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: [email protected] 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 ([email protected]). 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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