Blue Light Negatively Regulates Tolerance to Phosphate Deficiency in Arabidopsis

bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Blue Light Negatively Regulates Tolerance to Phosphate 2 Deficiency in Arabidopsis 3 4 Chuan-Ming Yeh1,2, Koichi Kobayashi3, Sho Fujii3, Hidehiro Fukaki4, Nobutaka Mitsuda2, 5 Masaru Ohme-Takagi1,2* 6 7 1 Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan 8 2 Bioproduction Research Institute, National Institute of Advanced Industrial Science and 9 Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan 10 3 Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902, Japan 11 4 Graduate School of Sciences, Kobe University, Kobe 657-8501, Japan 12 13 *Correspondence and material requests should be addressed to Masaru Ohme-Takagi. 14 (mtakagi@mail.saitama-u.ac.jp) 15 16 Abbreviations 17 B, blue; DGDG, digalactosyldiacylglycerol; FR, far-red; hps, hypersensitive to phosphate 18 starvation; 19 monogalactosyldiacylglycerol; NPC4, novel phospholipase C; PC, phosphatidylcholine; 20 PHL1, PHR1-like 1; PHO1, PHOSPHATE1; PHR1, PHOSPHATE STARVATION 21 RESPONSE 1; Pi, inorganic phosphate; PR, primary root; PSI, phosphate starvation-induced; 22 PSR, 23 sulfoquinovosyldiacylglycerol; TF, transcription factor; WT, wild type. HY5, phosphate ELONGATED starvation HYPOCOTYL response; R, 24 25 1 red; 5; LR, slr-1, lateral root; solitary-root-1; MGD, SQDG, bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 Abstract 27 Plants have evolved mechanisms to improve utilization efficiency or acquisition of inorganic 28 phosphate (Pi) in response to Pi deficiency, such as altering root architecture, secreting acid 29 phosphatases, and activating the expression of genes related to Pi uptake and recycling. 30 Although many genes responsive to Pi starvation have been identified, transcription factors 31 that affect tolerance to Pi deficiency have not been well characterized. We show here that 32 defect in the ELONGATED HYPOCOTYL 5 (HY5) transcription factor gene results in 33 tolerance to Pi deficiency in Arabidopsis. The primary root length of hy5 was only slightly 34 inhibited under Pi deficient condition and its fresh weight was significantly higher than that of 35 wild type. The Pi deficiency-tolerant phenotype of hy5 was similarly observed when grown 36 on the medium without Pi. In addition, a double mutant, hy5slr1, without lateral roots also 37 showed tolerance to phosphate deficiency, indicating that the tolerance of hy5 does not result 38 from increase of external Pi uptake and may be related to internal Pi utilization or recycling. 39 Moreover, we found that blue light negatively regulates tolerance to Pi-deficiency and that 40 hy5 exhibits tolerance to Pi deficiency due to blockage of blue-light responses. Collectively, 41 this study points out light quality may play an important role in the regulation of internal Pi 42 recycling and utilization efficiency. Also, it may contribute to reducing Pi fertilizer 43 requirements in plants through a proper illumination. 44 45 Keywords 46 HY5, light, phosphate deficiency, recycling, root architecture, transcription factor 47 48 49 50 2 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 51 Introduction 52 Inorganic phosphate (Pi) is an essential constituent of ATP, nucleic acids and membrane 53 phospholipids. In addition, it is crucial to various cellular metabolic pathways, including 54 photosynthesis, glycolysis, respiration, signal transduction and carbohydrate metabolism 55 (Ticconi AND Abel 2004, Péret et al. 2011, Niu 2013). However, Pi is easily chelated by soil 56 particles or formed insoluble complexes with aluminum or iron at acid pH and with calcium 57 at alkaline pH leading to a low mobility and availability in soils (Wissuwa 2003, Gaxiola et al. 58 2011). Therefore, available soil Pi concentrations are often less than the requirement for 59 optimal crop production (Nussaume et al. 2011, Péret et al. 2011, Niu 2013). Plants have 60 evolved adaptive mechanisms to acquire and recycle Pi in response to Pi deficiency. 61 Alteration of root architecture, such as enhancement of lateral root growth and root hair 62 formation, increases root surface areas for Pi absorption (Ticconi AND Abel 2004, Péret et al. 63 2011). Induction of high-affinity Pi transporter genes increases uptake of soluble Pi, while 64 activation or secretion of acid phosphatases, ribonucleases, and organic acids enhances 65 scavenging of extracellular Pi from insoluble organic complexes. In addition, the activities of 66 acid phosphatases and ribonucleases also help release Pi from intracellular organic 67 Pi-containing molecules (Raghothama 2000, Poirier and Bucher 2002, Nussaume et al. 2011). 