The lipid flippases ALA4 and ALA5 play critical roles in cell … · 2020. 2. 12. · 65 dwarfism,...

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Short Title: ALA4/5 are important for vegetative growth 1 2 Author for Contact: Harper, J.F. ([email protected]) 3 4 Title: The lipid flippases ALA4 and ALA5 play critical roles in cell expansion and plant 5 growth 6 7 Authors: Davis, J.A.1, Pares, R.B. 1, Bernstein, T.3, McDowell, S.C. 1, Brown, E.1, Stubrich, J. 1, 8 Rosenberg, A. 1, Cahoon, E.B.2, Cahoon, R.E.2, Poulsen, L.R.3, Palmgren, M.3, López-Marqués, 9 R.L.3, Harper, J.F.1 10 11 Affiliations: 12 1Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Reno, NV 13 89557, USA. 14 2Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, 15 Lincoln, Lincoln, NE 68588, USA. 16 3Department of Plant and Environmental Sciences, University of Copenhagen, 1871, 17 Frederiksberg, Denmark. 18 19 James A. Davis [email protected] 20 Randall B. Pares [email protected] 21 Tilde Bernstein [email protected] 22 Stephen C. McDowell [email protected] 23 Elizabeth Brown [email protected] 24 Jason Stubrich [email protected] 25 Alexa Rosenberg [email protected] 26 Edgar B. Cahoon [email protected] 27 Rebecca E. Cahoon [email protected] 28 Lisbeth R. Poulsen [email protected] 29 Michael Palmgren [email protected] 30 Rosa L. López-Marqués [email protected] 31 Jeffrey F. Harper [email protected] 32 33 One-sentence summary: A subclass of lipid flippases contribute to glycerolipid and 34 sphingolipid homeostasis in Arabidopsis leaves, and play critical roles in cell expansion and 35 vegetative growth. 36 37 Footnotes: 38 Author contributions: J.D. authored the manuscript. R.P. and J.D. analyzed vegetative 39 physiology. S.M. bred the double-knockout lines and identified dwarf mutants. E.B., A.R., and 40 J.S. performed subcellular localization experiments. L.P. and R.L. were involved in the cloning 41 of ALA5 for yeast expression. T.B. performed lipid uptake and functional complementation 42 assays. R.L. analyzed the data and carried out statistical analysis for yeast experiments. M.P. 43 and R.L. supervised yeast experiments. E.C. and R.C. performed sphingolipid profiling of ala4/5 44 mutants. J.H. performed all plant crosses, supervised in planta experiments, and assisted J.D. 45 in writing. J.H. agrees to serve as author responsible for contact. 46 47 Funding information: 48 This work was supported by grants to JFH from USDA Hatch NEV00384 and NSF IOS 49 1656774, to RLL from Villum Fonden project number 13234, to MP from Innovation Fund 50 Denmark project number 8053-00035B, to EBC from NSF MCB 1818297. Microscopy was 51

Plant Physiology Preview. Published on February 12, 2020, as DOI:10.1104/pp.19.01332

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supported by the National Institute of General Medical Sciences of the National Institutes of 52 Health under grant number P20 GM103554. The funders had no role in the designing the 53 research, data collection, analysis, or manuscript preparation. 54 55 Email address of Author for Contact: Harper, J.F. ([email protected]) 56 57 58 Abstract: 59 Aminophospholipid ATPases (ALAs) are lipid flippases involved in transporting specific lipids 60 across membrane bilayers. Arabidopsis thaliana contains 12 ALAs in five phylogenetic clusters, 61 including four in cluster 3 (ALA4, -5, -6, and -7). ALA4/5 and ALA6/7 are expressed primarily in 62 vegetative tissues and pollen, respectively. Previously, a double knockout (KO) of ALA6/7 was 63 shown to result in pollen fertility defects. Here we show that a double KO of ALA4/5 results in 64 dwarfism, characterized by reduced growth in rosettes (6.5-fold), roots (4.3-fold), bolts (4.5-fold), 65 and hypocotyls (2-fold). Reduced cell size was observed for multiple vegetative cell types, 66 suggesting a role for ALA4/5 in cellular expansion. Members of the 3rd ALA cluster are at least 67 partially interchangeable, as transgenes expressing ALA6 in vegetative tissues partially rescued 68 ala4/5 mutant phenotypes, and expression of ALA4 transgenes in pollen fully rescued ala6/7 69 mutant fertility defects. ALA4-GFP displayed plasma membrane (PM) and endomembrane 70 localization patterns when imaged in both guard cells and pollen. Lipid profiling revealed ala4/5 71 rosettes had perturbations in glycerolipid and sphingolipid content. Assays in yeast revealed 72 that ALA5 can flip a variety of glycerolipids and the sphingolipid sphingomyelin across 73 membranes. These results support a model whereby the flippase activity of ALA4/5 impacts the 74 homeostasis of both glycerolipids and sphingolipids and is important for cellular expansion 75 during vegetative growth. 76 77 Introduction: 78 Lipid flippases of the P4-type ATPase family are enzymes that hydrolyze ATP to flip lipids 79 across a membrane bilayer towards cytosolic facing leaflets, whether that be from the outer 80 leaflet of the plasma membrane (PM) or luminal leaflet of internal membranes (López-Marqués 81 et al., 2014). Flippase activity and proper subcellular localization requires a β-accessory subunit 82 (e.g. cell division control protein 50 in yeast) (Saito et al., 2004). Flippases are proposed to be 83 involved in multiple processes, such as flipping specific lipids to generate an asymmetric 84 distribution between the leaflets of a membrane bilayer, generating membrane curvature, 85 assisting vesicle formation, importing lipids from the environment, and facilitating lipid 86 metabolism (Seigneuret and Devaux, 1984; Gall et al., 2002; Pomorski et al., 2003; Poulsen et 87 al., 2015; Roland et al., 2019). 88 89 In plants, flippases are referred to as Aminophospholipid ATPases (ALAs). The β-accessory 90 subunits of ALAs are referred to as ALA interacting subunits (ALISs) (Poulsen et al., 2008). The 91 Arabidopsis thaliana genome contains 12 ALAs that form five phylogenetic clusters (Baxter et 92 al., 2003). Representatives from all five ALA clusters have been studied with either loss-of-93 function knockout (KO) mutants or RNAi lines, revealing phenotypes that include increased 94 sensitivity to cold stress (ALA1 RNAi – cluster 1) (Gomès et al., 2000), disrupted stomatal 95 regulation and lipid desaturation levels (ala10 KO – cluster 2) (Poulsen et al., 2015; Botella et 96 al., 2016), increased sensitivity to heat and heavy metals as well as constitutive male fertility 97 defects (ala6 KO, ala4 KO, ala6/7 double KO – cluster 3) (McDowell et al., 2015; Niu et al., 98 2017; Sanz-Fernández et al., 2017), impaired pathogen defenses and reduced growth in 99 rosettes and trichomes (ala3 KO – cluster 4) (Zhang and Oppenheimer, 2009; McDowell et al., 100 2013; Underwood et al., 2017), and impaired viral defenses (ala2 KO – cluster 5) (Zhu et al., 101 2017; Guo et al., 2017). 102



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103 Lipid transport preferences have been characterized for four of the five ALA clusters, with 104 fluorescent lipid uptake studies in yeast providing evidence that ALAs from different clusters 105 have distinct transport activities. For example, ALA1 from cluster 1 transports 106 phosphatidylserine (PS) > phosphatidylethanolamine (PE) (Gomès et al., 2000), ALA10 from 107 cluster 2 transports phosphatidylcholine (PC) > PE > PS > phosphatidylglycerol (PG) > 108 lysophosphatidylcholine (LysoPC) (Poulsen et al., 2015; Jensen et al., 2017), ALA3 from cluster 109 4 transports PE > PC > PS (Poulsen et al., 2008), and ALA2 from cluster 5 transports only PS 110 (López-Marqués et al., 2010). Additionally, ALA10 (cluster 2) has been shown to facilitate the 111 uptake of sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer) (Jensen 112 et al., 2017). To date, the lipid transport activities for a cluster-3 ALA has not been reported. 113 114 In this study, we created a double-KO mutant for the two cluster-3 ALAs expressed in vegetative 115 tissues, specifically ALA4 and ALA5. The ala4/5 mutations resulted in dwarfed plants with 116 underlying defects in cellular expansion. This growth reduction could be restored by the 117 expression of either ALA4 or ALA5, and partially restored by the ectopic expression of ALA6, 118 another cluster-3 ALA normally expressed only in pollen. Lipidomic profiling of ala4/5 rosettes 119 revealed perturbations in both glycerolipids and sphingolipids, including relative increases in the 120 abundance of phosphatidic acid (PA), lysophosphatidylethanolamine (LysoPE), and GlcCers, as 121 well as decreases in glycosylinositolphosphoceramides (GIPCs). Transport assays using ALA5 122 expressed in yeast provided evidence that cluster-3 ALAs can transport both glycerolipids (PC > 123 PE > PS) and a phosphate-containing sphingolipid SM. Together, these results support a model 124 whereby ALA4/5 function as lipid flippases that contribute to lipid homeostasis, cellular 125 expansion, and plant growth. 126 127 Results: 128 In A. thaliana, transcript expression profiles of genes encoding cluster-3 ALAs (ALA4, -5,- 6, and 129 -7) generated from public databases (Winter et al., 2007; Huang et al., 2017) indicate that ALA4 130 and ALA5 are expressed primarily in vegetative structures, whereas ALA6 and ALA7 are 131 expressed primarily in pollen (Fig. 1a-b). A double KO of ALA6 and ALA7 was previously shown 132 to result in pollen tubes that were short, slow growing, and hypersensitive to a heat stress 133 (McDowell et al., 2015). To investigate the functions of ALA4 and ALA5 in vegetative tissues, 134 plant lines harboring T-DNA disruptions in these genes were obtained from publicly available 135 mutant collections (Fig. 1c) (Sessions et al., 2002; Alonso et al., 2003; Woody et al., 2007). 136 137 A double genetic disruption of ALA4 and ALA5 results in dwarfism. 138 Two independent sets of ala4/5 mutants, namely ala4-2/5-3 and ala4-3/5-1, were generated as 139 homozygous double disruptions (Fig. 1c). The T-DNA insertions for ala4-3, ala5-1, and ala5-3 140 were located within exon-coding sequences and are predicted to disrupt the expression of full-141 length proteins (Fig. 1d), with any truncated protein products lacking essential features of a 142 typical ATPase (e.g., active site, transmembrane (TM) domains). By contrast, the ala4-2 143 insertion in the second set of alleles (ala4-2/5-3) was located within an intron, which has the 144 potential to be spliced out to allow production of full-length proteins. A qualitative reverse 145 transcription-PCR (RT-PCR) analysis indicated that the ala4-3/5-1 allele set provides a 146 complete KO for both ala4-3 and ala5-1 (Fig. 2g). However, the ala4-2/5-3 set represents a 147 mixed combination of a KO for ala5-3 but only a knockdown (KD) for ala4-2. The ala4-2/5-3 set 148 is hereafter referred to as ala4/5 KD, whereas ala4/5 KO refers to ala4-3/5-1. 149 150 Plants with either ala4/5-KD or -KO allele sets showed a similar dwarf phenotype. Compared to 151 wild-type, the rosette diameters were 4-fold and 6.5-fold smaller for KD and KO plants (p < 1E-152 11), respectively (Fig. 2a-b). KO mutants were also 1.6-fold smaller than KD mutants (p < 1E-153



