RESEARCH ARTICLE 1 2

The Tip-Localized Phosphatidylserine Established by Arabidopsis ALA3 3 is Crucial for Rab GTPase-Mediated Vesicle Trafficking and Pollen Tube 4 Growth 5

6 Yuelong Zhou1#, Yang Yang1#, Yue Niu1#, TingTing Fan1, Dong Qian1, 7 Changxin Luo1, Yumei Shi1, Shanwei Li2, Lizhe An1, Yun Xiang1* 8

9 1 MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life 10 Sciences, Lanzhou University, Lanzhou 730000, China 11 2 State Key Laboratory of Crop Biology, College of Life Sciences, Shandong 12 Agricultural University, Tai’an 271018, China 13 # These authors contributed equally to this work. 14 * Corresponding author: Yun Xiang, E-mail: xiangy@lzu.edu.cn15 Short title: PS regulates polar growth of pollen tubes 16 One-sentence summary: Arabidopsis ALA3 plays a key role in the 17 inverted-cone distribution of PS at the pollen tube tip, and PS is crucial for the 18 distribution and activity of certain Rab GTPases as well as pollen tube growth. 19

20 The authors responsible for distribution of materials integral to the findings 21 presented in this article in accordance with the policy described in the 22 Instructions for Author (www.plantcell.org) is Yun Xiang (xiangy@lzu.edu.cn). 23

24 ABSTRACT 25

RabA4 subfamily proteins, the key regulators of intracellular transport, are 26 vital for tip growth of plant polar cells, but their unique distribution in the apical 27 zone and role in vesicle targeting and trafficking in the tips remain poorly 28 understood. Here, we found that loss of Arabidopsis thaliana 29 AMINOPHOSPHOLIPID ATPASE 3 (ALA3) function resulted in a marked 30 decrease in YFP-RabA4b/ RFP-RabA4d- and FM4-64-labeled vesicles from 31 the inverted-cone zone of the pollen tube tip, misdistribution of certain 32 intramembrane compartment markers and an obvious increase in pollen tube 33 width. Additionally, we revealed that phosphatidylserine (PS) was abundant in 34 the inverted-cone zone of the apical pollen tube in wild-type Arabidopsis and 35 was mainly colocalized with the trans-Golgi network/early endosome, certain 36 post-Golgi compartments, and the plasma membrane. Loss of ALA3 function 37 resulted in loss of polar localization of apical PS and significantly decreased 38 PS distribution, suggesting that ALA3 is a key regulator for establishing and 39 maintaining the polar localization of apical PS in pollen tubes. We further 40 demonstrated that certain Rab GTPases colocalized with PS in vivo and bound 41 to PS in vitro. Moreover, ALA3 and RabA4d collectively regulated pollen tube 42 growth genetically. Thus, we propose that the tip-localized PS established by 43 ALA3 is crucial for Rab GTPase-mediated vesicle targeting/trafficking and 44 polar growth of pollen tubes in Arabidopsis. 45

Plant Cell Advance Publication. Published on August 18, 2020, doi:10.1105/tpc.19.00844

©2020 American Society of Plant Biologists. All Rights Reserved

INTRODUCTION 46

Pollen tube growth is an indispensable event during double fertilization in 47

flowering plants. This peculiar polarized cell growth, in which the elongation 48

occurs only at the tip area and the growth rate is exceedingly high, is distinct 49

from that of most other plant cells. Notably, rapid tip growth requires 50

intracellular transport machinery to continuously deliver essential cargoes to a 51

defined growth site, and this complex process is governed precisely (Hepler et 52

al., 2001; Cheung and Wu, 2008; Johnson et al., 2019). Indeed, there are 53

mainly two kinds of crucial material transport patterns during tip growth: 54

cytoplasmic streaming in the shank region and polarized vesicle trafficking 55

at the apical area of the pollen tube (Hepler et al., 2001; Cheung and Wu, 2008; 56

Chebli et al., 2013). In angiosperms, vesicles with different diameters 57

aggregate into an inverted-cone zone at the apical area of the pollen tube, 58

where large organelles such as endoplasmic reticulum (ER) and Golgi are 59

absent (Cheung and Wu, 2008; Chebli et al., 2013). Because cytoplasmic 60

streaming cannot reach the apical area of the pollen tube, tip-polarized vesicle 61

trafficking is indispensable. Numerous studies have also shown that many 62

cellular activities, including secretion, endocytosis, vesicle recycling and 63

degradation, play influential and distinct roles in tip growth (Hepler et al., 2001; 64

Zhang et al., 2010; Grebnev et al., 2017). Unfortunately, the precise regulatory 65

mechanism of tip-polarized vesicle trafficking remains unclear. 66

Rab GTPases are a key class of conserved proteins responsible for 67

vesicle trafficking in eukaryotes, participating in a variety of endomembrane 68

trafficking processes: vesicle budding, transportation, tethering, and 69

membrane fusion (Zerial and McBride, 2001; Stenmark, 2009; Bhuin and Roy, 70

2014). In Arabidopsis thaliana, 57 members of Rab GTPases are divided into 71

eight functional subfamilies (named AtRabA - AtRabH) (Rutherford and Moore, 72

2002; Vernoud et al., 2003). Several members of plant Rab GTPases 73

participate in growth and development as well as biotic and abiotic stress 74

responses via vesicle-mediated endomembrane trafficking pathways (Mazel et 75

al., 2004; Woollard and Moore, 2008; Bottanelli et al., 2011; Ebine et al., 2014; 76

Ellinger et al., 2014; Uemura and Ueda, 2014; Yin et al., 2017). Additional 77

evidence of the relationship between Rab GTPases and pollen development 78

and polarized pollen tube growth has been obtained from research on 79

Arabidopsis RabA and RabD (Szumlanski and Nielsen, 2009; Peng et al., 80

2011). For instance, an Arabidopsis Rab family member, RabA4d, which is 81

homologous to the mammalian Rab11 subfamily, is typically localized at the 82

inverted-cone zone of the pollen tube and is responsible for targeted polar 83

transport of vesicles; additionally, a lack of RabA4d activity leads to severe 84

defects in the polarized growth phenotype of pollen tubes, such as slow growth 85

and increased width of pollen tubes as well as swelling of pollen tubes; 86

RabA4d also participates in the transport of the cell wall and membrane 87

components required for pollen tube growth (Szumlanski and Nielsen, 2009). 88

RabA4b, another member of this subfamily, localizes to the root hair tip (which 89

also exhibits polarized cell growth) and is widely used to label vesicles at the 90

pollen tube tip (Preuss et al., 2004; Zhang et al., 2010). Similarly, alteration in 91

tobacco Rab11b function also results in abnormal pollen tube growth, 92

suggesting that the role of the RabA4 subfamily in vesicle trafficking is 93

important for polarized growth in plants (de Graaf et al., 2005). However, little 94

is known regarding the detailed regulatory mechanisms that establish and 95

maintain the inverted-cone distribution of RabA4 in pollen tubes or root hairs. 96

In eukaryotic cells, anionic phospholipids, including phosphatidylinositol 97

phosphates (PIPs), phosphatidylserine (PS), and phosphatidic acid (PA), are 98

among the minor lipids in biomembranes (Caillaud, 2019). In addition to being 99

components of the membrane, anionic phospholipids act as key signaling 100

molecules. For example, the contribution of these lipids to the surface charge, 101

curvature and lipid packing of the membrane can effectively control the 102

localization and activity of Rab GTPases (Bigay and Antonny, 2012; Simon et 103

al., 2016; Kulakowski et al., 2018). Among these phospholipids, 104

phosphatidylinositol-4-phosphate (PI4P), phosphatidylinositol-4,5-phosphate 105

(PI(4,5)P2) and PA are three well-studied anionic lipids that localize to the 106

plasma membrane (PM) of pollen tubes (Kost et al., 1999; Zhao et al., 2010; 107

Potocký et al., 2014; Hempel et al., 2017; Noack and Jaillais, 2017), but 108

another lipid, PS, is mainly enriched in the inverted-cone zone of the tobacco 109

pollen tube apex (Platre et al., 2018), suggesting that PS might be involved in 110

vesicle trafficking in the apical area of pollen tubes. The asymmetric 111

distribution of PS contributes to the biological function of this lipid and relies on 112

P4-type ATPases (P4-ATPases), which flip phosphatidylcholine (PC), 113

phosphatidylethanolamine (PE) and PS across the biomembrane from the 114

extracellular/luminal leaflet to the cytosolic leaflet powered by ATP hydrolysis 115

in eukaryotes (Roland and Graham, 2016). The Arabidopsis P4-ATPase family 116

consists of 12 members, termed aminophospholipid ATPases 1-12 117

(ALA1-ALA12). In recent years, progress in the research on flippases in plants 118

has shown that P4-ATPases have pleiotropic effects on physiological functions, 119

including plant growth and development, reproduction, and adaptation to biotic 120

and abiotic stress (Gomès et al., 2000; Poulsen et al., 2008; Zhang and 121

Oppenheimer, 2009; McDowell et al., 2013; Poulsen et al., 2015; Botella et al., 122

2016; Guo et al., 2017; Niu et al., 2017; Underwood et al., 2017; Zhang et al., 123

2020). However, whether ALAs are involved in the establishment of the 124

asymmetric distribution of PS in pollen tubes remains unclear. Further 125

research is also required to clarify the subcellular localization and biological 126

function of PS in pollen tubes. 127

To better understand the mechanisms of the inverted-cone distribution of 128

apical vesicles and the biological functions of PS in pollen tube tips, we 129

screened Arabidopsis ALA mutants with abnormal distributions of apical 130

vesicles and Rab GTPases as well as PS in pollen tubes. Further, we explored 131

the physiological function of PS in the polar growth of pollen tubes. The 132

findings of this study indicate that PS in the apical area of pollen tubes might 133

function as a phospholipid signaling molecule, regulating the distribution of 134

certain Rab GTPases as well as pollen tube growth. 135

RESULTS 136

Mutation in ALA3 Affects the Polarized Distribution of Apical Vesicles 137

and Pollen Tube Width 138

Bioinformatics analysis shows that six Arabidopsis ALA members are 139

abundantly expressed in pollen, including ALA1, ALA3, ALA6, ALA7, ALA9 140

and ALA12 (Supplemental Figure 1A). To determine whether and how the ALA 141

family participates in the polarized transport of vesicles at the apical region of 142

pollen tube, we used the lipophilic dye FM4-64 to label the pollen tubes of 143

pollen-expressed-ALA mutants and then identified the mutants with abnormal 144

tip-polarized vesicles. In WT pollen tubes, FM4-64-labeled vesicles were 145

abundantly located at the inverted-cone zone of the pollen tube (Figure 1A), 146

which was consistent with the previous studies (Szumlanski and Nielsen, 2009; 147

