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Plant Physiology Preview. Published on August 18, 2016, as DOI:10.1104/pp.16.00464 1 Running head: ADT3, ROS homeostasis and cotyledon development 2 3 Corresponding Author: Katherine M. Warpeha, University of Illinois at Chicago, 4 Biological Sciences, 900 S. Ashland Ave, Chicago, IL 60607, 312 996 7646, 5 [email protected] 6 7 Research Area: Cell Biology 8 Secondary Research Area: Biochemistry and Metabolism or Signaling and 9 Response Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Copyright 2016 by the American Society of Plant Biologists 1 10 11 The dehydratase ADT3 affects ROS homeostasis and cotyledon development 12 Alessia Para1, DurreShahwar Muhammad2, Danielle A. Orozco-Nunnelly2, Ramis Memishi2, 13 Sophie Alvarez3, Michael J. Naldrett3, Katherine M. Warpeha*2 14 15 1. Weinberg College of Art and Science, Northwestern University, Evanston, IL. 16 2. Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL. 17 3. Proteomics and Mass Spectrometry Facility, Donald Danforth Plant Science Center, St. 18 Louis, MO. 19 20 21 22 23 24 25 26 27 28 29 30 Arabidopsis seedlings mutated in phenylalanine biosynthetic enzyme ADT3 cannot buffer reactive oxygen species, produce a defective cuticle, and display impaired cotyledon development Original project conceived by KMW; KMW supervised the experiments; KMW performed lab experiments (except proteome) with technical assistance from DM, DO-N, RM, with SA and MJN conducting the proteome on KMW samples, and AP handling data analysis of all proteomic data; KMW designed the experiments with interpretation with input from the authors especially AP, and AP and KMW analyzed the data; AP wrote the article with input on proteome from SA and MJN, with editing and oversight from KMW. 31 32 33 This work herein has been supported by National Science Foundation grant MCB-0848113 34 (NSF: http://www.nsf.gov/div/index.jsp?div=MCB), awarded to Katherine M. Warpeha and 35 Lon S. Kaufman. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 2 36 This work herein has been supported by National Science Foundation grant MCB-0848113 37 (NSF: http://www.nsf.gov/div/index.jsp?div=MCB), awarded to Katherine M. Warpeha and 38 Lon S. Kaufman. 39 40 Corresponding author: 41 42 Katherine Warpeha 43 Biological Sciences, Molecular Cell and Developmental Biology Group 44 University of Illinois at Chicago 45 900 S. Ashland Ave 46 Chicago, IL, 60612. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 3 47 ABSTRACT 48 49 During the transition from seed to seedling, emerging embryos strategically balance the 50 available resources between building up defenses against environmental threats, and 51 initiating the developmental program that promotes the switch to autotrophy. We present 52 evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE 53 DEHYDRATASE 3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and 54 cotyledon development in etiolated Arabidopsis thaliana seedlings. We show that ADT3 is 55 expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and the 56 epidermis of adt3 cotyledons contains higher levels of ROS. Genome-wide proteomics of 57 adt3 mutant revealed a general downregulation of plastidic proteins and ROS scavenging 58 enzymes, corroborating the hypothesis that ADT3 supply of Phe is required to control of ROS 59 concentration and distribution to protect cellular components. In addition, loss of ADT3 60 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement 61 cells and stomata cell fate specification; we also observed severe alterations in mesophyll 62 cells, which lack oil bodies and normal plastids. Interestingly, upregulation of the pathway 63 leading to cuticle production is accompanied by an abnormal cuticle structure and/or 64 deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and 65 provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an 66 additional role of Phe in supplying nutrients to the young seedling. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 4 67 INTRODUCTION 68 During the transition from seed to seedling, the coordination of defense and 69 development is critical for early survival (Finch-Savage and Leubner-Metzger, 2006; 70 Holdsworth et al., 2008). After emerging from the seed coat, the embryo pushes through the 71 soil to reach the surface, and at this time it is more vulnerable to biotic and abiotic stresses 72 (Raven et al., 2005), where underlying actors of this transition are relatively unstudied 73 (Warpeha 74 phenylpropanoids, play an important role in the first line of defense by contributing to the 75 reinforcement of the external cuticle layer and by conferring UV protection properties to 76 epicuticular waxes (Steyn et al., 2002; Pollard et al., 2008); in addition, phenylpropanoids 77 influence wax production in response to UV exposure (Rozema et al., 2002; Pollard et al., 78 2008; Warpeha et al., 2008). The activity of the phenylpropanoid pathway provides an 79 additional line of defense as phenolic compounds take part in a non-enzymatic mechanism to 80 efficiently scavenge reactive oxygen species (ROS), whose levels increase as a result of 81 metabolic reactions, and when plants initiate a stress response (Sharma et al., 2012; Agati et 82 al., 2013). Moreover, by influencing the cell’s ability to balance and modulate ROS production 83 and scavenging, phenylpropanoids allow fluctuations in ROS levels that are required to elicit 84 stress signaling pathways for specific defense strategies (Apel and Hirt, 2004; Mittler et al., 85 2011). and Montgomery 2016). Phenylalanine (Phe)-derived compounds, the 86 AROGENATE DEHYDRATASE 3/PREPHENATE DEHYDRATASE 1 (ADT3/PD1) 87 belongs to the arogenate dehydratase (ADT) protein family, whose members catalyze the last 88 steps of the biosynthesis of Phe (Warpeha et al., 2006; Cho et al., 2007; Bross et al., 2011). 89 Activation of ADT3 leads to an increase in Phe content and in the production of 90 phenylpropanoids (Warpeha et al., 2006). Accordingly, loss of ADT3 results in an enhanced 91 sensitivity to UV irradiation in etiolated seedlings due to reduced synthesis of photoprotective 92 compounds and UV-scattering epicuticular waxes (Warpeha et al., 2008). However, the 93 physiological and molecular basis of this phenotype and the function of ADT3 in the seed-to- 94 seedling transition remain to be elucidated. 95 We sought to understand the role of ADT3 post-germination, in the seed-to-seedling 96 transition. ADT3 is expressed early in seedling growth (Warpeha et al., 2006; Hruz et al., 97 2008). Localization studies in Arabidopsis using protoplasts from cell suspension and light 98 grown rosette leaves have placed this enzyme within the chloroplast (Rippert et al., 2009), 99 while we have reported ADT3 activity in the cytosolic fraction in young etiolated seedlings Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 5 100 (Warpeha et al., 2006). These reports differ likely due to the different age and growth 101 conditions of the studied plant material. Cytosolic forms of chorismate mutase, which act at 102 the first committed step in the Phe and Tyr biosynthesis pathway, were found in Arabidopsis 103 and other plants (d'Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996), 104 suggesting the possibility of extra-plastidic Phe biosynthesis. 105 Herein, we report in transgenic complementation experiments that ADT3 is expressed 106 widely in the young shoot and largely accumulates in the cytosol. Based on the role of 107 phenylpropanoids in plant defense, we hypothesize that ADT3 regulation of Phe supply is 108 required to coordinate defense and development at the seed-to-seedling transition. We found 109 that, without ADT3, the cells of the epidermis cannot buffer and restrict ROS; moreover, adt3 110 cotyledons enter an aberrant developmental program that results in abnormal morphology 111 and patterning as well as several alterations at the subcellular level. Proteomic analysis of 112 adt3 seedlings provided insights into the molecular basis of adt3 phenotypes as it uncovered 113 a chronic inability to buffer an excess of ROS and maintain plastid integrity. It also revealed a 114 failed attempt to control cell rheology through up-regulation of the biochemical pathway for 115 cuticle biosynthesis and assembly, as indicated by the increase in the permeability of adt3 116 epidermal cells; we propose that this could also be the cause of the defecting epidermal 117 patterning in adt3 cotyledons. In addition, we suggest an additional role of Phe in nutrient 118 supply in etiolated seedlings. 119 120 RESULTS 121 122 ADT3 is expressed in cotyledons of young etiolated seedlings, where its loss causes 123 an imbalance in ROS homeostasis. 124 To investigate the impact of loss of ADT3, we generated transgenic plants where 125 native ADT3 fused to CFP was driven by its native promoter (ADT3::ADT3-CFP) in the adt3-1 126 mutant background to examine ADT3 spatial expression pattern in 4-d-old dark-grown 127 seedlings (Fig. 1). ADT3 is expressed in the shoot in developing mesophyll, and SAM (leaf 128 primordial and dome) (Fig. 1A and S2) and epidermal cells, especially pavement cells (Fig. 129 1B). In particular, ADT3 appeared to accumulate mainly in the cytosol (inset Fig,1A and 1B). 130 An additional transgenic line harboring the ADT3::ADT3-GFP fusion in adt3-1 mutant 131 background 132 complementation of all adt3 defects reported herein (Fig. S1; see next section), and recapitulated the ADT3::ADT3-CFP expression pattern, Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. and showed 6 133 elsewhere (Warpeha et al., 2008). A similar expression pattern of ADT3::ADT3-GFP was 134 observed in the WT background, where a more intense GFP signal is observed compared to 135 the mutant background. ADT3 expression was also observed in the developing guard cells 136 (Fig. S2). 