68 To improve Pi use efficiency, plants substitute bypass pathways that do not require Pi for 69 metabolic processes requiring Pi (Plaxton and Tran 2011). Replacing membrane 70 phospholipids with non-P-containing glycolipids also plays an important role in the supply of 71 free Pi during Pi deficiency (Kobayashi et al. 2006). 72 Many efforts have been made to unravel the molecular mechanisms that regulate Pi 73 starvation responses (PSRs). An array of Pi starvation-induced (PSI) genes have been 74 identified by transcriptome studies (Wu et al. 2003, Misson et al. 2005, Thibaud et al. 2010, 75 Woo et al. 2012) and a series of hypersensitive to phosphate starvation (hps) mutants have 3 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 been isolated and characterized (Yeh et al. 2017). Although various plant transcription factors 77 (TFs) affect PSRs, the transcriptional regulation of these processes is not yet well elucidated. 78 AtPHR1 (PHOSPHATE STARVATION RESPONSE 1) is the first Arabidopsis TF gene shown 79 to mediate diverse PSRs (Rubio et al. 2001). Although AtPHR1 is not Pi starvation-inducible, 80 PHR1 regulates a subset of PSI genes through the miR399-PHO2 (an ubiquitin-conjugating 81 E2 enzyme) signaling pathway (Bari et al. 2006, Chiou et al. 2006). AtPHR1, AtPHL1 82 (PHR1-like 1), and their two rice orthologues, OsPHR1 and OsPHR2, have been identified as 83 having partially redundant functions (Zhou et al. 2008, Bustos et al. 2010, Liu et al. 2010). In 84 addition, several TFs have been identified as negative regulators of PSRs in Arabidopsis. 85 BHLH32, a basic helix-loop-helix TF, negatively regulates anthocyanin accumulation, root 86 hair formation, and induction of the PSI genes (Chen et al. 2007). AtMYB62 is 87 low-Pi-inducible and mediates its negative effects on PSRs through modulation of gibberellin 88 metabolism (Devaiah et al. 2009). WRKY6 and WRKY42 negatively regulate the expression 89 of PHOSPHATE1 (PHO1), which is responsible for Pi translocation from root to shoot in 90 Arabidopsis (Hamburger et al. 2002, Chen et al. 2009). AtWRKY75 and AtZAT6 have been 91 reported to regulate root development and Pi acquisition, although they may not be specific to 92 PSRs due to their responsiveness to multiple nutrient deficiencies (Devaiah et al. 2007a and 93 2007b). In recent years, several Arabidopsis TF genes, such as AtERF070, APSR1, AtMYB2 94 and AL6, have been shown to be involved in the regulation of root growth and architecture 95 under Pi deficiency (Yeh and Ohme-Takagi 2015). 96 Adding Pi fertilizer can improve soil Pi levels; however, the world’s Pi rock reserves 97 may be exhausted within 120 years (Gilbert 2009; Nussaume et al. 2011) and the demand for 98 Pi fertilizers will likely increase to support crop productivity for the growing global 99 population (Nussaume et al. 2011, Péret et al. 2011). In addition, the low solubility of Pi in 100 soils often causes over-application of chemical fertilizers, subsequently, leading to potential 4 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 threats to the environment and the ecosystem (Gaxiola et al. 2011, Péret et al. 2011). 102 Therefore, proper utilization of the remaining Pi reserves is important to reduce Pi resource 103 depletion and environmental threaten. To this end, development of crops with tolerance to Pi 104 deficiency is required, especially if crops can be manipulated to possess higher ability for Pi 105 recycling or Pi utilization efficiency. 106 In this study, we identified a Pi deficiency-tolerant hy5-215 mutant with defect in the 107 Arabidopsis bZIP TF ELONGATED HYPOCOTYL 5 (HY5). Under Pi-deficient conditions, 108 primary root length and seedling fresh weight were reduced to a lesser extent in the hy5-215 109 mutant compared to the wild type (WT). The Pi-deficiency tolerance phenotype of hy5-215 110 did not change in plants grown on medium without Pi, indicating that this tolerance may be 111 related to an enhanced internal Pi utilization but not uptake of external Pi. Furthermore, we 112 found that continuous blue light accelerate sensitivity to Pi deficiency in WT and elimination 113 from blue light improve WT tolerance to Pi deficiency. Our results indicate that blue light 114 plays a negative role in Pi deficiency tolerance and hy5-215 exhibits tolerance to Pi deficiency 115 probably due to blockage of blue-light responses. 