Zhang et al., 2010). Interestingly, we only observed obviously decreased 148

accumulation of vesicles in the inverted-cone zone at the apical region of 149

pollen tube in the ala3 mutant; the intensity of the fluorescence signal within 150

the apical and shank regions of the pollen tube also decreased but was 151

especially high in the subapical region of the ala3 pollen tube (Figures 1A and 152

1B; Supplemental Figures 1B to 1G). 153

We used RabA4b (a RabA4 subfamily member expressed in 154

vegetative organs and widely used to label tip vesicles in pollen tubes) to verify 155

whether ALA3 affected the localization of RabA4 GTPases (Zhang et al., 2010). 156

As expected, the distribution of YFP-RabA4b-labeled vesicles in ala3 was 157

consistent with the above results (Figures 1C to 1E). Based on time-lapse 158

imaging analysis of the movement of vesicles in living WT and ala3 pollen 159

tubes, FM4-64- and YFP-RabA4b-labeled vesicles in WT pollen tubes could 160

move rapidly through an inverted-cone zone at the tip of the pollen tube (see 161

Supplemental Movies 1 and 2 online), but the vesicles in the ala3 pollen tube 162

were scarcely enriched in the apical area, unlike those in the WT, and moved 163

only in a disordered manner between two flanks of the subapical region (see 164

Supplemental Movies 3 and 4 online). In addition, although the cytoplasmic 165

streaming in the shank region of the pollen tube of ala3 was not notably 166

changed, the velocity of the vesicles in the apical and subapical regions 167

decreased observably (Figures 1F to 1H). Previous studies have reported that 168

the pollen tube growth rate was decreased in ala3 (Zhang and Oppenheimer, 169

2009; McDowell et al., 2013), and we also demonstrated that the pollen tube 170

growth of ala3 was obviously slower than that of WT in vitro (Figures 1I and 1J) 171

and in vivo (Supplemental Figures 2C and 2D). In addition, we found a 172

significant increase in pollen tube width in ala3 (8.26 ± 0.96 μm), which was in 173

contrast to the WT plants (6.95 ± 0.83 μm) (Figures 1I and 1J), thereby 174

revealing that ALA3 might also be involved in the formation and maintenance 175

of the polar growth of pollen tubes. 176

To ascertain the relationship between the above phenotypes and the lipid 177

flipping activity of ALA3, a complementation experiment was performed in an 178

ala3 mutant, in which the full-length cDNAs of ALA3 and ALA3 with a point 179

mutation in the conserved functional site of the ALA family, named D413N, 180

were used. We found that only full-length ALA3 cDNA could rescue the 181

abnormal phenotypes of ala3 related to the growth and width of pollen tubes 182

(Figures 1I and 1J; Supplemental Figures 2A to 2D), suggesting that loss of 183

function of ALA3 probably led to the above phenotypes and that the lipid 184

flipping activity of ALA3 played a crucial role in determining the growth rate and 185

width of pollen tubes. Moreover, brefeldin A (BFA, a vesicle trafficking inhibitor), 186

wortmannin (Wort, a PI3K inhibitor) and latrunculin B (LatB, an actin 187

depolymerization drug) are widely used to inhibit the polarized tip growth of 188

pollen tubes (Gibbon et al., 1999; Zhang et al., 2010). Treatment of ala3 and 189

WT pollens with the above inhibitors resulted in a decrease in pollen tube 190

growth; however, compared to WT, the negative effect of BFA on pollen tube 191

length significantly decreased in ala3 (Figure 1K; Supplemental Figure 2E), 192

whereas both Wort and LatB showed no obvious differences (Supplemental 193

Figures 2F to 2I). Together, our results suggested that loss of function of ALA3 194

induced the disordered distribution of vesicles and the altered trajectory of 195

vesicular trafficking at the pollen tube tip, which ultimately caused an increase 196

in pollen tube width and defective polar growth of the pollen tube. 197

198

ALA3 is Abundant in the Apical Region of Pollen Tube and Colocalizes 199

with Certain Endomembrane Compartments 200

Next, the distribution patterns of ALA3 in tissues and organelles were 201

determined. ALA3 could be abundantly expressed in mature pollen and pollen 202

tubes (Supplemental Figure 3). Using an ALA3-GFP transgenic plant, in which 203

the defective phenotype of ala3 was almost completely complemented, we 204

found that GFP signals were largely distributed in the apical and subapical 205

regions of the pollen tube and dotted at the tip and shank regions of the pollen 206

tube (Figure 2A). Then, based on time-lapse imaging analysis, the ALA3-GFP 207

moved into the inverted-cone zone at the apical region of the pollen tube and 208

was present in the cytoplasmic streaming in the shank region of the pollen tube 209

(see Supplemental Movie 5 online). Importantly, ALA3-GFP colocalized with 210

FM4-64 in the apical and shank regions of the pollen tube, suggesting that 211

ALA3 was distributed in both vesicles and PM (Figures 2B to 2D). Furthermore, 212

the colocalization between ALA3-GFP and various organelle markers was 213

identified and quantified, showing that ALA3 chiefly colocalized with markers of 214

the trans-Golgi network (TGN)/early endosome (EE) (Pearson correlation 215

coefficient (rp) was 0.05 and 0.43 in the tip and the shank, respectively), 216

post-Golgi (rp was 0.55 and 0.43 in the tip and the shank, respectively) and 217

recycling endosome (RE) (rp was 0.29 and 0.53 in the tip and the shank, 218

respectively), but not the Golgi (rp was 0.08 and 0.08 in the tip and the shank, 219

respectively) and late endosome (LE) (rp was 0.10 and 0.11 in the tip and the 220

shank, respectively) (Figures 2E to 2J). Kymograph analysis showed the 221

localization of spots in the two channels over time (Figures 2K to 2M; 222

Supplemental Figure 4). In addition, the treatment of ALA3-GFP transgenic 223

plants with endomembrane inhibitors (including BFA, Wort and LatB) resulted 224

in a significant decrease of the polarized distribution of ALA3-GFP at the pollen 225

tube tip (Supplemental Figure 5), indicating that the tip-polarized localization of 226

ALA3 itself might also rely on polarized tip membrane trafficking. 227

228

ALA3 and RabA4d Genetically Regulate the Polar Growth of Pollen Tubes 229

There are four members of the RabA4 protein subfamily, but only RabA4d 230

can be specifically expressed in pollen tubes (Preuss et al., 2004; Szumlanski 231

and Nielsen, 2009). It has been also reported that RabA4d is located at the 232

inverted-cone zone of the pollen tube and is responsible for targeted polar 233

transport of the TGN/EE-derived secretory vesicles (Szumlanski and Nielsen, 234

2009). We found that RFP-RabA4d and ALA3-GFP exhibited a similar 235

distribution pattern and colocalized in pollen tube tip in certain extent (rp = 0.52) 236

(Figures 3A and 3B). In addition, ALA3-GFP partially colocalized with 237

RFP-RabA4b at the pollen tube tip (rp = 0.50) (Supplemental Figure 6). 238

Moreover, in ala3, vesicles labeled with RFP-RabA4d were also mislocalized, 239

which is similar to the result obtained for YFP-RabA4b (Figures 3C to 3E; see 240

Supplemental Movies 6 and 7 online), indicating that the polar growth of the 241

pollen tube of ala3 might be directly associated with RabA4d. To further assess 242

whether there is a genetic correlation between ALA3 and RabA4d, the width 243

and length of the pollen tube of the double mutant ala3 raba4d were 244

determined. The results showed that the pollen tube of the double mutant ala3 245

raba4d had a similar width as that of the single mutant raba4d (Figures 3F to 246

3H), suggesting that these two genes may work in the same genetic pathway 247

in the regulation of the pollen tube width and polar growth. Next, we also 248

identified the vesicle distribution in raba4d labeled with FM4-64. The 249

abundance of apical vesicles labeled with FM4-64 visibly decreased in the 250

raba4d pollen tube, and this reduction was directly related to the swelling 251

degree of the pollen tube tip; the more swollen pollen tube tips the fewer apical 252

vesicles (Figures 3I and 3J). This finding showed that loss of function of 253

RabA4d caused anomalies in vesicle distribution in the apical region and polar 254

growth of pollen tubes. In summary, ALA3 might regulate the polar growth of 255

pollen tubes by affecting the function of RABA4d, which in turn controls the 256

vesicle distribution in the inverted-cone zone at the pollen tube tip. 257

258

Loss of Function of ALA3 Alters the Distribution Pattern and the Amount 259

of PS in Pollen Tubes 260

ALA3 was previously reported to flip phospholipids, including PE, PS and 261

PC, between two membrane leaflets (López-Marqués et al., 2010). However, 262

the total content of phospholipids in ala3 pollen was not obviously changed 263

compared to that in WT pollen; thus, it was proposed that the phenotypes of 264

ala3 were caused by the disordered distribution of lipids (McDowell et al., 265

2013). Here, the C2 domain of bovine lactadherin (Lact-C2), which could mark 266

PS on the cytoplasmic leaflet of the PM and the membrane of the organelles 267

(Yeung et al., 2008), was used to mark PS in Arabidopsis pollen tubes. As 268

shown in Figure 4, EGFP-Lact-C2-labeled PS was abundantly localized at the 269

inverted-cone zone of the pollen tube and partially dotted the shank region of 270

the pollen tube (Figure 4A). Next, by tracking its dynamic process, PS was 271

shown to move fast, resembling the movement pattern of RabA4b-labeled 272

vesicles (see Supplemental Movie 8 online). EGFP-Lact-C2 also colocalized 273

with the membrane dye FM4-64 at the apical and shank regions of the pollen 274

tube (Figures 4B to 4D). Furthermore, the colocalization between 275

EGFP-Lact-C2 and various organelle markers was identified and quantified, 276

and the results indicated that EGFP-Lact-C2 was mainly colocalized with 277

markers of the TGN/EE (rp was 0.18 and 0.42 in the tip and the shank, 278

respectively), post-Golgi (rp was 0.55 and 0.51 in the tip and the shank, 279

respectively) and endosome (rp was 0.56 and 0.54 in the tip and the shank, 280

respectively) (Figures 4E to 4K). Kymograph analysis showed the localization 281

of spots in the two channels over time (Figures 4N to 4P; Supplemental Figure 282