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04). Primary root lengths were shorter, ranging from 3.1-fold (KD) to 4.3-fold (KO) less than that 154 of wild-type after 7 days (Fig. 2c). Both ala4/5 mutants also exhibited hypocotyls that were 2-fold 155 shorter and 1.5-fold thicker than wild-type controls after 5 days of growth (Fig. 2d-f). 156 Additionally, the bolt length of mature ala4/5 plants was reduced 2.3-fold (KD) to 4.5-fold (KO) 157 relative to wild-type (Fig. S1). Importantly, neither of the ala4/5 mutants displayed reductions in 158 total chlorophyll content, suggesting that the plants were otherwise healthy (Fig. S2). 159 160 To confirm that the T-DNA disruptions were the cause of dwarfism in ala4/5 mutants, 161 transgenes encoding either ALA4 or ALA5 under the control of a Cauliflower mosaic virus-35S 162 promoter (35S promoter) were expressed in the mutants, which restored normal rosette sizes 163 (Fig. 2a-b). Thus, two independent sets of gene disruptions and transgene-rescue experiments 164 establish that a complete (or even partial) loss of ALA4/5 function results in severe plant 165 dwarfing. 166 167 ala4/5 mutants display reductions in cell size. 168 To determine if the dwarfism of ala4/5 plants was correlated with smaller cell size, cell borders 169 were stained with propidium iodide. Images showed ala4/5 mutants had significant reductions in 170 leaf pavement cell area, ranging from 1.5-fold (KD) to 2.7-fold (KO) less than wild-type (Fig. 3a-171 d). Hypocotyl cells from ala4/5 mutants grown in the dark were 1.7-fold shorter for both allele 172 sets, and 1.5-fold (KD) to 2-fold (KO) wider relative to wild-type (Fig. 3f-j). Guard cells were 173 reduced in size for both mutants (p < 1E-05) (Fig. 3e), though this difference was determined to 174 be less than 10%. 175 176 Trichomes and root hairs were also examined. Root hairs were clearly visible, but were shorter 177 by 2.5-fold (KD) to 2.6-fold (KO) compared to wild-type (Fig. S3a-d). Trichomes were nearly 178 absent in the mutants, and when detected showed either globular deformities or short pin-like 179 structures (Fig. S3e-g). The reduced cell sizes indicate that ALA4/5 flippase activity is important 180 for cellular expansion of multiple cell types in vegetative tissues, especially those highly 181 dependent on anisotropic growth. 182 183 Ectopic expression of ALA6 and ALA4 rescues the phenotypes of ala4/5 and ala6/7 184 mutants, respectively. 185 To determine if there was any biochemical redundancy between cluster-3 ALAs expressed in 186 vegetative tissues (ALA4/5) and pollen (ALA6/7), we tested the ability of ALA6 and ALA4 187 transgenic expression to rescue ala4/5 and ala6/7 mutant phenotypes, respectively. ALA6 188 expression driven by a promoter derived from ALA4 provided a partial reversal of the ala4/5 189 dwarf phenotype in all five independent transgenic lines evaluated (Fig. 4). 190 191 Conversely, ALA4 was transgenically expressed in ala6/7 mutants using a promoter from a 192 gene encoding the autoinhibited Ca2+-ATPase 9 (ACA9) that drives expression primarily in 193 pollen (Fig. 5). The ALA4 transgene was able to confer a full restoration of seed set in all 194 independent transgenic lines tested (Fig. 5a-b). Additionally, reciprocal crosses were used to 195 evaluate pollen transmission efficiencies (Fig. 5c) using the same three transgenic lines shown 196 in Figure 5b. To quantify differences in transmission efficiencies between mutant pollen with or 197 without an ACA9p::ALA4 rescue construct, F1 seedlings were scored for a hygromycin-198 resistance selectable marker associated with the transgene construct. A transmission efficiency 199 ratio (TEr) was calculated as the ratio of resistant to sensitive F1 offspring, with a TEr = 1 200 expected for normal Mendelian transmission in an outcross. For transmission of the rescue-201 transgene through the female gametes, none of the plants tested showed evidence of a 202 significant difference from the expected TEr of 1, which confirmed that each transgenic plant 203 was heterozygous for a single transgene insertion. By contrast, transmission of the transgene 204



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through pollen showed TEr values that were ~88-fold higher. This demonstrated that the ala6/7 205 mutant pollen harboring ALA4 transgenes were rescued and able to easily outcompete mutant 206 pollen without a transgene rescue construct. Thus, both ectopic expression studies indicate that 207 ALA6 and ALA4 are at least partially interchangeable when the corresponding transgene for 208 each is expressed ectopically in vegetative tissues or pollen, respectively. 209 210 ALA4-GFP fusions are localized to both the PM and endomembrane 211 To determine the subcellular localization of ALA4/5, confocal imaging was used to evaluate 212 vegetative cell types of ala4/5 mutants rescued by 35S promoter-driven expression of ALA4-213 GFP transgenes (Fig. 6). Comparisons were made to the following controls: cytosolic (GFP), 214 PM (ACA8-GFP), endoplasmic reticulum (ER) (ACA2-GFP), and chloroplast autofluorescence. 215 In guard cells, ALA4-GFP showed accumulation at both the PM and internal endomembrane 216 structures, including the ER that wraps around the nucleus. 217 218 Similar experiments were performed in pollen that compared the localization of an ACA9p-219 driven ALA4-GFP transgene. Comparisons were made to the following controls: cytosolic (YFP), 220 PM (ACA9-YFP), ER (ACA2-GFP), and ALA6-GFP controls (Fig. 7). In support of the vegetative 221 localization results, ALA4-GFP accumulated at both PM and endomembrane structures, which 222 was similar to the previously characterized ALA6-GFP control. 223 224 ala4/5 plants exhibit altered membrane lipid composition 225 To evaluate whether the loss of ALA4/5 might cause differences in the relative abundance of 226 specific cellular lipids, a lipidomic survey of glycerolipids was performed on total lipid extracts 227 taken from rosette tissues of 14-day-old ala4/5 seedlings (Fig. 8a-b, for profiles as percentage 228 of total glycerolipid, and Fig. S4 for data normalized to dry weight of tissue). The profiling 229 included quantification of the following “major” lipid components that comprise most of the 230 membrane content of rosette tissue: digalactosyldiacylglycerol (DGDG), 231 monogalactosyldiacylglycerol (MGDG), PG, PC, PE, and phosphatidylinositol (PI). The following 232 “minor” membrane components were also analyzed: PS, PA, lysophosphatidylglycerol 233 (LysoPG), LysoPC, and LysoPE. All glycerolipids monitored are detailed in Table S1. The ala4/5 234 mutants displayed PA concentrations that were increased 3.4-fold (KD) to 4.2-fold (KO), and 235 LysoPE levels that were increased 1.6-fold (KD) to 1.4-fold (KO) (Fig. 8). Whereas other 236 changes were detected when values were normalized to tissue dry weight, these changes were 237 attributable to a 1.4-fold (KD) and 1.5-fold (KO) reduction in total glycerolipid content (Fig. S4), 238 and were not observed when values were normalized to percent total glycerolipid. Importantly, 239 increases in PA were detected for both mutants regardless of the normalization parameter used. 240 241 The sphingolipid content of ala4/5 KO and KD rosettes was also evaluated to determine if the 242 abundances of these structurally distinct lipids were perturbed. The sphingolipidomic study 243 included quantification of long chain bases (LCBs), ceramides (Cers), hydroxyceramides 244 (hCers), GlcCers, and GIPCs. All sphingolipids monitored are detailed in Table S2. Both ala4/5 245 mutants displayed 1.4-fold increased GlcCers and 1.3-fold reduced GIPCs (Fig. 8c). GlcCer 246 increases and GIPC decreases were broadly distributed across sphingolipids containing C16–247 C26 fatty acid moieties (Fig. S5), and were not specific to any single GlcCer or GIPC species. In 248 contrast to GlcCers and GIPCs, there were no significant changes in LCBs, Cers, or hCers (Fig. 249 8c). Together, these results provide evidence that a deficiency in ALA4/5 flippase activity 250 impacts the relative abundance of both glycerolipid and sphingolipid membrane contents. 251 252 ALA4/5 are flippases that transport PC, SM, PE, and PS 253 To test the lipid transport capabilities associated with cluster-3 ALAs, ALA5 was expressed in 254 yeast either alone or together with three different ALIS β-subunits. The yeast host was a 255



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Saccharomyces cerevisiae mutant with negligible background flippase activity due to a triple 256 mutation of endogenous flippases (drs2Δdnf1,2Δ). Yeast transformants were incubated with 7-257 nitrobenz-2-oxa-1,3-diazole (NBD)-labeled lipid analogues of PG, PS, PC, PE, LysoPC, and SM 258 (Fig. 9a). Subsequent flow cytometry analyses of these yeast cells revealed that expression of 259 ALA5/ALIS complexes, but not ALA5 alone, increased the uptake of exogenous PC, PE, and 260 SM. By contrast, no increase in uptake was detected for PG, PS, or LysoPC. 261 262 To evaluate whether the perturbations in GlcCer content observed in ala4/5 mutants might 263 occur from a direct loss of GlcCer transport, an additional uptake assay was performed to 264 determine if ALA5 could transport NBD-labeled Cers or glycosphingolipids such as GlcCers, 265 lactosylCers (LacCers), or galactosylCers (GalCers) (Fig. 9b). A parallel analysis of GIPCs was 266 not performed because NBD-labeled GIPCs are not currently commercially available. 267 Comparisons were made to a positive control of yeast expressing Dnf1p, which was recently 268 described as showing GlcCer transport activity (Roland et al., 2019). Whereas the ALA5/ALIS-269 expressing lines failed to show any detectable transport of Cers, GlcCers, LacCers, or GalCers, 270 the Dnf1p control facilitated the uptake of GlcCers and GalCers (but not Cers or LacCers). 271 272 As a complementary approach to uptake assays, growth assays were employed to test survival 273 of the previously mentioned yeast lines in the presence of drugs that are toxic when they bind 274 specific surface exposed lipids (Fig. 9c). The flippase deficiencies of drs2Δdnf1,2Δ yeast cells 275 results in PS and PE accumulating on the outer leaflet of the PM, a trait that renders the cells 276 sensitive to cytotoxic drugs papuamide A and duramycin that respectively bind to PS and PE. 277 Only lines that expressed both ALA5 and an ALIS were able to survive high concentrations of 278 papuamide A or duramycin, suggesting that ALA5 was capable of flipping both PS and PE from 279 the exoplasmic to cytosolic membrane leaflet. It is noteworthy that despite the lack of NBD-PS 280 uptake from exogenous sources (Fig. 9a), the ability to convey resistance to papuamide A 281 provides evidence that ALA5 can mediate the flipping of PS already present in a membrane. 282 283 Additional yeast growth experiments were performed with miltefosine, an analogue of LysoPC 284 that is toxic when transported into a cell. In accordance to the previous uptake results (Fig. 9a), 285 none of the ALA5/ALIS-expressing lines showed sensitivity to high concentrations of miltefosine, 286 providing additional evidence that ALA5 is unable to transport LysoPC or its analogues. 287 288 Discussion: 289 ALA4 and ALA5 are important for cellular expansion and vegetative growth 290 In this study, we present genetic evidence that a double KO of ALA4 and ALA5 results in 291 dwarfism, characterized by reduced growth in roots, hypocotyls, bolts, and rosettes (Fig. 2, S1). 292 This dwarfism was observed in two independent double-mutant lines, and was reversed through 293 the expression of ALA4 or ALA5 transgenes driven by a 35S promoter (Fig. 2a-b). Despite the 294 reduced growth, ala4/5 plants were otherwise healthy with normal chlorophyll levels and no 295 obvious chlorotic lesions indicative of cell death or poor health (Fig. 2a, and Fig. S2). To date, 296 our observation of a 6.5-fold reduction in ala4/5 rosette diameter (Fig. 2b) is the most severe 297 growth defect associated with a plant flippase deficiency, in comparison to a ~1.2-fold reduction 298 for an ala3 KO (McDowell et al., 2013), a ~2.5-fold reduction for ALA1 RNAi lines grown at 8–299 12°C (Gomès et al., 2000), and no significant changes reported for an ala2 KO (Zhu et al., 300 2017), an ala6/7 KO (McDowell et al., 2015), or an ala10 KO (Poulsen et al., 2015; Botella et al., 301 2016). 302 303 The loss of ALA4/5 also resulted in cell size reductions across multiple cell types, including leaf 304 epidermal cells which showed a 2.7-fold reduction in surface area (Fig. 3). Whereas this 305 indicates a deficiency in cell expansion, it does not by itself explain the more severe 6.5-fold 306