116 117 118 119 120 121 122 123 124 125 5 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 126 Results and Discussion 127 Tolerant phenotypes of hy5-215 mutants under Pi deficiency 128 To identify transcription factors (TFs) that can be manipulated to allow plants growing well 129 under minimal Pi fertilization, we grew Arabidopsis mutants in Pi-deficient conditions and 130 screened for plant phenotypes indicative of tolerance to Pi deficiency: larger plant size, longer 131 primary root (PR) length, and lower anthocyanin accumulation than wild type (WT). The 132 hy5-215 mutant with a defect in HY5, which encodes a bZIP TF that functions in 133 photopmophogenesis, exhibited a Pi deficiency-tolerant phenotype. The PR lengths of WT 134 were significantly reduced under Pi-deficient conditions (10 µM Pi) when compared with 135 those grown under Pi-sufficient conditions (625 µM Pi) while only slight inhibition of PR 136 growth was observed in the hy5-215 mutant between Pi-sufficient and Pi-deficient conditions 137 (Fig. 1A, B). WT fresh weight declined to 37% under Pi deficient-conditions compared to 138 Pi-sufficient conditions while hy5-215 fresh weight declined to 65% under Pi 139 deficient-conditions compared to Pi-sufficient conditions (Fig. 1C and Supplementary Fig. 140 S1). We also confirmed the tolerance of hy5-215 to Pi deficiency by examination of several 141 well-known PSRs including expression of ribonuclease, purple acid phosphatase and 142 anthocyanin biosynthesis genes (Supplementary Note 1 and Supplementary Fig. S2-4). 143 144 Alteration of root architecture in hy5-215 is not responsible to Pi-deficiency tolerance 145 Plant root architecture, the spatial arrangement of a root system, is highly plastic in 146 response to depletion of mineral nutrients. Modifications of RA through altering the number, 147 length, angle and diameter of roots or root hairs enable plants to cope with nutrient shortages 148 (Gruber et al. 2013). The “topsoil foraging” strategy is employed to get immobile Pi from the 149 Pi-enriched upper-layer soil under Pi deficiency; in topsoil foraging, plants inhibit PR growth 150 but enhance lateral root (LR) growth and root hair formation, thus increasing the surface area 6 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 151 available for Pi uptake (Péret et al. 2011, Sato and Miura 2011, Niu 2013). In this study, a 152 great number of root hairs were initiated in the WT under Pi-deficient conditions, whereas 153 hy5-215 formed fewer and shorter root hairs (Fig. 2A), suggesting that hy5-215 may not show 154 as strong of a response to Pi deficiency as WT. However, LR numbers and lengths were not 155 enhanced by low-Pi treatment in both WT and hy5-215. Instead, LR growth was repressed by 156 our Pi deficiency condition (Fig. 2B-D). This inconsistency may result from different Pi 157 concentrations and experimental conditions used in the different studies. Plants grown at 158 relatively higher levels of Pi (> 1 mM) in Pi-sufficient media form fewer or almost no LRs 159 (Pérez-Torres et al. 2008, Lei et al. 2011). However, Pi-sufficient treatment (625 µM) in this 160 work induces much more LR formation and growth. This is in agreement with some previous 161 reports that use relative lower concentrations for Pi-sufficient treatments (Devaiah et al. 2007a, 162 Pérez-Torres et al. 2008, Devaiah et al. 2009, Lei et al. 2011, Gruber et al. 2013). 163 Although LR growth was not enhanced by Pi starvation in this study, a root system 164 possessing more and longer LRs was found in hy5-215 in both Pi-sufficient and Pi-deficient 165 conditions (Fig. 2B-D). To examine whether the increased LR number and lengths contribute 166 to the Pi-deficiency tolerance in hy5-215, a double mutant constructed with hy5-215 and 167 solitary-root-1 (slr-1), a gain-of-function mutant of IAA14 (a repressor of auxin signaling) 168 that produces no LRs, was examined under Pi deficiency (Fukaki et al. 2002; Kobayashi et al. 169 2012). The hy5-215 slr-1 double mutant showed a long-hypocotyl phenotype similar to that of 170 hy5-215 and a PR lacking LR growth similar to the slr-1 phenotype (Fig. 2E). Interestingly, 171 the PR elongation of hy5-215 slr-1 seedlings was only slightly inhibited by Pi deficiency, 172 although the PR of hy5-215 slr-1 was shorter than that of hy5-215 in the respective conditions. 173 The results revealed that LR growth is beneficial for growth on Pi-deficient medium, but the 174 change in hy5-215 root architecture does not appear to be responsible for the observed 175 tolerance to Pi deficiency in hy5-215. Auxin signaling was reported to be enhanced in 7 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 176 Arabidopsis hy5 mutants (Oyama et al. 1997, Cluis et al. 2004), whereas it may be repressed 177 in hy5-215 slr-1 mutants due to the gain-of-function mutation of SLR/IAA14. Therefore, the 178 similar tolerance phenotypes between hy5-215 slr-1 and hy5-215 also suggest that auxin 179 signaling may not be responsible for the Pi-deficiency tolerance in hy5-215. 