7). The polarized localization of EGFP-Lact-C2 at the apical region could be 283

seriously affected by endomembrane inhibitors (Supplemental Figure 8), 284

suggesting that apical PS distribution also depended on endomembrane 285

transport. 286

To identify the direct effect of ALA3 on PS distribution in pollen tubes, we 287

crossed the ala3 mutant with the above PS marker lines. In the ala3 mutant, 288

the polarized distribution of PS in the apical region of pollen tube dramatically 289

decreased, chiefly accumulating in the subapical region, and PS fluorescent 290

signals in both the apical and shank regions of the pollen tube were observably 291

decreased (Figures 4L and 4M; see Supplemental Movies 9 and 10 online). In 292

addition, the distributions of PS in WT and ala3 were almost consistent with 293

those of vesicles labeled with FM4-64 or YFP-RabA4b (Figures 1A to 1D), and 294

PS was also colocalized with RabA4b, RabD1, RabA1g and FM4-64 in pollen 295

tubes (Figures 4B to 4K). Thus, these results suggested that ALA3 plays a key 296

role in the polarized distribution of PS at the pollen tube tip, and PS might 297

regulate the localization and related vesicle trafficking of certain Rab 298

GTPases. 299

300

Loss of Function of ALA3 Has a Negative Effect on TGN/EE Distribution 301

in Pollen Tubes 302

In the ala3 mutant, the fluorescence signals of vesicles labeled with 303

FM4-64 and RabA4b/RabA4d were obviously decreased (Figures 1A to 1D; 304

Figures 3C to 3E), suggesting that ALA3 might also affect vesicle formation at 305

the TGN/EE. To further clarify whether ALA3 affects TGN specially or regulates 306

other subcellular compartments, we crossed the ala3 mutant with different 307

organelle markers and then quantified the fluorescence intensity of these 308

markers. As shown in Figure 5 and Supplemental Figure 9, compared with the 309

WT, the distribution pattern and fluorescence intensity of the ER and Golgi 310

markers HDEL-GFP and mCherry-SYP32 in ala3 were not changed, 311

suggesting that loss of function of ALA3 had no effect on these two organelles 312

(Supplemental Figure 9). Next, VHAa1-RFP and mCherry-VTI12, two 313

well-documented TGN/EE markers, were used. We found that both the density 314

and fluorescence intensity of these TGN/EE markers were observably 315

decreased within the shank region of the pollen tube of ala3 (Figures 5A to 5E). 316

In addition, the movement velocities of these markers were comparable in the 317

WT and ala3 mutant (Supplemental Figure 10). Interestingly, we found that 318

there was no signal at the apical region of pollen tube with VHAa1-RFP in WT, 319

but the TGN/EE labeled with mCherry-VTI12 still exhibited some fast-moving 320

small particles at the pollen tube tip, which were similar to FM4-64-labeled 321

vesicles (Figures 5A and 5C; see Supplemental Movies 11 and 12 online). 322

Additionally, the mCherry-VIT12 signal at the apical region of the pollen tube 323

dramatically decreased and moved to the subapical region in ala3, similar to 324

the markers FM4-64, RabA4b/4d and EGFP-Lact-C2 (Figures 5C and 5E, see 325

Supplemental Movies 13 and 14 online). Thus, our data provide evidence that 326

VTI12 could also mark other vesicles and endomembrane compartments in 327

addition to the TGN/EE. Next, we performed fluorescence recovery after 328

photobleaching (FRAP) analysis to examine the fluorescence recovery of 329

YFP-RabA4b and mCherry-RabA1e. As shown in Figures 5F to 5I, the 330

recovery rates of YFP-RabA4b and mCherry-RabA1e in ala3 were much 331

slower than those in WT, indicating that lack of ALA3 function had a significant 332

influence on secretory vesicle recycling. Based on these results, it is possible 333

that ALA3 was also involved in regulating the TGN/EE to reduce the number of 334

secretory vesicles and had an effect on their polarized distribution in the apical 335

region of pollen tube. 336

337

Loss of Function of ALA3 Ultimately Leads to Aberrant Composition of 338

the Cell Wall of Pollen Tubes 339

RabA4d-mediated vesicle trafficking delivers indispensable cargoes, such 340

as cell wall components, for the polar growth of pollen tubes (Szumlanski and 341

Nielsen, 2009). Therefore, we determined the cell wall composition of the 342

pollen tube of the ala3 mutant. As shown in Figure 6, compared with WT, the 343

levels of arabinogalactan-proteins (AGPs), xyloglucan and 344

low-methyl-esterified homogalacturonan (HG), marked by LM2, LM15 and 345

JIM5, were sharply reduced in the ala3 pollen tube, while there was no 346

significant change in the levels of rhamnogalacturonan I (RG-I) and 347

high-methyl-esterified HG between WT and the mutants labeled with LM6 and 348

JIM7, respectively (Figures 6A and 6B). Additionally, the activity of callose 349

synthase was mainly present at the PM near the apical region and 350

spread to the shank of the pollen tube, but the cell wall stained with 351

aniline blue was not changed (Figures 6C and 6D). Another enzyme 352

expressed in pollen grains and pollen tubes is the pectin methylesterase 353

VANGUARD1 (VGD1), the function of which could affect the ratio of low HG to 354

high HG to promote pollen tube growth. In our work, the VGD1-GFP signal in 355

the ala3 pollen tube also dramatically decreased (Figures 6E and 6F). Taken 356

together, these results indicated that loss of function of ALA3 caused a defect 357

in cell wall transport, which in turn induced alterations in the cell wall 358

components of the pollen tube and finally had an adverse effect on the polar 359

growth of the pollen tube. 360

361

ALA3 Principally Affects the Polarized Localization and Distribution of 362

Secreted Rab GTPases at the Pollen Tube Tip 363

To further determine whether and how loss of function of ALA3 affects Rab 364

GTPases in a wide variety of endomembrane transport pathways, we identified 365

the localization pattern of Rab GTPases involved in different transport 366

pathways in WT and ala3. RabA1e and RabA1g, which are known to 367

specifically label recycling endosomes (REs), were located in the shank region 368

and a smaller region close to the apical membrane of the pollen tube, and they 369

also moved in an ordered and rapid manner between the shank and apical 370

regions (Figures 7A to 7D; see Supplemental Movies 15 and 16 online). 371

However, in ala3, the polarized localization of these two proteins dramatically 372

decreased, and the fluorescence intensity prominently decreased; these 373

proteins mainly accumulated in the two flanks of the subapical region 374

with irregular activities (Figures 7A to 7D; see Supplemental Movies 17 and 18 375

online). The results indicate that ALA3 had a marked influence on both the 376

polarized localization and distribution of RabA1e and RabA1g. Interestingly, 377

although RabA5d also colocalized with REs, the fluorescence intensity of 378

mCherry-RabA5d decreased in both the apical and shank regions in the ala3 379

mutant (Figures 7E and 7F), but its distribution pattern was not changed. In 380

addition, RabE1d and RabC1, which are post-Golgi markers, were located in 381

the whole pollen tube and moved rapidly in the apical region. We found that 382

the fluorescence intensity of RabE1d was reduced in ala3 (Figures 7G and 7H). 383

However, the fluorescence intensity of RabC1, as well as the distribution 384

pattern of these two markers, were unchanged in ala3 compared with the WT 385

(Figures 7K and 7L). Likewise, ARA6 and RabG3f are two specific LE markers 386

that were limited at the shank region of the pollen tube. The fluorescence 387

intensity of ARA6 was obviously weakened, but its distribution pattern was not 388

changed in ala3 (Figures 7I and 7J). Unlike ARA6, both the fluorescence 389

intensity and distribution pattern of RabG3f were unchanged (Figures 7M and 390

7N). In conclusion, we found that the functional deficiency of ALA3 affected 391

multiple Rab GTPase-mediated vesicle transport pathways, but the regulatory 392

mechanisms for Rab GTPases in various transport pathways were different. 393

394

DISCUSSION 395

PS is Abundantly Localized in the Inverted-cone Zone of the Arabidopsis 396

Pollen Tube. 397

The asymmetric distribution of PS between the two biomembrane leaflets 398

has a profound influence on the surface charge, curvature and lipid packing of 399

the PM and specific endomembrane, as well as the electrostatic gradient 400

among different intracellular compartments (Yeung et al., 2008; Muthusamy et 401

al., 2009; Xu et al., 2013; Platre et al., 2018). Thus, PS plays a crucial role in 402

endomembrane trafficking. Here, we found that EGFP-Lact-C2 labeled PS was 403

mainly located in the inverted-cone zone of the pollen tube in Arabidopsis, 404

suggesting a potential correlation between the polarized distribution of PS and 405

an accumulation of large amounts of post-Golgi-derived vesicles in the apical 406

region of pollen tube. We also observed the localization of mCITRINE-Lact-C2 407

labeled PS in the pollen tube of Arabidopsis thaliana (Platre et al., 2019), and 408

found that its localization pattern was similar to that of the EGFP-Lact-C2 409

labeled PS (Supplemental Figure 11). In addition, this polarized PS distribution 410

in Arabidopsis pollen tubes was quite similar to that in root hairs as well as in 411

tobacco pollen tubes (Platre et al., 2018). We therefore determined the 412

subcellular localization of PS and found that PS was primarily enriched in the 413