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reduction detected in overall rosette diameter. The disparity between organ and cell size 307 reductions suggests that ala4/5 plants might also have a deficiency in generating or responding 308 to growth signals, such as growth hormones. 309 310 ALA4/5 and ALA6/7 are functionally interchangeable flippases 311 ALA4/5/6/7 all belong to the cluster-3 ALA subfamily (Fig. 1a) that is conserved between dicots 312 and monocots (Baxter et al., 2003). Examination of expression profiles in public databases 313 indicated that the genes encoding ALA4/5 are expressed primarily in vegetative tissues and 314 ALA6/7 in pollen (Fig. 1b). Despite their expression in different cell types, ectopic expression of 315 a “pollen” ALA6 transgene in vegetative tissues was capable of partially rescuing growth defects 316 associated with ala4/5 mutants (Fig. 4). Additionally, a “vegetative” ALA4 ectopically expressed 317 in pollen fully rescued the male-fertility defects associated with ala6/7 mutants (Fig. 5). ALA4-318 GFP and ALA6-GFP also show similar endomembrane and PM localization patterns (Fig. 7). 319 These results support a model whereby cluster-3 ALAs are at least partially redundant in 320 biochemical and cellular functions. In support of this contention, the Oryza sativa genome 321 contains only one cluster-3 ALA (Baxter et al., 2003), OsALA7, which presumably provides the 322 equivalent ALA4/5/6/7-dependent functions to all tissue types. 323 324 Cluster-3 ALAs are flippases capable of transporting PC, SM, PE, and PS 325 Using a fluorescent lipid uptake assay in yeast, expression of ALA5 (with an ALIS β-subunit) 326 was shown to mediate the transport of NBD-labeled PC > SM > PE, but not PS, PG, LysoPC, 327 Cers, GlcCers, LacCers, or GalCers (Fig. 9a-b). Whereas SM is not made in plants, the ability to 328 transport SM indicates a potential for ALA4/5 to transport at least one type of sphingolipid. It is 329 also noteworthy that ALA5 transported both NBD-PC and NBD-SM, which share an identical 330 choline head group. This is consistent with reports that ALAs contain central cavities that 331 accommodate the passage of lipids with specific headgroups (Jensen et al., 2017). However, no 332 transport was detected for choline-containing LysoPC (i.e. PC lacking an sn-2 acyl group), 333 indicating that a common choline head group is not the only feature of importance to ALA4/5 334 substrate specificity. 335 336 Whereas no uptake of exogenous NBD-PS was detected, a potentially more sensitive transport 337 assay suggested that ALA5 can still flip endogenous PS already present within the membrane. 338 In this alternative assay, ALA5/ALIS expression restored growth of a yeast flippase mutant in 339 the presence of the toxin papuamide A (Fig. 9c), a depsipeptide thought to have a membrane 340 pore-forming property that is potentiated when the toxin binds to surface-exposed PS (Parsons 341 et al., 2006; Andjelic et al., 2008). Thus, ALA5 flippase activity was still able to remove 342 endogenous PS from the outer leaflet of the PM and alleviate the toxicity of papuamide A, 343 despite the lack of detectable uptake activity for an externally supplied NBD-labeled PS 344 substrate. This suggests that the fluorescent NBD-lipid uptake assays might lack the sensitivity 345 to detect very low levels of flippase activity, and leaves open the possibility that ALA4/5 provide 346 functionally relevant levels of transport for some lipids that nevertheless fail to show a strong 347 uptake in yeast assays. 348 349 Whereas ALA5 transport specificity (PC > SM > PE > PS) (Fig. 9) was distinct from those 350 reported for flippases from other ALA clusters (Gomès et al., 2000; López-Marqués et al., 2010; 351 Poulsen et al., 2015; Jensen et al., 2017), the ability to flip PC and SM makes ALA5 activity 352 most similar to that of ALA10 from cluster 2 (SM > PC > PE > PS > PG > LysoPC > GlcCers). 353 Cluster-2 ALAs (ALA8, -9, -10, -11, and -12) are also the most closely related to cluster-3 ALAs 354 in terms of overall protein sequence similarities, and the two groups might have conserved 355 structural features related to substrate specificity. Nevertheless, the dwarfism observed in ala4/5 356 plants indicates that cluster-2 ALAs do not share functional redundancy with ALA4/5, despite 357



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their overlap in lipid transport preferences and similar levels of expression in all major vegetative 358 tissues (Fig. S6). Further research will be needed to identify and understand the unique 359 biochemical or functional features associated with these two closely related ALA groups. 360 361 PA and LysoPE accumulate in ala4/5 mutants 362 Whereas glycerolipid profiling revealed that ala4/5 rosettes display increased levels of two low-363 abundance phospholipids, specifically PA (3.4–4-fold) and LysoPE (1.4–1.6-fold) (Fig. 8b), it is 364 not yet clear how these changes are related to the loss of ALA4/5 activity. PA is a diacyl lipid 365 with a simple phosphate headgroup that is known to play important roles in signaling pathways 366 which can impact plant growth (Hong et al., 2009). Increases in PA can result from multiple 367 causes, including increased phospholipase D (PLD) activity removing headgroups from more 368 complex phospholipids (PC, PE, or PI), increased phosphorylation of diacylglycerol by 369 diacylglycerol kinases (DGKs), or decreases in the conversion of PA into other glycerolipids 370 such as PC, PE, PI, PG, or PS (Athenstaedt and Daum, 1999; Testerink and Munnik, 2011). It is 371 possible that the loss of ALA4/5 could directly or indirectly change one or more of these 372 metabolic pathways. It is also noteworthy that the loss of ALA6/7 (from cluster 3) results in 373 pollen grains with a similar ~2-fold increase in PA (McDowell et al., 2015), indicating a 374 consistent link between the ALA4/5/6/7 clade of flippases and the regulation of PA homeostasis. 375 376 The small increase in LysoPE, a monoacyl PE molecule, can also be from multiple causes, 377 including increased phospholipase A (PLA) activity removing a fatty acid from the sn-2 position 378 of the PE glycerol backbone (Bahn et al., 2003), or a decrease in the conversion of LysoPE to 379 PE via LysoPE acyltransferases (LPEATs) (Jasieniecka-Gazarkiewicz et al., 2017). LysoPE 380 also has connections to growth signaling, as mutants deficient in LPEATs display LysoPE 381 accumulation and growth inhibition. Interestingly, LysoPE is a known inhibitor of PLD-dependent 382 PA production in plants (Ryu et al., 1997), which raises the possibility that an increase in 383 LysoPE might be part of a feed-back mechanism in ala4/5 mutants to try and renormalize PA 384 concentrations. 385 386 Glycosphingolipid metabolism is perturbed in ala4/5 mutants 387 In addition to glycerolipid perturbations, sphingolipid profiling revealed that ala4/5 mutants had 388 relative increases in GlcCers (1.4-fold) which were offset by decreases in GIPCs (1.3-fold) (Fig. 389 8c). GlcCers are sphingolipids with a simple glucose headgroup, whereas GIPCs have a 390 headgroup containing phosphate, inositol, and a variable chain of carbohydrates. Importantly, 391 both glycosphingolipids are critical for plant growth and development (Msanne et al., 2015; 392 Tartaglio et al., 2017; Fang et al., 2016; Wang et al., 2008; Mortimer et al., 2013). As with PA 393 and LysoPE, it is not yet clear if changes in GlcCers or GIPCs result from changes in their 394 biosynthesis or degradation. When considering an underlying cause, it is difficult to posit 395 specific increases in the biosynthesis of GlcCers, as published examples show that increased 396 production of GlcCers are accompanied by parallel increases in accumulation of GIPCs 397 (Luttgeharm et al., 2015), which contrasts with the observed GIPC decreases in ala4/5 profiles 398 (Fig. 8c). Reductions in GIPC biosynthesis also seems unlikely, as mutant lines defective in 399 GIPC production did not show any accumulation of GlcCers (Tartaglio et al., 2017), which is 400 also in contrast to what was observed in ala4/5 profiles. A simpler alternative is that the loss of 401 ALA4/5 specifically decreases catabolism of GlcCers, or increases the relative catabolism of 402 GIPCs. 403 404 There are multiple direct and indirect models to explain the mechanisms through which ala4/5-405 dependent changes in catabolism could impact GIPC homeostasis. For example, it is possible 406 that ALA4/5 transport GIPCs across a membrane and away from degrading enzymes. An 407