137 adt3 mutation has been previously shown to make etiolated seedlings vulnerable to 138 UV irradiation and the defect was proposed to be due to a decrease in the production of 139 phenylpropanoids upon exposure to both blue light and UV (Warpeha et al., 2006; Warpeha 140 et al., 2008). To elucidate the physiological basis of UV sensitivity in relation to 141 phenylpropanoids, we examined the accumulation of ROS in the epidermis of adt3-1 142 cotyledons before and after UV treatment, as these compounds are produced in response to 143 short-wavelength light (Wituszyńska and Karpiński, 2013; Consentino et al., 2015). 6-d-old 144 dark-grown WT and adt3-1 seedlings were irradiated with a sublethal dose of UV-C radiation 145 and the CellRoxTM Deep Red cell permeable fluorescent probe was used to detect ROS 146 (Fig.2). This probe only fluoresces when in contact with ROS, and the signal can be captured 147 when excited by the Cy5 LED (false-colored pink). 148 Interestingly, probe fluorescence in the epidermis was detected in dark-grown 149 unirradiated adt3-1 compared to unirradiated WT (control), indicating that basal ROS levels 150 are elevated in adt3-1 (Fig. 2). Irradiation with a sublethal UV-C dose caused an increase in 151 CellRoxTM basal signal from both WT and adt3-1 pavement cells (Fig. 2, bar graph). An 152 additional ADT3 mutant (adt3-6) tested responded similarly to adt3-1 (Suppl. Fig. S3). Since 153 ADT3 catalyzes the last step of the biosynthesis of Phe (Maeda and Dudareva, 2012), we 154 assessed the requirement of Phe in counteracting ROS by transferring living, whole 6-d-old 155 WT, adt3-1 and adt3-6 seedlings to Phe-supplemented medium for 3 h before subjecting 156 them to a sublethal dose of UV-C radiation, immediately followed by incubation with 157 CellRoxTM, where pre-incubation with Phe restored the seedling to control (WT unirradiated) 158 levels of ROS in both adt3 alleles (Fig. 2; Suppl. Fig. S3). Another probe for ROS detection, 159 SOSGreenTM (SOSG), indicated similar results for adt3-1 compared to WT, confirming that 160 ROS production is significantly elevated as a consequence of loss of ADT3 (Suppl. Fig. S4), 161 whereas adt3-6 was not significant. 162 To test whether Phe metabolism is required to scavenge ROS, we UV-C irradiated 6- 163 d-old, dark-grown seedlings of the fah1-7/tt4-1 double mutant (Li et al., 1993; Landry et al., 164 1995; Peer et al., 2001), which cannot metabolize Phe to produce flavonoids. Indeed, we 165 observed that pretreatment with Phe was able to prevent UV-induced ROS production in the Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 7 166 fah1-7/tt4-1 background (Suppl. Fig. S3). Accordingly, pre-treatment with Phe had the same 167 effect as KI, a known ROS scavenger (Tsukagoshi et al., 2010), which was also able to 168 prevent a ROS increase upon UV-C irradiation (Suppl. Fig. S5). Likewise, treatment with a 169 non-metabolizable Phe analog, p-f-DL-Phenylalanine (Conway et al., 1963), caused a 170 significant decrease in Cy5 fluorescence in UV-C-irradiated pavement cells (but not in the 171 guard cells) in the adt3-1 epidermis (Suppl. Fig. S5). Taken together, these results indicate 172 that ROS homeostasis is altered in dark-grown adt3 seedlings. In addition, we observed that 173 Phe per se could prevent significant ROS accumulation as a result of UV-C irradiation. 174 175 Loss of ADT3 disturbs epidermis development in the cotyledons of dark-grown 176 seedlings. 177 To further investigate the consequences of loss of ADT3, we examined the cellular 178 makeup of the cotyledon’s epidermis. DAPI illumination of the epidermis of live 6-d-old dark- 179 grown WT and adt3-1 seedlings enabled a clear distinction of cell lineages (pavement cells 180 largely reflect, guard cell lineage largely absorbs DAPI), and revealed that adt3-1 mutants 181 produced fewer but larger pavement cells than WT (Fig. 3, A); however, adt3-1 cotyledons 182 are wider than the WT (Fig. 3, A and B). In addition, a reduced number of guard cells was 183 observed for the adt3 mutant cotyledons (Fig. 3, A and B). Addition of Phe to the growth 184 medium from sowing, rescued both the pavement cell and guard cell phenotypes in adt3-1 185 mutants (Fig. 3, A and B). adt3-6 seedlings also presented similar phenotypes which could be 186 rescued by Phe (Suppl. Fig. S6A). The defects persisted when exogenous L-tyrosine (L-Tyr), 187 or L-tryptophan (L-Trp) (which derive from the shikimate pathway like Phe) were supplied 188 instead of Phe (Suppl. Fig. S7). 189 To address whether the guard cell defect was due to failure to specify the stomatal 190 lineage, we supplied Phe to the mutants speechless and mute, which disrupt early and late 191 steps of the stomata lineage progression, respectively (MacAlister et al., 2007; Pillitteri et al., 192 2007). Phe did not prevent the guard cell defects in these mutant, indicating that Phe is 193 required upstream of these guard cell specification factors (Suppl. Fig. S7). 194 In summary, we observed ADT3 expression in the cytoplasm of epidermal cells of the 195 cotyledons and of the SAM. Loss of ADT3 severely affects the number and morphogenesis of 196 pavement cells and disrupts guard cell development. The defects were rescued by 197 supplementing Phe but not other amino acids, indicating that metabolism of ADT3-supplied 198 Phe impacts cotyledon patterning in etiolated seedlings. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 8 199 200 adt3 cotyledons exhibit abnormal subcellular features. 201 We also examined the ultrastructure of the epidermis and mesophyll cells in 202 cotyledons of 6-d-old, dark-grown adt3-1 seedlings by transmission electron microscopy 203 (TEM) (Fig. 4, A). A prominent feature we observed was the reduction in the number of 204 globular oil bodies in adt3-1 mesophyll cells compared to WT (Fig. 4, A, inset c and f). In 205 addition, adt3-1 cells were hyper-vacuolated (especially the epidermis), while most WT cells 206 contained a large central vacuole (Fig. 4, A). Developing plastids were identified in WT, as 207 they stain densely and contain prolamellar bodies with stromal strands extending from the 208 thylakoid membrane (Fig. 4, A, inset b), whereas the adt3-1 plastids displayed atypical 209 prolamellar bodies and loose stromal thylakoids (Fig. 4, A, inset e). The cytoplasm of adt3-1 210 epidermal cells had a blackened appearance, indicating possible accumulation of osmiophilic 211 material, and exhibited numerous round organelles, which are likely to be mitochondria, as 212 suggested by the ribbed structure (Fig. 4, A, inset a and c). 213 The distribution of oil bodies in WT and adt3-1 was further investigated using the 214 lipophilic stain Nile Red (Greenspan et al., 1985). Optical sections of adt3 cotyledons showed 215 a paucity of scattered oil bodies (Fig. 4, B), while in WT these organelles were more 216 abundant, particularly at the tip (Fig. 4, B). Addition of Phe to the germination medium 217 increased oil body production in adt3-1 (Fig. 4, B), rescuing the deficiency. This phenotype 218 was also observed for the adt3-6 allele (Suppl. Fig. S6). Thus, ADT3 activity is required to 219 preserve the subcellular structure of the cells of the cotyledons in etiolated seedlings. 220 221 adt3 proteome reveals the molecular basis of the adt3 mutant phenotypes. 222 In order to uncover the molecular phenotype underlying the adt3 phenotypes, we 223 obtained a comparative protein expression profile of the aerial portions (upper hypocotyl and 224 cotyledon) of adt3-1 and WT seedlings. We used the bioinformatics platform Virtual Plant for 225 Gene Ontology (GO) terms analyses (Katari et al., 2010) and KEGG database for metabolic 226 pathways (http://www.genome.jp/kegg/). 227 The proteomic analysis revealed that adt3-1 mutation caused alteration of the 228 expression of 1333 proteins, 608 of which were down-regulated, while 725 were up-regulated 229 (Table S1). The maximum fold change was 1.69 for up-regulation and 0.646 for down- 230 regulation, a dynamic range of expression in accordance with other proteomics studies 231 (Table S1)(Alvarez et al., 2011; Alvarez et al., 2014). Given the high number of statistically Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 9 232 significantly misregulated proteins in adt3-1 proteome, we investigated whether small 233 changes in expression could be biologically significant by asking whether they occur in a 234 specific tissue or at multiple steps within the same process, as it was conceptualized by 235 metabolic control analysis (Kacser and Burns, 1995; Fell and Cornish-Bowden, 1997). First, 236 we queried the Plant Ontology (PO) database in Virtual Plant to assess enrichment of 237 proteins of different cell and organ types. For both adt3-1 up-regulated and down-regulated 238 proteins, the most significant PO enrichment was observed for guard cell, shoot epidermal 239 cell and epidermal cell (Table 1), indicating that the changes in protein expression were 240 relevant to the cell types of the cotyledons where we observed ADT3 expression (Fig. 1, A). 241 Next, plant-specific overrepresented terms for cellular compartment, molecular function and 242 biological process were assessed by GO term analysis using a p value of 0.01 as the cutoff. 243 The results of this analysis are summarized in Table 1, Table 2, and Tables S2-S11. 244 GO term analysis of up-regulated proteins in adt3-1 proteome revealed enrichment 245 for plasma membrane and vacuole factors involved in transport as well as in cell wall 246 organization or biogenesis through the metabolism of the polysaccharides xylan and glucan 247 (Table 1). Accordingly, the most represented molecular function for the cell wall category is 248 “hydrolase activity”, since in these polymers the monosaccharide residues are joined by 249 glycosidic linkages. The involvement of the cell wall in adt3 cell morphology is also 250 underscored by the GO term “growth”, which showed one of the most significant enrichment 251 for the up-regulated proteins in adt3-1 proteome (Table 1). The proteins belonging to this 252 category are involved in cell expansion, like expansins (EXP3, 9, 11 and EXPL1), and DIM1 253 (Takahashi et al., 1995). Interestingly, a protein that showed the highest increase in the adt3- 254 1 mutant is AT14A, a protein that was shown to function like integrin in animals to regulate 255 cell shape and cell-to-cell adhesion (Lu et al., 2012) (Table S1). 