180 181 External Pi acquisition is not involved in Pi-deficiency tolerance of hy5-215 182 Enhancement of Pi influx through induction of high-affinity Pi transporter genes is one of 183 the conserved strategies evolved by plants to optimize their growth in response to Pi 184 limitation. There are nine genes encoding PHT homologs (PHT1;1–PHT1;9) in the 185 Arabidopsis genome. Most of the PHT1 family genes are strongly induced by low Pi 186 treatment within the first 12 hours (Bayle et al. 2011, Nussaume et al. 2011). Functional 187 studies show a major role for PHT1 in Pi acquisition in roots from Pi-deficient environment; 188 however, some of the PHTs are also required for Pi mobilization (PHT1;5), flower 189 development (PHT1;6) and Pi uptake in Pi replete condition (PHT1;1 and PHT1;4) 190 (Nussaume et al. 2011, Nagarajan et al. 2011). In this study, we found that expression of 191 PHT1 genes was lower in hy5-215 shoots than in the WT, suggesting hy5-215 may not be as 192 deficient as WT under low Pi treatment (Supplementary Fig. S5). However, several PHT1 193 genes were induced in a higher level in hy5-215 roots under both sufficient and deficient 194 conditions (Supplementary Table S1). To demonstrate whether the higher PHT1 gene 195 expression in hy5-215 roots can increase Pi uptake and subsequently contributes to 196 Pi-deficiency tolerance, the free Pi content were measured. A great reduction of Pi level was 197 found in hy5-215 shoots under Pi sufficient condition, although Pi content was slightly higher 198 in hy5-215 shoots than in WT shoots under Pi deficiency (Fig. 3A). There was no significant 199 difference between WT and hy5-215 in roots (Fig. 3B). The results indicated that the elevated 200 amounts of PHT1 transcripts in hy5-215 roots might not or only partially contribute to Pi 8 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 201 deficiency tolerance of hy5-215. To verify this finding, we cultured WT and hy5-215 plants on 202 Pi-free media. The hy5-215 plants exhibited similar growth on Pi-free medium and on 203 Pi-deficient medium containing 10 µM Pi. The PR length of hy5-215 grown on Pi-free 204 medium was only slightly diminished compared to that of plants grown on Pi-deficient 205 medium (Fig. 3C). Altogether, these results indicated that the tolerance of hy5-215 to Pi 206 deficiency was not related to extracellular Pi acquisition. Furthermore, it also suggested the 207 pre-accumulated Pi in seeds during seed development is sufficient to support hy5-215 growth 208 at the early stages of Pi deficiency. 209 210 Lower level of Pi deficiency-inducible membrane glycolipids in hy5-215 211 Since Pi deficiency tolerance of hy5-215 was not due to Pi acquisition, we investigated Pi 212 use efficiency in the mutant and wild type. Improvement of Pi utilization efficiency helps 213 plants to conserve internal Pi and can involve the recycling of Pi from senescent tissues and 214 the replacement of Pi from cellular structures or metabolic processes by alternative non-Pi 215 compounds (Kobayashi et al. 2006, Rose et al. 2013). Membrane lipid remodeling, in which 216 phospholipids are hydrolyzed and replaced by non-phosphorus glycolipids, such as 217 sulfoquinovosyldiacylglycerol (SQDG) and digalactosyldiacylglycerol (DGDG), is a 218 representative mechanism of Pi recycling, which improves Pi use efficiency (Kobayashi et al. 219 2006, Nakamura et al. 2013). Therefore, we analyzed the expression of genes involved in 220 hydrolysis of phospholipids, novel phospholipase C gene (NPC4), and synthesis of SQDG 221 and DGDG including SQD1, SQD2, MGD2 and MGD3 (monogalactosyldiacylglycerol 222 synthetic genes) in the WT and hy5-215. All the analyzed genes were induced by Pi deficiency, 223 but the expression levels were lower in hy5-215 than in the WT (Supplementary Fig. S6A-E). 224 The lipid composition calculated as the ratio of DGDG and PC (phosphatidylcholine), one of 225 the major membrane phospholipids, is used as a marker to indicate a Pi-deficient state 9 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 226 (Kobayashi et al. 2006). Enhancement of the DGDG/PC ratio represents an increase in 227 DGDG biosynthesis to replace membrane phospholipids in response to Pi deficiency. A lower 228 ratio of DGDG/PC was found in hy5-215 under Pi-deficient conditions (Supplementary Fig. 229 S6F), indicating that the increased tolerance to Pi deficiency in hy5-215 mutants is not caused 230 by increased free Pi from phospholipids. 