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alternative indirect model is that ALA4/5 are involved in the bulk flow of GIPCs through a plant 408 cell, perhaps by assisting in forming vesicles for removal away from a site of degradation. 409 Regardless of the mechanism, the loss of ALA4/5 would then increase GIPCs exposure to their 410 degrading enzymes, increase their rates of catabolism, and ultimately decrease their 411 abundances. 412 413 The possibility for a specific reduction in GlcCer catabolism is especially intriguing, as lipid 414 flippases have recently been implicated in GlcCer catabolism in mammals and fungi (Roland et 415 al., 2019). The proposed model involves the lipid flippases Dnf1p/Dnf2p (Yeast) and ATP10D 416 (Human) directly transporting GlcCers to cytosolic facing surfaces, thus exposing these lipids to 417 degrading enzymes. As to whether ALA4/5 might play a similar direct role in GlcCer catabolism 418 in plants, it is noteworthy that ALA5 failed to mediate the transport of NBD-labeled GlcCers in a 419 fluorescent lipid uptake assay in yeast (Fig. 9b). However, we cannot rule out that ALA4/5 have 420 the ability to transport low levels of endogenous GlcCers across a membrane bilayer, similar to 421 the ability of ALA5 to remove endogenous PS from the PM surface and provide resistance to 422 papuamide A without providing detectable uptake of externally supplied NBD-PS (Fig. 9a,c). 423 Similar to the above discussion concerning GIPCs, an alternative indirect model is that ALA4/5 424 are involved in the bulk flow of GlcCers through a plant cell, perhaps by assisting in forming 425 vesicles for delivery to a site of degradation. After delivery, the GlcCers are possibly flipped for 426 degradation by a non-ALA4/5 flippase, such as ALA1, which shows the highest levels of 427 sequence similarity to GlcCer-transporting Dnf1p and ATP10D (Poulsen et al., 2015), or ALA10, 428 which was previously shown to flip at least small amounts of GlcCers (Jensen et al., 2017). It is 429 also possible that ALAs might form hetero-multimers in plants, with ALA4/5 potentially 430 interacting with other ALAs or plant-specific proteins to form a more robust GlcCer flipping 431 complex. 432 433 Modeling the potential mechanisms behind dwarfism in ala4/5 mutants 434 Lipid flippases are involved in multiple cellular processes, which include maintaining the 435 asymmetric composition of membrane leaflets, creating membrane curvature for vesicular 436 trafficking, and facilitating lipid metabolism (Seigneuret and Devaux, 1984; Gall et al., 2002; 437 Pomorski et al., 2003; Roland et al., 2019). Thus, dwarfism in ala4/5 mutants could arise from a 438 deficiency in one or more of these important processes. 439 440 One possibility is that the accumulation of PA or LysoPE acts as a signal that inhibits cell 441 expansion and plant growth. For example, PA is a dynamic secondary messenger that plays 442 critical signaling roles in growth promotion (Hong et al., 2009). PA is also known to recruit and 443 activate specific proteins to the surfaces of membranes, some of which can result in growth 444 inhibition (Yao and Xue, 2018). Conversely, both PA and LysoPE have been shown to 445 accumulate after the application of various biotic and abiotic stresses (Welti et al., 2002; 446 Testerink and Munnik, 2005; Vu et al., 2015). Thus, PA or LysoPE accumulation in ala4/5 447 mutants could potentially disrupt growth signaling by instigating stress responses that inhibit 448 growth, or by PA accumulation disrupting the normal recruitment, activation, and accumulation 449 of important growth signaling proteins. However, whereas mutant lines that accumulate LysoPE 450 have been shown to have growth reductions (Jasieniecka-Gazarkiewicz et al., 2017), these 451 reductions were not as severe as those seen in ala4/5 mutants. Additionally, there are examples 452 of plant lines that accumulate PA and display normal growth (Nakamura et al., 2009; Du et al., 453 2013). Thus, increases in PA and LysoPE alone cannot fully explain the growth defects of 454 ala4/5 mutants. 455 456



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In contrast to the rather minor growth deficiencies linked to PA or LysoPE perturbations, there 457 are several examples of plant dwarfism correlated with sphingolipid accumulation (i.e. 458 sphingolipidosis) (Chen et al., 2008; Markham et al., 2011; Luttgeharm et al., 2015). Of the 459 three previously characterized sphingolipidosis dwarfs, all displayed increases in Cers, hCers, 460 GlcCers, and GIPCs containing C16 fatty acid moieties. Evidence suggests that these lines 461 exhibited dwarfing due to the mislocalization of auxin transporters (Markham et al., 2011) and/or 462 the accumulation of growth-inhibiting salicylic acid (Luttgeharm et al., 2015). As to whether 463 ala4/5 mutants are also inhibited by a sphingolipid accumulation, it is worth noting that there are 464 multiple features that make the sphingolipid profile of ala4/5 mutants distinct. First, the 465 sphingolipid increases in ala4/5 plants were limited to GlcCers, and not other sphingolipid 466 species (Fig. 8c). Additionally, the GlcCer increases in ala4/5 plants were more broadly 467 distributed across fatty acid moieties from C16 to C26 (Fig. S5), and were not restricted to C16 468 as reported for other mutants. Thus, it is not clear if ala4/5 mutants share a common underlying 469 mechanism for a sphingolipidosis-induced growth defect, or if the growth defects are unrelated. 470 Importantly, reductions in GIPC content are also associated with dwarfism (Fang et al., 2016; 471 Tartaglio et al., 2017), and we cannot rule out that both GlcCer increases and GIPC reductions 472 contribute independently to ala4/5 growth impairments. 473 474 Regardless of whether ala4/5 mutant dwarfism is caused by an imbalance in the concentration 475 of one or more specific lipid, or a pleiotropic defect related to a general impairment to vesicular 476 trafficking, this study demonstrates that the ALA4/5 lipid flippases are of critical importance for 477 cellular expansion and plant growth. 478 479 480 Materials and Methods: 481 Plant Materials 482 ALA4 (At1g17500) and ALA5 (At1g72700) mutant alleles were introgressed to generate two 483 independent double T-DNA insertion mutants: ala4-2 (SALK_129551)/5-3 (SALK_049232) 484 (seed stock ss948) and ala4-3 (WiscDsLox_435D3)/5-1 (SAIL_613_F05) (ss1270) (Sessions et 485 al., 2002; Alonso et al., 2003; Woody et al., 2007). The ala6-1 (SALK_150173)/7-2 486 (SALK_125598) double mutant (ss1351) was generated as previously described (McDowell et 487 al., 2015). Mutant lines were obtained from the Arabidopsis Biological Resource Facility at Ohio 488 State University and were backcrossed to the ecotype Columbia. Double-mutant lines were 489 PCR genotyped to ensure homozygosity of both alleles using primers listed in Table S3. 490 491 Transgenic events were produced by a floral dip method (Clough and Bent, 1998). Transgenic 492 plants were selected for hygromycin resistance (25 mg/L) or kanamycin resistance (50 mg/L) 493 provided by plant expression vectors used in this study. Rescues of ala4-3/5-1 were generated 494 by transforming with constructs containing either 35S::ALA4 (Rescue 1, ss2145, transformed 495 with plasmid stock ps1712), 35S::ALA5 (Rescue 2, ss2146, ps1713), or ALA4p::ALA6 496 (Rescue1-2, ss2504-5, ps2809). Rescues of ala6-1/7-2 contained ACA9p::ALA4 (TG1-3: 497 ss2149-51, all transformed with ps1967). Imaging was performed on plant lines transformed 498 with the following transgenes: 35S::GFP (ss1811, ps346), 35S::ACA8-GFP (ss248, ps396), 499 35S::ACA2-GFP (ss2216, ps660), 35S::ALA4-GFP (ss2145, ps1712), ACA9p::YFP (ss2228, 500 ps532), ACA9p::ACA9-YFP (ss2229, ps580), ACA9p::ACA2-GFP (ss2254, ps585), 501 ACA9p::ALA4-GFP (ss2161, ps2096), and ACA9p::ALA6-GFP (ss2399, ps1958). 502 503 Growth Conditions 504 Seeds were spread on plates containing 0.5x Murashige and Skoog medium (Phytotechnology 505 laboratories) (pH 5.7) with 1% (w/v) agar and 0.05% (w/v) MES. Plates were stratified for 72 506 hours at 4°C and then transferred to room temperature (~23°C) under constant light conditions. 507



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Root growth measurements were made daily for 7 days. After 10 days of growth, seedlings 508 were transplanted into soil (Sunshine SMB-238, SunGro Horticulture) supplemented with 509 Marathon pesticide and Cleary Turf and Ornamental Systemic Fungicide according to 510 manufacturer’s directed use. Plants were grown to maturity in growth chambers (Percival 511 Scientific) under a long-day photoperiod (16 hours light at 22°C/8 hours dark at 22°C, 70% 512 humidity, and ~125 μmol m-2 s-1 light). Plate grown seedlings used in root hair assays were 513 grown as described above except on sterile cellulose acetate sheets (Research Products 514 International Corp.) and imaged after 7 days of growth. Seedlings used in dark-grown hypocotyl 515 experiments were imaged after 6 days of growth at room temperature in the absence of light. 516 Images were analyzed using Fiji (Schindelin et al., 2012) for size measurements. Student t-517 test’s were used to determine statistical significance for all assays unless specified otherwise. 518 F1 progeny from male and female outcrosses were scored for transgenes by germinating seeds 519 on growth media supplemented with 25 mg/L hygromycin B (Gold Biotechnology). Pearson's 520 chi-squared test (χ2) was used to determine statistical significance for transmission assays. 521 Selection for kanamycin-resistant seedlings was performed on growth media supplemented with 522 50 mg/L kanamycin. 523 524 Chlorophyll measurements 525 The rosettes of 10-day-old seedlings were dissected from root tissues and incubated in DMSO 526 at 65°C for 1 hour to extract total chlorophyll, as previously described (Hiscox and Israelstam, 527 1979). The absorbance at 645 nm and 663 nm was measured on an LKB Ultrospec II 528 spectrophotometer and the chlorophyll content was subsequently calculated using Arnon’s 529 equations and normalized to fresh weight. 530 531 Phylogenetic tree and expression profiles 532 Protein sequence similarity comparisons were generated using the online tool Arabidopsis Heat 533 Tree Viewer (http://arabidopsis-heat-tree.org) which utilizes clustalW sequence alignment 534 software (Thompson et al., 1994). Phylogenetic bootstrap values were presented as thick lines 535 for values greater than 0.98. Gene expression data for ALA4–12 was taken from Arabidopsis 536 eFP browser (Winter et al., 2007) for rosette, root, hypocotyl, and mature pollen. Root hair 537 expression data are from Huang et al., 2017. 538 539 Plasmid Construction 540 All primer sequences and plant expression plasmid sequences are available in Table S3 and 541 Supplemental File S1, respectively. For expression in plants, ALA4, ALA5, and ALA6 genomic 542 coding regions were PCR amplified from wild-type A. thaliana genomic DNA using Phusion 543 High-Fidelity DNA Polymerase (New England Biolabs) and moved into plant transformation 544 vectors derived from pGreen II (Hellens et al., 2000) that contained hygromycin-resistance 545 selectable markers, with the exception of 35S::ACA8-GFP (ps396) which contained a 546 kanamycin-resistance selectable marker. PCR-derived regions were sequence verified. 547 548 For expression in yeast, the full-length coding sequence of ALA5 was PCR amplified from a 549 wild-type A. thaliana cDNA library and moved into plasmid pENTR™/D-TOPO® using the 550 pENTR™/D-TOPO® Cloning Kit (Invitrogen™, Life Technologies), rendering plasmid pMP2035. 551 The ALA5 cDNA was subsequently cloned into a yeast expression plasmid by homologous 552 recombination, achieved by co-transforming yeast with EcoRI- and SalI-digested pMP4075 553 (López-Marqués et al., 2012) and ALA5 PCR fragments flanked by 25-bp region homologous to 554 the opened vector. Yeast cells were lysed with acid-washed 0.5-mm glass beads and the 555 recombinant plasmid product (pMP4842) was isolated using the GenElute™ Plasmid Miniprep 556 Kit (Sigma-Aldrich). The plasmid was bulked in E. coli and sequence verified. The final construct 557 contained an untagged version of ALA5 under the control of galactose-inducible GAL1 promoter 558