256 For adt3-1 up-regulated proteins assigned to epidermal cells according to the PO 257 database, we noticed an enrichment in proteins involved in carboxylic acid/oxoacid metabolic 258 process (Table 1), encompassing enzymes for fatty acids conjugation (LACS4, LACS8, 259 GPAT8 and GPAT4), and synthesis and elongation of very long chain fatty acids (VLCFAs) 260 [ACC1/PAS3, CER2, CER6, CER10, ATT1 (CYP86A2), FDH (KCS10)]. We also found up- 261 regulation of the 3-ketoacyl-CoA synthases (KCS) KCS8, 9, 16, 19 and CY86A4, as well as 262 transporters of wax molecules and cuticle building blocks (ABCG11/DSO/CER5 and LTPG1). 263 All of these functions were previously associated with cuticle formation (Kunst and Samuels, 264 2009; Nawrath et al., 2013 and specific references therein). Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 10 265 When considering down-regulated proteins, one of the most significant GO terms for 266 cellular components is “plastid”, and the affected proteins are associated with the stroma, 267 envelope and thylakoid membrane (Table 1). This is in agreement with the observations that 268 the integrity of the plastids is compromised by loss of ADT3 (Fig. 4, A). Interestingly, we 269 observed an overrepresentation of proteins associated with the stromule, an elusive plastidic 270 structure that has been previously implicated in GPA1 signaling (Huang et al., 2006; Hanson 271 and Sattarzadeh, 2011) (Table 1). 272 One of the top GO terms that describes the overrepresented molecular functions for 273 the group of down-regulated proteins in the epidermis of adt3-1 mutant is “oxidoreductase 274 activity” (Table 1) and includes proteins of both the enzymatic and non-enzymatic scavenging 275 system that controls ROS homeostasis like superoxide dismutases (MSD1, FSD2 and CSD2 276 and CSD3), catalases (CAT2), enzymes of ascorbate-glutathione (AsA-GSH) cycle ascorbate 277 peroxidase 278 dehydroascorbate reductase (DHAR2), and glutathione reductase (ATGR2) (Sharma et al., 279 2012). GDP-Mannose 3′,5′-epimerase (GME), an important enzyme in the ascorbate 280 biosynthetic pathway, is also down-regulated (Wheeler et al., 1998). In addition, of the 93 281 down-regulated proteins that were assigned to gene families, 7 are members of the 282 glutathione S-transferase family of proteins in adt3-1 (Table S1). (SAPX, APX1, APX3), monodehydroascorbate reductase (MDAR4), 283 In addition to the GO terms listed above, “primary metabolic process” is highly 284 significant for both up-regulated and down-regulated proteins in adt3-1 proteome (Table 1). 285 Hence, we investigated the metabolic pathways that were affected by loss of ADT3 using the 286 KEGG database. We observed down-regulation of key enzymes of the central carbohydrate 287 and energy metabolism (Table 2), including enzymes of the glycolysis/gluconeogenesis 288 pathway, the pentose phosphate pathway, and glyoxylate and dicarboxylate metabolism. 289 However, some of the enzymes involved in oxidative phosphorylation are up-regulated, 290 suggesting an intense mitochondrial activity. Also, the expression of proteins of starch and 291 sucrose metabolism, pentose and glucuronate interconversions as well as in amino sugar 292 and nucleotide sugar metabolism is increased in the adt3-1 mutant. The enrichment of 293 enzymes from these pathways provides a link between cell wall metabolism and central plant 294 metabolism (Seifert, 2004; Bar-Peled and O'Neill, 2011). 295 In summary, the overrepresentation of terms for specific biological functions, 296 localization or molecular components is consistent with the observed adt3 phenotypes, 297 linking the physiological and morphological alterations to changes in the expression of key Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 11 298 proteins or pathways. Moreover, in agreement with functional enrichment analysis, the 299 pathways analysis suggests that alteration of cell morphology and subcellular structure might 300 be linked to metabolic deficiencies as a result of loss of ADT3 activity. 301 302 Cuticle formation and cell permeability are altered in adt3 cells. 303 Many key proteins involved in the production of the cuticle were up-regulated in the 304 adt3-1 mutant (Table 1 and Table S3). Higher magnification of the outermost cell wall 305 revealed irregular deposition of electron-opaque material within the deeper layers (Fig. 5, A), 306 indicating that cuticle biosynthesis and/or deposition is somehow defective. To further 307 examine adt3-1 cuticular properties, we tested the permeability of adt3-1 cuticle under 308 hyposmotic conditions using toludine Blue (TB) staining (Tanaka et al., 2004). Interestingly, 309 diffuse TB staining was observed in adt3 seedlings after a 48 h incubation in water (Fig. 5, B) 310 compared to the WT in the same conditions, a sign of incomplete or reduced cuticle in the 311 mutant. TB staining of adt3-6 dark grown seedlings showed similar results (data not shown). 312 Since it was found that mutations in genes of the wax biosynthetic pathway that affect the 313 composition of the epicuticular waxes also perturb stomatal development and increase the 314 number of guard cells (Gray et al., 2000; Guseman et al., 2010), we inspected the epidermis 315 of white light grown adt3 seedlings and found adjacent stomata that were breaking the one- 316 cell spacing rule (Fig. 5, C). Our results indicates that the cuticle of adt3 epidermis does not 317 achieve a proper organization, and this causes a reduction in the isolating properties of the 318 epidermis and is likely to impact cell patterning of adt3 cotyledons. 319 320 Sucrose supply restores guard cell number while Asn and NH3Ac rescue pavement cell 321 cell morphology in adt3 cotyledons. 322 Proteomic analysis revealed that a significant number of proteins from primary 323 metabolic pathways – in particular for carbon metabolism — are down-regulated in adt3-1 324 seedlings (Table 1 and Table 2), so we investigated whether carbon input could alleviate the 325 defects in adt3-1 cotyledons by transferring 4-d-old adt3-1 seedlings to medium containing 326 1% sucrose. When we examined adt3-1 cotyledons 48 h later, we found that the pavement 327 cell phenotype was not rescued by exogenous sucrose (Fig. 6, A); however, the number of 328 guard cells was similar to the WT (Fig. 6, A). The rescue was not due to a change in osmotic 329 potential, as it was not ameliorated by exogenous supply of 1% mannitol (Suppl. Fig. S8). Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 12 330 Since ASPARAGINE SYNTHASE 1 (ASN1) is up-regulated in adt3-1 mutant (Table 331 S1), we next attempted to rescue adt3-1 phenotype by supplying exogenous asparagine 332 (Asn) from sowing. After 6 d of growth on Asn in darkness, we observed that the pavement 333 cell size and number as well as the cotyledon size were completely restored in the mutant 334 (Fig. 6, B). The same results were obtained when ammonium acetate (NH4Ac) was added to 335 the medium (Fig. 6, C). However, the guard cell lineage defect was not corrected by the 336 application of neither exogenous Asn nor NH4Ac (Fig. 6, B and C). To test whether the ability 337 of Asn and NH4Ac to rescue adt3-1 cell morphology phenotype could be linked to restored 338 fatty acid production (Fig. 4, B), we incubated Asn- and NH4Ac-supplied adt3-1 seedlings with 339 Nile Red (Fig. 6, C) and, similarly to the result we obtained by supplying Phe (Fig. 4, B), 340 addition of Asn and NH4Ac also resulted in an increase in Nile Red signal (Fig. 6, C), 341 indicating that these compounds could be used by adt3 to feed metabolic pathways that lead 342 to fatty acids production. These observations suggest that a nutrient imbalance could be part 343 of the defects in cell morphology and epidermal patterning of adt3 cotyledons (Fig. 3). 344 345 DISCUSSION 346 347 ADT3 accumulates in the cytosol and is required to maintain ROS homeostasis. 348 We previously reported that an arogenate dehydratase activity could be detected in the 349 cytosol of dark grown, young seedlings upon blue light irradiation. In this study, we used 350 transgenic lines expressing CFP tagged ATD3 to further investigate in vivo ADT3 localization 351 in 4-d-old dark-grown seedling. Here ADT3 expression was detected in the shoot, including 352 cotyledons and SAM (Fig.1; S Figure S2). We also examined the subcellular localization of 353 ADT3 and confirmed that the enzyme accumulates mainly in the cytosol at this stage of 354 seedling development (Fig 1). This observation adds to the growing body of evidence 355 supporting the existence of extra-plastidial pathways for Phe biosynthesis (Yoo et al., 2013). 356 Phe production in the cytosol has important implications since cytosolic Phe could be directly 357 channeled into secondary metabolism, as the enzymes of the pathways that utilize Phe as a 358 precursor are found outside the plastids. In particular, cytosolic synthesis of Phe would 359 ensure timely production of antioxidants and photoprotective molecules, which are required to 360 contain the damages caused by high-frequency radiations when etiolated seedlings are 361 exposed to solar light. Accordingly, we previously reported an overall reduction in UV and 362 Blue light-absorbing compounds in the cotyledon when Phe supply is compromised by loss of Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 13 363 ADT3 (Warpeha et al., 2006). In addition to providing protection from photodamage, we show 364 herein that Phe metabolism could counteract the increase in ROS concentration as part of 365 the stress response to high frequency radiations (Fig. 2) (Mackerness et al., 2001; Kalbina 366 and Strid, 2006; Consentino et al., 2015). 