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Plant Physiology Preview. Published on August 4, 2015, as DOI:10.1104/pp.15.00991 1 Running Head: ES1 affects water loss 2 3 Corresponding authors: 4 Dali Zeng 5 6 State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 7 310006, China 8 9 Tel: (+0571)6337 0537 10 Fax: (+0571)6337 0389 11 E-mail: [email protected] 12 13 Research Area: Genes, Development and Evolution 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Copyright 2015 by the American Society of Plant Biologists 14 15 EARLY SENESCENCE 1 Encodes a SCAR-like Protein2 that Affects Water 16 Loss in Rice 17 Yuchun Rao†, Yaolong Yang†, Jie Xu†, Xiaojing Li, Yujia Leng, Liping Dai, Lichao Huang, 18 Guosheng Shao, Deyong Ren, Jiang Hu, Longbiao Guo, Jianwei Pan, Dali Zeng* 19 20 State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 21 310006, China (Y.R., Y.Y., J.X., Y.L., L.D., L.H., G.S., D.R., J.H., L.G. and D.Z.); College of 22 Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China (Y.R., X.L 23 and J.P.); Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of 24 Education, Jiangxi Agricultural University, Nanchang 330045, China (Y.Y. and J.X.) 25 26 27 One sentence summary: 28 The SCAR/WAVE-like protein2 affects water loss by regulating stomatal density in rice 29 30 Total word count: 10652 31 Total characters (including space): 67804 32 2 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 33 34 35 Footnotes 36 37 1 38 (31201183, 31221004, 31171531, 31171520, and 91435105), the State Key Basic Research 39 Program (2013CBA01403), the Ministry of Agriculture of China for transgenic research 40 (No.2013ZX08009003-001), and the China Postdoctoral Science Foundation 41 (2014M561108). This work was supported by grants from the National Natural Science Foundation of China 42 43 44 *Corresponding author e-mail: [email protected] 45 46 47 48 1 49 this article in accordance with the policy described in the Instructions for Authors 50 (www.plantphysiol.org) is: Dali Zeng ([email protected]). 51 † These authors contributed equally to this work. The author responsible for the distribution of materials integral to the findings presented in 52 53 54 55 56 57 58 59 3 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 60 61 62 Abstract 63 The global problem of drought threatens agricultural production and constrains the 64 development of sustainable agricultural practices. In plants, excessive water loss causes 65 drought stress and induces early senescence. In this study, we isolated a rice (Oryza sativa) 66 mutant, designated as early senescence 1 (es1), which exhibits early leaf senescence. The 67 es1-1 leaves undergo water loss at the seedling stage (as reflected by whitening of the leaf 68 margin and wilting) and display early senescence at the three-leaf stage. We used map-based 69 cloning to identify ES1, which encodes a SCAR-like protein 2, a component of the 70 SCAR/WAVE complex involved in actin polymerization and function. The es1-1 mutants 71 exhibited significantly higher stomatal density. This resulted in excessive water loss and 72 accelerated water flow in es1-1, also enhancing the water absorption capacity of the roots and 73 water transport capacity of the stems, as well as promoting in vivo enrichment of metal ions 74 co-transported with water. Expression of ES1 is higher in the leaves and leaf sheaths than in 75 other tissues, consistent with its role in controlling water loss from leaves. GFP-ES1 fusion 76 proteins were ubiquitously distributed in the cytoplasm of plant cells. Collectively, our data 77 suggest that ES1 is important for regulating water loss in rice. 78 79 Key words: ES1; water loss; SCAR; stomatal density 80 81 82 INTRODUCTION Rice is a major world-wide food crop, but it consumes more water than most crops 83 (Bruce et al., 2015), with water consumption for rice cultivation accounting for 84 approximately 65% of agricultural water usage. Rice provides a staple food for about 3 85 billion people, while using an estimated 24–30% of the world's developed freshwater 86 resources (Bouman et al., 2007). Severe water shortages restrict the expansion of rice 87 production and hinder the irrigation of existing paddy fields (Zhu et al., 2013). One effective 88 way to overcome water shortages is to reduce water loss in rice plants, thus allowing 89 cultivation of this key crop in environments with less water (Nguyen et al., 1997). 90 In plants, the stomata on the leaf surface work as the main channels for discharge of 4 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 91 water and entry of carbon dioxide, and thus strongly affect physiological processes such as 92 transpiration and photosynthesis. Previous studies showed that mutations in some genes 93 could affect stomatal density or differentiation, such as AtTMM (Yang et al., 1995), 94 AtSCRM2 (Kanaoka et al., 2008), AtSDD1(Von Groll et al., 2002), AtYODA2(Bergmann et 95 al.,2004). Stomata show a regular distribution on rice leaves (Huang et al., 2009) during plant 96 growth and development, various environmental factors affect the density and size of 97 stomata, as well as the chlorophyll contents of rice leaves. For example, rice leaves that 98 develop under water stress show substantial fewer stomata compared with leaves that develop 99 under well-watered conditions (Huang et al., 2009). Changes in stomatal density and 100 morphology affect water loss (Boonrueng et al., 2013). In rice, OsSRO1 (Similar to RCD- 101 One) enhances drought tolerance by regulating stomatal closure (You et al., 2013) while the 102 zinc finger protein DST (Drought and Salt Tolerance) functions in drought and salt tolerance 103 by adjusting stomatal aperture (Huang et al., 2009). In Arabidopsis thaliana, overexpression 104 of the Mg-chelatase H subunit in guard cells confers drought tolerance by promoting stomatal 105 closure (Tsuzuki et al., 2013). Thus, stomatal closure and low stomatal density enhance 106 drought tolerance by reducing water loss in plants. 107 The epicuticular wax layer in plants acts as the first barrier to environmental conditions 108 by reducing water loss due to transpiration and preventing plant damage due to excessively 109 strong sunlight (Riederer et al., 2001). Leaf transpiration involves both stomatal and cuticular 110 transpiration. Stomatal conductance controls stomatal transpiration, but the physicochemical 111 properties of the leaf surface mainly control cuticular transpiration. For example, the 112 composition, thickness, and microstructure of the cuticular wax affect water permeability and 113 transport (Buschhaus et al., 2012; Svenningsson, 1988; Xu et al., 1995). OsDWA1 (rice 114 Drought-induced Wax Accumulation 1), OsGL1 (GLOSSY1), and OsWR1 (rice Wax 115 synthesis Regulatory gene 1) affect drought tolerance by regulating deposition or 116 biosynthesis of cuticular wax (Islam et al., 2009; Wang et al., 2012; Zhou et al., 2013; Zhu et 117 al., 2013). 118 Besides, leaf trichomes can also affect water loss (Konrad et al., 2015), and leaf 119 trichomes closely linked with actin cytoskeleton. The involvement of the actin cytoskeleton 120 in controlling directional cell expansion in trichomes has received much attention (Zhang et 121 al., 2005). Generally, genes that affect cytoplasmic organization can be studied by screening 122 leaf trichome mutants (Qiu et al., 2002). In Arabidopsis, a reproducible morphogenetic 123 program directs the polarized development of trichome branches (Le et al., 2006; Mathur et 124 al., 1999; Syzmanski et al., 1999). Some of these genes affect the cytoskeleton and also affect 5 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 125 the morphology of normal plant cells, especially epidermal cells. For example, mutation of 126 SPIKE1 in Arabidopsis causes epidermal cells to show simple arrangement and 127 morphologies, i.e., all cells dividing along a single axis (Qiu et al., 2002). 128 To study the molecular mechanisms underlying water loss in rice, we isolated and 129 characterized the es1-1 (early senescence 1-1) rice mutant, which showed excessive water 130 loss and early senescence phenotypes. Map-based cloning data showed that ES1 encodes a 131 SCAR-like protein 2, and its Arabidopsis thaliana homolog affects the polymerization of 132 actin. The es1-1 mutants showed obvious changes in leaf trichomes, similar to Arabidopsis. 