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linked to a HIS3 selection marker. Cloning of ALIS into yeast plasmids containing a URA3 559 marker was described previously (Poulsen et al., 2008). 560 561 Qualitative RT-PCR 562 Total RNA was extracted from 9-day-old seedlings using an RNeasy Plant Mini Kit (QIAGEN) 563 followed by reverse transcription to cDNA using an iScript cDNA Synthesis Kit (Bio-Rad). 564 Amplification for qualitative RT-PCR was performed using ExTaq Polymerase (Takara) and 565 primer combinations listed in Table S3. PCR products were separated on a 1.5% (w/v) agarose 566 gel and stained with ethidium bromide prior to imaging. Gels were analyzed using the Fiji 567 software package (Schindelin et al., 2012) for pixel density measurements. Mean pixel densities 568 were normalized to actin7 control. 569 570 Fixation and Propidium Iodide staining 571 Intact hypocotyls and leaf sections were fixed using methods previously described (Kalantidis et 572 al., 2000). Fixed tissues were incubated in sterile water with 7.48 μM propidium iodide (Sigma-573 Aldrich) at room temperature overnight prior to imaging. Images analyzed with Fiji (Schindelin et 574 al., 2012) for cell size measurements. 575 576 Confocal and light microscopy 577 Dark-grown hypocotyls, trichomes, and root hairs were imaged using a 4x objective lens on a 578 Leica S6D dissection microscope. For subcellular localization and cell size determination, 579 images were acquired using an Olympus IX81 FV1000 confocal microscope with an Olympus 580 FluoView 1.07.03.00 software package. A 40x objective lens (numerical aperture 1.30) was 581 used for PI-stained leaf epidermal cell and hypocotyl cell images, and a 60x objective lens 582 (numerical aperture 1.42) for guard cell and pollen tube images. An Argon-Ion laser was used 583 for excitation at 488 nm (eGFP and propidium iodide) and 515 nm (eYFP), and a HeNe laser 584 was used for excitation at 543 nm (chloroplasts). Spectral emission windows used for imaging 585 vegetative cell types were 500–545 nm (eGFP), 687–787 nm (chloroplasts), and 597–637 nm 586 (propidium iodide). Spectral emission windows for pollen imaging were 500–600 nm (eGFP) and 587 545–595 nm (eYFP). 588 589 In-vitro Pollen Tube Growth 590 The pollen growth germination media was previously described (Boavida and McCormick, 2007) 591 and contained: 5 mM CaCl2, 0.01% (w/v) H3 BO3, 5 mM KCl, 10% (w/v) sucrose, 1 mM MgSO4, 592 pH 7.8, and 1.5% (w/v) low melting agarose (Nusieve). Pollen from stage 13–14 flowers was 593 applied to 400 μL of solidified germination media on a microscope slide. Slides were incubated 594 at room temperature (~23°C) in a humidity chamber. Pollen tubes were grown for 1–2 hours 595 prior to imaging. 596 597 Glycerolipid profiling 598 Plant tissue was obtained by dissecting 14-day-old rosettes and immediately performing lipid 599 extractions as described previously (McDowell et al., 2015), with the exception of no NaCl wash 600 and four additional 15-minute extractions at 60°C using the lower phase of a solvent consisting 601 of isopropanol/hexane/water (50:20:25, v/v/v) (Markham and Jaworski, 2007). Extracts from five 602 replicates were analyzed by the Kansas Lipidomics Research Center (https://www.k-603 state.edu/lipid/) using a routine polar lipid analysis in which 156 polar lipids were quantified 604 using precursor and neutral loss electrospray ionization tandem mass spectrometry (ESI-605 MS/MS), as previously described (Shiva et al., 2013). Electrospray ionization voltage was set at 606 -17V for LysoPG quantification, and 40V for all other glycerolipids. Compound formula, mass 607 transition, mode, scan function, and ion detected for all profiled glycerolipids are provided in 608 Table S1. 609



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610 Sphingolipid profiling 611 Rosettes were dissected from 14-day-old seedlings grown on an agar surface and flash frozen 612 in liquid nitrogen, followed by freeze drying at -40°C and 133 x 10-3 mBAR for 3 hours in a 613 Freezone 18 Freeze Dry System (Labconco). Sphingolipid extraction was performed as 614 described previously (Markham and Jaworski, 2007). Lyophilized root tissue was extracted in 615 triplicate using 2–5 mg of tissue per sample. The subsequent LC-ESI-MS/MS profiling of total 616 sphingolipid was performed using a Shimadzu Prominence ultra-high performance liquid 617 chromatograph (UHPLC) feeding into a QTRAP4000 mass spectrometer (AB SCIEX). 618 Electrospray ionization voltage was set at 5000 V. Sphingolipids were separated on a Zorbax 619 Eclipse Plus narrow bore RRHT C18 column, 2.1 x 100 mm, 1.8-µm particle size (Agilent) at 620 40°C and a flow rate of 0.2 mL/min using a binary gradient as previously described (Kimberlin et 621 al., 2013). Sphingolipids from three replicates were profiled and quantified using the Multiple 622 Reaction Monitoring (MRM) method described in Markham and Jaworski (2007), with 623 instrument-specific modifications as described by Kimberlin et al. (2013). Compound formula, 624 mass transitions (Q1/Q3), mode, scan function, and ion detected for all profiled sphingolipids 625 are provided in Table S2. Data analysis was performed using Analyst 1.5 and Multiquant 2.1 626 software (AB SCIEX) as previously described (Markham and Jaworski, 2007). 627 628 Yeast strain and culture 629 Saccharomyces cerevisiae mutant strain ZHY709 (MATα his3 leu2 ura3 met15 dnf1Δ dnf2Δ 630 drs2::LEU2) (Hua et al., 2002) was used as a host. For co-expression of ALA5 with ALIS genes, 631 cells were simultaneously transformed by a lithium acetate method (Gietz and Woods, 2002) 632 with two individual plasmids bearing the desired genes and histidine (ALA5) or uracil (ALIS) 633 auxotrophic markers. For growth assays, transformants were grown in Synthetic Complete 634 Galactose (SG) medium (0.7% (w/v) Yeast Nitrogen Base, 2% (w/v) galactose,1.4 g/L yeast 635 synthetic dropout medium lacking histidine and uracil (Sigma-Aldrich)) at 30°C with 150 r.p.m. 636 shaking for 4 hours to induce overexpression. Cultures were diluted with water to 0.1 OD600/mL, 637 and either 5 or 3 µL was spotted onto solid SG (2% (w/v) agar added) or synthetic complete 638 glucose (SD) medium plates (SG with 2% (w/v) glucose instead of galactose). SG solid media 639 containing toxin gradients were prepared as previously described (Liu et al., 2011), except that 640 they were stored at 4°C for 2 days before use to allow for diffusion of the toxins. Gradients 641 contained the following maximum concentrations: 0.3 µg/mL of papuamide A (Flintbox, Lynsey 642 Huxham), 1.6 µM of duramycin (Sigma-Aldrich), or 2.5 µg/mL of miltefosine 643 (hexadecylphosphocholine, Calbiochem). Plates were incubated at 30°C for 4 days prior to 644 imaging. For NBD-lipid uptake assays, transformants were grown as previously described 645 (López-Marqués et al., 2010). 646 647 NBD-lipid uptake assays 648 Fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-lipids were purchased from Avanti Polar 649 Lipids, including palmitoyl-(NBD-hexanoyl)-PS (NBD-PS), palmitoyl-(NBD-hexanoyl)-PE (NBD-650 PE), palmitoyl-(NBD-hexanoyl)-PC (NBD-PC), palmitoyl-(NBD-hexanoyl)-phosphatidylglycerol 651 (NBD-PG), NBD-dodecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (NBD-LysoPC), N-652 hexanoyl-NBD-sphingosine-1-phosphocholine (NBD-SM), NBD-hexanoyl-D-erythro-sphingosine 653 (NBD-Cer), NBD-hexanoyl-D-glucosyl-ß1-1'-sphingosine (NBD-GlcCer), NBD-hexanoyl-D-654 galactosyl-ß1-1'-sphingosine (NBD-GalCer), and NBD-hexanoyl-D-lactosyl-ß1-1'-sphingosine 655 (NBD-LacCer). All NBD-lipid stocks (4 mM) were prepared in DMSO. Uptake experiments in 656 yeast were performed as described previously (Poulsen et al., 2015). Cells were resuspended 657 to 10 OD600/mL and incubated in selective SG medium supplemented with 32 µM NBD-lipids for 658 30 min at 30°C with periodic mixing. The cells were washed three times in ice-cold selection 659 media that lacked galactose but contained 2% (w/v) sorbitol, 3% (w/v) bovine serum albumin, 660



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and 20 mM NaN3. Flow cytometry was performed on a Becton Dickinson FACS equipped with 661 an argon laser using Cell Quest software. Prior to analysis, 107 cells were labeled with 1 µL of 1 662 mg/mL propidium iodide for staining of non-viable cells. Twenty thousand cells were analyzed. 663 Data were analyzed using Cyflogic (CyFlo, Ltd). Viable yeast cells were selected based on 664 forward/side-scatter gating and propidium iodide exclusion. NBD-fluorescence of living cells was 665 plotted on a histogram and the geometric-mean fluorescence intensity was used for further 666 statistical analysis. Statistical significance was determined by a Tukey’s Honest Significant 667 Difference test. 668 669 Accession numbers: 670 Genes: ALA4 (At1g17500), ALA5 (At1g72700), ALA6 (At1g54280), ALA7 (At3g13900). A. 671 thaliana Mutants: ala4-2 (SALK_129561), ala4-3 (WiscDsLox_435D3), ala5-1 (SAIL_613_F05), 672 ala5-3 (SALK_049232), ala6-1 (SALK_150173), ala7-2 (SALK_125598). 673 674 Supplemental Data: 675 Supplemental Figure S1. Bolt length is reduced in ala4/5 mutants. 676 Supplemental Figure S2. Chlorophyll content is unchanged in ala4/5 mutants. 677 Supplemental Figure S3. Loss of ALA4 and ALA5 results in shortened root hairs and impaired 678 trichome formation. 679 Supplemental Figure S4. Loss of ALA4 and ALA5 results in severe perturbations in 680 glycerolipid content 681 Supplemental Figure S5. Profiling of GlcCers and GIPCs in ala4/5 mutants by fatty acid 682 composition. 683 Supplemental Figure S6. ALA4/5 and ALAs from cluster 2 are expressed broadly in vegetative 684 tissues. 685 Supplemental Table S1. Analytic settings for quantification of profiled glycerolipids. 686 Supplemental Table S2. Analytic settings for quantification of profiled sphingolipids. 687 Supplemental Table S3. PCR primers used in genotyping, cloning, and qualitative RT-PCR. 688 Supplemental File S1. Sequences of plant expression vectors. 689 690 Acknowledgements: 691 This work was supported by grants to JFH from USDA Hatch NEV00384 and NSF IOS 692 1656774, to RL from Villum Fonden project number 13234, to MP from Innovation Fund 693 Denmark project number 8053-00035B, to EBC from NSF MCB 1818297. Microscopy was 694 supported by the National Institute of General Medical Sciences of the National Institutes of 695 Health under grant number P20 GM103554. The glycerolipid analyses described in this work 696 were performed at the Kansas Lipidomics Research Center Analytical Laboratory. Instrument 697 acquisition and glycerolipidomics method development was supported by National Science 698 Foundation (EPS 0236913, MCB 1413036, MCB 0920663, DBI 0521587, DBI1228622), Kansas 699 Technology Enterprise Corporation, K-IDeA Networks of Biomedical Research Excellence 700 (INBRE) of National Institute of Health (P20GM103418), and Kansas State University. The 701 funders had no role in the designing the research, data collection, analysis, or manuscript 702 preparation. 703 704 Figure Legends: 705 Figure 1. Expression patterns of A. thaliana cluster-3 ALAs and genetic organization of 706 ALA4 and ALA5 T-DNA disruptions. (A) Phylogenetic tree of A. thaliana ALA family and other 707 P-type ATPases generated in Arabidopsis Heat Tree Viewer (http://arabidopsis-heat-tree.org), 708 with ALA clusters (Baxter et al., 2003) bracketed by grey rectangles and thicker lines 709 representing bootstrap values of 98 or greater. (B) Database expression profile of ALA4/5/6/7 710 genes in rosette, root, hypocotyl, trichomes, and mature pollen, generated from Arabidopsis 711