231 232 Identification of possible candidate genes responsible for Pi-deficiency tolerance in 233 hy5-215 234 To determine the Pi-deficiency tolerance mechanism of hy5-215, we performed a 235 transcriptome study using microarray. Consistent with previous reports, the well-known PSI 236 genes were up-regulated in the WT under Pi deficiency. However, the expression levels of 237 most PSI genes were significantly lower in hy5-215, including genes encoding high-affinity Pi 238 transporters, ribonucleases, acid phosphatases, lipid remodeling and anthocyanin synthesis 239 enzymes (Supplementary Table S1). Previously reported Pi deficiency-responsive TF genes in 240 Arabidopsis mainly belong to the MYB and WRKY families (Rubio et al. 2001, Bustos et al. 241 2010, Yeh and Ohme-Takagi 2015). In this study, various TF genes, including MYB, WRKY, 242 AP2/ERF, bHLH, C2H2ZnF and MADS-box, were up-regulated or down-regulated in hy5-215 243 under Pi-deficient conditions (Supplementary Table S2), suggesting possible roles in the 244 tolerance of hy5-215 to Pi deficiency. 245 Liu et al. (2017) recently reported that HY5 negatively regulates expression of PHR1 and 246 its downstream PSI genes, and hy5 mutant increases Pi and anthocyanin contents. According 247 to their results, the longer root phenotype of hy5 to phosphate starvation may result from the 248 increased PSRs and Pi content. Although the root phenotypes of hy5 are similar to our results, 249 the expression of PHR1 and PSI genes, and Pi and anthocyanin content were lower in the 250 hy5-215 mutant in our study (Fig. 3, Supplementary Fig. S2, S3, Table S1), which are 10 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 251 consistent with previous reports that the expression of anthocyanin biosynthesis genes and 252 anthocyanin accumulation are reduced in hy5 (Lee et al. 2007, Jeong et al. 2010, Shin et al. 253 2013). Our results clearly show that the hy5 tolerant phenotype to phosphate starvation is 254 unlikely to be related to external Pi uptake because of similar growths of hy5 on Pi-deficient 255 and Pi-free conditions (Fig. 3C). Further information is required to address whether these 256 inconsistencies result from different growth conditions and different plant tissues. 257 Unexpectedly, a significant number of photosynthesis-related and chlorophyll synthesis 258 genes were down-regulated in roots but not shoots of hy5-215 (Supplementary Fig. S7 and 259 Table S3). Plant roots can accumulate chlorophyll and turn green under light illumination. The 260 green roots are supposed to have photosynthetic ability as green leaves (Kobayashi et al. 261 2012). We therefore analyzed whether the Pi-deficiency tolerance of hy5-215 is related to 262 down-regulation of photosynthesis-related and chlorophyll synthesis genes, which may induce 263 lower Pi consumption by decreasing photosynthesis in hy5-215 roots. GLK1 and GLK2 have 264 been shown to regulate expression of various photosynthetic genes in Arabidopsis roots 265 (Kobayashi et al. 2012, Kobayashi et al. 2013). In addition, it was reported the roots of 266 35S:GLK1 accumulates much chlorophyll and are hypersensitive to Pi deficiency (Kang et al. 267 2014).We thus examined whether the glk mutants also show tolerance to Pi deficiency. The 268 similar PR lengths between WT and glk mutants indicate GLK1 and GLK2 may not be 269 involved in Pi-deficiency tolerance (Supplementary Fig. S8A, C). We further investigated the 270 overexpression lines of GLK1 and GLK2 in hy5-215 background (35S:GLK1 hy5-215 and 271 35S:GLK2 hy5-215), which have a recovered chlorophyll content as WT (Kobayashi et al. 272 2012). The 35S:GLK1 hy5-215 and 35S:GLK2 hy5-215 plants exhibited longer PR lengths 273 under Pi deficiency similar to hy5-215 (Supplementary Fig. S8B), suggesting that tolerance of 274 hy5-215 to Pi deficiency may not be related to chlorophyll content and photosynthetic 275 activity. 11 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 276 To confirm this finding, the photosynthetic ability of hy5-215 and WT plants was 277 measured and compared, although photosynthetic gene expression in shoots was not 278 significantly different between hy5-215 and WT under Pi-sufficient or Pi-deficient conditions. 279 As shown in Supplementary Fig. S9, the maximum quantum yield of photosystem II (Fv/Fm) 280 and the actual quantum yield of photosystem II under light (YII) were reduced in the 281 cotyledons of both WT and hy5-215 in response to Pi deficiency. Although the measurement 282 of Fv/Fm and YII of hy5-215 under Pi sufficient treatment were lower than those of WT, there 283 was no significant difference between WT and hy5-215 in response to Pi depletion. In 284 addition, Fv/Fm and YII in the true leaves of WT and hy5-215 were not affected by our low Pi 285 treatment. These data indicate that the tolerance of hy5-215 to Pi deficiency is not related to 286 photosynthetic ability (Supplementary Fig. S9). All together, these results indicate a novel 287 mechanism other than the well-known PSRs may account for hy5-215 tolerance to Pi 288 deficiency. 