133 However, few studies have reported a connection between the actin cytoskeleton and water 134 loss in Arabidopsis. Our results demonstrated a critical role of the actin cytoskeleton in 135 regulating water loss in rice. 136 RESULTS 137 Identification and Characterization of Early Senescence Mutants 138 To study the mechanisms of senescence in rice, we screened a large pool of mutants 139 generated in the japonica rice cultivar Nipponbare (NPB) background by mutagenesis using 140 ethyl methanesulfonate (EMS). From this pool, we identified two mutants with an early 141 senescence phenotype. The two mutants showed similar phenotypes under the same growth 142 conditions and F1 hybrid individuals produced by the two parental mutants exhibited 143 phenotypes like the parental line with the weaker phenotype (subsequently named es1-1, see 144 below), indicating that the two mutants are allelic (Supplemental Table 1); therefore, we 145 termed these mutants early senescence 1 (es1-1 and es1-2). Under normal growth conditions, 146 es1-1 plants showed severely retarded development. At the seedling stage, the es1-1 mutants 147 displayed whitish and yellowish leaf tips (Figure 1A), a hallmark of early senescence (Li et 148 al., 2014); this phenotype increased with increasing leaf age, becoming more severe at the 149 heading stage (Figure 1B). Newly developed leaves showed yellowing margins that rolled 150 inwards or formed a spiral and old leaves showed spots with a rusty color and water 151 deficiency phenotypes, including wilting (Figures 1A and 1B). Histochemical analysis 152 showed that concentrations of senescence-related substances, including hydrogen peroxide 153 (H2O2), superoxide radical (O2•-), and malondialdehyde (MDA), were higher in es1-1 leaves 154 than in the wild-type leaves (Figures 1C and 1D), indicating that the mutant does show 155 senescence phenotypes (Moradi et al., 2007). Moreover, we measured the expression of two 6 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 156 senescence marker genes, Staygreen (Sgr) and Os185, in NPB and es1-1 (Supplemental 157 Figures 1F and 1G). We found that the transcript levels of these two genes were substantially 158 higher in es1-1 than in NPB, suggesting that es1-1 caused senescence (Park et al., 2007; Lee 159 et al., 2001). We also measured plant height, finding that es1-1 plants were much shorter than 160 the wild-type plants from the seedling to the mature stages (Figures 1A and 1B, Supplemental 161 Table2). At the mature stage, es1-1 mutants showed degraded or white panicles with a low 162 seed setting rate (only 2.4%; Figure 1E, Supplemental Table 2), short internodes (Figure 1F, 163 Supplemental Table2), and brown, open glumes (Figure 1G). Scanning transmission electron 164 microscopy showed that es1-1 mutants had disordered thylakoids, compared with the neat 165 and well-ordered thylakoids observed in wild-type plants; moreover, es1-1 mutants had lower 166 chlorophyll contents than wild type NPB. 167 Compared with es1-1, the es1-2 plants had more tillers (Supplemental Figures 1A and 168 1C), higher plants (Supplemental Figures 1A and 1D), and showed a stronger early 169 senescence phenotype in the leaves (Supplemental Figure 1B). In this study, we mainly 170 focused on es1-1 mutants, unless otherwise specified. 171 172 Map-Based Cloning of ES1 To clarify whether es1-1 phenotypes were caused by a single or multiple gene(s) and 173 dominant or recessive genes, we crossed the mutant plants directly and reciprocally with 174 three wild-type cultivars. All the F1 plants showed no symptoms of early senescence. In the 175 F2 segregating populations of these three crosses, normal and early senescent plants showed a 176 typical segregation ratio of 3:1 (Supplemental Table 3). This result suggested that a single 177 recessive nuclear gene controls the early senescence phenotype of es1-1. 178 For cloning ES1, we crossed the mutant es1-1 (japonica) with a typical indica cultivar 179 (NJ6). Plants with the es1-1 phenotype were identified from the F2 generation and used as the 180 initial mapping population. For primary chromosome mapping, we used 164 pairs of simple 181 sequence repeat (SSR) primers distributed on the 12 chromosomes of rice at a distance of 10– 182 30 cM from each other to detect differences between es1-1 and NJ6. A significant 183 amplification difference was detected on chromosome 1 using the SSR marker M1, showing 184 linkage with a small population in the F2. The M1 marker was further used to analyze 320 185 mutants in the F2 population, which detected 15 recombinants. Genetic linkage analysis 186 mapped the M1 marker to a distance of 2.3 cM from ES1 (Figure 2A). 7 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 187 For fine mapping of ES1, the mutation in ES1 must be flanked by two molecular 188 markers. Thus, we designed multiple pairs of primers at positions relatively far from M1 189 (Supplemental Table 4). The marker M6 also showed polymorphisms between es1-1 and NJ6. 190 Screening of the primary mapping population (320 mutants) identified 11 recombinants that 191 differed from those identified using M1 (Figure 2A). Linkage analysis mapped the M6 192 marker to a distance of 1.8 cM from ES1. Next, we used M1 and M6 to screen the entire 193 population (2850 plants), which identified 50 and 39 recombinants, respectively (Figure 2A). 194 To further narrow the ES1 region, we developed a large number of sequence-tagged site 195 (STS) and cleaved amplified polymorphic sequence (CAPS) markers between M3 and M4. 196 Primers C1–C9 showed good polymorphisms between es1-1 and TN1. Thus, we used these 197 primers to analyze the recombinants obtained by preliminary screening and to construct fine 198 genetic and physical maps of ES1 (Figure 2B; Supplemental Table 4). The number of 199 recombinants detected by C1 and C10 decreased to 28 and 35, respectively. Similarly, the 200 number of recombinants screened by C2 and C9 decreased to 10 and 12, respectively, and 201 those detected by C3 and C8 further decreased to three and four, respectively. The 202 recombinants detected by C3 and C8 corresponded to different individual plants. 203 We also designed primers for the region between C3 and C8 to further screen the 204 recombinants. Finally, these new primers detected only one recombinant and thus these 205 markers tightly co-segregate with ES1 (Figure 2B). The physical distance between the end 206 markers C5 and C6 was 13.1 kb, which included two predicted open reading frames. 207 Sequencing analysis detected a unique mutation in LOC-Os01g11040 within the 13.1 kb 208 region in es1-1 compared with the wild-type sequence. This single-base mutation replaced G 209 with A in the first nucleotide of the third intron (nucleotide 2275; Figure 2C). To test whether 210 this mutation alters the normal splicing of the third intron, we generated es1-1 and wild-type 211 genomic cDNAs using reverse transcription. Sequencing showed that the es1-1 cDNA was 62 212 bases longer than the wild-type cDNA (Figure 2D). This increase in length coincided with the 213 length of the third intron, which contains a stop codon that results in the premature 214 termination of protein translation (Figure 2E). Similarly, sequencing analysis showed that the 215 mutation in es1-2 was a single-base substitution (G to A) at nucleotide 4660. This mutation 216 changed the tryptophan (Trp) codon into a stop codon, thus resulting in the premature 217 termination of protein translation (Figures 2E). 218 219 We then used genetic complementation to verify the identity of ES1. A 10.6-kb fragment containing ES1 (including the upstream 2-kb and downstream 1-kb sequences) was 8 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 220 introduced into es1-1 and es1-2 mutants, and 52 and 36 transgenic plants, respectively, were 221 recovered. Exogenous ES1 rescued the phenotypes of es1-1 and es1-2 (Figure 2F), and the 222 complementation reversed the mutant phenotype of es1-1. Taken together, these data 223 demonstrate that LOC-Os01g11040 (Os01g0208600) is ES1. 224 ES1 Encodes a SCAR-Like Protein of the Arp2/3-SCAR Pathway 225 We performed a BLAST search to identify ES1 homologs in more than 20 different 226 plant species. ES1 showed high amino acid sequence similarity to proteins in other species 227 such as Oryza brachyantha (85%), Brachypodium distachyon (68%), maize (60%), and 228 Setaria italica (64%) (Figure 3E). ES1 also shares high amino acid sequence similarity to 229 proteins in both monocots and dicots, suggesting that ES1 and its homologs may perform 230 similar functions. Examination of ES1 homologs in Arabidopsis showed that ES1 231 (Os01g0208600) encodes a SCAR-like protein 2; its closest homolog in Arabidopsis forms 232 part of the actin-related 2/3 (ARP2/3) complex, which functions in regulating actin filament 233 polymerization and has been examined in detail in Arabidopsis. ARP2/3 alone is inactive in 234 Arabidopsis. A domain formed by the SCAR-like family proteins, and members of other 235 protein families (e.g., SPA1 and ABI families) are responsible for binding to and activation of 236 the Arp2/3 complex (Zhang et al., 2005). In Arabidopsis, mutation of the gene encoding 237 SCAR-like protein 2 leads to changes in leaf trichomes and stomata (Basu et al., 2005). In 238 our study, we observed substantial changes in leaf trichomes of es1-1, mainly reflected by the 239 serious degradation of the trichome tip (Figures 3A and 3B). The leaves of es1-1 had smooth 240 leaf margins without a jagged pattern (Figure 3A). 