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eFP browser (Winter et al., 2007), with root hair data from Huang et al., 2017. Error bars 712 represent ± SD; n=3 expression replicates. ND; not detected. (C) Gene diagram of ALA4 and 713 ALA5 with exons in black, introns in white, and triangles indicating position of T-DNA insertion. 714 Allele name and accession number are detailed above gene schematics. (D) Protein topology 715 representing ALA4/5 with TM domains labeled and represented by black boxes. Predicted 716 truncated protein products are detailed below the protein schematic. 717 718 Figure 2. Loss of ALA4 and ALA5 results in reduced vegetative growth. (A) Representative 719 image of a growth comparison assay and (B) rosette diameter quantification of wild-type Col-0, 720 ala4-2/5-3 KD (ss948), ala4-3/5-1 KO (ss1270), Rescue 1 (35S::ALA4 in KO, ss2145, ps1712), 721 and Rescue 2 (35S::ALA5 in KO, ss2146, ps1713) plants; n=8 growth assays. (C) Root length 722 quantification from light-grown seedlings sampled over 7 days; n=8 seedlings total from four 723 growth assays. (D) Representative images of dark-grown hypocotyls and quantifications of (E) 724 length and (F) width; n=39 hypocotyls total from three growth assays. Arrows indicate root-shoot 725 transition. Scale bars: 1 cm. Different letters indicate statistically significant difference (p < 0.05; 726 Student’s t-test). (G) Expression of ALA4 and ALA5 transcripts in wild-type, ala4-2/5-3 KD, and 727 ala4-3/5-1 KO mutants. ND; not detected. Error bars represent ± SE; n=3 expression replicates. 728 729 Figure 3. Loss of ALA4 and ALA5 results in impaired cellular expansion in vegetative 730 tissues. Confocal images of propidium iodide-stained leaf epidermal cells (A-C) and dark-grown 731 hypocotyl cells (F-H) for wild-type Col-0, ala4-2/5-3 KD, and ala4-3/5-1 KO, with a single cell 732 shaded grey in each image. Scale bars: 50 μm. Images were analyzed with Fiji (Schindelin et 733 al., 2012) to quantify leaf cell area (D), guard cell length (E), hypocotyl cell length (I), and 734 hypocotyl cell width (J). Different letters indicate statistically significant difference (p < 0.01; 735 Student’s t-test). Error bars represent ± SE; n=20 cells total from 3 plants. 736 737 Figure 4. Ectopic expression of ALA6 in vegetative tissues restores growth in an ala4/5 738 mutant. (A) Representative image of a growth comparison assay and (B) rosette diameter 739 quantification of wild-type Col-0, ala4-3/5-1 KO mutant (ss1270), and Rescue 1–2 740 (ALA4p::ALA6 in KO, ss2504-5, ps2809) plants. Scale bar: 1 cm. Shown are two out of five 741 independent transgenic rescue lines with similar results. Different letters indicate statistically 742 significant difference (p < 1E-06; Student’s t-test). Error bars represent ± SD; n=5 growth 743 assays. 744 745 Figure 5. Ectopic expression of ALA4 in pollen restores seed set and normal pollen 746 transmission in an ala6/7 double KO. (A) De-stained silique images and (B) seeds per silique 747 counts of wild-type Col-0, ala6/7 mutants (ss1351), and ala6/7 lines rescued by a pollen specific 748 ACA9p::ALA4 construct with a hygromycin-resistance selectable marker (Transgene Gene 749 rescues: TG-1, TG-2, TG-3, ss2149-2151, ps1967). Scale bar: 5 mm. Silique orientation shown 750 with arrow pointing from stigma end to base. Asterisk indicates statistically significant difference 751 compared to wild-type (p < 1E-11; Student’s t-test). Error bars represent ± SD; n=10-15 siliques 752 total from at least two plants, specified in figure. (C) Hygromycin resistance and sensitivity 753 counts of male to female outcrosses performed between Col-0, ala6/7 mutants, and rescue lines 754 were used to calculate transmission efficiency ratios after selection of F1 offspring germinated 755 on hygromycin (TEr = #HygR/#HygS). Observed (Obs) and Expected (Exp) TErs are shown. 756 Statistical confidence p-values were calculated from a Pearson's chi-squared test (χ2). 757 758 Figure 6. Expression of ALA4-GFP in guard cells shows endomembrane and PM 759 localization. Fluorescence confocal images of guard cells. Images taken from stable transgenic 760 plants expressing transgenes under the control of a 35S promoter. An arrow marks the nucleus. 761 Cells shown are expressing: cytosolic GFP (ss1811, ps346), PM-localized ACA8-GFP (ss248, 762



16

ps396), ER-localized ACA2-GFP (ss2216, ps660), and ALA4-GFP (ss2145, ps1712). Scale bar: 763 5 µm. Similar results were observed in at least three independent plants. 764

765 Figure 7. Ectopic expression of ALA4-GFP in pollen shows endomembrane and PM 766 localization. Fluorescence confocal images of growing pollen tubes 1 hour after germination. 767 Pollen were from stable transgenic plants expressing transgenes under the control of an ACA9 768 promoter. Pollen shown are expressing: cytosolic YFP (ss2228, ps532), PM-localized ACA9-769 YFP (ss2229, ps580), ER-localized ACA2-GFP (ss2254, ps585), ALA4-GFP (ss2161, ps2096), 770 and ALA6-GFP (ss2399, ps1958). Scale bar: 5 µm. Similar results were observed in at least 771 three independent plants. 772 773 Figure 8. Loss of ALA4 and ALA5 results in perturbations in the abundance of membrane 774 glycerolipids and sphingolipids in rosettes. The relative abundance of (A, B) glycerolipids 775 and (C) sphingolipids in rosettes from wild-type Col-0, ala4-2/5-3 KD, and ala4-3/5-1 KO. 776 Analyzed glycerolipids include major components DGDG, MGDG, PG, PC, PE, and PI, as well 777 as minor components PS, PA, LysoPG, LysoPC, and LysoPE, presented as percentage of total 778 glycerolipid signal; n=5 independent seedling pools profiled for glycerolipids. Analyzed 779 sphingolipids include LCBs, Cers, hCers, GlcCers, and GIPCs, presented as percentage of total 780 sphingolipid; n=3 independent seedling pools profiled for sphingolipids. Single asterisk indicates 781 statistically significant difference for only KD or KO mutants and double asterisk indicates 782 statistically significant difference for both ala4/5 mutants compared to wild-type (p < 0.05; 783 Student’s t-test). Error bars represent ± SD. 784 785 Figure 9. ALA5 can transport PC, PE, SM, and PS membrane lipids. The uptake of NBD-786 labeled (A) PG, PS, PE, PC, LysoPC, and SM, as well as (B) SM, PC, Cer, GlcCer, GalCer, and 787 LacCer in drs2Δ dnf1,2Δ S. cerevisiae lines expressing ALA5 alone or in combination with β-788 subunits ALIS1, ALIS3, or ALIS5, normalized to empty vector (e.v.) values. Data are averages ± 789 SD from at least three independent experiments. A two-factor ANOVA followed by a Tukey’s 790 Honest Significant Difference test was used for statistical analysis. Asterisk indicates statistically 791 significant difference compared to e.v. (p < 0.05). (C) The same lines were dropped onto media 792 containing glucose (Glc) and onto galactose plates containing no added toxins (ø) or increasing 793 concentrations of papuamide A, duramycin, or miltefosine (direction of gradient indicated by 794 triangles). 795 796

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Figure 1. Expression patterns of A. thaliana cluster-3

ALAs and genetic organization of ALA4 and ALA5 T-

DNA disruptions. (A) Phylogenetic tree of A. thaliana

ALA family and other P-type ATPases generated in

Arabidopsis Heat Tree Viewer (http://arabidopsis-heat-

tree.org), with ALA clusters (Baxter et al., 2003) bracketed

by grey rectangles and thicker lines representing bootstrap

values of 98 or greater. (B) Database expression profile of

ALA4/5/6/7 genes in rosette, root, hypocotyl, trichomes,

and mature pollen, generated from Arabidopsis eFP

browser (Winter et al., 2007), with root hair data from

Huang et al., 2017. Error bars represent ± SD; n=3

expression replicates. ND; not detected. (C) Gene

diagram of ALA4 and ALA5 with exons in black, introns in

white, and triangles indicating position of T-DNA insertion.

Allele name and accession number are detailed above

gene schematics. (D) Protein topology representing

ALA4/5 with TM domains labeled and represented by

black boxes. Predicted truncated protein products are

detailed below the protein schematic.

1500

700

ALA #: 4 5 6 7 4 5 6 7 4 5 6 7 4 5 6 7 4 5 6 7 4 5 6 7

500

100

Arb

itra

ry E

xpre

ssio

n U

nit

s

0

N–

100aa

C

ALA5

ala5-1

SAIL_613_F05 ala5-3

SALK_049232

5’– –3’

500bp

500bp

ALA4

ala4-3

WiscDsLox_435D3

ala4-2

SALK_129561

5’– –3’

-C

ALA4/5

B

TM: 1 2 3 4 5 6 7 8 9 10

allele

4-3

5-1

D

ALA4 ALA5 ALA6 ALA7

A

Truncation points in

protein structure

ALA11 ALA10 ALA12 ALA9 ALA8 ALA4 ALA5 ALA6 ALA7 ALA3 ALA1 ALA2 MIA (P5) HMA2 (P1) ECA3 (P2A) AHA1 (P3) ACA9 (P2B)

P4-type ATPases

Other P-type

ATPases

At1g13210 At3g25610 At1g26130 At1g68710 At3g27870 At1g17500 At1g72700 At1g54280 At3g13900 At1g59820 At5g04930 At5g44240 At5g23630 At4g30110 At1g10130 At2g18960 At3g21180

Cluster

2

4

3

5 1

Gene

ND

* *



0

10

20

30

40

50

60

70

80

Root

Length

(m

m)

Day0 2 4 6

100

50

0

KO

KD KOCol-0

Col-

0

4-2

/5-3

4-3

/5-1

Rescue 1

Rescu

e2

0

20

40

60

80

100

120

Rosette D

iam

ete

r (%

WT

)

A

B C

0

5

10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

DE

F

a

Col-0

KD

Figure 2. Loss of ALA4 and ALA5 results in reduced vegetative growth. (A) Representative

image of a growth comparison assay and (B) rosette diameter quantification of wild-type Col-0, ala4-

2/5-3 KD (ss948), ala4-3/5-1 KO (ss1270), Rescue 1 (35S::ALA4 in KO, ss2145, ps1712), and

Rescue 2 (35S::ALA5 in KO, ss2146, ps1713) plants; n=8 growth assays. (C) Root length

quantification from light-grown seedlings sampled over 7 days; n=8 seedlings total from four growth

assays. (D) Representative images of dark-grown hypocotyls and quantifications of (E) length and (F)

width; n=39 hypocotyls total from three growth assays. Arrows indicate root-shoot transition. Scale

bars: 1 cm. Different letters indicate statistically significant difference (p < 0.05; Student’s t-test). (G)

Expression of ALA4 and ALA5 transcripts in wild-type, ala4-2/5-3 KD, and ala4-3/5-1 KO mutants.