367 However, ROS levels were significantly elevated in unirradiated adt3 cotyledon cells 368 compared to WT cells (Fig. 2; Suppl. Fig. S3-5), indicating that ADT3 activity is required to 369 maintain a low basal level of ROS in etiolated seedlings. Because of the high number of 370 mitochondria in the adt3 mutant cells and the decrease in oil bodies (Fig. 4), we suggest that 371 ROS over-production could be due to a combination of normal reoxygenation of the 372 germinating tissues when the embryo emerges from the seed coat (Sattler et al., 2004), and 373 the intense metabolic activity that is likely required for the catabolism of the oil bodies. 374 Increased sensitivity to light and oxidative stress has been reported previously for a 375 mutant of the ROS-scavenging enzyme ASCORBATE PEROXIDASE 1 (APX1) gene, which 376 exhibited a low cytosolic content of the antioxidant ascorbic acid (Davletova et al., 2005, 377 Miller et al., 2007). Similarly to apx1, the adt3 mutant phenotype could be a consequence of 378 high steady-state of cytosolic ROS levels, In Arabidopsis it has been shown that cytosolic 379 scavenging enzymes are induced upon light stress (Karpinski et al., 1997; Karpinski et al., 380 1999; Pnueli et al., 2003; Davletova et al., 2005), even though ROS are thought to be 381 generated in chloroplasts and/or peroxisomes (Mittler, 2002). In addition, buffering ROS in 382 the cytosol was shown to be essential for protecting chloroplast from oxidative damage 383 (Davletova et al., 2005). Indeed, these chemical species can damage membranes of 384 organelles (Gill and Tuteja, 2010), in particular, plastids (Asada, 2000). Since we observed 385 localization of ADT3 in the cytosol of the cells in etiolated seedlings (Fig. 1), we concluded 386 that cytosolic Phe-derived antioxidants, and even cytosolic Phe per se, may be required to 387 protect the integrity of the plastids from the cytosolic side, a notion that is supported by the 388 abnormal structure of these organelles in adt3 plastids (Fig. 4), and to preserve their 389 functionality, as indicated by down-regulation of essential processes that take places in these 390 organelles such as nitrogen metabolism and response to oxidative stress (Table 1). Indeed, 391 when we irradiated dark-grown WT seedlings with high energy UV-B (300nm), we noticed 392 that the structure of the plastids was visibly affected by the treatment and inclusion of Phe to 393 the growth medium from sowing restored the characteristic pro-lamellar body organization 394 that was observed before irradiation (Suppl. Fig. S9). The effect is even more dramatic in 395 adt3-1 mutant, where plastids were hardly discernable upon UV-B irradiation, but were Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 14 396 resistant to the treatment in presence of Phe (Suppl. Fig. S9). This is consistent with the 397 ability of the phenylpropanoids such as flavonols to bind to non-bilayer lipids such as 398 monogalactosyl diacyl glycerol (MGDG) (Erlejman et al., 2004). The outer membrane of 399 plastids is reported to be deficient in MGDG (Erlejman et al., 2004; Scheidt et al., 2004). Part 400 of the acute sensitivity of adt3 seedling to UV-C irradiation doses that are tolerated by WT 401 (Warpeha et al., 2008) could also be explained by the observation that other enzymes 402 involved in ROS removal are down-regulated in adt3 (Table 1 and Table S1). This could lead 403 to a chronic imbalance between the production of ROS upon high-energy UV treatment and 404 the ROS buffering capacity of adt3 seedlings. Failure to activate additional ROS-scavenging 405 pathways as part of the physiological response to increase in ROS concentration (Sharma et 406 al., 2012) would suggest that, without the contribution of ADT3 function, ROS would 407 accumulate above the threshold toxic levels in young etiolated seedlings, resulting in 408 irreversible cellular damage (Sharma et al., 2012). 409 Thus, Phe biosynthesis through ADT3 activity is required to protect the cotyledons of 410 etiolated seedlings from the deleterious effects of ROS by maintaining a low concentration of 411 these toxic compounds at the steady-state, and by buffering ROS increase as a consequence 412 of photostress. Control of ROS homeostasis is still poorly understood, and advancing the 413 knowledge of Phe and related metabolism will help elucidating the cellular signaling and 414 metabolic processes involved in this important mechanism (Geigenberger and Fernie, 2014). 415 416 A role for ADT3 supplied Phe in morphogenesis and cell proliferation through cuticle 417 assembly. 418 ADT3 was previously shown to participate in the production of epicuticular waxes 419 (Warpeha et al., 2008). In the present study, we have shown that adt3 epidermis bears signs 420 of alterations in the deposition process (Fig. 5, A). This is likely to cause a severe alteration in 421 the rheological properties of adt3 epidermal cells, particularly when the permeability of adt3 422 seedlings is challenged (Fig. 5, B). Increased levels of known factors involved in cuticle 423 production and transport indicates that the molecular machineries that act in this process are 424 in place in the adt3 epidermis (Table 1), so the defect might be due to a shortage of 425 substrates (like VLCFA), or impairment in cuticle assembly. Both hypotheses are plausible as 426 the VLCFA are produced by plastids (Yeats and Rose, 2013), whose structure and function 427 appear to be altered in adt3 (Fig. 4 and Table 1), or could derive from the degradation of 428 triacylglycerols stored in oil bodies, which are nearly absent in adt3 mutants (Fig. 4, A and B, Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 15 429 Suppl. Fig. S6). The presence of osmiophilic compounds in the cytoplasm of adt3 epidermis 430 might suggest that a high amount of lipid substrates is available for the formation of cuticle 431 monomers (Fig. 4, B), but the observation that deposition of electron-opaque material occurs 432 within the deeper layers of the cell wall is a sign that the process is disrupted (Fig. 5, A). A 433 similar phenotype was previously seen in the bodyguard (bdg) mutant, a putative alpha-beta 434 hydrolase, which is thought to function in cutin biosynthesis, or as a cross-linking enzyme 435 (Kurdyukov et al., 2006). Similarly, Phe-derived phenolics components of cutin and cuticular 436 waxes (Nawrath et al., 2013) could participate in cuticle assembly as a cross-linker by 437 reacting with cutin monomers (Pollard et al., 2008). The involvement of phenylpropanoids in 438 cuticle formation has been previously reported for transparent testa mutants like ttg1, which 439 are regulators of the phenylpropanoid biosynthetic pathway (Koornneef, 1990; Xia et al., 440 2010), strengthening the hypothesis that decreased Phe availability is linked to the adt3 441 defect in cuticle formation. TTG1 is also involved in epidermal development, as it participates 442 in trichomes and stomata patterning (Bean et al., 2002). Interestingly, mutations in other 443 genes involved in cuticle formation and up-regulated in adt3 mutant, like FIDDLEHEAD, also 444 affect the number of trichomes (Yephremov et al., 1999; Wellesen et al., 2001), while 445 different genes involved in the same process, like CER1 and CER6, affect stomatal number 446 (Bird and Gray, 2003). Thus, Phe derived compounds – and, hence, ADT3 function – could 447 play a role in cell differentiation by modulating cuticle assembly. Indeed, phenylpropanoids 448 appear to be more abundant in the cuticle of the guard cells, where the natural blue 449 fluorescence that they emit under UV light is higher than in the pavement cells because of 450 either the thickness or the richness of wax-bound phenolics in the epicuticular waxes of the 451 guard cells (Karabourniotis et al., 2001). Cuticle composition influences diffusion rates 452 (Kerstiens, 1996; Schreiber and Riederer, 1996), so tightening of the cuticular structure could 453 confine the effect of developmental cues within the stomatal cell lineage (Guseman et al., 454 2010; Engineer et al., 2014), while a loose cuticle could facilitate exchanges of signaling 455 molecules to prevent pavement cells from acquiring stomatal identity at later stages of 456 stomatal development. Change of permeability due to a decrease in cell wall resistance could 457 then cause the arrest of stomatal development at the meristemoid stages in adt3, with a 458 consequent reduction in the number of stomata, or even at an earlier stage, when the initial 459 precursor stomatal lineage, the mother cells (MMC), first differentiate from the protoderm. 460 This would explain not only the reduced number of stomata in adt3 cotyledons, but also the 461 decreased number of pavement cells, as the MMCs divides asymmetrically to generate one Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 16 462 pavement cell and one meristemoid (Pillitteri and Torii, 2012). The defective cuticle could also 463 contribute to the enlargement observed for adt3 pavement cells, as they are incapable of 464 maintaining a correct water balance (Fig. 4, B). Interestingly, enlarged pavement cells are 465 also observed in stomata development mutants spch and mute (Tanaka et al., 2013), 466 suggesting that communication signals from the stomata cell lineage can influence the 467 morphology of proximal pavement cells. Hence, our work provides evidence that ADT3 468 function is required during early stages of seedling establishment to regulate the 469 development of the epidermal layer by controlling the size of pavement cells and stomatal cell 470 fate acquisition though the modulation of cuticle formation. Further analysis is required to 471 establish the chemical composition of the cuticle at this stage of cotyledon development. 472 473 Phe role in nutrient availability. 