241 To examine the possible role of ES1 in regulating actin filaments, we examined the 242 organization of F-actin in the roots of wild type and es1-1 mutants by Alexa Fluor 488- 243 phalloidin staining. The actin filaments were well organized in wild type (Figure 3C). The 244 actin filaments in wild-type were complete, orderly arranged, while es1-1 showed irregular 245 arrangement. Moreover, the actin filaments in es1-1 were shorter than that in wild type, as 246 indicated by arrows (Figure 3D), which indicated that ES1 likely functions in the Arp2/3- 247 SCAR pathway in rice and that a mutation in ES1 affects the actin cytoskeleton in rice cells 248 (Dyachok et al., 2011). 9 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 249 250 Expression of ES1 and Subcellular Localization of ES1 To monitor the tissue-specific expression of ES1, we generated an ES1:GUS (β- 251 glucuronidase) reporter construct containing the 2.0-kb genomic region upstream of the start 252 codon of ES1. We detected high GUS activity in leaves, leaf sheaths, and root tips, and also 253 detected lower GUS activity in stamens and vascular bundles (Figures 4A–4E). Reverse- 254 transcription quantitative polymerase chain reaction (RT-qPCR) assays showed that ES1 was 255 ubiquitously expressed in various tissues at the tillering and heading stages, including the 256 roots, culms, leaves, sheaths, and spikelets. We observed the highest levels of ES1 expression 257 in the leaf and leaf sheath (Figure 4F). Consistent with the RT-qPCR data, ES1:GUS 258 expression was higher in rice leaves and leaf sheaths than in other plant tissues. 259 To investigate the subcellular localization of ES1, we constructed a construct to express 260 eGFP-ES1 (35S::eGFP-ES1 DNA) and introduced it into onion and tobacco epidermal cells. 261 Fluorescence signals were detected in both cell types, and the signals surrounded the 262 cytoplasm, nucleus, and cell membrane (Figures 4G, 4H, 4I, and 4J). Consistent results were 263 obtained by examining protoplasts extracted from tobacco leaves after the expression of the 264 fusion protein (Figures 4K and 4L). These results indicated that ES1 was ubiquitously 265 distributed in the cytoplasm of plant cells. 266 ES1 Affects the Density of Leaf Stomata in Rice 267 Scanning electron microscopy of es1-1 leaves showed that these leaves had smoother 268 surfaces than the leaves of wild-type plants. In addition, these leaves showed substantial 269 smaller siliceous protrusions at the seedling stage and substantially more stomata per unit leaf 270 area compared with wild-type leaves. The number of semi-open stomata in es1-1 was also 271 larger than that in wild-type (Figures 5A and 5B). Moreover, the tips of trichomes and the 272 waxy layer were severely degraded on the leaf surface of es1-1 mutants (Figure 5A). 273 Statistical data showed that, regardless of the position, stomatal density was significantly 274 higher in es1-1 than that in the wild-type. While both stomatal conductance and transpiration 275 rate were also detected, they were also significantly higher in es1-1, these data indirectly 276 suggested stomatal density in es1-1 was higher (Figure 5B). The maximum and minimum 277 stomatal apertures were greater in the wild-type compared with es1-1(Figure 5C). At the 278 seedling stage, the transcriptional level of several genes which could affect the increase of 10 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 279 stomatal density were analyzed by the real-time PCR, including OsTMM, OsSCRM, 280 OsSDD1, OsYODA2 (Du et al., 2014). The expression level of OsTMM, OsSCRM and 281 OsSDD1 were significantly higher in es1-1 than that in the wild-type, and ES1 gene also 282 showed higher expression level in es1-1(Figure 5E). 283 Leaves of wild-type and es1-1 at seedling stage were sprayed with 300 umol / L ABA, 284 after 3 hours, quantitative analysis of OsTMM, OsSCRM, OsSDD1, OsYODA2 was taken. In 285 the wild-type, the expression level of these genes almost didn’t change; but in es1-1, the 286 majority of these genes had shown a significantly declined expression level, this indicated in 287 es1-1 those genes which could affect the increase of stomatal density were sensitive to ABA. 288 We also detected the rate of vitro water loss (RWL) of seedling leaves from the same position 289 of the wild-type and es1-1 before and after ABA treatment. The results showed that, no 290 matter with or without ABA treatment, the vitro RWL value was always higher in es1-1 than 291 that in the wild-type. The vitro RWL value in the wild-type was reduced after ABA 292 treatment, and the value in es1-1 was also reduced after ABA treatment, but it was not so 293 obvious compared with the wild-type (Figure 5D). SEM assay on the leaves of wild-type and 294 es1-1 with ABA treatment showed that nearly all stomata of both wild-type and es1-1 were 295 closed. Moreover, we also examined the expression level of multiple ABA response-related 296 genes (ABA signaling genes) in wild-type and es1-1 before and after ABA treatment (Shang 297 et al., 2010), and discovered that after ABA treatment, OsDREB2A gene was up-regulated, 298 while OsABI4 gene was down-regulated. These results suggest that both wild-type and es1-1 299 were sensitive to ABA (Figure 5E). 300 301 302 The es1-1 Plants Showed Higher Water Loss Rates than Wild Type In vitro, the rate of water loss of es1-1 was higher than that of NPB at the seedling 303 stage (Figure 6A), as the detached leaves of es1-1 shrunk into a line at 30 min, but the NPB 304 leaves remained nearly normal in width (Figure 6A). To confirm this phenomenon, we also 305 measured water loss per unit of time (g cm-2.10-3) at the tillering stage in vivo and in vitro 306 (Figures 6B and 6C), which gave similar results: the water loss per unit of time in vitro 307 (detached leaves) in different periods was uniformly higher in es1-1 than in NPB (Figure 6B). 308 The water loss per unit of time in vivo was also uniformly higher in es1-1 than in NPB at 4, 8, 309 and 14 days after tillering (Figure 6C). 310 To verify excessive water loss in es1-1, we performed a guttation experiment, where we 11 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 311 looked for the exudation of drops of xylem sap on the leaf periphery in plants grown at 312 different humidities. Hydroponic seedlings were cultured in light, in an incubator at different 313 humidities, from 30% and 90%, and the es1-1 and wild-type plants were examined for 314 guttation. At the seedling stage (two-leaf), both the wild-type plants and es1-1 were capable 315 of guttation at 30% humidity. However, higher humidity was required for guttation with plant 316 growth. Until the strong seedling stage (two-tiller), guttation was observed in wild-type 317 plants but not in es1-1 (Supplemental Figure 2). This result indicated that water loss occurred 318 more rapidly in es1-1 mutants than in the wild-type plants. 319 320 Water Absorption and Transport Capacity was Increased in es1-1 321 Mutants 322 It is unknown whether excessive water loss through leaves affects water absorption in 323 the root. To address this, we measured root traits in es1-1 and wild-type plants (Figures 7A, 324 7B, 7C, and 7D). The es1-1 plants had smaller primary and total root lengths (Figures 7E and 325 7F) but higher average root diameters, total root volumes, and total numbers of fibrous roots, 326 compared with the wild-type plants (Figures 7G, 7H, 7I, and 7J). As fibrous roots are a major 327 tissue for water adsorption in rice (Smith et al., 2012), an increase in fibrous root number 328 indicated an increase in the water absorption capacity of the roots in es1-1 mutants. 329 330 Examination of cross sections of the culm and primary root from es1-1 and NPB showed 331 that the es1-1 stems had 3–4 additional vascular bundles (Figures 8A, 8B, and 8C) and the 332 es1-1 primary roots had 2 to 3 additional conduits, compared with NPB (Figures 8D, 8E, and 333 8F). Vascular bundles and conduits are necessary for water transport (Kim et al., 2014). 334 Moreover, the water potential values were all higher in es1-1 than that in wild-type in various 335 tissues, including roots, stems, leaves, and sheath, which indicated that the ability to transport 336 water was stronger in es1-1 than in WT (Figure 8G) (Silva et al., 2011). Therefore, our results 337 indicate that es1-1 mutants have an enhanced ability to transport water. 