TGN/EE, vesicles, endosomes and the PM in pollen tubes (Figures 4B to 4K), 414

which was consistent with the localization in root cells (Platre et al., 2018), 415

indicating that the subcellular localization of PS in different cell types in planta 416

are similar. Based on previous studies on anionic lipids in pollen tubes, it was 417

known that LE/multivesicular body (MVB)-localized PtdIns(3)P was involved in 418

vesicle degradation (Vermeer et al., 2006; Jean and Kiger, 2012); PtdIns(4)P 419

mainly accumulated at the PM of subapical and shank regions of pollen tubes 420

and contributed to the endocytic process (Zhao et al., 2010); PtdIns(4,5)P2 421

was also localized at the PM in both the apical and subapical regions and was 422

reported to control the growing site and direction of pollen tube growth (Kost et 423

al., 1999; Hempel et al., 2017); PA was also a PM-localized lipid in tobacco 424

pollen tubes (Potocký et al., 2014). Furthermore, we noticed that PS was not 425

only distributed chiefly in the inverted-cone zone of vesicle enrichment at the 426

apical region of pollen tube but also colocalized with the vesicular markers 427

RabA4b, RabA1g and RabE1 or with the endocytic dye FM4-64 (Figures 4B to 428

4K), confirming that anionic lipids participate in the regulation of the polarized 429

distribution and trafficking of vesicles at the apical region of pollen tube in 430

various ways. In addition, it was reported that PI4P and PA were also located 431

at the PM, while PS was accumulated at both the PM and endomembrane in 432

dividing root cells (Platre et al., 2018). Taken together, these results show that 433

PS probably plays an important role in endomembrane trafficking in different 434

cell types in plants. 435

436

ALA3 is a Key Regulator of PS Distribution and the Polarized Growth of 437

Arabidopsis Pollen Tubes. 438

Each member of the Arabidopsis ALA protein has its own tissue 439

expression and subcellular localization patterns as well as specific lipid flipping 440

activity (Gomès et al., 2000; Poulsen et al., 2008; López-Marqués et al., 2010). 441

However, which ALAs are involved in the polarized distribution and localization 442

of PS in pollen tubes remains unknown to date. We revealed that ALA3 was 443

abundantly expressed in pollen grains (Supplemental Figure 3), and its 444

distribution, movement trajectory and subcellular localization were extremely 445

similar to those of PS in the apical region of pollen tubes (Figures 2 and 4A to 446

4K). Additionally, a lack of ALA3 activity resulted in a significant decrease of 447

PS in the apical region but aggregation in the subapical region and an 448

apparent decrease in PS in the shank of the pollen tube (Figures 4L and 4M). 449

Lact-C2 marks PS only on the cytoplasmic leaflet of the PM and the 450

membranes of organelles (Yeung et al., 2008). In addition, ALA3 had been 451

reported to have PS flipping activity (López-Marqués et al., 2010), and the total 452

content of PS in ala3 pollen was not obviously changed (McDowell et al., 2013). 453

Therefore, the main reason for the decrease of PS signal in the ala3 mutant 454

may be the lack of the PS flipping activity of ALA3, leading to the failure of PS 455

to effectively flip to the cytoplasmic side of the PM and organelles. Thus, we 456

propose that ALA3 is a key member of the ALA family involved in establishing 457

and maintaining the polarized distribution of PS in pollen tubes. Indeed, ALA3 458

might translocate PS from the extracellular/luminal leaflet of the membrane 459

into the cytosolic leaflet when transported in a polarized pattern, causing 460

polarized distribution of PS at the apical region of pollen tube with a highly 461

dynamic pattern. 462

Previous studies have shown that ALA3 participates in many crucial 463

physiological activities via multiple mechanisms, including root and pollen tube 464

growth (Poulsen et al., 2008; McDowell et al., 2013; Zhang et al., 2020), 465

trichome development (Zhang and Oppenheimer, 2009), and resistance to 466

pathogens (Underwood et al., 2017). For example, in Arabidopsis root and 467

tobacco leaf cells, ALA3 was reported to be localized mainly in the Golgi 468

apparatus and only partially in the TGN/EE, and loss of function of ALA3 led to 469

a defect in the production of secretory vesicles in root peripheral columella 470

cells, which thereby inhibited root growth (Poulsen et al., 2008).The latest 471

research has reported that ALA3, which is located mainly in the Glogi, TGN/EE, 472

and PM, functions together with the ARF GTPase exchange factors (GNOM 473

and BIG3) in regulating PIN polarity, trafficking, and auxin-mediated 474

development (Zhang et al., 2020). In addition, it was recently shown that loss 475

of ALA3 function had a negative impact on the cycling of the defense protein 476

PENETRATION3 between the PM and TGN/EE and subsequently affected 477

plant immune responses against powdery mildews (Underwood et al., 2017). 478

In addition, the reason for the slow growth rate of the ala3 mutant pollen tube 479

was proposed to be a change in the trajectory of cytoplasmic streaming 480

(McDowell et al., 2013). Here, we found that loss of ALA3 resulted in increased 481

pollen tube width and an absence of apical vesicle distribution, suggesting that 482

ALA3 also played another important role in the polarity of pollen tube growth. 483

In contrast to the subcellular localization within root cells, ALA3 in pollen 484

tubes is mainly located at the TGN/EE, vesicles, RE and the PM but is scarcely 485

colocalized with Golgi markers (Figure 2). This result was highly consistent 486

with the fact that no ER and Golgi were present in the apical region of pollen 487

tube (Cheung and Wu, 2008; Chebli et al., 2013) (Supplemental Figure 9). 488

Importantly, in the ala3 pollen tube, the fluorescence intensity of the TGN/EE 489

was observed to be significantly reduced; in addition, the amounts of secretory 490

vesicles also decreased, and the vesicle secretion rate correspondingly 491

decreased (Figure 5). Moreover, the transport direction of apical vesicles in the 492

ala3 pollen tube was severely affected; PS and other endomembrane-derived 493

vesicles could not arrive at the apical region and moved in a disordered 494

manner within the subapical region of the pollen tube. In addition, the polarized 495

distribution of ALA3 and PS in the apical region of pollen tube was highly 496

sensitive to several trafficking inhibitors, including BFA, Wort and LatB 497

(Supplemental Figures 5 and 8). Furthermore, we also measured the uptake of 498

FM4-64 in Col-0 and ala3 pollen tubes over time, and we found that the levels 499

of internalized FM4-64 in Col-0 and ala3 were similar at 5 minutes, indicating 500

that the endocytosis is not affected in the ala3 mutant (Supplemental Figure 501

12). 502

In summary, ALA3 was confirmed to be closely associated with 503

post-Golgi-mediated vesicular secretion and polarized transport at the apical 504

region of pollen tube, and the altered transport direction of apical vesicles was 505

suggested to be the primary reason for the defects in the polar growth of the 506

ala3 pollen tube. Interestingly, Zhang and Oppenheimer (2009) reported that 507

the pollen tubes in ala3 grew slowly, but the root hair length was relatively high, 508

indicating that ALA3 had pleiotropic effects in different types of polar cells. 509

Based on these results, we proposed that the subcellular localization and 510

functional mechanism of ALA3 varied among different cell types in plants. 511

512

PS Might Be a Potential Phospholipid Signaling Molecule Regulating 513

Polarized Transport at the Apical Region of Pollen Tubes. 514

Rab GTPases constitute a key functional protein family that contributes to 515

endomembrane trafficking in eukaryotes and can recognize and bind to 516

specific organelles and the PM to participate in multifarious transport pathways 517

via varying molecular mechanisms (Nielsen et al., 2008; Woollard and Moore, 518

2008; Kjos et al., 2018). Therefore, the specificity of Rab protein localization 519

is the basis for its biological function. However, the molecular mechanisms 520

regulating Rab GTPases localization have proved tricky issues to address. 521

Numerous studies have shown that the localization and distribution of Rabs 522

are directly regulated by multiple factors, including 523

guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins 524

(GAPs), effector proteins, interacting proteins, and phosphoinositides (Zerial 525

and McBride, 2001; Grosshans et al., 2006; Thomas et al., 2019). Here, the 526

vesicles labeled with FM4-64, RabA4b/4d, RabA1e and RabA1g could not 527

reach the apical region of pollen tubes of the ala3 mutant; and the vesicles 528

clustered in the subapical region of the pollen tube in a disordered manner 529

(Figures 1A to 1D, 3A to 3E, and 7A to 7D). These results showed that ALA3 530

was involved in establishing the polarized localization of these vesicles in the 531

apical region of pollen tubes. Moreover, we found that PS colocalized with 532

RabA4b, RabD1, and RabA1g in pollen tubes (Figures 4E to 4K). The 533

evidence from genetic analysis showed that ALA3 and RabA4d were 534

collectively responsible for regulation of the polar growth of pollen tubes 535

(Figure 3). Based on these results, we propose that PS likely acts as a 536

signaling molecule to participate in the polarized distribution and transport of 537

Rab GTPase-mediated vesicles at the apical region of pollen tube. 538

Recently, Platre et al. (2019) reported that PS directly binds to another 539

important small GTPase, termed Rho of Plants 6 (ROP6), in vitro and in vivo; 540

PS could recruit ROP6 into nanodomains in specific membrane regions and 541

had a notable effect on polar auxin transport and gravitropism in roots. We 542

also conducted the same lipid overlay experiments in vitro and found that 543

ROP6, RabA4b, RabA4d, RabD1 and RabA1g could bind to PS and certain 544

other anionic lipids in vitro (Supplemental Figures 13D to 13H). In addition, to 545

verify whether different forms of Rab GTPases have different phospholipid 546

binding activity, we also improved the experiment by expressing the 547

constitutively active and dominant negative forms of RabA4d (RabA4dQ74L and 548

RabA4dT29N), RabA4b (RabA4bQ76L and RabA4bS31N) and RabD1 (RabD1Q67L 549

and RabD1S22N). The results of lipid overlay experiments showed that either 550

constitutively active or dominant negative forms of these Rab GTPases could 551

bind to PS and the other anionic lipids (Supplemental Figures 13I to 13N). 552

Then, a liposome cosedimentation assay was performed to quantify the 553

phospholipid binding activity of different forms of RabA4d. We found that 554

RabA4dQ74L and RabA4d exhibited comparable binding activities to PS; 555

however, RabA4dT29N exhibited slightly increased binding activity to PS 556

compared to RabA4dQ74L and RabA4d (Supplemental Figures 14A to 14E). 557

Moreover, the binding activity of RabA4d to PI4P was higher than that to PS 558

(Supplemental Figures 14F and 14G). Thus, we hypothesized that the 559

polarized distribution of PS might have a direct influence on the polarized 560

localization of these Rab GTPases as well as of their targeted vesicles at the 561

pollen tube tip. These findings indicate the existence of a new mechanism by 562

which PS regulates vesicle trafficking. However, Rop1 was found to be 563

localized at the apical PM of the pollen tube, which was chiefly involved in 564

regulating the growth direction of the pollen tube (Fu et al., 2001; Luo et al., 565