474 Our findings that loss of ADT3 compromises ROS homeostasis and alters cuticle 475 formation indicate that ADT3 function is required to coordinate defense and development in 476 the seed-to-seedling transition. Moreover, we speculate that Phe synthesis by ADT3 could be 477 implicated in energy production during early stages of plant growth. We have shown that 478 supply of Phe restores lipid synthesis in oil body-depleted adt3 cotyledons (Fig. 3, B, 479 Suppl.Fig. S6). As fatty acid biosynthesis takes place in chloroplasts (Yeats and Rose, 2013), 480 which are severely affected by loss of ADT3, Phe treatment of adt3 seedlings could support 481 this process by shielding the developing plastid membranes from the damage caused by 482 increased ROS levels at steady-state, therefore preserving their functionality. Alternatively, 483 de novo synthesis of fatty acids could be carried out using Phe as a substrate of the 484 tricarboxylic acid (TCA) cycle through Tyr conversion; however, resupplying adt3 mutant with 485 Tyr did not rescue adt3 phenotypes (Fig. S7). Although we cannot exclude that this may be 486 due to the inability of adt3 to uptake or metabolize Tyr in the form we supplied, it is tempting 487 to speculate that Phe could be metabolized by a pathway that does not encompass the Tyr 488 conversion step (Wang et al., 2013). 489 We could also rescue fatty acid biosynthesis by supplying the adt3-1 mutant with Asn 490 and NH4Ac (Fig. 5), which also led to the restoration of the pavement cell phenotype (Fig. 5). 491 Curiously, the pavement cell defect could not be ameliorated by addition of sucrose, which 492 was instead successful in rescuing the guard cell phenotype in adt3 cotyledons. Asn is known 493 to be an important nitrogen carrier for transport and storage and an increase in Asn through 494 up-regulation of ASN1 in dark-grown seedling was shown to be part of a metabolic strategy Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 17 495 for the remobilization of metabolites in low energy states (Lam et al., 1998; Lam et al., 2003). 496 This seems to be the case for XANTHINE DEHYDROGENASE 1 (XHD1), whose activity 497 provides a source of nitrogen through purine catabolism (Brychkova et al., 2008). As both 498 proteins are significantly up-regulated in adt3 proteome (Table S1), we put forward that Phe 499 metabolism may provide an alternative source of nitrogen in etiolated seedlings. Indeed, 500 deamination by Phenylalanine Ammonia Lyase (PAL) is the first step of Phe metabolism and 501 the ammonia is thought to be recycled back into Phe biosynthesis (Singh et al., 1998; Corea 502 et al., 2012); however, under nitrogen shortage, glutamate from Phe metabolism could be 503 used as alternative nitrogen source (Lee et al., 2007) or as nitrogen donor for other 504 pathways, as observed for other organisms (Marusich et al., 1981; Sikora and Marzluf, 1982; 505 Vuralhan et al., 2003). Nitrogen requirement in adt3 seedlings could be needed to restore an 506 imbalance in carbon/nitrogen ratio that is caused by an increase in carbon availability as a 507 result of cell wall metabolism, as suggested by up-regulation of SUGAR TRANSPORTER 508 PROTEIN 1 (STP1) and the enrichment in enzymes involved in cell wall remodeling (Table 1 509 and Table S1)(Stadler et al., 2003; Baena-Gonzalez et al., 2007; Schofield et al., 2009). 510 511 ADT3 in signaling and development. 512 In Arabidopsis, α, β and γ subunits of G-proteins and their effectors are part of 513 complex signal transduction pathways that regulate defense mechanisms and developmental 514 processes (Huang et al., 2006; Pandey et al., 2006; Lee et al., 2013; Urano et al., 2013; 515 Urano and Jones, 2014). G-protein signaling is thought to participate in the developmental 516 program underlying the seed to seedling transition by influencing the structure of the 517 cotyledons. Mutations in GPA1, the sole Gα subunit found to date in Arabidopsis, result in low 518 stomata density, and in an increase in the size of epidermal cells in young seedlings (Ullah et 519 al., 2001; Zhang et al., 2008; Nilson and Assmann, 2010). These observations indicate a 520 defect in cell division and elongation as the possible cause of gpa1 morphological 521 phenotypes, which could also explain the short hypocotyl and open apical hook of gpa1 522 etiolated seedlings (Lease et al., 2001; Ullah et al., 2002; Ullah et al., 2003). Interestingly, the 523 assembly of a G-protein interactome has revealed that GPA1 interacts with proteins that are 524 involved in the modulation of cell wall composition or structure (Klopffleisch et al., 2011), like 525 those we reported misregulated in the adt3 proteome (Table I), providing a possible link 526 between G proteins and morphogenesis. ADT3 is part of a signaling module that includes 527 GPA1 and GCR1, whose activation by blue light leads to Phe synthesis (Warpeha et al., Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 18 528 2006; 2008). gpa1 and gcr1 mutants are deficient in the production of Phe-derived 529 compounds, and share a high degree of phenotypic similarity with adt3, including reduced 530 number of stomata, broader cotyledons and poorly developed chloroplasts (Ullah et al., 2001; 531 Zhang et al., 2008; Nilson and Assmann, 2010), which can be rescued by supplying 532 exogenous Phe (Fig. 3; Suppl. Fig. S10). We therefore conclude that ADT3-derived Phe 533 could function as liaison between signaling and development at the early stages of seed 534 establishment. 535 536 CONCLUSIONS 537 In this study, We propose a simple model for ADT3 action, and the subsequent Phe produced 538 (Fig. 7, inset epidermis). We show that ADT3 localizes mainly in the cytosol of cotyledon cells 539 at the transition from seed to seedling, therefore placing Phe biosynthesis outside the plastid. 540 The existence of an extra-plastidic pathway for Phe biosynthesis was previously speculated 541 in Arabidopsis (d'Amato et al., 1984; Benesova and Bode, 1992; Eberhard et al., 1996) and 542 evidence of a cytosolic route for Phe production was recently reported in petunia (Yoo et al., 543 2013). Later in development, ADT3 appears to be localized in the chloroplast of green 544 tissues, suggesting that ADT3 localization appears to be age- and light-dependent; further 545 investigation will be required to unravel the molecular dynamics of ADT3 function. 546 Our work opens a window into an elusive phase of plant life, when the seedling has 547 emerged from the seed coat but is not yet capable to carry on photosynthesis. We 548 demonstrated that cytosolic Phe plays a critical role during the transition from heterotrophy to 549 autotrophy by protecting the cells from oxidative damage, providing substrates for defense 550 and, possibly, by fueling energy pathways. In laboratory conditions this transition is quite fast, 551 since germination is routinely achieved by planting seeds on synthetic medium in light 552 conditions; however, in natural environments variables such as light limitation due to dense 553 vegetation could extend the transition as well as the agricultural practice of burying seeds 554 deeper than natural dispersal, making accumulation of cytosolic Phe critical for survival. 555 Recently, a molecular switch that regulates allocation of resources to growth and to defense 556 has been described where the increase in uncharged tRNAPhe accumulation as a 557 consequence of the imbalance in Phe metabolism that accompanies pathogen challenge 558 switches off primary growth and development by repressing the expression of chloroplast 559 proteins and switch on the cellular program that lead to systemic acquired resistance (SAR) 560 (Pajerowska-Mukhtar et al., 2012). Through a similar mechanism, Phe availability could play Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 19 561 a critical role in etiolated seedlings by providing a physical, metabolic and defense support to 562 the emerging embryo. 563 564 MATERIALS AND METHODS 565 Chemicals. All chemicals, unless otherwise noted, were obtained from Sigma (St. Louis, MO). 566 Plant materials, seed stocks and accessions. Seeds of wild type (WT) Columbia (Col) 567 Arabidopsis thaliana, T-DNA insertion lines for AROGENATE DEHYDRATASE_3 (ADT3, 568 AT2G27820) adt3-1 (SALK_029949) and adt3-6 (SALK_071907) were obtained from the 569 Arabidopsis Biological Resource Center (Columbus, OH)(Alonso et al., 2003). Double mutant 570 (acc. CS8602) of CYTOCHROME P450 84A1/FERULIC ACID 5 HYDROXYLASE 1 (FAH1-7; 571 At4g36220) (Landry et al., 1995) and CHALCONE SYNTHASE/TRANSPARENT TESTA 4 572 (TT4-1; At5g13930) (Li et al., 1993) were an appreciated gift from Wendy Peer, University of 573 Maryland College Park. The mutant lines are homozygous null for the reported insertions. 574 The mutant speechless (SPCH; At5g53210) was an appreciated gift from Keiko Torii, 575 University of Washington, Seattle; mute (At3g06120) was an appreciated gift from Dominique 576 Bergmann, Stanford University. 577 Plant growth conditions for experiments. Seeds of Arabidopsis thaliana WT or T-DNA 578 insertion mutants were sown on 0.5X MS medium with no added sugars, hormones, vitamins 579 or other nutrients in complete darkness using a dim green safelight to prevent 580 photomorphogenesis (Mandoli and Briggs, 1982; Orozco-Nunnelly et al., 2014). For some 581 experiments, seeds were sown on top agarose supplemented with chemical (500μM NH3Ac, 582 500μM L-Phe, 500μM L-Tyr, 500μM L-Trp, L-Asparagine) then grown for 6 d. For other 583 experiments, 4-d post-sowing on 0.5X MS top agarose, disks bearing about 30 seedlings 584 were moved to filter paper soaked with 0.5X MS (control) or 1% sucrose or 1% mannitol (final 585 concentration in 0.5X MS), then analyzed after 48 h. Most replicates n=30 unless specified. 586 Contrast microscopy: deconvoluting. Living seedlings were mounted in sterile water to 587 be viewed on a Zeiss Observer.Z1 deconvoluting microscope utilizing the 20X objective or 588 63X (oil immersion) with DAPI excitation to view traditional fluorescence, or optical apotome 589 utilizing XCite 120 LED/Lumen Dynamics DAPI, FITC, CY5 LEDs, as managed by Zen pro 590 software (2012). Images were captured on a high resolution Axio503 mono camera. At least 591 30 seedlings were viewed per experimental condition for experiments unless described 592 otherwise (20-40 seedlings per experimental condition for analysis). A 150 μm square Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 20 593 superimposed on images was analyzed to quantitate cell-type numbers, and cell area was 594 quantified using Image J (http://imagej.nih.gov/ij/). 