338 339 340 Excessive Water Loss Causes in Situ Enrichment of Minerals in es1-1 In es1-1 mutants, excessive water loss through the leaves enhances the water absorption 12 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 341 capacity of the roots and the water transportation capacity of the roots and culms. This in turn 342 accelerates water circulation in the plant, thus leading to predicted in situ enrichment of metal 343 ions cotransported with water (Kim et al., 2014). To test this hypothesis in this mutant, we 344 determined the mineral concentrations of different tissues of es1-1 and wild-type plants at the 345 mature stage. Consistent with the hypothesis, the concentrations of all four minerals tested 346 (Cu, Fe, Mn, and Zn) in the examined tissues were much higher in es1-1, especially in the 347 roots and new leaves, than in wild-type plants (Table 1). The manganese (Mn) concentration 348 in es1-1 leaves (719 mg kg-1) was seven times higher than that in wild-type plants (100 mg 349 kg-1). The concentrations of the four minerals were also higher in es1-1 seeds than in NPB 350 (Table 1). These results suggested that mutation in ES1 promoted the accumulation of 351 minerals in plants. The substantial accumulation of metal ions in es1-1 plants was likely due 352 to excessive water loss and higher transpiration pull in the upper part of the plant. 353 DISCUSSION 354 In this study, we characterized es1-1, an early senescence mutant that experienced 355 excessive water loss. Map-based cloning showed that ES1 encodes a SCAR-like protein 2, a 356 component of the SCAR/WAVE complex that is involved in actin polymerization. This 357 suggests that a mutation in ES1 affects polymerization of actin and causes an abnormality in 358 the cytoskeleton that promotes a higher stomatal density (Huang et al., 2000; Xiao et al., 359 2003; Yu et al., 2001; Zhang et al., 2012), thus leading to excessive water loss. 360 Under stress conditions, plants develop a wax layer on the leaf surface or moderately roll 361 their leaves to reduce transpiration, thereby protecting themselves from excessive water loss 362 (Hu et al., 2012; Jäger et al., 2014). Although stomata account for only 1–2% of the leaf area, 363 approximately 90% of water loss occurs through stomata (Buckley et al., 2005). Under 364 drought conditions, rice cultivars that can maintain relatively high water potential and low 365 transpiration rate in their leaves have strong drought resistance (Luo et al., 2001). These 366 drought-resistant cultivars commonly retain water in the plant body by closing their stomata. 367 Small stomatal aperture and large diffusion resistance can jointly reduce water loss due to 368 transpiration, thus avoiding the adverse effects of drought. Grill and Ziegler (1998) proposed 369 that controlling the stomata is the most important way to improve the efficiency of water use 370 in plants. In the present study, scanning electron microscopy showed that the es1-1 mutants 371 had substantially more stomata than wild-type plants. The expression level of OsTMM, 372 OsSCRM and OsSDD1 were significantly higher in es1-1 than that in the wild-type (Figure 13 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 373 5E), which was closely related to stomatal density. Stomatal density was significantly higher 374 in es1-1 than that in the wild-type. While both stomatal conductance and transpiration were 375 also significantly higher in es1-1 than that in the wild-type (Figure 5B), these data suggested 376 that es1-1 definitely lose water more quickly than wild-type. Nearly all stomata of both wild- 377 type and es1-1 were closed after ABA treatment, and the vitro RWL value in the wild-type 378 was reduced after ABA treatment, the value in es1-1 was also reduced after ABA treatment, 379 but it was not so obvious compared with the wild-type (Figure 5D). Moreover, the wax layer 380 on the leaf surface of es1-1 mutants was considerably degraded. So, we thought the excessive 381 water loss in es1-1 mainly due to increased stomatal density, meanwhile the degradation of 382 waxy layer and leaves hair may aggravate the rate of water loss. 383 Excessive water loss in the leaves inevitably depletes water from the body of es1-1 384 plants. Thus, more water needs to be absorbed through the roots to meet the metabolic 385 requirements of es1-1 plants. In this study, we analyzed the roots of es1-1 and found that the 386 total volume of root and the number of fibrous roots increased compared to wild type 387 (Figures 7I and 7J), indicating that the water absorption capacity of the roots was enhanced in 388 es1-1. Cross-sections of the primary root and stems also showed that the number of vascular 389 bundles in the stems increased by four (Figure 8C) and those in the roots increased by two to 390 three in es1-1 compared to the wild-type plants (Figure 8F). Moreover, the water potential 391 values were all higher in es1-1 than in wild-type in various tissues (Figure 8G). These 392 observations confirmed that the water transport capacity of the roots was enhanced in es1-1. 393 All the above changes inevitably accelerate water circulation and cause minerals 394 commonly cotransported with water to accumulate in the various tissues of es1-1 plants. 395 Indeed, in different tissues, the concentrations of four minerals were higher in es1-1 than in 396 NPB (Table 1). For example, the Cu concentration in es1-1 (446.3 mg kg-1) was 18-times 397 higher than in wild-type plants (23.5 mg kg-1), which could cause Cu overload in es1-1 398 plants. Enrichment of low-mobility metal elements (e.g., Cu, Fe, Mn, and Zn) in various 399 tissues of es1-1 may in turn accelerate the onset of senescence. Moreover, the higher 400 concentrations of mineral nutrients in seeds from the es1-1 mutants might produce more- 401 nutritious food; however, the accumulation of toxic elements such as arsenic might produce 402 food that could adversely affect human health. 403 In our study, we found that actin filaments were shorter in es1-1, and the continuity was 404 poor compared with wild-type (Figures 3C and 3D). Actin filaments function in many 405 endomembrane processes, such as endocytosis of plasma membrane, vacuole formation, 406 plasma membrane protrusion in crawling cells, and vesicle transport from the Golgi complex 14 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 407 (Svitkina et al., 1999; Eitzen et al., 2002; Kaksonen et al., 2003; Mathur et al., 2003; Chen et 408 al., 2004). The ARP2/3 complex functions as a key regulator of actin filament nucleation in 409 plants (Zhang et al., 2008). The ARP2/3 is inactive by itself, as actin polymerization requires 410 activation by the SCAR/WAVE complex, formed by proteins of the SCAR family. The 411 SCAR/WAVE complex is highly conserved in plants. A deficiency of the SCAR/WAVE 412 complex often leads to morphological changes in leaf trichomes and epidermal cells. This 413 phenomenon has been reported in several studies in Arabidopsis (Li et al., 2003; Mathur et 414 al., 2003; Saedler et al., 2004) but not in rice. In our study, es1-1 showed evident 415 morphological changes in leaf trichomes (Figures 3A, 3B, and 5C), confirming that the 416 function of the SCAR/WAVE complex is conserved in different species. Additional 417 components of the SCAR/WAVE complex can be identified by screening for mutants that 418 affect the leaf trichomes of the es1-1 mutant. 419 Actin polymerization occurs in the cytoplasm. In the present study, the fluorescent 420 signal of eGFP-ES1 was detected in the cytoplasm and plasma membrane of tobacco and 421 onion epidermal cells, a localization consistent with the predicted function of ES1 in affecting 422 the actin cytoskeleton. After fusing the ES1 promoter with the GUS reporter gene, GUS 423 staining was observed in various tissues, including the roots, root hairs, stems, leaves, leaf 424 sheath, and vascular bundles, which are associated with water absorption, transportation, and 425 loss. The ES1 expression level was relatively high in the leaves and leaf sheaths—the two 426 major tissues involved in water loss. All these results are consistent with the proposed 427 function of ES1. 428 The increased number of stomata would increase water loss and induce drought stress 429 in es1-1 plants. Drought stress affects photosynthetic capacity, respiration, membrane 430 permeability, osmotic adjustment, enzyme activity, and hormone levels in rice leaves, thus 431 reducing leaf area, leaf area index, and dry weight. Drought stress also prevents the 432 accumulation of dry matter and accelerates senescence (Hu et al., 2010; Ding et al., 2014; 433 Sellammal et al., 2014). In the present study, es1-1 plants showed early senescence 434 characterized by yellowish leaf color and wilting of leaf margins at the seedling stage. 