2017), indicating that PS probably had specific regulatory mechanisms for 566

distinct small GTPases (Figure 7O). 567

Furthermore, lipid binding assays demonstrated that Rab GTPases could 568

also bind to other anionic lipids, such as PA and PIPs, in addition to PS 569

(Supplemental Figure 13). Liposome cosedimentation assays also 570

demonstrated that RabA4d cannot bind to PE and PC in vitro, which is 571

consistent with the results of lipid overlay experiments (Supplemental Figure 572

14). Because PIPs have been confirmed to affect the localization of Rab 573

GTPases by regulating many effector proteins (Jean and Kiger, 2012; Noack 574

and Jaillais, 2017), it was suggested that the localization and function of Rab 575

GTPases in pollen tubes were also likely regulated by these anionic lipids. 576

Based on these results, we proposed that Rab GTPases might bind to these 577

anionic lipids for preliminary localization, and Rab GEFs or other effector 578

proteins likely affect the subsequent precise localization of Rab GTPases. In 579

addition, PI4Ks have been proven to be important effectors of RabA4 family, 580

which plays an important role in the polar growth of pollen tubes and root hairs 581

(Thole et al., 2008; Szumlanski and Nielsen, 2009). The polar distribution of 582

RabA4d/RabA4b in the tip of pollen tube of ala3 mutant decreased significantly, 583

so the distribution of PI4Ks in ala3 mutant may also be affected, which 584

indirectly/directly leads to changes in the distribution and content of membrane 585

phospholipids, such as PtdIns(4)P and PtdIns(4,5)P2. Deciphering the 586

underlying mechanism of these intracellular activities should be an important 587

direction for future research. 588

589

METHODS 590

Plant Materials 591

Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the WT. 592

Transgenic plants used in this study were in the Col-0 background. The ala3 593

(SALK_082157), raba4d (CS360297), ala1 (SALK_056947), ala6 594

(SALK_150173), ala7 (SALK_125598), ala9 (SALK_128495) and ala12 595

(SALK_111498) T-DNA insertion mutant seeds were obtained from the 596

Arabidopsis Biological Resource Center (ABRC). The T-DNA insertion in ala3 597

was identified by PCR using the primers ALA3LP, ALA3RP and LBb1.3. The 598

T-DNA insertion in raba4d was identified by PCR using the primers RabA4d-F, 599

RabA4d-R and LB1. Primers are described in Supplemental Table 1. The 600

single mutants ala3 and raba4d were crossed to obtain the double mutant ala3 601

raba4d. Selection of transformed lines with an ala3 background was 602

conducted as previously described (Zhu et al., 2017). The previously reported 603

transgenic lines used in this study included UBQ10: mCherry-RabC1 (Geldner 604

et al., 2009), UBQ10: mCherry-RabG3f (Geldner et al., 2009), UBQ10: 605

mCherry-Rha1 (Geldner et al., 2009), UBQ10: mCherry-VTI12 (Geldner et al., 606

2009), UBQ10: mCherry-Got1p homolog (Geldner et al., 2009), UBQ10: 607

mCherry-SYP32 (Geldner et al., 2009), UBQ10: mCherry-RabA5d (Geldner et 608

al., 2009), UBQ10: mCherry-RabD1 (Geldner et al., 2009), UBQ10: 609

mCherry-RabE1d (Geldner et al., 2009), UBQ10: mCherry-RabA1e (Geldner 610

et al., 2009), UBQ10: mCherry-RabA1g (Geldner et al., 2009), UBQ10: 611

mCherry-MEMB12 (Geldner et al., 2009), LAT52:YFP-RabA4b (Zhang et al., 612

2010), UBQ10:mCITRINE-Lact-C2 (Platre et al., 2019) and 613

VHAa1:VHAa1-RFP (Dettmer et al., 2006). 614

615

Growth Conditions 616

Seeds were vernalized for three days at 4 °C after surface sterilization and 617

then grown on Murashige & Skoog (MS) medium with 1% agar. Seven-day-old 618

seedlings grown on MS medium were transferred to mixed soil in a 619

greenhouse with a 16 h light/8 h dark photoperiod with white fluorescent light 620

(bulb type: TCL, TCLMY-28, 28W, with five tubes; 100 μmol m–2 s–1) and 65% 621

relative humidity at 22 ± 2 °C. 622

623

GUS Staining 624

The ALA3 putative promoter, including the 1895-bp fragment upstream of the 625

start codon, was amplified from Col-0 genomic DNA by PCR. Primers are 626

described in Supplemental Table 1. The fragment was cloned into the 627

pDONR/zeo entry vector, and then the fragment was transferred into 628

pBIB-BASTA-GWR-GUS binary vectors using LR Clonase II (Invitrogen). The 629

T2-generation homozygous transgenic seedlings of ALA3:GUS were 630

subjected to GUS (β-glucuronidase) staining. GUS staining procedures were 631

performed as previously described (Jia et al., 2013). Images were obtained 632

using a Carl ZEISS ZEN2 microscope equipped with ZEN 2.3 software. 633

634

RT-PCR 635

Mature pollen grains of Col-0, ala3, the LAT52:ALA3-GFP;ala3 complemented 636

line, and the ALA3:ALA3D413N;ala3 complemented line were collected. For 637

mature pollen grain collection, at least 2 mL of fresh opened flowers were 638

collected in a 5-mL tube, and then, 2 mL of 2% sucrose solution was added 639

and vortexed for 1 min. The samples were centrifuged at 6000 g for 2 min. The 640

flowers and solutions were removed, and then, the tubes with pellets (pollen 641

grains) were saved in liquid nitrogen. Total RNA of the pollens was extracted 642

using the MiniBEST Plant RNA Extraction Kit (TaKaRa). Total cDNA was 643

obtained through reverse transcription using M-MLV (RNase H-) reverse 644

transcriptase (TaKaRa). To confirm the ALA3 expression levels in Col-0, ala3, 645

and the complemented lines, the ALA3 fragment was amplified from the total 646

cDNA template by PCR. EF4A cDNA was amplified as the internal control. 647

Primers are described in Supplemental Table 1. 648

649

Pollen Tube Growth Analysis 650

For in vitro pollen tube growth, pollen from freshly opened flowers was 651

smeared onto solid pollen germination medium as described previously (Zhu 652

et al., 2017). Pollen tube length and width were measured using ImageJ 653

software (http://rsbweb.nih.gov/ij/). To confirm the pollen tube growth rate in 654

vivo, fresh pollen grains were pollinated onto the Col-0 pistils, the pistils were 655

fixed for 2 h with ethanol/acetic acid (3:1) after pollination for 5 or 9 h, and a 656

graded ethanol series of 80%, 50%, 30%, and ddH2O were used to rehydrate 657

the pistils. For softening, the pistils were incubated with 8 M NaOH overnight at 658

22 ºC and then washed at least three times with ddH2O. Samples were then 659

stained with aniline blue staining solution (0.1% aniline blue in 100 mM 660

phosphate buffer, pH 8.0) for at least three hours in the dark. Images were 661

obtained using a spinning-disk confocal microscope (Andor). Pollen tube 662

length was measured using ImageJ software. 663

664

Gene Cloning and Plasmid Construction 665

The coding sequences of ALA3, ARA6, RabA4d and RabA4b and the 666

full-length genomic sequence of VGD1 were amplified from the Col-0 cDNA 667

library or genomic DNA by PCR. The coding sequence of ALA3 with a point 668

mutation (ALA3D413N) was amplified by fusion PCR. The C2 domain of the 669

lactadherin (Lact-C2) fragment with a stop codon was artificially synthesized. 670

The coding sequence of Lact-C2 was fused at the C terminus of EGFP to 671

generate EGFP-Lact-C2. The coding sequences of ALA3 and EGFP-Lact-C2 672

were cloned into the pDONR/zeo entry vector and then transferred into the 673

pBIB-BASTA-LAT52-GWR-GFP vector. The coding sequence of ALA3D413N 674

with a stop codon was cloned into the pDONR/zeo entry vector and then 675

transferred into the pBIB-HYG-pALA3-GWR vector. The coding sequence of 676

ARA6 was cloned into the pDONR/zeo entry vector and then transferred into the 677