595 Cellular stress assays. 4 d after moving to 20oC, dark-grown seedlings were placed on 596 sterile filter paper for 3 h with either 0.5X MS (control), potassium iodide (KI), Phe, or the 597 analog para-DL-flouro-Phenylalanine (Conway et al., 1963). Seedlings received an empircally 598 determined sublethal (3 min) dose of 254nm (UV methods described in Warpeha et al., 599 2008). Young cotyledons emit natural fluorescence after absorption of UV (DAPI)(Warpeha et 600 al., 2008) or blue/blue-green (FITC)(Tattini et al., 2004; Warpeha et al., 2008) irradiation, so 601 these LEDs were used to collect fluorescence information. The specific cell permeable dye 602 CellRox Deep RedTM (Life Technologies, Grand Island, NY) emitting in the red/far red region 603 was used as recommended by the manufacturer to detect ROS species dark-grown 604 seedlings (Cy5 channel, false-colored pink) (Ghura et al., 2016). In brief, dark-grown 605 seedlings of WT or adt3 (each replicate 30 seedlings) on d 6 were exposed to a sublethal 606 treatment of UV-C (254nm), then cut mid hypocotyl to be immediately immersed in 400 μL of 607 5 μM CellRoxTM in sterile DMSO in a glass well dish in darkness. Samples were orbitally 608 rotated (50 rpm) for 30 min. At that time CellRoxTM reagent was removed by p200 pipet tip to 609 avoid removing cut seedlings, then solution immediately replaced by sterile 1.0X PBS (pH 610 7.2) for 3 x 3 min washes under the same rotation. After washing, the PBS was removed and 611 seedlings were rinsed 1 time in sterile water for 3 min, then mounted in sterile water on glass 612 slides for immediate microscopy. Cotyledons were imaged at 63X (oil immersion), and for 613 some experiments 20X objectives with DAPI, FITC and Cy5 LEDs in 1 μm optical sections by 614 apotome (microscope described above). At least 20 seedlings were viewed per replicate, and 615 all exposure settings were based on the WT untreated cotyledons (that setting was used for 616 all seedling conditions and genotypes). For seedlings exposed to Phe, seedlings were moved 617 on d 6 to 500μM L-Phe for 3 h, then experienced the same conditions for CellRoxTM reagent 618 experiments. All growth conditions and seedling manipulations were identical for Singlet 619 Oxygen Sensor GreenTM (SOSG)(Thermo Fisher, Rockford, IL), however the preparations of 620 the reagent were as follows. SOSG is provided as a solid, and was dissolved as directed in 621 methanol to 5 mM. The working solution was utilized at 10 μM in 1X PBS (pH 7.2). Identically 622 to CellRox experiments, seedlings were immersed into the reagent immediately post- 623 irradiation, then washed in the same way, then mounted in sterile water for microscopy. 624 Microscopy was handled in the same way as for CellRox reagent, except that SOSG 625 responds to singlet oxygen by causing a large fluorescence that can be captured by FITC. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 21 626 The LEDs utilized for these experiments were DAPI, FITC and Texas Red. As for CellRox, 627 the WT unirradiated was the replicate used to determine the best exposure upon which all 628 other samples used the identical exposure settings. From all fluorescence experiments 629 epidermal pavement cells were traced in ImageJ and the fluorescence level (artificial units) 630 was determined, then compared (Ghura et al., 2016). A minimum of 6 cells per cotyledon of 631 4-6 seedlings per replicate was assessed utilizing Prism (see Statistics). 632 Nile Red staining. 6-d-old dark-grown seedlings were cut into 1mM Nile Red stain 633 (saturated solution in acetone as directed by manufacturer (Sigma); working solution in 1X 634 PBS pH 7.2 made immediately before use, maintained in darkness throughout) and 635 incubated for 30 min under rotation, with all same experimental handling, timing as SOSG. 636 Seedlings then were mounted in sterile water as described for SOSG, then visualized by 637 optical sectioning (described above) at 20X magnification using FITC (false-colored gold). 638 n=30 each replicate. 639 Cloning. Standard molecular biology techniques and the Gateway system (Invitrogen) 640 were used for all cloning procedures as described (Orozco-Nunnelly et al., 2014). The 819 bp 641 ADT3 promoter fragment was cloned into pDONR P4-P1R (Invitrogen) and the ADT3 ORF 642 fragment was cloned into pENTR/D-TOPO (Invitrogen). pGreen (Hellens et al., 2000) was 643 used as the destination vector for the construct ADT3::ADT3-GFP or ADT3::ADT3-CFP. T3 644 homozygous transformed seedlings were used in experiments. 645 Visualization of transgene expression on spinning disk confocal. Transgenic and 646 untransformed 4-d-old dark-grown seedlings were mounted live and immediately viewed on 647 an Andor WD Spinning Disk confocal system using iQ3 software as described (Orozco- 648 Nunnelly et al., 2014). 1nm width LEDs used included 405nm (DAPI, Blue), 488nm (GFP, 649 Green), 445nm (CFP, magenta), or 561nm (Red). Images were captured on 30X or 60X as 650 described, with 1μm optical section thickness. Images were prepared with ImageJ software 651 (NIH, Bethesda, MD). n=20 each replicate. 652 TEM. Seedlings were harvested into 0.5X Karnovsky’s fixative for overnight storage at 653 4oC, buffer washed then fixed in 1% OSO4 for 2 h. After washes in 0.1M Cacodylate buffer, 654 the samples were embedded in 3% SeaPrep agar, dehydrated in a 10-100% ethanol series 655 and infiltrated with Spurr’s resin, which was let to polymerize at 70oC for 2 d. Thin sections 656 (between 70-80nm) of at least 5 seedlings for each sample were cut on a Leica UC7 657 ultramicrotome, collected on a 200 mesh formvar/carbon coated copper grid (Ted Pella, Inc.) Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 22 658 and stained with aq UA for 2 h and lead citrate for 5 min. The grids were viewed in a JEOL 659 JEM 1400 TEM and images taken at 120KV. 660 Toludine Blue staining. On d 4, dark-grown seedling islands of top agarose were 661 transplanted to 0.5XMS in phytatrays with disk annealed by 200μL top agarose drop to the 662 tray. The bottom of the phytatray was covered with sterile water only or 0.5XMS medium and 663 on d 6, the phytatray was filled with Toludine Blue solution to cover seedlings (Tanaka et al., 664 2004). After 3 min seedlings were washed and immediately imaged on bright field Zeiss V.2 665 Stereo dissecting scope. 666 Statistics. Data shown in figures were entered into Prism v. 5.0 (GraphPad Software, 667 Inc., GraphPad.com), where mean and SE were calculated for relevant experiments. 668 Unpaired T test with Welch’s correction were used to assess significance. 669 Protein Extraction, Digestion, Labeling and Sub-fractionation. Protein was extracted 670 from each biological replicate of 6-d-old dark-grown WT and adt3 liquid nitrogen ground 671 samples, as previously described (Alvarez et al., 2011). The pellets were then dissolved in 672 150 µL of 3 M urea, 0.5 M triethylammonium bicarbonate (TEAB), pH 8.5, and assayed using 673 the CB-X™ protein assay kit (G-Biosciences, St Louis, MO). Each sample was reduced in a 674 final concentration of 5 mM tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) for 1 h at 675 37 °C and alkylated using 10 mM iodoacetamide for 30 min at 22 °C in the dark. Aliquots 676 containing 40 µg of protein were diluted to 1 M urea and digested with trypsin at a 1:20 677 enzyme:protein ratio for 16 h at 37 °C. Samples were desalted using 1 cc Sep-Pak® Vac 678 C18 solid-phase extraction (SPE) columns (Waters Ltd, Milford, MA) and vacuum centrifuged 679 (Centrivap™, Labconco, Kansas City, MO) to dryness. Stable isotope labeling was carried 680 out with TMT-6plex reagents (Thermo Fisher Scientific, Rockford, IL) according to the 681 manufacturer’s instructions and the six samples were combined, vacuum centrifuged and 682 desalted using SPE. Sub-fractionation was carried out on a 3100 OFFGEL™ fractionator 683 (Agilent Technologies, Santa Clara, CA) using pH 3-10 gradient strips and ampholytes 684 according to the manufacturer’s guidelines, but without the incorporation of glycerol. Focusing 685 was carried out for 50 kWh with a current limit of 50 µA and voltage limit of 8000 V. Fractions 686 were fully dried. 687 Protein identification and quantification by liquid chromatography–tandem mass 688 spectrometry (LC-MS/MS). LC-MS/MS was carried out on a LTQ-Orbitrap Velos Pro 689 (ThermoFisher Scientific, Waltham, MA) as previously described (Alvarez et al., 2013; 690 Alvarez et al., 2014) coupled with a U3000 RSLCnano HPLC (ThermoFisher Scientific, Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 23 691 Waltham, MA). The dried OFFGEL fractions were dissolved in 5% acetonitrile, 0.1% formic 692 acid and a portion loaded onto a C18 trap column (PepMap100, 300µm ID × 5mm, 5µm 693 particle size, 100Å; ThermoFisher Scientific, Waltham, MA) at a flow rate of 15 µL/min for 4 694 min equilibrated with 2% acetonitrile, 0.1% formic acid. Peptide separation was carried out on 695 a C18 column (Acclaim PepMap RSLC, 15cm × 75μm nanoViper™, C18, 2μm, 100 Å, 696 ThermoFisher Scientific) at a flow rate of 0.3μL/min and the following gradient: Time = 0-3 697 min, 2% B isocratic; 3-12 min, 2-15% B; 12-84 min, 15-38% B; 84-93 min, 38-50% B; 93-97 698 min 50-90% B. Mobile phase A, 0.1% formic acid; mobile phase B, 0.1% formic acid in 80:20 699 acetonitrile:water. The Orbitrap mass analyzer was operated as previously described 700 (Alvarez et al, 2014) except for the exclusion duration, which was set at 90 s, and the 701 minimum MS ion count for triggering MS/MS to 50,000 counts. 