ND; not detected. Error bars represent ± SE; n=3 expression replicates.

ALA #: 4 5 4 5 4 5

ND

Col-0 ala4-3/5-1

ND ND

ala4-2/5-3

Tran

scri

pt

leve

l(%

Co

l-0

)

G

Col-

0

KD

KO

Rescue 1

Rescue 2

Col-0

KD

KO

Hypocoty

l

Wid

th (

mm

)H

ypocoty

l

Length

(m

m)

ala4-2/5-3 (KD)

ala4-3/5-1 (KO)

Col-0

Rescue 1

KD

KO

Col-0

KD

KO

b

a,d

d

c

a

b c

bc

a



0

50

100

150

200

250

Cell

Wid

th (

% C

ol-

0)

Cell Width

KD

F Col-0

Figure 3. Loss of ALA4 and ALA5 results in impaired

cellular expansion in vegetative tissues. Confocal

images of propidium iodide-stained leaf epidermal cells

(A-C) and dark-grown hypocotyl cells (F-H) for wild-type

Col-0, ala4-2/5-3 KD, and ala4-3/5-1 KO, with a single

cell shaded grey in each image. Scale bars: 50 μm.

Images were analyzed with Fiji (Schindelin et al., 2012)

to quantify leaf cell area (D), guard cell length (E),

hypocotyl cell length (I), and hypocotyl cell width (J).

Different letters indicate statistically significant difference

(p < 0.01; Student’s t-test). Error bars represent ± SE;

n=20 cells total from 3 plants.

0

20

40

60

80

100

Cell

Len

gth

(%

Co

l-0

)

Guard cells

0

20

40

60

80

100

Cell

Are

a (

% C

ol-

0)

Epidermal cells

0

20

40

60

80

100

Cell

Len

gth

(%

Co

l-0

)

Cell Length

A

KD KO

D

E

I

J

Col-0

B C

G

H KO

a

b

c

a b c

b

a

a

b

c

b



B

Figure 4. Ectopic expression of ALA6 in vegetative

tissues restores growth in an ala4/5 mutant. (A)

Representative image of a growth comparison assay and

(B) rosette diameter quantification of wild-type Col-0,

ala4-3/5-1 KO mutant (ss1270), and Rescue 1–2

(ALA4p::ALA6 in KO, ss2504-5, ps2809) plants. Scale

bar: 1 cm. Shown are two out of five independent

transgenic rescue lines with similar results. Different

letters indicate statistically significant difference (p < 1E-

06; Student’s t-test). Error bars represent ± SD; n=5

growth assays.

0

20

40

60

80

100

Col-0 KO ALA6Rescue 1

ALA6Rescue 2

Rosette D

iam

ete

r (%

wild

-type)

a

c c

b

A ala4-3/5-1 KO

Rescue 2

Col-0

Rescue 1



ACA9p::ALA4

in ala6/7

0

20

40

60

80

100

Se

ed

s/S

iliq

ue

(%

Co

l-0

)

A

B

C

*

n=15

n=10

n=10n=10n=10

Figure 5. Ectopic expression of ALA4 in pollen

restores seed set and normal pollen transmission in

an ala6/7 double KO. (A) De-stained silique images and

(B) seeds per silique counts of wild-type Col-0, ala6/7

mutants (ss1351), and ala6/7 lines rescued by a pollen

specific ACA9p::ALA4 construct with a hygromycin-

resistance selectable marker (Transgene Gene rescues:

TG-1, TG-2, TG-3, ss2149-2151, ps1967). Scale bar: 5

mm. Silique orientation shown with arrow pointing from

stigma end to base. Asterisk indicates statistically

significant difference compared to wild-type (p < 1E-11;

Student’s t-test). Error bars represent ± SD; n=10-15

siliques total from at least two plants, specified in figure.

(C) Hygromycin resistance and sensitivity counts of male

to female outcrosses performed between Col-0, ala6/7

mutants, and rescue lines were used to calculate

transmission efficiency ratios after selection of F1

offspring germinated on hygromycin (TEr =

#HygR/#HygS). Observed (Obs) and Expected (Exp)

TErs are shown. Statistical confidence p-values were

calculated from a Pearson's chi-squared test (χ2).

Col-0 ala6/7 TG-1 TG-2 TG-3

Male X Female n

Transmission

Efficiency ratio

(TEr) of TG

Obs / Exp

p-Value

Col-0TG-1 (+/-) in

ala6/7184 1.09 / 1 0.65

Col-0TG-2 (+/-) in

ala6/7147 0.92 / 1 0.56

Col-0TG-3 (+/-) in

ala6/7283 1.22 / 1 0.08

TG-1 (+/-)

in ala6/7ala6/7 140 >140 / 1 <0.0001

TG-2 (+/-)

in ala6/7ala6/7 124 >124 / 1 <0.0001

TG-3 (+/-)

in ala6/7ala6/7 88 >88 / 1 <0.0001

Col-0 ala6/7 TG-1 TG-2 TG-3

Stigma end

Silique base



Figure 6. Expression of ALA4-GFP in guard cells

shows endomembrane and PM localization.

Fluorescence confocal images of guard cells. Images

taken from stable transgenic plants expressing

transgenes under the control of a 35S promoter. An arrow

marks the nucleus. Cells shown are expressing: cytosolic

GFP (ss1811, ps346), PM-localized ACA8-GFP (ss248,

ps396), ER-localized ACA2-GFP (ss2216, ps660), and

ALA4-GFP (ss2145, ps1712). Scale bar: 5 µm. Similar

results were observed in at least three independent

plants.

DIC GFP Chloroplast Overlay

GFP

ACA8 (PM)

ACA2 (ER)

ALA4



Figure 7. Ectopic expression of ALA4-GFP in pollen

shows endomembrane and PM localization.

Fluorescence confocal images of growing pollen tubes 1

hour after germination. Pollen were from stable transgenic

plants expressing transgenes under the control of an

ACA9 promoter. Pollen shown are expressing: cytosolic

YFP (ss2228, ps532), PM-localized ACA9-YFP (ss2229,

ps580), ER-localized ACA2-GFP (ss2254, ps585), ALA4-

GFP (ss2161, ps2096), and ALA6-GFP (ss2399,

ps1958). Scale bar: 5 µm. Similar results were observed

in at least three independent plants.

YFP

ALA6-GFP

ACA9-YFP (PM)

ALA4-GFP

ACA2-GFP (ER)



0

10

20

30

40

50

60

70

% t

ota

l sp

hin

go

lip

ids

Sphingolipids

0

0.2

0.4

0.6

0.8

1

1.2

1.4

% t

ota

l g

lycero

lip

ids

Minor Lipids

0

10

20

30

40

50

60

70

% t

ota

l g

lycero

lip

ids

Major Lipids

Col-0

KD

KO

A

Figure 8. Loss of ALA4 and ALA5 results in

perturbations in the abundance of membrane

glycerolipids and sphingolipids in rosettes. The

relative abundance of (A, B) glycerolipids and (C)

sphingolipids in rosettes from wild-type Col-0, ala4-2/5-

3 KD, and ala4-3/5-1 KO. Analyzed glycerolipids

include major components DGDG, MGDG, PG, PC,

PE, and PI, as well as minor components PS, PA,

LysoPG, LysoPC, and LysoPE, presented as

percentage of total glycerolipid signal; n=5 independent

seedling pools profiled for glycerolipids. Analyzed

sphingolipids include LCBs, Cers, hCers, GlcCers, and

GIPCs, presented as percentage of total sphingolipid;

n=3 independent seedling pools profiled for

sphingolipids. Single asterisk indicates statistically

significant difference for only KD or KO mutants and

double asterisk indicates statistically significant

difference for both ala4/5 mutants compared to wild-

type (p < 0.05; Student’s t-test). Error bars represent ±

SD.

**

**

B

C

*

*

*

** **



Figure 9. ALA5 can transport PC, PE, SM, and PS

membrane lipids. The uptake of NBD-labeled (A) PG,

PS, PE, PC, LysoPC, and SM, as well as (B) SM, PC,

Cer, GlcCer, GalCer, and LacCer in drs2Δ dnf1,2Δ S.

cerevisiae lines expressing ALA5 alone or in combination

with β-subunits ALIS1, ALIS3, or ALIS5, normalized to

empty vector (e.v.) values. Data are averages ± SD from

at least three independent experiments. A two-factor

ANOVA followed by a Tukey’s Honest Significant

Difference test was used for statistical analysis. Asterisk

indicates statistically significant difference compared to

e.v. (p < 0.05). (C) The same lines were dropped onto

media containing glucose (Glc) and onto galactose plates

containing no added toxins (ø) or increasing

concentrations of papuamide A, duramycin, or miltefosine

(direction of gradient indicated by triangles).

PG

P

S

PE

P

C

Lyso

PC

S

M

ALA5 ALA5

+ALIS1

ALA5

+ALIS3

ALA5

+ALIS5

Glc ø papuamide A duramycin miltefosine

Induced gene expression

drs

2Δ

dnf1

,2Δ

e.v.

ALA5

ALA5+ALIS1

ALA5+ALIS3

ALA5+ALIS5

A

C

NB

D-f

luo

res

ce

nc

e (

% e

. v.)

400

300

200

100

0

*

*

*

*

*

*

*

* *

0

100

200

300

400 B

ALA5 ALA5

+ALIS1

ALA5

+ALIS3

ALA5

+ALIS5 Dnf1p

NB

D-f

luo

res

ce

nc

e (

% e

. v.)