pBIB-BASTA-LAT52-RFP-GWR vector. The coding sequences of RabA4b and 678

RabA4d were cloned into the pDONR/zeo entry vector and then transferred into 679

the pBIB-BASTA-LAT52-RFP-GWR vector. The full-length genomic sequence 680

of VGD1 was cloned into the pDONR/zeo entry vector and then transferred into 681

the pBIB-HYG-GWR-GFP vector. To construct LAT52:HDEL-GFP, the ER 682

retention signal HDEL was subcloned from the 683

LAT52:AtbCH:EYFP:HDEL:NOS vector by digesting the HindIII and XbaI sites 684

and fused to the pCAMBIA1300-LAT52:GFP vector. These constructs were 685

then transformed by Agrobacterium tumefaciens strain GV3101-mediated 686

transformation into ala3 homozygous mutants or the WT. Transgenic plants 687

were selected on hygromycin or Basta. All PCR amplifications were performed 688

using PrimeSTAR GXL DNA polymerase (TaKaRa). Sequencing analyses 689

were performed to confirm the amplified fragments. The primers used for the 690

above cloning are described in Supplemental Table 1. 691

692

Confocal Microscopy and Image Analysis 693

Confocal images of pollen tubes on solid pollen germination medium were 694

acquired using a spinning-disk confocal microscope equipped with an iXON 695

Ultra EMCCD camera (Andor) according to previously reported (Qu et al., 696

2013). Samples were observed using a 63 × NA oil immersion lens. Image 697

acquisition was carried out using Andor IQ3 software. Time-lapse images were 698

captured every 0.5 s–1 s for 20 s–2 min. GFP was excited with a 488-nm laser 699

and observed using a 514-nm emission filter. YFP, mCITRINE was excited with 700

a 514-nm laser and observed using a 542-nm emission filter. RFP, mCherry 701

and FM4-64 were excited with a 561-nm laser and observed using a 607-nm 702

emission filter. Images were quantified with ImageJ software. For 703

colocalization evaluations, ImageJ with the Colocalization Finder plugin was 704

used to calculate the Pearson correlation coefficients. 705

706

Quantification of the Velocity and Trajectory of Vesicle Movement 707

Imaris software was used to analyze the average trajectory speed. The 708

parameters of the Imaris program were defined as follows: vesicles with an 709

average diameter of 0.5 μm were labeled, the maximum distance between 710

frames was 2 μm, the maximum gap size was 3, and the trajectory duration 711

was 3 s. 712

713

Pharmacological Treatments 714

To determine the sensitivity of pollen tube growth to BFA, LatB or Wort, a final 715

concentration of 0.4 or 0.5 μM BFA, 1 or 2 nM LatB, or 0.6 or 1 μM Wort was 716

added to the pollen germination medium. The same dilution ratio of DMSO 717

was added as a control. After growth for 3 h, paraformaldehyde at a final 718

concentration of 4% in pollen germination medium was added to fix the 719

samples before measuring pollen tube length. To determine the effects of 720

inhibitors on ALA3-GFP and EGFP-Lact-C2 distribution in pollen tubes, a final 721

concentration of 0.4 μM BFA, 1 nM LatB, or 1 μM Wort corresponding to 5 μM 722

FM4-64 was added. 723

724

Aniline Blue Staining of Pollen Tubes 725

Pollen germinated for 3 h in vitro was fixed by using a fixative (4% 726

paraformaldehyde in liquid pollen germination medium, pH 7.0) and then 727

washed three times with 100 mM phosphate buffer. Samples were stained 728

directly with aniline blue staining solution for 5 min before microscopy. 729

730

FM4-64 Staining 731

After germination for 3 h on pollen germination medium, a final concentration 732

of 5 μM FM4-64 was added. Pollen tubes were subjected to time-lapse 733

imaging at the appropriate time point. 734

735

Immunolabeling 736

Immunolabeling of pollen tube cell wall epitopes was performed with LM2, LM6, 737

LM15, JIM5 and JIM7 as described previously (Fabrice et al., 2018). Pollen 738

tubes grown for 3 h on solid pollen germination medium were fixed by using a 739

fixative (4% paraformaldehyde, 50 mM PIPES buffer (pH 7.0), 2 mM CaCl2, 3 740

mM MgSO4, 18% sucrose) for 0.5 h at room temperature (RT). Samples were 741

washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 742

and 2 mM KH2PO4) for 5 min each and then blocked with 3% BSA in PBS for 1 743

h at RT or overnight at 4 °C. Samples were incubated for 1 h at RT with 1% rat 744

primary antibody (LM2, LM6, LM15, JIM5, JIM7) in PBS with 3% BSA. 745

Samples were washed three times with the same buffer and incubated with 746

0.5% anti-rat IgG (whole molecule)-FITC antibody (Sigma; F1763) in PBS with 747

3% BSA in the dark for 1 h at RT. Samples were washed five times for 5 min 748

each with PBS, and then slides were mounted with mounting solution (50% 749

glycerol, 0.1% p-phenylenediamine, and PBS) and observed with a 750

spinning-disk confocal fluorescence microscope (Andor). 751

752

FRAP Analysis 753

FRAP experiments were performed using a spinning-disk confocal 754

fluorescence microscope (Andor) equipped with a 63 × NA oil immersion lens. 755

The region of interest was bleached with a 488-nm argon laser at 100% 756

transmission for 5 s. Fluorescence recovery was recorded at 5 s intervals for 757

up to 5 min. The recovery percentage of each time point was calculated using 758

the following equation: (It – Is) / (Ia – Is) × 100, where It is the fluorescence 759

intensity at each time point, Is is the initial fluorescence intensity after 760

bleaching, and Ia is fluorescence intensity before bleaching (Luu et al., 2012). 761

762

Immunoblot Analysis 763

Ten microliters (100 ng) of purified protein was used to analyze protein purity 764

by immunoblotting using 1:10000 anti-GST primary antibody (Abmart, 765

M20007), and incubated at RT for 1 h. Then, 1:10000 secondary anti-mouse 766

IgG (whole molecule)-peroxidase antibody (Sigma, A9044) was applied at RT 767

for 1 h. Images were obtained using chemiluminescence detection. 768

769

Protein Expression, Purification and Lipid-Protein Overlay Assay 770

The cDNAs of RabA4d, RabA4b, RabD1, ROP6, and RabA1g were amplified 771

from the Col-0, with the primers listed in Supplemental Table 1. The primers 772

used for site mutation of the constitutively active and dominant negative forms 773

of the Rab GTPases are also listed in Supplemental Table 1. The PCR 774

products were cloned into the pMD19-T vector and then cloned into the pGEX 775

4T-1 destination vector. The expression plasmids were transformed into 776

Escherichia coli Rosetta cells for protein expression. Purification of 777

GST-tagged recombinant proteins was performed as previously described 778

(Xiang et al., 2007). For lipid-protein overlay assays, PIP strips containing 779

fifteen purified lipids (P-6001, Echelon Bioscience) were blocked in 3% BSA in 780

TBS-T (150 mM NaCl, 50 mM Tris, and 0.05% Tween 20; pH 8.0) at RT for 3 h.781

The strips were then incubated with 2 μg/mL purified proteins in 10 mL of 782

TBS-T with 3% BSA at RT for 2 h. After incubation, the strips were washed 783

three times for 10 min each with TBS-T, then incubated with 1:10000 anti-GST 784

primary antibody (Abmart, M20007) in 10 mL TBS-T at RT for 1 h, then washed 785

three times for 10 min each with TBS-T and incubated with 1:10000 secondary 786

anti-mouse IgG (whole molecule)-peroxidase antibody (Sigma, A9044) in 10 787

mL of TBS-T at RT for 1 h. Images were obtained at RT using 788

chemiluminescent signals. 789

790

Liposome Cosedimentation Assay 791

The liposome cosedimentation experiments were performed as previously 792

described, with slight modifications (Zhu et al., 2014). Liposomes were 793

prepared from 50% PC and 50% of the indicated lipids [PS, PE or PI4P]. The 794

lipid mixtures (dissolved in chloroform) were dried by nitrogen gas, and the 795

dried lipid was resuspended in liposome buffer and sonicated for 20 min. For 796

each protein, a final concentration of 2 μM, 3 μM or 4 μM protein solution was 797

incubated with or without 100 μg of lipid mixture at RT for 30 min. Then, the 798

samples were centrifuged at 20,000 ×g at 4 °C for 30 min. Then, the pellets 799

and supernatants were separated, subjected to SDS-PAGE and stained with 800

Coomassie blue. The percentage of the bound proteins was analyzed by 801

ImageJ software. 802

803

Statistical Analysis 804

Statistical analysis was performed via the SPSS software package (version 805

22.0; IBM). The data represent the means ± SDs based on three independent 806

biological replicates. Two-tailed Student’s t tests (t test) were performed to 807

determine statistical significance, and the threshold was set at 0.05. 808

809

Accession Numbers 810

The sequence data from this article can be found in The Arabidopsis 811

Information Resource (https://www.arabidopsis.org/) or GenBank 812

(http://www.ncbi.nlm.nih.gov/genbank/) databases under the following 813

accession numbers: At-ALA3, AT1G59820; At-ALA1, AT5G04930; At-ALA2, 814

AT5G44240; At-ALA4, AT1G17500; At-ALA5, AT1G72700; At-ALA6, 815

AT1G54280; At-ALA7, AT3G13900; At-ALA8, AT3G27870; At-ALA9, 816

AT1G68710; At-ALA10, AT3G25610; At-ALA11, AT1G13210; At-ALA12, 817

AT1G26130; At-RabA4d, AT3G12160; At-RabA4b, AT4G39990; At-RabD1, 818

AT3G11730; At-RabA1g, AT3G15060; At-VGD1, AT2G47040; At-ARA6, 819

AT3G54840; MFGE8 (lactadherin), NM_176610, At-ROP6, AT4G35020. 820

821

Supplemental Data 822

Supplemental Figure 1. The distribution pattern of the FM4-64-labeled 823

compartment in ala1, ala6, ala7, ala9, and ala12 mutant pollen tubes was 824

comparable to that in Col-0 (Supports Figure 1). 825

Supplemental Figure 2. The pollen tube growth of the ala3 mutant is less 826

sensitive to BFA than that of Col-0 (Supports Figure 1). 827

Supplemental Figure 3. ALA3 is abundantly expressed in inflorescences, 828

mature pollen grains and pollen tubes (Supports Figure 2). 829

Supplemental Figure 4. Kymograph showing ALA3-GFP not colocalized with 830

Golgi or LE (Supports Figure 2). 831

Supplemental Figure 5. The exogenous inhibitors BFA, LatB and Wort can 832

significantly perturb the polar localization of ALA3-GFP in pollen tubes 833

(Supports Figure 2). 834

Supplemental Figure 6. ALA3-GFP colocalizes with RFP-RabA4b at the 835

pollen tube tip (Supports Figure 3). 836

Supplemental Figure 7. Kymograph showing EGFP-Lact-C2 colocalized or 837

not colocalized with certain endomembrane compartments (Supports Figure 838

4). 839

Supplemental Figure 8. The exogenous inhibitors BFA, LatB and Wort can 840

significantly perturb the polar localization of EGFP-Lact-C2 in pollen tubes 841

(Supports Figure 4). 842

Supplemental Figure 9. The distribution of the ER and Golgi is not affected by 843