289 290 Light quality is involved in regulation of Pi deficiency response 291 Because HY5 acts as an integrator of different light signaling pathways downstream of 292 multiple photoreceptor families and regulates photomorphogenesis (Cluis et al. 2004), we 293 examined the effect of light on hy5-215 tolerance to Pi deficiency. When the seedlings were 294 grown in Pi-deficient conditions under continuous white light, WT and hy5-215 PR lengths 295 were 28% and 46% of PR lengths under Pi-sufficient conditions, respectively (Fig. 4A). 296 Under continuous dark, there were no significant differences in PR growth between WT and 297 hy5-215 (Fig. 4B). These results, together with the results from long-day treatments (16 h 298 light/8 h dark; Fig. 1B), indicate that increased light irradiation time inhibits Arabidopsis PR 299 growth in Pi-deficient conditions. Therefore, light may play a role in hy5-215 tolerance to Pi 300 deficiency. 12 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 301 To better understand light effects on Pi-deficiency tolerance, Arabidopsis plants were 302 grown under continuous blue (B), red (R) and far-red (FR) light. PR growth was inhibited by 303 Pi deficiency in the WT under continuous B light to a similar extent as was observed in white 304 light. In contrast, the same level of inhibition by Pi deficiency under B light was not observed 305 in hy5-215 (Fig. 5A). Interestingly, PR growth was not inhibited by Pi deficiency in both WT 306 and hy5-215 when grown under continuous R and FR irradiation (Fig. 5B-D and 307 Supplementary Fig. S10). These results indicate that the tolerance of hy5-215 to Pi deficiency 308 is negatively regulated by B light and is not related to R and FR light. To further confirm this 309 finding, the B light receptor mutants, cry1 cry2 and phot1 phot2, were examined under Pi 310 deficiency. Indeed, a tolerant phenotype to Pi deficiency was found in these two mutants (Fig. 311 5E-F). Therefore, the tolerance of hy5-215 to Pi deficiency likely results from blockage of B 312 light responses, and the tolerance mechanism may be related to enhancement of internal Pi 313 recycling or utilization efficiency but not external Pi acquisition due to the tolerant phenotype 314 of hy5-215 under Pi-free condition. Our findings may provide valuable insights for 315 developing Pi deficiency-tolerant crops in the future. Furthermore, light quality-regulated 316 responses to Pi deficiency may allow indoor plant growers to reduce Pi fertilizer application 317 through proper illumination. 318 319 320 321 322 323 324 325 13 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 326 Materials and Methods 327 Plant materials and growth conditions 328 The surface-sterilized seeds of Arabidopsis thaliana wild type [ecotypes Columbia 329 (Col-0)] and mutants (hy5-215, slr-1, hy5-215 slr-1, glk1, glk2, glk1 glk2, cry1 cry2, phot1 330 phot2), and transformants (35S:GLK1 hy5-215 and 35S:GLK2 hy5-215) were sown on 1/2 331 Murashige and Skoog (MS) agar plates containing 625 µM KH2PO4 (Pi sufficient) or 10 µM 332 KH2PO4 (Pi deficient). Each experiment used 10 plants and was replicated three to four times. 333 The seedlings were grown at 22°C and illuminated with 100-125 µmol m-2 s-1 white light for 334 16 hours per day or with blue (B), red (R) and far-red (FR) light for 24 hours. For 335 determination of primary root (PR) length and fresh weight, the seedlings were cultured on 336 vertical and horizontal plates for 10 and 14 days, respectively. The seedlings were then 337 collected for photographs, measurement of PR length and fresh weight, and further 338 experiments. 339 340 Quantification of anthocyanin content 341 The shoots of 10-day-old seedlings were frozen in liquid nitrogen, ground into a powder, 342 and then re-suspended in an extraction buffer containing 45% methanol and 5% acetic acid. 343 The supernatant was taken after centrifugation at 12,000 rpm for 10 minutes. Anthocyanin 344 content was calculated by the absorbance at 530 and 637 nm as described previously (Matsui 345 et al. 2004). 346 347 Determination of acid phosphatase activity 348 The histochemical staining of acid phosphatase activity was performed according to the 349 method described by Yu et al. (2012) with some modifications. The roots of 10-day-old 350 seedlings were overlaid with a 0.1% 14 agar solution containing 0.01% bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 351 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The acid phosphatase activity indicated by 352 blue color on the root surface was observed and photographed after 6 to 24 hours. 