cell Physiol 54: 838-847


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http://www.ncbi.nlm.nih.gov/pubmed?term=Park M%2C Song K%2C Reichardt I%2C Kim H%2C Mayer U%2C Stierhof Y%2DD%2C Hwang I%2C Jürgens G %282013%29 Arabidopsis μ%2Dadaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth%2E Proc Nat Acad Sci USA&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Singh MK%2C Kruger F%2C Beckmann H%2C Brumm S%2C Vermeer JE%2C Munnik T%2C Mayer U%2C Stierhof YD%2C Grefen C%2C Schumacher K%2C Jurgens G %282014%29 Protein delivery to vacuole requires SAND protein%2Ddependent Rab GTPase conversion for MVB%2Dvacuole fusion%2E Curr Biol 24%3A 1383%2D1389&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Steinhorst L%2C Kudla J %282013%29 Calcium %2D a central regulator of pollen germination and tube growth%2E Biochim Biophys Acta 1833%3A 1573%2D1581&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Steinhorst L%2C Mahs A%2C Ischebeck T%2C Zhang C%2C Zhang X%2C Arendt S%2C Schultke S%2C Heilmann I%2C Kudla J %282015%29 Vacuolar CBL%2DCIPK12 Ca2%2B%2Dsensor%2Dkinase complexes are required for polarized pollen tube growth%2E Curr Biol 25%3A 1475%2D1482&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Teh OK%2C Shimono Y%2C Shirakawa M%2C Fukao Y%2C Tamura K%2C Shimada T%2C Hara%2DNishimura I %282013%29 The AP%2D1 � adaptin is required for KNOLLE localization at the cell plate to mediate cytokinesis in Arabidopsis%2E Plant Cell Physiol 54%3A 838%2D847&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Uemura T%2C Ueda T %282014%29 Plant vacuolar trafficking driven by RAB and SNARE proteins%2E Curr Opin Plant Biol 22%3A 116%2D121&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Van Damme D%2C Coutuer S%2C De Rycke R%2C Bouget FY%2C Inze D%2C Geelen D %282006%29 Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE%2C which has domains similar to coat proteins%2E Plant Cell 18%3A 3502%2D3518&dopt=abstract

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Hillmer S, Grebe M, Schumacher K (2013) The Endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuolein Arabidopsis. Plant Cell 25: 3434-3449


Wang JG, Li S, Zhao XY, Zhou LZ, Huang GQ, Feng C, Zhang Y (2013) HAPLESS13, the Arabidopsis µ1 adaptin, is essential for proteinsorting at the trans-Golgi network/early endosome. Plant Physiol 162: 1897-1910


Wang T, Liang L, Xue Y, Jia PF, Chen W, Zhang MX, Wang YC, Li HJ, Yang WC (2016) A receptor heteromer mediates the maleperception of female attractants in plants. Nature


Wolfenstetter S, Wirsching P, Dotzauer D, Schneider S, Sauer N (2012) Routes to the tonoplast: the sorting of tonoplast transporters inArabidopsis mesophyll protoplasts. Plant Cell 24: 215-232


Wudick MM, Luu DT, Tournaire-Roux C, Sakamoto W, Maurel C (2014) Vegetative and sperm cell-specific aquaporins of Arabidopsishighlight the vacuolar equipment of pollen and contribute to plant reproduction. Plant Physiol 164: 1697-1706


Yamaoka S, Shimono Y, Shirakawa M, Fukao Y, Kawase T, Hatsugai N, Tamura K, Shimada T, Hara-Nishimura I (2013) Identification anddynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development. Plant Cell 25: 2958-2969


Zhang Y, Li S, Zhou L-Z, Fox E, Pao J, Sun W, Zhou C, McCormick S (2011) Overexpression of Arabidopsis thaliana PTEN causedaccumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics. Plant J 68: 1081-1092


Zhang YL, Li E, Feng QN, Zhao XY, Ge FR, Zhang Y, Li S (2015) Protein palmitoylation is critical for the polar growth of root hairs inArabidopsis. BMC Plant Biol 15: 50


Zhou LZ, Li S, Feng QN, Zhang YL, Zhao X, Zeng YL, Wang H, Jiang L, Zhang Y (2013) PROTEIN S-ACYL TRANSFERASE10 is critical fordevelopment and salt tolerance in Arabidopsis. Plant Cell 25: 1093-1107


Zwiewka M, Feraru E, Moller B, Hwang I, Feraru MI, Kleine-Vehn J, Weijers D, Friml J (2011) The AP-3 adaptor complex is required forvacuolar function in Arabidopsis. Cell Res 21: 1711-1722



http://www.ncbi.nlm.nih.gov/pubmed?term=Viotti C%2C Kruger F%2C Krebs M%2C Neubert C%2C Fink F%2C Lupanga U%2C Scheuring D%2C Boutte Y%2C Frescatada%2DRosa M%2C Wolfenstetter S%2C Sauer N%2C Hillmer S%2C Grebe M%2C Schumacher K %282013%29 The Endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis%2E Plant Cell 25%3A 3434%2D3449&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Viotti C, Kruger F, Krebs M, Neubert C, Fink F, Lupanga U, Scheuring D, Boutte Y, Frescatada-Rosa M, Wolfenstetter S, Sauer N, Hillmer S, Grebe M, Schumacher K (2013) The Endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis. Plant Cell 25: 3434-3449