the ala3 mutation (Supports Figure 5). 844

Supplemental Figure 10. The velocities of the TGN/EE in Col-0 and ala3 are 845

comparable (Supports Figure 5). 846

Supplemental Figure 11. Localization pattern of mCITRINE-Lact-C2 PS 847

biosensor is similar to the EGFP-Lact-C2 PS biosensor in Col-0 and ala3. 848

(Supports Discussion). 849

Supplemental Figure 12. The levels of internalized FM4-64 in Col-0 and ala3 850

are similar. (Supports Discussion). 851

Supplemental Figure 13. Different forms of some Rab GTPases can directly 852

bind to PS in vitro. (Supports Discussion). 853

Supplemental Figure 14. Different forms of RabA4d can directly bind to PS 854

but not PE or PC in vitro. (Supports Discussion). 855

Supplemental Table 1. Primers used in this study. 856

857

Supplemental Movie 1. FM4-64 distributes in inverted-cone zone of the Col-0 858

pollen tube (Supports Figure 1). 859

Supplemental Movie 2. YFP-RabA4b localizes inverted-cone zone of the 860

Col-0 pollen tube (Supports Figure 1). 861

Supplemental Movie 3. FM4-64 moved in a disordered manner of the 862

subapical region of ala3 mutant pollen tube (Supports Figure 1). 863

Supplemental Movie 4. YFP-RabA4b moved in a disordered manner of the 864

subapical region of ala3 mutant pollen tube (Supports Figure 1). 865

Supplemental Movie 5. ALA3-GFP signals were largely distributed in the 866

apical and subapical regions of the pollen tube (Supports Figure 2). 867

Supplemental Movie 6. RFP-RabA4d localizes inverted-cone zone of the 868

Col-0 pollen tube (Supports Figure 3). 869

Supplemental Movie 7. RFP-RabA4d moved in a disordered manner of the 870

subapical region of ala3 mutant pollen tube (Supports Figure 3). 871

Supplemental Movie 8. EGFP-Lact-C2 PS biosensor was abundantly 872

localized at the inverted-cone zone of Col-0 pollen tube (Supports Figure 4). 873

Supplemental Movie 9. EGFP-Lact-C2 PS biosensor was abundantly 874

localized at the inverted-cone zone of Col-0 pollen tube (Supports Figure 4). 875

Supplemental Movie 10. EGFP-Lact-C2 PS biosensor moved in a disordered 876

manner of the subapical region of ala3 mutant pollen tube (Supports Figure 4). 877

Supplemental Movie 11. VHAa1-RFP TGN/EE marker localizes at the shank 878

region of Col-0 pollen tube (Supports Figure 5). 879

Supplemental Movie 12. mCherry-VTI12 TGN/EE marker localizes at the 880

apical, subapical and shank regions of Col-0 pollen tube (Supports Figure 5). 881

Supplemental Movie 13. VHAa1-RFP TGN/EE marker localizes at the shank 882

region of ala3 mutant pollen tube (Supports Figure 5). 883

Supplemental Movie 14. mCherry-VTI12 TGN/EE marker was abundantly 884

accumulate at the subapical region of ala3 mutant pollen tube (Supports 885

Figure 5). 886

Supplemental Movie 15. mCherry-RabA1e RE marker localizes at the apical 887

and shank region of Col-0 pollen tube (Supports Figure 7). 888

Supplemental Movie 16. mCherry-RabA1g RE marker localizes at the apical 889

and shank region of Col-0 pollen tube (Supports Figure 7). 890

Supplemental Movie 17. mCherry-RabA1e RE marker was abundantly 891

accumulate at the subapical region of ala3 mutant pollen tube (Supports 892

Figure 7). 893

Supplemental Movie 18. mCherry-RabA1g RE marker was abundantly 894

accumulate at the subapical region of ala3 mutant pollen tube (Supports 895

Figure 7). 896

897

ACKNOWLEDGMENTS 898

We thank Dr. Tonglin Mao (China Agricultural University), Dr. Xiangfeng Wang 899

(China Agricultural University) and Dr. Ruixi Li (Southern University of Science 900

and Technology) for valuable comments on the manuscript. We are grateful to 901

Dr. Lijia Qu (Peking University), Dr. Genji Qin (Peking University), Dr. Yan 902

Zhang (Shangdong Agricultural University) and Dr. Jianwei Pan (Lanzhou 903

University) for sharing seeds. We thank the Core Facility of the School of Life 904

Sciences, Lanzhou University, for technical assistance. This work was 905

supported by grants from the National Natural Science Foundation of China 906

(31722005, 31970195, 31670180) and the Fundamental Research Funds for 907

the Central Universities (lzujbky-2019-75 and lzujbky-2020-sp04). 908

909

AUTHER CONTRIBUTIONS 910

Y.X. and Y.N. conceived the study and designed the research; Y.Z., Y.Y., D.Q. 911

and T.F. performed the research; Y.Z., Y.Y., C.L., Y.S. S.L. and L.A. analyzed 912

the data; Y.X., Y.N., Y.Z. and Y.Y. wrote the manuscript. 913

914

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Figure Legends 1135

Figure 1. The distribution pattern and the velocity of apical vesicles, as 1136

well as the width of the pollen tube, are altered in the ala3 mutant. 1137

(A) Visualization of the FM4-64 distribution in the pollen tubes from the wild 1138

type (Col-0) and ala3. Confocal images captured after FM4-64 staining for 20 1139

min. Bar = 10 μm. The arrowhead and arrow indicate the apical and subapical 1140

zones, respectively. The apical, subapical and shank regions used for 1141

fluorescence intensity measurement in here and the following pollen tubes are 1142

shown on the right. 1143

(B) Quantitative analysis of the relative fluorescence intensity of FM4-64 in the 1144

apical, subapical and shank zones of the pollen tube. The results represent the 1145

means ± SDs (n = 30). **P < 0.01 (t test compared to wild-type values). 1146

(C) Representative confocal images of growing pollen tubes from Col-0 and 1147

ala3 expressing YFP-RabA4b from the same transgenic line. Bar = 10 μm. The 1148

arrowhead and arrow indicate the apical and subapical zones, respectively. 1149

(D) Quantitative analysis of the relative fluorescence intensity of YFP-RabA4b 1150

in the apical, subapical and shank zones of the pollen tube. The results 1151

represent the means ± SDs (n = 30). **P < 0.01 (t test compared to wild-type 1152

values). 1153

(E) Quantitative analysis of the percentage of apical-localized and 1154

subapical-localized YFP-RabA4b and the relative length of the growing pollen 1155

tube. The percentage of apical localization and subapical localization of 1156

YFP-RabA4b was calculated as the sum of the pixel intensities in the apical 1157

and subapical zones of the pollen tube compared with the sum of the pixel 1158

intensities in the entire pollen tube. 1159

(F) and (G) Representative time-lapse trajectories of YFP-RabA4b at 5 s for 1160

Col-0 and ala3 in the tip (F) and shank (G) zones of pollen tubes obtained by 1161

Imaris image analysis. Bars = 10 μm. 1162

(H) Quantitative analysis of the relative vesicle velocity of YFP-RabA4b at the 1163

pollen tube tip (F) and shank (G) zones from Col-0 and ala3. The results 1164

represent the means ± SDs (n = 15). **P < 0.01 (t test compared to wild-type 1165

values). 1166

(I) Growth of Col-0, ala3, LAT52:ALA3-GFP;ala3, and ALA3:ALA3D413N;ala31167

pollen tubes in vitro for 3 h. Bar = 30 μm. 1168

(J) Quantitative analysis of the relative pollen tube width and length in Col-0,1169

ala3, LAT52:ALA3-GFP;ala3, and ALA3:ALA3D413N;ala3. The results represent 1170

the means ± SDs (n = 100). **P < 0.01 (t test compared to wild-type values). 1171

(K) Quantitative analysis of the relative length of Col-0 and ala3 pollen tubes1172

treated with BFA. The results represent the means ± SDs (n = 300). **P < 0.01 1173

(t test compared to wild-type values). 1174

1175

Figure 2. ALA3 exhibits polarized distribution in pollen tubes and is 1176

localized to the endomembrane and plasma membrane. 1177

(A) Representative growing pollen tube from the ala3 mutant expressing1178

ALA3-GFP from the LAT52:ALA3-GFP transgene. Bar = 10 μm. 1179

(B) Representative confocal images of a growing pollen tube expressing1180

ALA3-GFP (green) with FM4-64 staining (magenta). Merged images are 1181

shown on the right. Bar = 10 μm. 1182

(C) Line scan measurements (white dotted arrow 1) of the confocal image in (B)1183

are plotted to show colocalization profiles. 1184

(D) Line scan measurements (white dotted arrow 2) of the confocal image in (B)1185

are plotted to show colocalization profiles. PM represents plasma membrane 1186

signals. 1187

(E) to (I) Representative confocal images of growing pollen tubes1188

coexpressing ALA3-GFP (green) with mCherry-SYP32 (Golgi) (E), 1189

VHAa1-RFP (TGN/EE) (F), mCherry-RabD1 (post-Golgi) (G), 1190

mCherry-RabA1e (RE) (H) or mCherry-RHA1 (LE) (I) (white). Merged images 1191

are shown on the right. Bars = 10 μm. 1192

(J) Measurement of the Pearson correlation coefficient to reflect the1193

colocalization level of ALA3-GFP with compartment markers. The tip and 1194

shank regions used for Pearson correlation coefficient measurement in here 1195

and the following pollen tubes are shown in (E). The results represent the 1196

means ± SDs (n = 15). 1197

(K) to (M) Kymograph representation of non-colocalized foci (green and 1198

magenta foci) and colocalized foci (white foci). The region used for Kymograph 1199

analysis in here and the following pollen tubes are shown on the left (white 1200

arrow). 1201

1202

Figure 3. ALA3 and RabA4d likely collaborate to regulate the polarized 1203

growth of pollen tubes. 1204

(A) Representative confocal images of growing pollen tubes coexpressing 1205

ALA3-GFP (green) with RFP-RabA4d (magenta). Merged images are shown 1206

on the right. Bar = 10 μm. 1207

(B) Kymograph representation of non-colocalized foci (green and magenta foci) 1208

and colocalized foci (white foci). 1209

(C) Representative confocal images of growing pollen tubes from Col-0 and 1210

ala3 expressing RFP-RabA4d from the same transgenic line. Bar = 10 μm. 1211

(D) Quantitative analysis of the relative fluorescence intensity of RFP-RabA4d 1212

in the apical, subapical and shank zones of the pollen tube. The results 1213

represent the means ± SDs (n = 30). **P < 0.01 (t test compared to wild-type 1214

values). 1215

(E) The percentage of apical-localized and subapical-localized RFP-RabA4d 1216

and the relative length of the growing pollen tube were measured. The 1217

percentage of apical localization and subapical localization of RFP-RabA4d 1218

was calculated as the sum of the pixel intensities in the apical and subapical 1219

zones of the pollen tube compared with the sum of the pixel intensities in the 1220

entire pollen tube. 1221

(F) Growth of the Col-0, ala3, raba4d, and ala3 raba4d pollen tubes in vitro for 1222