*

* *

*

* *

* * *

NBD-

PG

P

S

PE

P

C

Lyso

PC

S

M

PG

P

S

PE

P

C

Lyso

PC

S

M

PG

P

S

PE

P

C

Lyso

PC

S

M

NBD- NBD- NBD-

SM

P

C

Cer

Glc

Cer

Ga

lCe

r L

acC

er

NBD-

SM

P

C

Cer

Glc

Cer

Ga

lCe

r L

acC

er

NBD-

SM

P

C

Cer

Glc

Cer

Ga

lCe

r L

acC

er

NBD-

SM

P

C

Cer

Glc

Cer

Ga

lCe

r L

acC

er

NBD-

SM

P

C

Cer

Glc

Cer

Ga

lCe

r L

acC

er

NBD-



Parsed CitationsAlonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al. (2003)Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana. Science, 301: 653–657

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Bahn SC, Lee HY, Kim HJ, Ryu SB, Shin JS (2003) Characterization of Arabidopsis secretory phospholipase A2-γ cDNA and itsenzymatic properties. FEBS Letters, 553(1–2): 113–118


Baxter I, Tchieu J, Sussman MR, Boutry M, Palmgren MG, Gribskov M, Harper JF, Axelsen KB (2003) Genomic Comparison of P-TypeATPase Ion Pumps in Arabidopsis and Rice. Plant Physiol, 132: 618–628


Boavida LC, McCormick S (2007) TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitropollen germination in Arabidopsis thaliana. Plant J, 52: 570–582


Botella C, Sautron E, Boudiere L, Michaud M, Dubots E, Yamaryo-Botté Y, Albrieux C, Marechal E, Block MA, Jouhet J (2016) ALA10, aPhospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Compositionin Arabidopsis thaliana. Plant Physiol, 170: 1300–14


Chen M, Markham JE, Dietrich CR, Jaworski JG, Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important for growthand regulation of sphingolipid content and composition in Arabidopsis. Plant Cell, 20: 1862–78


Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J,16: 735–43


Du ZY, Chen MX, Chen QF, Xiao S, Chye ML (2013) Arabidopsis acyl-CoA-binding protein ACBP1 participates in the regulation of seedgermination and seedling development. Plant J, 74(2): 294–309


Fang L, Ishikawa T, Rennie EA, Murawska GM, Lao J, Yan J, Tsai AY, Baidoo EE, Xu J, Keasling JD, et al. (2016) Loss of InositolPhosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis.Plant Cell, 28(12): 2991–3004


Gall WE, Geething NC, Hua Z, Ingram MF, Liu K, Chen SI, Graham TR (2002) Drs2p-Dependent Formation of Exocytic Clathrin-CoatedVesicles In Vivo. Curr Biol, 12: 1623–1627


Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.Methods Enzymol, 350: 87–96


Gomès E, Jakobsen MK, Axelsen KB, Geisler M, Palmgren MG (2000) Chilling tolerance in Arabidopsis involves ALA1, a member of anew family of putative aminophospholipid translocases. Plant Cell, 12: 2441–2454


Guo Z, Lu J, Wang X, Zhan B, Li W, Ding S-W (2017) Lipid flippases promote antiviral silencing and the biogenesis of viral and hosthttps://plantphysiol.orgDownloaded on December 10, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

https://www.ncbi.nlm.nih.gov/pubmed?term=Alonso JM%2C Stepanova AN%2C Leisse TJ%2C Kim CJ%2C Chen H%2C Shinn P%2C Stevenson DK%2C Zimmerman J%2C Barajas P%2C Cheuk R%2C et al%2E %282003%29 Genome%2DWide Insertional Mutagenesis of Arabidopsis thaliana%2E Science%2C 301%3A 653%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Alonso&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Andjelic CD%2C Planelles V%2C Barrows LR %282008%29 Characterizing the Anti%2DHIV Activity of Papuamide A%2E Mar Drugs%2C 6%3A 528%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Characterizing the Anti-HIV Activity of Papuamide A.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Characterizing the Anti-HIV Activity of Papuamide A.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Andjelic&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Athenstaedt K%2C Daum G %281999%29 Phosphatidic acid%2C a key intermediate in lipid metabolism%2E European Journal of Biochemistry%2C 266%281%29%3A 1%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Phosphatidic acid, a key intermediate in lipid metabolism.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Phosphatidic acid, a key intermediate in lipid metabolism.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Athenstaedt&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Bahn SC%2C Lee HY%2C Kim HJ%2C Ryu SB%2C Shin JS %282003%29 Characterization of Arabidopsis secretory phospholipase A2%2Dγ cDNA and its enzymatic properties%2E FEBS Letters%2C 553%281%26%23x2013%3B2%29%3A 113%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Characterization of Arabidopsis secretory phospholipase A2-�?³ cDNA and its enzymatic properties.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization of Arabidopsis secretory phospholipase A2-�?³ cDNA and its enzymatic properties.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bahn&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Baxter I%2C Tchieu J%2C Sussman MR%2C Boutry M%2C Palmgren MG%2C Gribskov M%2C Harper JF%2C Axelsen KB %282003%29 Genomic Comparison of P%2DType ATPase Ion Pumps in Arabidopsis and Rice%2E Plant Physiol%2C 132%3A 618%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baxter&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Boavida LC%2C McCormick S %282007%29 TECHNICAL ADVANCE%3A Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana%2E Plant J%2C 52%3A 570%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Boavida&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Botella C%2C Sautron E%2C Boudiere L%2C Michaud M%2C Dubots E%2C Yamaryo%2DBott� Y%2C Albrieux C%2C Marechal E%2C Block MA%2C Jouhet J %282016%29 ALA10%2C a Phospholipid Flippase%2C Controls FAD2%2FFAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana%2E Plant Physiol%2C 170%3A 1300%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Botella&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chen M%2C Markham JE%2C Dietrich CR%2C Jaworski JG%2C Cahoon EB %282008%29 Sphingolipid long%2Dchain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis%2E Plant Cell%2C 20%3A 1862%26%23x&dopt=abstract

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

https://scholar.google.com/scholar?as_q=Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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McDowell SC, López-Marqués RL, Cohen T, Brown E, Rosenberg A, Palmgren MG, Harper JF (2015) Loss of the Arabidopsis thalianaP4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane. Front Plant Sci, 6: 197


McDowell SC, López-Marqués RL, Poulsen LR, Palmgren MG, Harper JF (2013) Loss of the Arabidopsis thaliana P4-ATPase ALA3Reduces Adaptability to Temperature Stresses and Impairs Vegetative, Pollen, and Ovule Development. PLoS One, 8: e62577


Mortimer JC, Yu X, Albrecht S, Sicilia F, Huichalaf M, Ampuero D, Michaelson LV, Murphy AM, Matsunaga T, Kurz S, et al. (2013).Abnormal Glycosphingolipid Mannosylation Triggers Salicylic Acid–Mediated Responses in Arabidopsis. Plant Cell, 25(5): 1881–1894


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Niu Y, Qian D, Liu B, Ma J, Wan D, Wang X, He W, Xiang Y (2017) ALA6, a P4-type ATPase, Is Involved in Heat Stress Responses inArabidopsis thaliana. Front Plant Sci, 8: 1732


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Pomorski T, Lombardi R, Riezman H, Devaux PF, van Meer G, Holthuis JC (2003) Drs2p-related P-type ATPases Dnf1p and Dnf2p arerequired for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis. Mol Biol Cell, 14: 1240–54


Poulsen LR, López-Marqués RL, Mcdowell SC, Okkeri J, Licht D, Schulz A, Pomorski T, Harper JF, Palmgren MG (2008) TheArabidopsis P4-ATPase ALA3 Localizes to the Golgi and Requires a b-Subunit to Function in Lipid Translocation and Secretory VesicleFormation W. Plant Cell, 20: 658–76


Poulsen LR, López-Marqués RL, Pedas PR, McDowell SC, Brown E, Kunze R, Harper JF, Pomorski TG, Palmgren M (2015) Aphospholipid uptake system in the model plant Arabidopsis thaliana. Nat Commun, 6: 7649


Roland BP, Naito T, Best JT, Arnaiz-Yépez C, Takatsu H, Yu RJ, Shin H-W, Graham TR (2019) Yeast and human P4-ATPases transportglycosphingolipids using conserved structural motifs. Journal of Biological Chemistry, 294(6): 1794–1806


Ryu SB, Karlsson BH, Ozgen M, Palta JP (1997) Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derivedsenescence retardant. Proc Natl Acad Sci, 94(23): 12717–12721

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https://www.ncbi.nlm.nih.gov/pubmed?term=Luttgeharm KD%2C Chen M%2C Mehra A%2C Cahoon RE%2C Markham JE%2C Cahoon EB %282015%29 Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth%2C Sphingolipid Metabolism%2C Programmed Cell Death%2C and Mycotoxin Resistance%2E Plant Physiol%2C 169%3A 1108%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Markham JE%2C Molino D%2C Gissot L%2C Bellec Y%2C H�maty K%2C Marion J%2C Belcram K%2C Palauqui J%2DC%2C Satiat%2DJeunema�tre B%2C Faure J%2DD %282011%29 Sphingolipids containing very%2Dlong%2Dchain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis%2E Plant Cell%2C 23%3A 2362%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=McDowell SC%2C L�pez%2DMarqu�s RL%2C Poulsen LR%2C Palmgren MG%2C Harper JF %282013%29 Loss of the Arabidopsis thaliana P4%2DATPase ALA3 Reduces Adaptability to Temperature Stresses and Impairs Vegetative%2C Pollen%2C and Ovule Development%2E PLoS One%2C 8%3A e&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Mortimer JC%2C Yu X%2C Albrecht S%2C Sicilia F%2C Huichalaf M%2C Ampuero D%2C Michaelson LV%2C Murphy AM%2C Matsunaga T%2C Kurz S%2C et al%2E %282013%29%2E Abnormal Glycosphingolipid Mannosylation Triggers Salicylic Acid%26%23x2013%3BMediated Responses in Arabidopsis%2E Plant Cell%2C 25%285%29%3A 1881%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Nakamura Y%2C Koizumi R%2C Shui G%2C Shimojima M%2C Wenk MR%2C Ito T%2C Ohta H %282009%29 Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation%2E Proc Natl Acad Sci%2C 106%2849%29%3A 20978%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Pomorski T%2C Lombardi R%2C Riezman H%2C Devaux PF%2C van Meer G%2C Holthuis JC %282003%29 Drs2p%2Drelated P%2Dtype ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis%2E Mol Biol Cell%2C 14%3A 1240%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Poulsen LR%2C L�pez%2DMarqu�s RL%2C Mcdowell SC%2C Okkeri J%2C Licht D%2C Schulz A%2C Pomorski T%2C Harper JF%2C Palmgren MG %282008%29 The Arabidopsis P4%2DATPase ALA3 Localizes to the Golgi and Requires a b%2DSubunit to Function in Lipid Translocation and Secretory Vesicle Formation W%2E Plant Cell%2C 20%3A 658%26%23x&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Poulsen LR%2C L�pez%2DMarqu�s RL%2C Pedas PR%2C McDowell SC%2C Brown E%2C Kunze R%2C Harper JF%2C Pomorski TG%2C Palmgren M %282015%29 A phospholipid uptake system in the model plant Arabidopsis thaliana%2E Nat Commun&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Saito K%2C Fujimura%2DKamada K%2C Futura N%2C Kato U%2C Umeda M%2C Tanaka K %282004%29 Cdc50p%2C a protein required for polarized growth%2C associates with the Drs2p P%2Dtype ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae%2E Molecular Biology of the Cell%2C 15%287%29%3A 3418%26%23x&dopt=abstract

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The lipid flippases ALA4 and ALA5 play critical roles in cell … · 2020. 2. 12. · 65 dwarfism,...

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