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Viotti&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The Endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The Endoplasmic reticulum is the main membrane source for biogenesis of the lytic vacuole in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Viotti&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wang JG%2C Li S%2C Zhao XY%2C Zhou LZ%2C Huang GQ%2C Feng C%2C Zhang Y %282013%29 HAPLESS13%2C the Arabidopsis �1 adaptin%2C is essential for protein sorting at the trans%2DGolgi network%2Fearly endosome%2E Plant Physiol 162%3A 1897%2D1910&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wang JG, Li S, Zhao XY, Zhou LZ, Huang GQ, Feng C, Zhang Y (2013) HAPLESS13, the Arabidopsis �1 adaptin, is essential for protein sorting at the trans-Golgi network/early endosome. Plant Physiol 162: 1897-1910

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang&hl=en&c2coff=1


http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wang T%2C Liang L%2C Xue Y%2C Jia PF%2C Chen W%2C Zhang MX%2C Wang YC%2C Li HJ%2C Yang WC %282016%29 A receptor heteromer mediates the male perception of female attractants in plants%2E Nature&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wang T, Liang L, Xue Y, Jia PF, Chen W, Zhang MX, Wang YC, Li HJ, Yang WC (2016) A receptor heteromer mediates the male perception of female attractants in plants. Nature

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A receptor heteromer mediates the male perception of female attractants in plants.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A receptor heteromer mediates the male perception of female attractants in plants.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wolfenstetter S%2C Wirsching P%2C Dotzauer D%2C Schneider S%2C Sauer N %282012%29 Routes to the tonoplast%3A the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts%2E Plant Cell 24%3A 215%2D232&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wolfenstetter S, Wirsching P, Dotzauer D, Schneider S, Sauer N (2012) Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts. Plant Cell 24: 215-232

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wolfenstetter&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wolfenstetter&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wudick MM%2C Luu DT%2C Tournaire%2DRoux C%2C Sakamoto W%2C Maurel C %282014%29 Vegetative and sperm cell%2Dspecific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and contribute to plant reproduction%2E Plant Physiol 164%3A 1697%2D1706&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wudick MM, Luu DT, Tournaire-Roux C, Sakamoto W, Maurel C (2014) Vegetative and sperm cell-specific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and contribute to plant reproduction. Plant Physiol 164: 1697-1706

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wudick&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Vegetative and sperm cell-specific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and contribute to plant reproduction&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Vegetative and sperm cell-specific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and contribute to plant reproduction&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wudick&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Yamaoka S%2C Shimono Y%2C Shirakawa M%2C Fukao Y%2C Kawase T%2C Hatsugai N%2C Tamura K%2C Shimada T%2C Hara%2DNishimura I %282013%29 Identification and dynamics of Arabidopsis adaptor protein%2D2 complex and its involvement in floral organ development%2E Plant Cell 25%3A 2958%2D2969&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Yamaoka S, Shimono Y, Shirakawa M, Fukao Y, Kawase T, Hatsugai N, Tamura K, Shimada T, Hara-Nishimura I (2013) Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development. Plant Cell 25: 2958-2969

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yamaoka&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yamaoka&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang Y%2C Li S%2C Zhou L%2DZ%2C Fox E%2C Pao J%2C Sun W%2C Zhou C%2C McCormick S %282011%29 Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3%2Dphosphate dynamics%2E Plant J 68%3A 1081%2D1092&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhang Y, Li S, Zhou L-Z, Fox E, Pao J, Sun W, Zhou C, McCormick S (2011) Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics. Plant J 68: 1081-1092

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang YL%2C Li E%2C Feng QN%2C Zhao XY%2C Ge FR%2C Zhang Y%2C Li S %282015%29 Protein palmitoylation is critical for the polar growth of root hairs in Arabidopsis%2E BMC Plant Biol 15%3A 50&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhang YL, Li E, Feng QN, Zhao XY, Ge FR, Zhang Y, Li S (2015) Protein palmitoylation is critical for the polar growth of root hairs in Arabidopsis. BMC Plant Biol 15: 50

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=Protein palmitoylation is critical for the polar growth of root hairs in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhou LZ%2C Li S%2C Feng QN%2C Zhang YL%2C Zhao X%2C Zeng YL%2C Wang H%2C Jiang L%2C Zhang Y %282013%29 PROTEIN S%2DACYL TRANSFERASE10 is critical for development and salt tolerance in Arabidopsis%2E Plant Cell 25%3A 1093%2D1107&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhou LZ, Li S, Feng QN, Zhang YL, Zhao X, Zeng YL, Wang H, Jiang L, Zhang Y (2013) PROTEIN S-ACYL TRANSFERASE10 is critical for development and salt tolerance in Arabidopsis. Plant Cell 25: 1093-1107

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhou&hl=en&c2coff=1

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http://scholar.google.com/scholar?as_q=PROTEIN S-ACYL TRANSFERASE10 is critical for development and salt tolerance in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhou&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zwiewka M%2C Feraru E%2C Moller B%2C Hwang I%2C Feraru MI%2C Kleine%2DVehn J%2C Weijers D%2C Friml J %282011%29 The AP%2D3 adaptor complex is required for vacuolar function in Arabidopsis%2E Cell Res 21%3A 1711%2D1722&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zwiewka M, Feraru E, Moller B, Hwang I, Feraru MI, Kleine-Vehn J, Weijers D, Friml J (2011) The AP-3 adaptor complex is required for vacuolar function in Arabidopsis. Cell Res 21: 1711-1722

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zwiewka&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The AP-3 adaptor complex is required for vacuolar function in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The AP-3 adaptor complex is required for vacuolar function in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zwiewka&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1


University, Tai’an, 271018, hinaMar 09, 2018 · 58. gametophytic d. evelopment, interactions...

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