4 h. Bar = 30 μm. 1223

(G) and (H) Quantification of pollen tube width (G) and relative pollen tube 1224

length (H) in Col-0, ala3, raba4d, and ala3 raba4d. The results represent the 1225

means ± SDs (n = 100). **P < 0.01 (t test). 1226

(I) Representative confocal images of Col-0 and raba4d pollen tubes stained 1227

with FM4-64 for 20 min. Bars = 10 μm. 1228

(J) Quantification of the fluorescence intensity of FM4-64 at the apical region 1229

and plasma membrane in Col-0 and raba4d pollen tubes. The regions used for 1230

fluorescence intensity measurement are marked in (I). The apical region is 1231

marked in yellow, and the PM is marked in white. The results represent the 1232

means ± SDs (n = 30). **P < 0.01 (t test compared to wild-type values). 1233

1234

Figure 4. Loss of function of ALA3 alters the distribution pattern of PS. 1235

(A) Representative growing pollen tube from Col-0 expressing EGFP-Lact-C2 1236

from the LAT52:EGFP-Lact-C2 transgene. Bar = 10 μm. 1237

(B) Confocal images of a growing pollen tube expressing EGFP-Lact-C2 1238

(green) stained with FM4-64 (magenta) for 20 min. Merged images are shown 1239

on the right. Bar = 10 μm. 1240

(C) Line scan measurements (white dotted arrow 1) of the confocal image in (B) 1241

are plotted to show colocalization profiles. 1242

(D) Line scan measurements (white dotted arrow 2) of the confocal image in (B) 1243

are plotted to show colocalization profiles. PM represents plasma membrane 1244

signals. 1245

(E) to (J) Representative confocal images of growing pollen tubes 1246

coexpressing EGFP-Lact-C2 or mCITRINE-Lact-C2 (green) with the 1247

mCherry-Got1p homolog (Golgi) (E), mCherry-VTI12 (TGN/EE) (F), 1248

mCherry-RabD1 (post-Golgi) (G), mCherry-RabA1g (RE) (H), mCherry-RHA1 1249

(LE) (I) or RFP-RabA4b (rp = 0.42) (J) (magenta). Merged images are shown 1250

on the right. Bars = 10 μm. 1251

(K) Measurement of the Pearson correlation coefficient to reflect the 1252

colocalization level of EGFP-Lact-C2 with compartment markers. The results 1253

represent the means ± SDs (n = 15). 1254

(L) Representative confocal images of growing pollen tubes from Col-0 and 1255

ala3 expressing EGFP-Lact-C2 from the same transgenic line. Bar = 10 μm. 1256

(M) Quantification of the relative fluorescence intensity of EGFP-Lact-C2 in 1257

Col-0 and ala3 pollen tubes. The results represent the means ± SDs (n = 30). 1258

**P < 0.01 (t test compared to wild-type values). 1259

(N) to (P) Kymograph representation of non-colocalized foci (green and 1260

magenta foci) and colocalized foci (white foci). 1261

1262

Figure 5. Loss of ALA3 leads to abnormal distribution of the TGN/EE in 1263

pollen tubes. 1264

(A) Representative confocal images of growing pollen tubes from Col-0 and 1265

ala3 expressing VHAa1-RFP from the same transgene. Bar = 10 μm. 1266

(B) Quantification of the relative puncta density, diameter, and fluorescence 1267

intensity marked by VHAa1-RFP in Col-0 and ala3 pollen tubes. The results 1268

represent the means ± SDs (n = 30). *P < 0.05; **P < 0.01 (t test compared to 1269

wild-type values). 1270

(C) Representative confocal images of growing pollen tubes from Col-0 and 1271

ala3 expressing mCherry-VTI12 from the same transgenic line. The arrowhead 1272

and arrow indicate the apical and subapical zones, respectively. Bar = 10 μm. 1273

(D) Quantification of the relative puncta density and diameter marked by 1274

mCherry-VTI12 in Col-0 and ala3 pollen tubes. The results represent the 1275

means ± SDs (n = 30). **P < 0.01 (t test compared to wild-type values). 1276

(E) Quantification of the relative fluorescence intensity of mCherry-VTI12 in 1277

Col-0 and ala3 pollen tubes. The results represent the means ± SDs (n = 30). 1278

**P < 0.01 (t test compared to wild-type values). 1279

(F) Fluorescence recovery after photobleaching (FRAP) analysis of 1280

YFP-RabA4b in growing pollen tubes. The time points after photobleaching are 1281

labeled in each image. The bleached area is marked by the box. Bar = 10 μm. 1282

(G) FRAP analysis of mCherry-RabA1g in growing pollen tubes. The time 1283

points after photobleaching are labeled in each image. The bleached area is 1284

marked by the box. Bar = 10 μm. 1285

(H) Measurement of the mean YFP fluorescence intensity of the bleached area1286

in (F). The results represent the means ± SDs (n = 7). 1287

(I) Measurement of the mean mCherry fluorescence intensity of the bleached1288

area in (G). The results represent the means ± SDs (n = 7). 1289

1290

Figure 6. The abundance of cell wall components in the pollen tube is 1291

changed in ala3. 1292

(A) Immunolabeling of cell wall components in Col-0 and ala3 pollen tubes1293

using LM2, LM6, LM15, JIM5 and JIM7. Bar = 10 μm. 1294

(B) Quantitative analysis of the relative fluorescence intensity of the different1295

cell wall epitopes indicated in Col-0 and ala3 pollen tubes. The results 1296

represent the means ± SDs (n = 30). **P < 0.01 (t test compared to wild-type 1297

values). 1298

(C) Aniline blue staining of the pollen tubes in Col-0 and ala3. Bar = 10 μm.1299

(D) Quantitative analysis of the relative fluorescence intensity of aniline blue in1300

Col-0 and ala3 pollen tubes. The results represent the means ± SDs (n = 30). 1301

(E) Representative confocal images of growing pollen tubes from Col-0 and1302

ala3 expressing VGD1-GFP from the same transgenic line. Bar = 10 μm. 1303

(F) Quantitative analysis of the relative fluorescence intensity of VGD1-GFP in1304

Col-0 and ala3 pollen tubes. The results represent the means ± SDs (n = 30). 1305

**P < 0.01 (t test compared to wild-type values). 1306

1307

Figure 7. The localization and distribution of secreted Rab GTPases in 1308

the pollen tube are changed in ala3. 1309

(A), (C), (E), (G), (I), (K), (M) Representative confocal images of growing 1310

pollen tubes from Col-0 and ala3 expressing mCherry-RabA1e (RE) (A), 1311

mCherry-RabA1g (RE) (C), mCherry-RabA5d (RE) (E), mCherry-RabE1d 1312

(post-Golgi) (G), RFP-ARA6 (LE) (I), mCherry-RabC1 (post-Golgi) (K) or 1313

mCherry-RabG3f (LE) (M) from the same transgene. The arrowhead and 1314

arrow indicate the apical and subapical zones, respectively. Bars = 10 μm. 1315

(B), (D), (F), (H), (J), (L), (N) Quantitative analysis of the relative fluorescence 1316

intensity of mCherry-RabA1e (RE) (B), mCherry-RabA1g (RE) (D), 1317

mCherry-RabA5d (RE) (F), mCherry-RabE1d (post-Golgi) (H), RFP-ARA6 (LE) 1318

(J), mCherry-RabC1 (post-Golgi) (L) and mCherry-RabG3f (LE) (N) in Col-0 1319

and ala3 pollen tubes. The results represent the means ± SDs (n = 30). **P < 1320

0.01 (t test compared to wild-type values). 1321

(O) A model was proposed for the role of ALA3 in the polarized distribution of1322

PS and vesicle trafficking. In Col-0 pollen tubes, ALA3 is required for the 1323

polarized distribution of PS, which regulates the distribution and activity of 1324

certain Rab GTPases. In ala3 pollen tubes, the total PS level at the cytosolic 1325

leaflet decreases significantly, and PS accumulates in the subapical zone, 1326

causing reduced vesicle formation and accumulation of vesicles in the 1327

subapical zone. The mislocalization of vesicles causes failed secretion, 1328

leading to pollen tube growth defects.1329

DOI 10.1105/tpc.19.00844; originally published online August 18, 2020;Plant Cell

Li, Lizhe An and Yun XiangYuelong Zhou, Yang Yang, Yue Niu, Tingting Fan, Dong Qian, Changxin Luo, Yumei Shi, Shanwei

GTPase-mediated Vesicle Trafficking and Pollen Tube GrowthThe Tip-localized Phosphatidylserine Established by Arabidopsis ALA3 is Crucial for Rab

 This information is current as of January 7, 2021

 

Supplemental Data /content/suppl/2020/08/18/tpc.19.00844.DC1.html

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