435 Transmission electron microscopy showed that the volume percentage of chloroplasts in the 436 es1-1 cells was lower than that in wild-type cells; however, the number of chloroplasts 437 (irregular and fine prolate) was higher in es1-1 cells than in wild-type cells (Figure 1H). This 438 might have caused the decline in chlorophyll content in es1-1, causing the leaves to turn 439 yellowish. In es1-1, the chloroplasts contained disorganized thylakoids (Figure 1H) as 15 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 440 opposed to the well-organized thylakoids in wild-type plants. The disordered thylakoids may 441 have reduced the capacity to absorb light energy, which in turn would decrease the 442 photosynthetic rate, thus causing early senescence in plant leaves. 443 Early senescence in rice (especially in functional leaves) is extremely unfavorable for 444 rice growth and production because of its serious effects on yield and quality (Zhu et al., 445 2012). Therefore, rice breeders generally avoid lines showing early senescence. Similarly, the 446 stay-green trait in rice also does not improve yield; although the stay-green trait promotes the 447 ability of source tissues to fix carbon, it does not necessarily enhance the contents of sink 448 tissues in plants. For example, a study on a stay-green mutant of rice (sgr[t]) reported that 449 late photosynthesis and yield traits (seed-setting rate and thousand-grain weight) were not 450 enhanced in sgr[t] mutants (Cha et al., 2002). In the present study, the early senescing mutant 451 es1-1 showed other phenotypes such as decreased number of tillers and substantially reduced 452 seed-setting rate. Our data provide new insights into the molecular mechanisms underlying 453 early senescence in plants, which will help genetically improve rice yield. 454 MATERIALS AND METHODS 455 Plant Growth and Sampling 456 The es1-1 and es1-2 mutants were isolated from the japonica variety Nipponbare that 457 was subjected to EMS treatment. After multiple generations of inbreeding, the phenotypes of 458 these two mutants were stably inherited, and homozygous mutant strains were obtained. The 459 homozygous mutant es1-1 was directly and reciprocally crossed with two typical japonica 460 cultivars (Zhonghua 11 and Chunjiang 06) and one typical indica rice cultivar (Nanjing 6) 461 providing a relatively clear genetic background to construct the genetic population. The 462 obtained seeds of the F1 generation were sown and transplanted as individual plants to 463 generate the F2 generation by inbreeding. The numbers of individuals showing wild-type and 464 early senescence phenotypes were counted at the seedling stage. The segregation ratio was 465 calculated using statistical methods. Genetic analysis was performed on es1-1, and recessive 466 individuals were retrieved for preliminary mapping of the mutation. Plant materials were bred 467 in experimental field of the China National Rice Research Institute, Hangzhou, China. 16 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 468 469 Detection of Reactive Oxygen Species and Malondialdehyde H2O2 and O2•- were detected using nitroblue tetrazolium (NBT) and diaminobenzidine 470 (DAB), respectively, according to (Li et al., 2010; Wi et al., 2010) with some modifications. 471 Leaves of 3-week-old plants that were grown in growth chambers were incubated in 0.05% 472 NBT (Duchefa) or 0.1% DAB (Sigma) at room temperature in darkness with gentle shaking 473 for 12 hrs. Chlorophyll was cleared by treating with 90% ethanol at 80°C. The concentration 474 of malondialdehyde (MDA) was measured according to the method described by Moradi and 475 Ismail (2007) with minor modifications (Moradi et al., 2007). 476 Determination of Water Potential 477 Ten seeds of NPB and es1-1 were selected, germinated for 3 days, then cultured in an 478 incubator at 28°C for 60 days. The WP4-T Dewpoint PotentiaMeter was preheated for 30 479 min until the temperature remained stable. Various tissues such as leaf, sheath, culm, and root 480 of NPB and es1-1 were harvested from the same position on each plant and cut into short 481 pieces. After making sure the sample temperature was lower than room temperature, the 482 samples were put in the measuring apparatus, which showed the water potential value of the 483 sample. Every sample measurement had six independent repeats, and the weight of the 484 samples was equal. 485 Map-Based Cloning of ES1 486 The mutation in es1-1 was mapped in the F2 population of a cross between es1-1 and the 487 indica variety NJ6. To finely map the ES1 locus, new molecular markers were developed 488 using public databases (Supplemental Table 4). Further, ES1 was sequenced using specific 489 primers (Supplemental Table 4). 490 491 492 Fluorescence Microscopy of Actin Filaments For F-actin observation, plants were grown in a culture chamber with 16/8 hrs 493 photoperiod at 30°C. Seedling roots were used to observe actin organization. F-actin was 494 stained using the previously described method (Olyslaegers and Verbelen, 1998; Zhang et al., 17 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 495 2011). Seven-day-old roots were fixed in PEM buffer (100 mM PIPES, 10 mM EGTA, 0.3 M 496 mannitol, 5 mM MgSO4, pH 6.9) with 3% paraformaldehyde for 30 min and washed 3 times 497 with PEM. After washing, roots were incubated in PEM buffer with 0.66 mM Alexa Fluor 498 488-phalloidin (Life Technologies). After overnight incubation at 4°C, samples were washed 499 three times with PEM buffer and then observed in 50% glycerol with a confocal microscope 500 (Olympus FLUOVIEW FV1000). 501 502 503 Phylogenetic Analyses A BLAST search of the SCAR-like 2 amino acid sequence was performed 504 against the database (http://www.ebi.ac.uk/Tools/sss/ncbiblast/). The phylogenetic tree was 505 constructed based on the amino acid sequences. Maximum likelihood (ML) phylogenetic 506 analysis was performed by MEGA6.0 (Tamura et al., 2013). ML analysis of the SCAR-like 2 507 family was based on the amino acid sequences using the Jones-Taylor-Thornton model, 508 which was chosen after model testing. Support for the ML trees was evaluated by 1000 509 bootstrap replicates. 510 511 512 Plasmid Construction and Plant Transformation The cDNA sequence of ES1 was amplified using the primers cES1-F and cES1-R (Table 513 S3). This cDNA was fused in frame with eGFP in pCambia1300 (http://www.cambia.org) 514 with minor modifications to generate the transient expression vector 35S::eGFP:cES1. The 515 35S::eGFP-cES1 plasmids were transferred into Agrobacterium tumefaciens strain EHA105 516 by electroporation; the cells of rice ES1, Nicotiana tabacum, and onion were transformed 517 according to a previously described method (Hiei et al., 1994; Lv, 2010). 518 For the molecular complementation assay, a 10.6-kb genomic fragment comprising the 519 ES1 promoter region, coding sequence, and 3′ untranslated region was PCR amplified from 520 the genomic DNA of wild-type plants using pES-F and pES-R primers (Table S3). The PCR 521 amplification product was cloned into pCAMBIA1300 to generate the ES1:ES1 construct. 522 Briefly, the pCambia1300::ES1 vector was first digested with the restriction enzymes PstI 523 and XbaI. Next, the fragment containing ES1 was digested with restriction enzymes Sse83871 18 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 524 and NheI. All the restriction enzymes used above are isocaudomers. The pCambia1300::ES1 525 plasmids were transformed into A. tumefaciens strain EHA105 by electroporation. The es1-1 526 and es1-2 plants were transformed according to a published method (Hiei et al., 1994). 527 To generate the ES1:GUS fusion construct, a 2.0-kb upstream genomic region of the 528 ES1 start codon was amplified with the primers ES1-gus-F and ES1-gus-R (Table S3). The 529 amplified fragment was linked with the reporter GUS gene and together inserted in the 530 pCambia1305 vector (http://www.cambia.org). 531 GUS Staining 532 Samples (proES1::GUS) were stained with a solution containing 50 mM NaPO4 buffer, 1 533 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid, 0.4 mM K3Fe(CN)6 , 0.4 mM 534 K4Fe(CN)6, and 0.1% (v/v) Triton X-100. The samples were then incubated at 37°C in the 535 dark for 10 hrs. Chlorophyll was removed with 70% ethanol for observation. 536 RNA Extraction and RT-qPCR 537 Total RNA was extracted from the roots, culms, leaves, sheaths, and spikelets of wild- 538 type and es1-1 plants at the seedling, tillering and heading stages using an RNeasy Plant Mini 539 Kit (Qiagen). Reverse transcription was handled with the ReverTra Ace qPCR-RT Kit 540 (TOYOBO), the primers are described in Supplemental Table 4. RT-qPCR analysis was 541 conducted with the StepOnePlus System (ABI) using THUNDERBIRD qPCR Mix 542 (TOYOBO), reaction procedure was 95°C 30 s; 95°C 5 s, 55°C 10 s, 72°C 15s for 40 cycles. 543 OsActin was used as a control. The 2-ΔΔCt method was used for quantification (Livak et al., 544 2001). 