353 354 Determination of lipid composition 355 Seedlings were grown on 1/2 MS medium with 625 µM Pi for 10 days and then 356 transferred to 1/2 MS medium with 625 µM Pi or 10 µM Pi for 10 days. Samples were 357 collected and immediately frozen in liquid nitrogen. Lipids were then extracted and analyzed 358 by the method described by Kobayashi et al. (2006). 359 360 RNA isolation, reverse-transcription quantitative PCR (RT-qPCR), and microarray 361 analyses 362 Total RNA was extracted by using the RNeasy Plant Mini kit (QIAGEN, Hilden, 363 Germany) following the manufacturer’s instructions. One µg of total RNA was subjected to 364 first-strand cDNA synthesis using the PrimeScript RT reagent kit (Takara). Quantitative 365 RT-qPCR was performed by the SYBR green method using the ABI7300 real-time PCR 366 system (Applied Biosystems) as described previously (Mitsuda et al. 2005). The UBQ1 gene 367 was used as an internal control. The microarray experiments and the data analysis were 368 conducted by the method described by Mitsuda et al. (2005). Three or four biological 369 replicates were included in each experiment. 370 371 Measurement of photosynthetic activity 372 The maximum quantum yield of photosystem II (Fv/Fm) and actual quantum yield of 373 photosystem II in light (YII) of cotyledons and true leaves were measured according to the 374 method described by Kobayashi et al. (2013). 375 15 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 376 Statistical analysis 377 All the experiments were performed in a completely randomized design. Data on root 378 length (cm) and seedling fresh weight (mg) were recorded after growth for 10 and 14 days, 379 respectively. Analysis of variance (ANOVA) and mean comparisons using least significant 380 difference (LSD) tests were conducted. Data represent means of three or four independent 381 experiments. Different letters above bars indicate statistically significant differences (P 382 <0.05). 383 384 Accession numbers 385 Arabidopsis Genome Initiative numbers described in this article are as follows: ACP5 386 (At3g17790), CHS (At5g13930), DFR (At5g42800), GLK1 (At2g20570), GLK2 (At5g44190), 387 HY5 (At5g11260), LDOX (At4g22880), MGD2 (At5g20410), MGD3 (At2g11810), MYB75 388 (At1g56650), MYB90 (At1g66390), NPC4 (At3g03530), PHT1;2 (At5g43370), PHT1;3 389 (At5g43360), PHT1;4 (At2g38940), PHT1;5 (At2g32830), PHT1;7 (At3g54700), PHT1;8 390 (At1g20860), PHT1;9 (At1g76430), RNS1 (At2g02990), SLR/IAA14 (At4g14550), SQD1 391 (At4g33030), SQD2 (At5g01220) and UF3GT (AT5G54060). 392 393 Funding 394 This work was supported by grants from The Japan Society for the Promotion of Science 395 (JSPS) and The Japan Science and Technology Agency (JST). 396 397 Disclosures 398 The authors declare no competing financial interests. 399 400 Acknowledgements 16 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 401 We thank Professor Kiyotaka Okada (National Institute for Basic Biology, Japan) for 402 providing hy5-215 seeds, Professor Shigo Takagi (Osaka University, Japan) and Professor 403 Hirokazu Tsukaya (The University of Tokyo, Japan) for phot1, phot2 and phot1 phot2 seeds, 404 Professor Christian Fankhauser (University of Lausanne, Switzerland) for cry1 cry2 seeds and 405 Dr. Yukari Nagatoshi (JIRCAS, Japan) for glk1, glk2 and glk1 glk2 seeds. We also thank Dr. 406 Sumire Fujiwara and Dr. Yukari Nagatoshi for discussion on the research, and Naomi Ujiie, 407 Machiko Onuki, Yukie Kimura and Sumiko Takahash for technical assistance. This work was 408 supported by grants from The Japan Society for the Promotion of Science (JSPS) and The 409 Japan Science and Technology Agency (JST). 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 17 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 426 References 427 Bari R., Datt Pant B., Stitt M., Scheible W.R. (2006) PHO2, microRNA399, and PHR1 define 428 a phosphate-signaling pathway in plants. Plant Physiol. 141: 988-999. 429 Bayle V., Arrighi J.F., Creff A., Nespoulous C., Vialaret J., Rossignol M. et al. (2011) 430 Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of 431 posttranslational regulation. Plant Cell 23: 1523-1535. 432 Bustos R., Castrillo G., Linhares F., Puga M.I., Rubio V., Pérez-Pérez J. et al. 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Bot. 63: 558 4527-4538. 559 Zhou J., Jiao F., Wu Z., Li Y., Wang X., He X. et al. (2008) OsPHR2 is involved in 560 phosphate-starvation signaling and excessive phosphate accumulation in shoots of plants. 561 Plant Physiol. 146: 1673-1686. 