702 Database search and Data mining for proteome. Data processing was automated 703 using Mascot Daemon (Matrix Science, London, UK). Mascot Distiller, version 2.4.3.3 64 bit 704 (Matrix Science, London, UK) was used to create mgf files which Mascot Server, version 2.4 705 (Matrix Science, London, UK) searched against the TAIR10 Arabidopsis thaliana database 706 (35,386 entries) to identify and quantify the proteins with a false discovery rate of 1 %. The 707 search parameters used have been previously described (Alvarez et al., 2014) except for the 708 fixed modifications which were set as TMT (N terminal) and TMT (K). Only proteins with at 709 least two peptides were used for the analysis. Peptide and protein ratios were calculated 710 using the weighted method in Mascot where the intensity values of the set of peptides are 711 summed and the protein ratio(s) calculated from the summed values. Normalization of the 712 ratios was performed using the total labeling intensity of each species (summed intensities). 713 The ratios and standard deviations for all combinations of the replicates adt3/WT were 714 reported. An overall ratio for a protein hit was only reported if the minimum number of two 715 peptide matches was achieved. If the ratios for the peptide matches were not consistent with 716 a sample from a normal distribution, the ratios were not used further in the analysis. Only 717 protein ratios statistically different from 1 (p value < 0.05), detected from the replicates, were 718 averaged. Only proteins for which there were at least 3 protein ratios, of which at least one 719 was associated with each control for each of the three mutant replicates, were used for 720 further analyses. For these proteins, the standard deviation and coefficient of variation were 721 calculated. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 24 722 The proteome was analyzed using the bioinformatics platform Virtual Plant 723 (http://virtualplant.bio.nyu.edu/cgi-bin/vpweb/)(Katari et al., 2010) and the KEGG database 724 (http://www.genome.jp/kegg/). 725 726 727 FIGURE LEGENDS 728 729 Figure 1. ADT3 localizes predominantly in the cotyledon of young, dark grown 730 seedlings. 731 Expression of ADT3::ADT3-CFP was analyzed in live 4-d-old adt3 seedlings by SD confocal 732 microscopy. A. (Top panels) In this view, CFP fluorescence (445 nm, false-colored magenta) 733 is detected in developing mesophyll cells of the cotyledon and in the SAM (yellow arrow) 734 (1μm thick optical slice). Autofluorescence from developing plastids (448nm, false-colored 735 green) is visible. Merged images of 445 and 488 indicate that ADT3 localizes mainly in the 736 cytosol. B. In the epidermal layer view, ADT3::ADT3-CFP accumulates in the pavement cells 737 (PC) and young guard cell (GC) lineage but not in mature GC. The signal captured by 488 is 738 due to accumulation of polyphenols (flavonoids) autofluorescence in the epidermis. Images 739 are representative, 60X. n=20. Scale bar = 50μm. 740 741 Figure 2. ROS levels are elevated in adt3 mutant seedlings compared to WT. 742 The cotyledon epidermis cells of live 6-d-old dark-grown seedlings incubated with 743 CellRoxTM Deep Red cell-permeable fluorescent dye are shown (merged DAPI, FITC and 744 Cy5). WT (top panels) and adt3-1 (middle panels) seedlings were either mock irradiated 745 (unirradiated control)(left panels) or irradiated with 254nm (+UV-C)(right panels), then 746 immediately immersed in CellRoxTM reagent, which emits fluorescence in Cy5 in presence of 747 ROS. adt3-1 were also treated with 500 μM Phe (+Phe) for 3h and then irradiated with 748 254nm (+UV-C) (bottom left panel). After washes and mounting in sterile water, seedlings 749 were imaged at 1μm optical slice thickness on a deconvoluting microscope in the epidermal 750 plane. CellRoxTM signal is low in untreated WT cells, and increases as a result of UV-C 751 irradiation, while signal is evident in unirradiated adt3-1 cells. Feeding Phe to adt3-1 752 seedlings before irradiation prevented ROS accumulation (bottom left panel and bar graph). 753 Scale bar= 10μm. Cy5 fluorescence from excited CellRoxTM (false-colored pink) was 754 quantified in PC (relative fluorescence, i.e. artificial units) (bottom right panel). In the bar Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 25 755 graph each value represents a minimum of 6 cells of 4 replicates, minimum 20 representative 756 seedlings viewed per replicate. Error bars are SEM. * = p<0.05. 757 758 Figure 3. Epidermal defects of adt3-1 seedlings. 759 A. Epidermis of WT (top) and adt3-1 live cotyledons (Cot; bottom) viewed on a deconvoluting 760 microscope (DAPI contrast). GC lineage (meristemoid and GC) cells appear dark gray, and 761 PCs appear light gray. Defects are prevented by inclusion of Phe in medium (right panels). 762 Scale bar = 25μm. B. Measurement of PC area and number, cotyledon width and GC number 763 in WT and adt3-1 without, and with added Phe. Cartoon indicates structure measured. Error 764 bars are SEM. ****<.0001. n=4 replicates (20-40 seedlings per experimental condition). 765 766 Figure 4. Ultrastructural phenotype of adt3-1 epidermal and mesophyll cells. 767 A. (Left) TEM micrographs of sections perpendicular to the adaxial surface of the cotyledons 768 of 6-day old, dark-grown WT (top) and adt3-1 (bottom) seedlings. (Right) Details of WT (a, b 769 and c) and adt3-1 (d, e and f) micrograph: cytoplasm of epidermal cells (a and c), plastids (b 770 and e) and cytoplasm of mesophyll cells (note the absence of oil bodies in f). n=5 seedlings 771 (~100 cells). Scale bar = 5 μm. 772 B. Adaxial side view of mesophyll cells in the cotyledons of 6-d-old dark grown WT (left), 773 adt3-1 (middle) and adt3-1 + Phe (right) seedling after staining with Nile Red (false-colored 774 gold). Merges of DAPI (blue), FITC (gold) and CY5 (pink) are shown. n=3 (30 seedlings). 775 Scale bar = 50 μm. 776 777 Figure 5. Defective cuticle formation, altered permeability and aberrant patterning of 778 GC in adt3-1 seedlings. 779 A. Ultrastructure of the cuticle of adt3-1 epidermis. TEM micrographs of WT (top) and adt3-1 780 (bottom) epidermis show deposition of electron-opaque material within adt3-1 cell wall (red 781 triangles). 782 B. Increased permeability of adt3-1 epidermis under hyposmotic treatment. TB penetration in 783 dark grown 6-d-old WT (top), and adt3-1 (bottom) seedlings after 48 h incubation in sterile 784 water. Scale bar = 50μm. 785 C. 16:8 light-grown, 6-d-old adt3-1 seedlings exhibit abnormal stomatal development; GC in 786 focal plane are circled. Scale bar = 25μm. 787 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 26 788 Figure 6. Effect of exogenous sucrose, Asn, and NH3Ac supply on adt3-1 epidermis. 789 A. GC number phenotype is rescued by sucrose, but not cotyledon width and PC defects of 790 adt3-1 seedlings. 4-d-old, dark grown seedlings were transferred to 1% sucrose and 791 epidermal phenotypes were quantified 48 h later (6-d-old). n=20-25. **** <.0001. 792 B. Asn supply rescues cotyledon width and PC number phenotypes but not the GC lineage in 793 adt3 seedlings. L-asparagine (Asn) was included in the top agarose at planting. Epidermal 794 phenotypes were measured on d 6. n=20-25. **** <.0001. 795 C. Asn and NH3Ac supply restores cotyledon morphology and oil bodies in adt3 cotyledons. 796 DAPI contrast images (top; Scale bar = 25μm), and Nile Red signal (bottom; Scale bar = 797 50μm) of 6-d-old, dark grown adt3 seedlings. Nile Red signal was false-colored gold. Merge 798 of DAPI (blue), FITC (gold) and CY5 (pink) is shown. 799 800 Figure 7. Model indicating multiple potential roles of Phe, mainly produced in the cytosol by 801 ADT3 in Arabidopsis cotyledons at the seed-to-seedling transition. 802 803 SUPPLEMENTAL FILES (SFigures and STables) 804 805 SFIGURES 806 SFigure S1. ADT3::ADT3-GFP restores adt3-1 cotyledon epidermis to WT appearance, and 807 confers survival to UV stress. 808 SFigure S2. Expression of ADT3-GFP fusions in adt3-1 and WT background and 809 ADT3::ADT3-GFP in the SAM. 810 SFigure S3. ROS detection in adt3-6 mutant by CellRoxTM. 811 SFigure S4. Singlet Oxygen Species (SOS) detection in adt3 mutant seedlings incubated 812 with Singlet Oxygen sensor GreenTM (SOSG). 813 SFigure S5. ROS levels detected by CellRoxTM from unirradiated and UV-C irradiated WT 814 and adt3-1 seedling cotyledons before and after pretreatment with Phe, the ROS scavenger 815 KI and a Phe analog. 816 SFigure S6. Epidermal defects in adt3-6 seedlings are rescued by Phe. 817 SFigure S7. 818 stomatal lineage mutants do not respond to Phe like adt3-1 mutants. 819 SFigure S8. Experiments indicate mannitol does not rescue GC lineage progression. Phenotypic defects of adt3-1 seedlings not complemented by Tyr or Trp; Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 27 820 SFigure S9. Developing chloroplast ultrastructural phenotype rescue in adt3 mesophyll cells 821 by inclusion of Phe in planting medium, 822 SFigure S10. gpa1 and gcr1 mutants have cotyledon phenotypes that can be rescued by 823 Phe. 824 825 S TABLES 826 STable 1. Proteome data of 6-d dark-grown adt3-1 mutant compared to WT. 827 STable 2. Plant Ontology analysis for for upregulated proteins in adt3 proteome. 828 STable 3. GO term analysis for biological process for adt3 upregulated proteins in the 829 "epidermal cell" Plant Ontology category. 830 STable 4. GO term analysis for cell components for upregulated proteins in adt3 proteome. 831 STable 5. GO term analysis for biological process for adt3 upregulated proteins in the 832 “plasma membrane” category. 833 STable 6. GO term analysis for biological process for adt3 upregulated proteins in the 834 “vacuole” category. 835 STable 7. GO term analysis for biological process for adt3 upregulated proteins in the “cell 836 wall” category. 837 STable 8. Plant Ontology analysis for for downregulated proteins in adt3 proteome. 838 STable 9. GO term analysis for molecular function for adt3 downregulated proteins in the 839 “epidermal cell” Plant Ontology category. 840 STable 10. GO term analysis for cell components for downregulated proteins in adt3 841 proteome. 842 STable 11. GO term analysis for biological process for downregulated proteins in adt3 843 proteome. 