545 Microscopy 546 In different periods, especially at the noon when the sunlight was the strongest (and thus 547 the stomata are likely to be closed), fresh leaf specimens were collected from the same 548 position ( 1/4, 1/2 and 3/4 of leaf blade ) in the mutant es1-1 and the control Nipponbare, 549 from plants that showed consistent growth. The specimens were cut into approximately 1 × 2 19 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 550 551 mm pieces and processed as follows for microscopic examination. Dual fixation: The specimens were fixed in 2.5% glutaraldehyde solution, vacuumed in a 552 vacuum apparatus for approximately 20–30 min (which allowed the specimens to be 553 immersed in the fixative as completely as possible), and kept at 4°C overnight. Thereafter, 554 the fixative was discarded, and the specimens were rinsed with 0.1 M phosphate-buffered 555 saline (PBS), pH 7.0. The specimens were rinsed 3–4 times (15 min each). The specimens 556 were then fixed in 1% osmium tetroxide for at least 1 h. The fixative was discarded, and the 557 specimens were rinsed with 0.1 M PBS three times (15 min each) as described above. 558 Dehydration: The specimens were first dehydrated with a gradient of ethanol (50%, 70%, 559 80%, 90%, and 95%; 15 min to 20 min at each gradient), processed in anhydrous ethanol for 560 20 min, and treated in pure acetone for 20 min. 561 After dehydration, the specimens were immersed in a mixture of ethanol and isoamyl 562 acetate esters (volume ratio 1:1) for 30 min and were then treated with pure isoamyl acetate 563 for 1-2 h. The specimens were dried, coated, and examined using a Hitachi TM-1000 564 scanning electron microscope. 565 For transmission electron microscopy of the leaves and cross-sectional examination of 566 the leaves and roots, the specimens were pretreated as described above. After dehydration, 567 the specimens were treated with a mixture of embedding agent and pure acetone at a volume 568 ratio of 1:1 for 1 h, followed by treatment with a mixture of embedding agent and pure 569 acetone at a volume ratio of 3:1 for 3 h and pure embedding agent overnight. The specimens 570 were then embedded in small boxes and heated at 70°C for approximately 9 h. The obtained 571 embedded samples were sliced into 70-90 nm sections using a Reichert ultramicrotome and 572 stained with lead citrate solution and saturated uranyl acetate solution in 50% ethanol for 15 573 min each. The sections were examined and photographed using a Hitachi H-7650 type 574 transmission electron microscope. 575 Determination of Minerals 576 Wild-type and es1-1 plants were cultured in complete hydroponic nutrient solution up 577 to the heading stage. Tissue specimens were taken from the roots, stems, old leaves, leaf 578 sheaths, and new leaves. The weight of fresh specimens was more than 10 g for the different 20 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 579 tissues. The specimens were oven dried at 80°C for 3 days to achieve a constant weight. After 580 grinding, the dry samples (0.25 g for es1-1 and NPB, respectively) were digested with a 581 mixture of concentrated nitric acid and perchloric acid (volume ratio 1:3). The volume of the 582 digestion solution was adjusted to 25 ml with single-distilled water. The treated samples were 583 then used for elemental analysis. Concentrations of elements were determined using a full- 584 spectrum direct reading inductively coupled plasma-atomic emission spectroscopy (IRIS 585 Intrepid II XSP ICP-OES PE-2100; Thermo Electron Co., Ltd, USA; Shao et al., 2008). 586 Detection of the Rate of Water Loss 587 In vitro detection: The same leaf parts were selected from NPB and es1-1 plants at the 588 seedling stage. These leaf parts were left at room temperature and observed and photographed 589 every 10 min to examine their dehydration. At the tillering stage, the same leaf parts of NPB 590 and es1-1 were quickly weighed (W1), and the total area (S) was measured. Moreover, the 591 weights of the leaf blade (W2, W3, and W4) were also measured every 1 h. The formula 592 (W1-W2) S-1 was used to calculate the rate of water loss. Accordingly, the rate of water loss 593 at different time points was obtained. 594 In vivo detection: After accelerating germination, the wild-type and es1-1 seeds were 595 cultured in fine sand and watered twice a day (in the morning and evening). At the 1–2-leaf 596 stage, rice seedlings were washed off and wrapped in a long sponge, then placed in a closed 597 bottle with clean water. At the 3-leaf stage, clean water was replaced by complete nutrient 598 solution. After the seedlings developed new roots, all the bottles were filled with a sufficient 599 volume of nutrient solution, and the level was marked for subsequent measurement. The 600 leaves were weighed on days 1 (W1), 4 (W2), 8 (W3), and 14 (W4). The corresponding leaf 601 areas were also measured (S2, S3, and S4). Water loss per unit of time in 4 days was 602 calculated as (W1 − W2) S2-1. Six plants were used for every time interval measurement (the 603 results are presented as mean values). Analogously, (W1 − W3) S3-1 was the amount of water 604 loss per unit of time in 8 days, and (W1 − W4) S4-1 was the amount of water loss per unit of 605 time in 14 days. 21 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 606 607 Measurement of Root Traits Hydroponic experiments were conducted using the wild-type and es1-1 plants. The seeds 608 were soaked for two days, during which time the water was changed once. Next, the seeds 609 were transferred into a germination box to accelerate germination. Three days later, seeds 610 with radicals breaking through the testa were transferred into fine sand for cultivation. After 611 incubation for 7 days, the seedlings were washed carefully to avoid damage to the roots. The 612 plants were transferred into a black bucket containing tap water. After the transition, the 613 plants were supplied with complete nutrient solution. The seedlings entered the two-leaf stage 614 after three days. At the 5-leaf stage, seedlings of the mutant and wild-type plants (10 each) 615 were taken out for root measurements. The hydroponic seedlings were rinsed and placed in a 616 transparent rectangular box containing single-distilled water and loaded onto a scanner 617 (ScanWizard Pro: ScanMarker 1000XL) for transparent scanning. The obtained images were 618 loaded into Win-RHIZO. Pro. V. 2002c for data analysis, and the results were exported to 619 MS Excel 2011 (Microsoft, USA) for analysis. 620 22 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 621 622 Accession Numbers 623 Sequence data from this article are available at the NBCI website 624 (http:www.ncbi.nlm.nih.gov) with the accession numbers: LOC_Os01g11040 625 (NP_001172227.1), OB01G1689 (XP_006643892.1), Bradi2g06610(XP_003565325.1), 626 Oryza_sativa_indica (OsI_00845), Setaria italica (Si000056m.g), Hordeum vulgare var. 627 distichum (M0XEC2), Aegilops tauschii (F775_07293), Triticum aestivum (W5CZS7), Zea 628 mays (ZEAMMB73_679274), Triticum urartu (TRIUR3_31093), Musa acuminata subsp. 629 Malaccensis (M0UB73), Medicago truncatula (MTR_8g086300), Theobroma cacao 630 (TCM_029698), Phaseolus vulgaris (PHAVU_002G238500g), Jatropha curcas 631 (JCGZ_21566), Glycine max (I1K3Z5), Populus trichocarpa (POPTR_0004s05600g), 632 Prunus persica (PRUPE_ppa000443mg), Citrus sinensis (CISIN_1g001053mg), Citrus 633 clementina (CICLE_v10014081mg), Eucalyptus grandis (EUGRSUZ_D00489), Solanum 634 lycopersicum (Solyc02g076840.2), Erythranthe guttata (MIMGU_mgv1a000504mg), Vitis 635 vinifera (VIT_10s0003g01270), Ricinus communis (RCOM_0850090), Amborella trichopoda 636 (AMTR_s00021p00061660), Morus notabilis (L484_002198), Arabidopsis lyrata 637 subsp.(Lyrata ARALYDRAFT_903078). 638 639 Supplemental Data 640 Supplemental Table 1. Allelic analysis of es1-1 and es1-2. 641 Supplemental Table 2: Phenotypic data of NPB and es1-1. 642 Supplemental Table 3. Reciprocal cross experiments between es1-1 and three crop strains 643 (ZH11, CJ06, and NJ6). 644 Supplemental Table 4. Primers used for PCR in this study. 645 Supplemental Figure 1. Phenotype of allelic mutants. 646 A, wild-type; bar = 10 cm. B, allelic mutants; bar = 10 cm (left, es1-1; right, es1-2). C, 647 Leaves of allelic mutants; bar = 5 cm (left, es1-1; right, es1-2). D, Difference in tiller number 648 between es1-1 and es1-2. E, Difference of plant height between es1-1 and es1-2. F-G, 649 Expression level of SGR and esl85 between NPB and es1-1. The results are given as means of 650 three independent assays, and error bars indicate SD. *, ** significant at the level of 5% and 651 1%, respectively. 652 Supplemental Figure 2. Comparison of guttation between wild-type and es1-1 plants. NPB 653 leaf showing guttation drops at the point of emergence (adaxial side). 