562 563 564 565 566 567 568 569 570 571 572 573 574 575 23 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 576 Figure legends 577 Figure 1. Primary root length and fresh weight of wild-type and mutant seedlings in 578 response to Pi treatment. (A) Wildtype (Col-0) and hy5-215 seedlings grown in Pi-sufficient 579 (625 µM Pi) and Pi-deficient (10 µM Pi) conditions. (B) Primary root (PR) lengths after 580 growth on vertical plates for 10 days. (C) Seedling fresh weights after growth on horizontal 581 plates for 14 days. Data represent the means ± standard error (SE) of four independent 582 experiments. Different letters above the bars indicate statistically significant differences 583 among the means based on ANOVA (Analysis of Variance) followed by Fisher’s LSD (Least 584 Significant Difference) tests (P <0.05). 585 586 Figure 2. Root hair formation and root architecture of wild-type and mutant seedlings in 587 response to Pi treatment. (A) Root hair formation of Col-0 and hy5-215 after growth of 7 588 days. (B) Root architecture of Col-0 and hy5-215 after growth of 10 days. (C) Increase of LR 589 number in hy5-215 plants. (D) Increase of LR length in hy5-215 plants. (E) PR length in 590 Col-0, hy5-215, slr, and hy5-215slr-1. All the seedlings were grown on 1/2 MS medium with 591 625 or 10 µM Pi for 7 to 10 days. Data represent means ± SE of four independent experiments. 592 Different letters above the bars indicate statistically significant differences among the means 593 based on ANOVA followed by Fisher’s LSD tests (P <0.05). 594 595 Figure 3. Pi content in wild-type and mutant seedlings in response to Pi treatment. (A) 596 Soot Pi content in Col-0 and hy5-215. (B) Root Pi content in Col-0 and hy5-215. (C) PR 597 length in Col-0 and hy5-215 when Pi was sufficient or absent. The seedlings were grown on 598 1/2 MS medium with 625, 10 or 0 µM Pi for 10 days. Data represent means ± SE of four 599 independent experiments. Different letters above the bars indicate statistically significant 600 differences among the means based on ANOVA followed by Fisher’s LSD tests (P <0.05). 24 bioRxiv preprint doi: https://doi.org/10.1101/235952. this version posted December 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 601 602 Figure 4. Effect of light on Pi-deficiency tolerance in Arabidopsis. The seedlings were 603 grown on 1/2 MS media with 625 or 10 µM Pi under continuous light (A) or dark (B) 604 treatments. The PR length was measured after 10 days of growth. Data represent means ± SE 605 of four independent experiments. Different letters above the bars indicate statistically 606 significant differences among the means based on ANOVA followed by Fisher’s LSD test (P 607 <0.05). 608 609 Figure 5. Effect of light quality on primary root length in Arabidopsis. The seedlings 610 were grown on 1/2 MS media with 625 or 10 µM Pi under continuous blue (B), red (R) or far 611 red (FR) light treatments, respectively (A-D). The blue light receptor mutants, cry1 cry2 (E) 612 and phot1 phot2 (F), were grown on Pi-sufficient and Pi-deficient media under long-day 613 condition (16 h light/8 h dark). PR length was measured after 10 days of growth. Data 614 represent means ± SE of four independent experiments. Different letters above the bars 615 indicate statistically significant differences among the means based on ANOVA followed by 616 Fisher’s LSD test (P <0.05). 617 25 A Pi sufficient (625 μM) Col-0 Pi deficient (10 μM) Col-0 hy5-215 B hy5-215 60 8 625  Pi 10  Pi a b 6 bc 4 d 2 Fresh weight (mg/seedling) Primary root length (cm/root) C 50 625  Pi 10  Pi a a 40 65% b 30 37% 20 c 10 0 0 Col-0 Col-0 Col-0 hy5-215 hy5-215 Fig. 1 hy5-215 hy5-215 A B Col-0 625 μM hy5-215 Col-0 10 μM hy5-215 Col-0 hy5-215 625 μM 10 μM C D 625  Pi 10  Pi 25 20 a a b 15 Lateral root length (cm) Number of lateral roots 30 b 10 5 a 2 a b 1 c 0 0 Col-0 Col-0 hy5-215 E Primary root length (cm/root) 625  Pi 10  Pi 3 6 a 625  Pi 10  Pi a b b b c 4 2 d d 0 Col-0 Col-0 hy5-215 slr hy5-215slr slr hy5-215slr Fig. 2 hy5-215 A Pi content (nmol/mg FW) Shoot 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 625  Pi 10  Pi a b c c 0 Col-0 Col-0 hy5-215 hy5-215 B Root Pi content (nmol/mg FW) 12 625  Pi 10  Pi 10 a a 8 6 b 4 b 2 0 Col-0 hy5-215 hy5-215 Primary root length (cm/root) C 625  Pi 0  Pi 8 a a 6 b 4 2 c 0 Col-0 Fig. 3 hy5-215 hy5-215 A B Dark 24 hr Light 24 hr 625  Pi 10  Pi 8 6 a a 46% 4 28% 2 b c 0 Primary root length (cm) Primary root length (cm) 6 625  Pi 10  Pi 5 4 a ab 3 ab b 2 1 0 Col-0 hy5-215 Col-0 Fig. 4 hy5-215 A B Continuous R 10 μM Pi 625 μM Pi Continuous B Primary root length (cm) 8 Col-0 625  Pi 10  Pi 6 hy5 Col-0 hy5 a a b 4 2 c 0 Col-0 hy5-215 C D Continuous R Continuous FR 6 a 6 625  Pi 10  Pi a b Primary root length (cm) Primary root length (cm) 8 b 4 2 0 5 a 625  Pi 10  Pi a 4 b 3 b 2 1 0 Col-0 hy5-215 Col-0 E hy5-215 F 625  Pi 10  Pi 8 a b 6 4 2 W light 16 h/ Dark 8 h 10 Primary root length (cm) Primary root length (cm) W light 16 h/ Dark 8 h c d 625  Pi 10  Pi 8 a a 6 b 4 c 2 0 0 Col-0 Col-0 cry1 cry2 Fig. 5 phot1 phot2