844 845 ACKNOWLEDGEMENTS: We thank Jennifer Baek, Ashley Williams, Yang Chen, Huini Wu, 846 and Nadia Kukuruza for assistance with experiments; Dr. Jeremy Lynch and Dr. Teresa 847 Orenic for assistance with confocal, and deconvoluting microscopy, respectively; and, Jack 848 Gibbons for EM services featured in Supplementary data. We thank Stephen MacFarlane, 849 Sharmon Knecht, and especially Bobbie Schneider for TEM (Fred Hutchinson Cancer Center, 850 Seattle, WA). We thank John Cason for artwork services herein, except Fig. 7. We thank Dr. 851 Lon Kaufman, now at CUNY Hunter College, and Dr. Gary Gardner, University of Minnesota, 852 for helpful discussion. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. 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Nature Communications 4: 2833, DOI: 10.1038/ncomms3833 Zhang L, Hu G, Cheng Y, Huang J (2008) Heterotrimeric G protein alpha and beta subunits antagonistically modulate stomatal density in Arabidopsis thaliana. Dev Biol 324: 68-75 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 38 1156 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 39 Table 1. Gene Ontology (GO) term analysis of UP-regulated and DOWN-regulated proteins in adt3 proteome. UP-regulated proteins in adt3 mutant Plant Ontology (PO) Term p-value guard cell 1.28*10-78 shoot epidermal cell 2.08*10-77 epidermal cell 1.33*10-76 Transport 1.17*10-16 carboxylic acid/oxoacid metabolic process 6.54*10-10 fatty acid metabolic process 3.42*10-06 GO Term plasma membrane Transport Vacuole Transport cell wall cell wall organization or biogenesis xylan catabolic process glucan metabolic process hydrolase activity Growth primary metabolic process p-value 5.70*10-109 5.92*10-27 2.05*10-34 2.03*10-15 7.98*10-32 3.76*10-06 1.18*10-3 4.37*10-3 5.39*10-14 2.78*10-11 1.94*10-14 DOWN-regulated proteins in adt3 mutant PO Term shoot epidermal cell guard cell epidermal cell p-value 1.19*10-86 4.38*10-86 3.35*10-84 oxidoreductase activity GO Term Plastid plastid stroma envelope thylakoid stromule plastoglobule nitrogen compound metabolic process primary metabolic process Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 1.92*10-14 p-value 5.61*10-128 6.38*10-127 2.77*10-50 6.28*10-46 1.18*10-19 3.15*10-10 1.8*10-17 7.25*10-25 40 Table 2. KEGG pathway analysis for up- and down-regulated proteins in adt3 mutant. Number of genes Total* DOWN- UP- regulated regulated Carbohydrate metabolism Glycolysis / Gluconeogenesis 29 19 10 Pyruvate metabolism 27 14 13 Starch and sucrose metabolism 27 6 21 Glyoxylate and dicarboxylate metabolism 26 23 3 19 4 15 Ascorbate and aldarate metabolism 15 7 8 Pentose and glucuronate interconversions 15 2 13 Citrate cycle (TCA) 14 9 5 Pentose phosphate pathway 11 10 1 Fructose and mannose metabolism 11 7 4 Inositol phosphate metabolism 7 2 5 Galactose metabolism 6 1 5 Carbon fixation in photosynthetic organisms 32 27 5 Oxidative phosphorylation (ATP synthase) 17 6 11 Photosynthesis 11 10 1 Nitrogen metabolism 9 8 1 Sulfur metabolism 6 6 0 Amino sugar and nucleotide sugar metabolism Energy metabolism * number of input genes assigned by the KEGG database. 1157 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 41 Para_Fig1 A 445 B 445 488 488 merge merge Figure 1. ADT3 localizes predominantly in the cotyledon of young, dark grown seedlings. Expression of ADT3::ADT3-CFP was analyzed in live 4-d-old adt3-1 seedlings by SD confocal microscopy. A. (Top panels) In this view, CFP fluorescence (445 nm, false-colored magenta) is detected in developing mesophyll cells of the cotyledon and in the SAM (yellow arrow) (1μm thick optical slice). Autofluorescence from developing plastids (448nm, false-colored green) is visible. Merged images of 445 and 488 indicate that ADT3 localizes mainly in the cytosol. B. In the epidermal layer view, ADT3::ADT3-CFP accumulates in the pavement cells (PC) and young guard cell (GC) lineage but not in mature GC. The signal captured by 488 is due to polyphenols (flavonoids) autofluorescence in the epidermis. Images are representative, 60X. n=20. Scale bar = 50μm. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Para_Fig2 WT WT + UV-C adt3-1 adt3-1 + UV-C adt3-1 + Phe + UV-C fluorescence intensity from CellRox Untreated 50 40 wt adt3-1 UV-C Phe+UV-C * * 30 20 10 0 Figure 2. ROS levels are elevated in adt3 mutant seedlings compared to WT. The cotyledon epidermis cells of live 6-d-old dark-grown seedlings incubated with TM Deep Red cell-permeable fluorescent dye are shown (merged DAPI, FITC and CellRoxTM Cy5). WT (top panels) and adt3-1 (middle panels) seedlings were either mock irradiated (unirradiated control)(left panels) or irradiated with 254nm (+UV-C)(right panels), then TM reagent, which emits fluorescence in Cy5 in presence immediately immersed in CellRoxTM of ROS. adt3-1 were also treated with 500μM Phe (+Phe) for 3h and then irradiated with 254nm (+UV-C) (bottom left panel). After washes and mounting in sterile water, seedlings were imaged at 1μm optical slice thickness on a deconvoluting microscope in the epidermal TM signal is low in untreated WT cells, and increases as a result UV-C irradiplane. CellRoxTM ation, while signal is evident in unirradiated adt3-1 cells. Feeding Phe to adt3-1 seedlings before irradiation prevented ROS accumulation (bottom left panel and bar graph). Scale bar TM (false-colored pink) was quantified in = 10μm. Cy5 fluorescence from excited CellRoxTM PC (relative fluorescence, i.e. artificial units) (bottom right panel). In the bar graph each value represents a minimum of 6 cells of 4 replicates, minimum 20 representative seedlings viewed per replicate. Error barsfrom areonSEM. = p<0.05. Downloaded June 17,*2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Para_Fig3 A WT + Phe WT **** 60 40 20 width of cotyledon day 6 0 **** 400 350 300 250 stomata 200 +P he ad t3 ad t3 0 adt3-1 adt3-1+Phe Ph e WT W T+ ad t3 +P he U nt r adt3-1 adt3-1+phe ad t3 w tU nt r WT **** 10 un tr 200 20 ad t3 ad t3 300 GC (number) ad t3 **** Ph e ce lls W T ce lls 400 W T 30 500 U nt Cot width (µm) PC (number) 80 W T B adt3-1 + Phe cell # PC area (µm2) adt3-1 Figure 3. Epidermal defects of adt3-1 seedlings. A. Epidermis of WT (top) and adt3-1 live cotyledons (Cot; bottom) viewed on a deconvoluting microscope (DAPI contrast). GC lineage (meristemoid and GC) cells are dark gray, and PCs appear light gray. Defects are prevented by inclusion of Phe in medium (right). Scale bar= 25µm. B. Measurement of PC area and number, cotyledon width and GC number in WT and adt3 without and with added Phe. Cartoon indicates structure measured. ****<. 0001. n=4 replicates (20-40 seedlings per experimental condition). Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Para_Fig4 A a WT b c d e f adt3-1 B WT adt3-1 adt3-1 + Phe Figure 4. Ultrastructural phenotype of adt3-1 epidermal and mesophyll cells. A. (Left) TEM micrographs of sections perpendicular to the adaxial surface of the cotyledons of 6-day old, dark-grown WT (top) and adt3-1 (bottom) seedlings. (Right) Details of WT (a, b and c) and adt3-1 (d, e and f) micrograph: cytoplasm of epidermal cells (a and c), plastids (b and e) and cytoplasm of mesophyll cells (note the absence of oil bodies in f). n=5 seedlings (~100 cells). Scale bar = 5µm. B. Adaxial side view of mesophyll cells in the cotyledons of 6-d-old dark grown WT (left), adt3-1 (middle) and adt3-1 + Phe (right) seedling after staining with Nile Red (false-colored gold). Merges of DAPI (blue), FITC (gold) and CY5 (pink) are shown. n=3 (30 seedlings). Scale bar= 50µm. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Para_Fig5 A WT adt3-1 B WT adt3-1 C WT adt3-1 Figure 5. Defective cuticle formation, altered permeability and aberrant patterning of GC in adt3-1 seedlings. A. Ultrastructure of the cuticle of adt3-1 epidermis. TEM micrographs of WT (top) and adt3-1 (bottom) epidermis show deposition of electron-opaque material within adt3-1 cell wall (red triangles). B. Increased permeability of adt3-1 epidermis under hyposmotic treatment. TB penetration in dark grown 6-d-old WT (top), and adt3-1 (bottom) seedlings after 48 h incubation in sterile water. Scale bars= 50µm. C. 16:8 light-grown, 6-d-old adt3-1 seedlings exhibit abnormal stomatal development; GC in focal plane are circled. Scale bar = 25µm. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Para_Fig6 A B C 450 **** 400 **** 300 300 250 20 20 **** +A sp ad t3 60 40 adt3-1 + Asn 0 0 30 15 **** **** ad t3 +a sp ad t3 un tr su + 20 W T **** ad t3 ad t3 un tr 25 W T adt3-1 + NH3Ac 10 10 5 0 0 WT adt3-1 adt3-1+Asn +a sp ad t3 un tr 4d ad t+ su c ad t3 W T WT adt3-1 adt3-1+Suc W T GC (number) un tr **** W T r nt U T **** ad t3 +S u 80 40 20 adt3-1 + NH3Ac 250 Data 10 W 60 adt3-1 + Asn **** 400 350 80 PC (number) 450 350 ad t3 Cot width (mm) Data 8 Figure 6. Effect of exogenous sucrose, Asn, and NH3Ac supply on adt3-1 epidermis. A. GC number defect phenotype is rescued by sucrose (Suc), but not cotyledon width and PC defects of adt3-1 seedlings. 4-d-old, dark grown seedlings were transferred to 1% sucrose and epidermal phenotypes were quantified 48 h later (6-d-old). n=20-25. **** <.0001. B. Asn supply rescues cotyledon width and PC number phenotypes but not the GC lineage in adt3-1 seedlings. L-asparagine (Asn) was included in the top agarose at planting. Epidermal phenotypes were measured on d 6. n=20-25. **** <.0001. C. Asn and NH3Ac supply restores cotyledon morphology and oil bodies in adt3-1 cotyledon. DAPI contrast images (top; Scale bar = 25µm), and Nile Red signal (bottom; Scale bar = 50µm) of 6-d-old, dark grown adt3-1 seedlings. Nile Red signal was falsecolored gold. Merge of DAPI (blue), FITC (gold) and CY5 (pink) is shown. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Para_Fig7 ADT3 (cytosol) Phe (cytosol) CELL FATE Figure 7. Model indicating multiple, potential roles of Phe, mainly produced in the cytosol by ADT3 in Arabidopsis cotyledons at the seed-to-seedling transition. Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 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