23 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 654 655 ACKNOWLEDGEMENTS 656 We sincerely thank Prof.Jianru Zuo (Institute of Genetics and Developmental Biology, 657 Chinese Academy of Sciences) for sharing unpublished data. This work was supported by 658 grants from the National Natural Science Foundation of China (31201183, 31221004, 659 31171531, 31171520, 91435105) and the State Key Basic Research Program 660 (2013CBA01403), the Ministry of Agriculture of China for transgenic research 661 (No.2013ZX08009003-001), China Postdoctoral Science Foundation (2014M561108). 662 663 LITERATURE CITED 664 665 Basu D, Le J, El-Essal SE, Huang S , Zhang C, Mallery EL, Koliantz G, Staiger CJ, 666 Szymanski DB (2005) DISTORTED3/SCAR2 is a putative Arabidopsis WAVE complex 667 subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis. 668 Plant Cell 17: 502-524 669 Boonrueng N, Anuntalabhochai S, Jampeetong A (2013) Morphological and anatomical 670 assessment of KDML 105 (Oryza sativa L. spp. indica) and its mutants induced by low- 671 energy ion beam. Rice Sci 20(3): 213-219 672 673 674 Bouman B, Humphreys E, Tuong T, Barker R (2007) Rice and water. 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J Amer Soc Hort Sci 120: 417-422 805 806 Yang M, Sack F (1995) The too many mouths and four lips mutations affect stomatal production in Arabidopsis. Plant Cell 7: 2227-2239 807 You J, Zong W, Li X, Ning J, Hu H, Li X, Xiao J, Xiong L (2013) The SNAC1-targeted 808 gene OsSRO1c modulates stomatal closure and oxidative stress tolerance by regulating 809 hydrogen peroxide in rice. J Exp Bot 64: 569-583 810 811 Yu R, Huang R, Wang X, Yuan M (2001) Microtubule dynamics are involved in stomatal movement of Vicia faba L. Protoplasma 216:113-118 812 Zhang C, Mallery EL, Schlueter J, Huang S, Fan Y, Brankle S, Staiger CJ, Szymanski 813 DB (2008) Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 814 activation thresholds during morphogenesis. Plant Cell 20: 995-1011 28 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 815 Zhang Q, Lin F, Mao T, Nie J, Yan M, Yuan M, Zhang W (2012) Phosphatidic Acid 816 Regulates Microtubule Organization by Interacting with MAP65-1 in Response to Salt 817 Stress in Arabidopsis. Plant Cell 24:4555-4576 818 Zhang X, Dyachok J, Krishnakumar S, Smith LG, Oppenheimer DG (2005) 819 IRREGULAR TRICHOME BRANCH1 in Arabidopsis encodes a plant homolog of the 820 actin-related protein2/3 complex activator Scar/WAVE that regulates actin and 821 microtubule organization. Plant Cell 17: 2314-2326 822 Zhou L, Ni E, Yang J, Zhou H, Liang H, Li J, Jiang D, Wang Z, Liu Z, Zhuang C 823 (2013) Rice OsGL1-6 is involved in leaf cuticular wax accumulation and drought 824 resistance. PLoS One 8: e65139 1-12 825 826 827 Zhu LF, Yu SM, Jin QY (2012) Effects of aerated irrigation on leaf senescence at late growth stage and grain yield of rice. Rice Sci 19(1): 44-48. Zhu X, Xiong L (2013) Putative megaenzyme DWA1 plays essential roles in drought 828 resistance by regulating stress-induced wax deposition in rice. Proc Natl Acad Sci USA 829 110: 17790-17795 830 831 832 29 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 833 834 Table 1. Comparison of mineral contents between wild-type and es1-1 plants. Cu(mg kg-1) Fe(mg kg-1) Mn(mg kg-1) 23.5±3.83 3757.7±122.55 17.75±1.75 NPB root 446.3±18.52** 18155.3±326.27** 58.97±3.88** es1-1 8.61±1.21 35.52±3.7 32.19±1.3 NPB culm 20.18±4.7** 46.05±3.54** 147.99±10.06** es1-1 6.59±1.15 136.2±8.59 92.331±5.9 NPB shealth 16.88±2.43** 240.2±14.65** 582.2±13.22** es1-1 9.32±2.1 228.8±15.1 100.01±7.1 old NPB leaf 18.27±3.88** 358.3±21.94** 719.05±34.8** es1-1 8.02±1.16 102.1±6.78 27.51±3.32 young NPB leaf 17.07±2.91** 223.1±11.4** 175.81±12.24** es1-1 4.26±0.64 4.05±0.35 31.62±2.86 NPB seed 5.91±0.72** 6.98±1.06** 50.36±4.1** es1-1 835 The results are given as mean ± SD of three independent assays. 836 Zn(mg kg-1) 28.49±1.12 91.26±5.88** 28.75±1.81 85.83±6.35** 42.52±3.4 65.36±7.92** 29.89±2.56 64.04±7.5** 30.48±3.44 62.22±4.28** 24.23±2.6 53.13±8.12** *, ** significant at the level of 5% and 1%, respectively 837 838 Figures and Legends 839 Table 1. Comparison of mineral contents between wild-type and es1-1 plants. 840 Figure 1. Phenotypes of wild-type and es1-1 plants. 841 A, Plants in the seedling stage. Wild-type (NPB, left) and es1-1 (right) plants; bar = 3 cm. B, 842 Plants at the heading stage. Wild-type (NPB, left) and es1-1 (right) plants; bar = 10 cm. C, 843 Nitroblue tetrazolium (NBT) staining (up: NPB, left; es1-1, right); Diaminobenzidine (DAB) 844 staining (down: NPB, left; es1-1, right). D, Malondialdehyde (MDA) contents at the seedling 845 stage. (NPB, left; es1-1, right). E, Panicle rachis (NPB, left; es1-1, right); bar = 5 cm. F, Leaf 846 at different parts (NPB, left; es1-1, right); from left to right, internode length of the first to 847 top, internode length of the second to top, internode length of third to top, and internode 848 length of fourth to top; bar = 3 cm. G, Grain size (NPB, up; es1-1, down); bar = 1 mm. H, 849 Chloroplasts, observed by transmission electron microscopy; (NPB, up; es1-1, down); bar = 850 0.5 μm. 851 852 Figure 2. Map-based cloning of ES1, and phenotype of es1-1 complementation transgenic 853 line. 30 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 854 A, Location of es1-1 on rice chromosome 1. B, Coarse linkage map of es1-1. C, es1-1 gene 855 structure. D, Reverse transcription of es1-1. E, Schematic diagram of es1-1 and mutated 856 proteins encoded by es1-1 and es1-2. F, Phenotype of complementation transgenic lines; es1- 857 1 plant (left), Wild-type (NPB, middle) and transgenic line (right), bar = 10 cm. 858 859 Figure 3. ES1 is a SCAR homolog. 860 A, Leaf margin (left, NPB; right, es1-1). B, Trichome phenotype (macro hairs; upper, NPB; 861 down, es1-1). C, D, F-actin organization was visualized with Alexa Fluor 488-phalloidin in 862 root cells of wild type and es1. Over 40 roots were used in this analysis. Bar =5 μm (C, NPB; 863 D, es1-1). Arrows indicate the continuity of F-actin. E, Phylogenetic tree of SCAR-like 2 864 protein in several species. 865 866 Figure 4. Expression of ES1 and subcellular localization of ES1. 867 A–E, GUS staining of leaf (A), leaf sheath (B), fibrous root (C), stamen (D), and culm 868 vascular bundles (E). F, Quantitative real-time PCR analysis of ES1 expression in different 869 tissues at the tillering stage (T) and heading (H) stage. The results are given as means of three 870 independent assays, and each error bar represents the percent standard deviation of the mean. 871 G–L, GFP fluorescence visualized by confocal microscopy. Transient expression of eGFP 872 (G) and eGFP–ES1 cDNA (H) in epidermal cells of onion. 35S::eGFP::ES1 cDNA and 873 35S::GFP were transiently expressed in onion epidermal cells. Transient expression of eGFP 874 in epidermal cells (I) and protoplasts (K) and expression of eGFP–ES1 in epidermal cells (J) 875 and protoplasts (L) of Nicotiana benthamiana leaves. 35S::eGFP::ES1 and 35S::GFP were 876 transiently expressed in N. benthamiana leaves. 877 878 Figure 5. Observation of stomata between NPB and es1-1. 879 A, Stomata density of the wild-type (left) and es1-1 (right) leaves. Bar = 30 µm. the red 880 triangle indicate the position of stomata. B, Statistical analysis of stomatal parameters, such 881 as stomata density, partially-open stomata, stomatal conductance, transpiration rate. C, 882 Morphological characteristics (left) and maximum opening size (right) of stomata between 883 wild-type (up) and es1-1 (down) plants. Bar = 5 µm D, Comparison of water loss rate 884 between wild-type and es1-1 plants with and without 300um ABA treatment at the 3-leaf- 885 stage. E, qRT-PCR analysis of stomata density related genes (OsTMM, OsSCRM, OsSDD1, 886 OsYODA2), ES1, and some ABA response-related genes (OsDREB1A, OsDREB2A, 887 OsMYB2,OsABI4, OsABI5, OsABF4) in WT and es1-1 leaves before and after 300um ABA 31 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. 888 treatment at the 3-leaf-stage. The values were normalized to the Actin1 gene. ** indicates 889 significance (t-test) at the P<0.01. Different letters indicate significant difference at the 1% 890 level (Duncan multiple range test) 891 892 893 Figure 6. Observation of water loss rate between wild-type and es1-1 plants. 894 A, Water loss rate of detached leaves at the seedling stage (left, NPB; right, es1-1). B, Water 895 loss rate of detached leaves at the tillering stage. C, Water loss per unit of time of living 896 leaves between NPB and es1-1 plants at the tillering stage. Error bars indicate SD (n = 15). 897 Asterisks indicate the significance of differences between NPB and es1-1 plants, as 898 determined by t-test. *0.01 ≤ P < 0.05, **P < 0.01. 899 900 Figure 7. Phenotype comparison of the roots between NPB and es1-1 plants. 901 A, Root scanning of NPB plants; bar = 5 cm. B, Root scanning of es1-1 plants; bar = 5 cm. 902 C, Enlarged figure of roots in NPB plants; bar = 5 cm. D, Enlarged figure of root in es1-1 903 plants; bar = 5 cm. E–J, Statistical analysis of the main root length (E), total root length (F), 904 total area of the root (G), average diameter of the root (H), total volume of the root (I), total 905 number of fibrous roots (J) of NPB and es1-1 plants. The results are given as means of three 906 independent assays, and error bars indicate SD. Asterisks indicate the significance of 907 differences between NPB and es1-1 plants, as determined by t-test. *0.01 ≤ P < 0.05, **P < 908 0.01. 909 910 Figure 8. Number of vascular bundles and conduits in wild-type and es1-1 plants. 911 A, Number of vascular bundles in NPB plants. B, Number of vascular bundles in es1-1 912 plants. C, Comparison of the number of vascular bundles between NPB and es1-1 plants. D, 913 Number of conduits in NPB plants. E, Number of conduits in es1-1 plants. F, Comparison of 914 the number of conduits between NPB and es1-1 plants. G, Detection of the water potential 915 value in WT and es1-1. Relative water potential value in different tissues at seedling stage of 916 WT and es1-1 analyzed by WP4-T Dewpoint PotentiaMeter. The results are given as means 917 of three independent assays, and error bars indicate SD. *, ** significant at the level of 5% 918 and 1%, respectively. 919 32 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 1. Phenotypes of wild-type and es1-1 plants. A, Plants in the seedling stage. Wild-type (NPB, left) and es1-1 (right) plants; bar = 3 cm. B, Plants at the heading stage. Wild-type (NPB, left) and es1-1 (right) plants; bar = 10 cm. C, Nitroblue tetrazolium (NBT) staining (up: NPB, left; es1-1, right); Diaminobenzidine (DAB) staining (down: NPB, left; es1-1, right). D, Malondialdehyde (MDA) contents at the seedling stage. (NPB, left; es1-1, right). E, Panicle rachis (NPB, left; es1-1, right); bar = 5 cm. F, Leaf at different parts (NPB, left; es1-1, right); from left to right, internode length of the first to top, internode length of the second to top, internode length of third to top, and internode length of fourth to top; bar = 3 cm. G, Grain size (NPB, up; es1-1, down); bar = 1 mm. H, Chloroplasts, observed by transmission electron microscopy; (NPB, up; es1-1, down); bar = 0.5 μm. 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 2. Map-based cloning of ES1, and phenotype of es1-1 complementation transgenic line. A, Location of es1-1 on rice chromosome 1. B, Coarse linkage map of es1-1. C, es1-1 gene structure. D, Reverse transcription of es1-1. E, Schematic diagram of es1-1 and mutated proteins encoded by es1-1 and es1-2. F, Phenotype of complementation transgenic lines; es1-1 plant (left), Wild-type (NPB, middle) and transgenic line (right), bar = 10 cm. 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 3. ES1 is a SCAR homolog. A, Leaf margin (left, NPB; right, es1-1). B, Trichome phenotype (macro hairs; upper, NPB; down, es1-1). C, D, F-actin organization was visualized with Alexa Fluor 488-phalloidin in root cells of wild type and es1. Over 40 roots were used in this analysis. Bar =5 μm (C, NPB; D, es1-1). Arrows indicate the continuity of F-actin. E, Phylogenetic tree of SCAR-like 2 protein in several species. 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 4. Expression of ES1 and subcellular localization of ES1. A–E, GUS staining of leaf (A), leaf sheath (B), fibrous root (C), stamen (D), and culm vascular bundles (E). F, Quantitative real-time PCR analysis of ES1 expression in different tissues at the tillering stage (T) and heading stage (H). The results are given as means of three independent assays, and each error bar represents the percent standard deviation of the mean. G–L, GFP fluorescence visualized by confocal microscopy. Transient expression of eGFP (G) and eGFP–ES1 cDNA (H) in epidermal cells of onion. 35S::eGFP::ES1 cDNA and 35S::GFP were transiently expressed in onion epidermal cells. Transient expression of eGFP in epidermal cells (I) and protoplasts (K) and expression of eGFP–ES1 in epidermal cells (J) and protoplasts (L) of Nicotiana benthamiana leaves. 35S::eGFP::ES1 and 35S::GFP were 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. transiently expressed in N. benthamiana leaves. 2 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 5. Observation of stomata between NPB and es1-1. A, Stomata density of the wild-type (left) and es1-1 (right) leaves. Bar = 30 µm. the red triangle indicate the position of stomata. B, Statistical analysis of stomatal parameters, such as stomata density, partially-open stomata, stomatal conductance, transpiration rate. C, Morphological characteristics (left) and maximum opening size (right) of stomata between wild-type (up) and es1-1 (down) plants. Bar = 5 µm D, Comparison of water loss rate between wild-type and es1-1 plants with and without 300um ABA treatment at the 3-leaf-stage. E, qRT-PCR analysis of stomata density related genes (OsTMM, OsSCRM, OsSDD1, OsYODA2) and ES1, and some ABA response-related genes (OsDREB1A, OsDREB2A, OsMYB2,OsABI4, OsABI5, OsABF4) in WT and es1-1 leaves before and after 300um ABA treatment at the 3-leaf-stage. The values were normalized to the Actin1 gene. ** 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. indicates significance (t-test) at the P<0.01. Different letters indicate significant difference at the 1% level (Duncan multiple range test) 2 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 6. Observation of water loss rate between wild-type and es1-1 plants. A, Water loss rate of detached leaves at the seedling stage (left, NPB; right, es1-1). B, Water loss rate of detached leaves at the tillering stage. C, Water loss per unit of time of living leaves between NPB and es1-1 plants at the tillering stage. Error bars indicate SD (n = 15). Asterisks indicate the significance of differences between NPB and es1-1 plants, as determined by t-test. *0.01 ≤ P < 0.05, **P < 0.01. 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 7. Phenotype comparison of the roots between NPB and es1-1 plants. A, Root scanning of NPB plants; bar = 5 cm. B, Root scanning of es1-1 plants; bar = 5 cm. C, Enlarged figure of roots in NPB plants; bar = 5 cm. D, Enlarged figure of root in es1-1 plants; bar = 5 cm. E–J, Statistical analysis of the main root length (E), total root length (F), total area of the root (G), average diameter of the root (H), total volume of the root (I), total number of fibrous roots (J) of NPB and es1-1 plants. The results are given as means of three independent assays, and error bars indicate SD. Asterisks indicate the significance of differences between NPB and es1-1 plants, as determined by t-test. *0.01 ≤ P < 0.05, **P < 0.01. 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Figure 8. Number of vascular bundles and conduits in wild-type and es1-1 plants. A, Number of vascular bundles in NPB plants. B, Number of vascular bundles in es1-1 plants. C, Comparison of the number of vascular bundles between NPB and es1-1 plants. D, Number of conduits in NPB plants. E, Number of conduits in es1-1 plants. F, Comparison of the number of conduits between NPB and es1-1 plants. G, Detection of the water potential value in WT and es1-1. Relative water potential value in different tissues at seedling stage of WT and es1-1 analyzed by WP4-T Dewpoint PotentiaMeter. The results are given as means of three independent assays, and error bars indicate SD. *, ** significant at the level of 5% and 1%, respectively. 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. All rights reserved. Table 1. Comparison of mineral contents between wild-type and es1-1 plants. root culm shealth old leaf young leaf seed NPB es1-1 NPB es1-1 NPB es1-1 NPB es1-1 NPB es1-1 NPB es1-1 Cu(mg kg-1) 23.5±3.83 446.3±18.52** 8.61±1.21 20.18±4.7** 6.59±1.15 16.88±2.43** 9.32±2.1 18.27±3.88** 8.02±1.16 17.07±2.91** 4.26±0.64 5.91±0.72** Fe(mg kg-1) 3757.7±122.55 18155.3±326.27** 35.52±3.7 46.05±3.54** 136.2±8.59 240.2±14.65** 228.8±15.1 358.3±21.94** 102.1±6.78 223.1±11.4** 4.05±0.35 6.98±1.06** Mn(mg kg-1) 17.75±1.75 58.97±3.88** 32.19±1.3 147.99±10.06** 92.331±5.9 582.2±13.22** 100.01±7.1 719.05±34.8** 27.51±3.32 175.81±12.24** 31.62±2.86 50.36±4.1** Zn(mg kg-1) 28.49±1.12 91.26±5.88** 28.75±1.81 85.83±6.35** 42.52±3.4 65.36±7.92** 29.89±2.56 64.04±7.5** 30.48±3.44 62.22±4.28** 24.23±2.6 53.13±8.12** The results are given as mean ± SD of three independent assays. *, ** significant at the level of 5% and 1%, respectively 1 Downloaded from on June 17, 2017 - Published by www.plantphysiol.org Copyright © 2015 American Society of Plant Biologists. 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