This is the accepted version of the article: Ortiz-Alcaide, Miriam; Llamas, Ernesto; Gómez-Cadenas, Aurelio; [et al.]. «Chloroplasts modulate elongation responses to canopy shade by retrograde pathways involving HY5 and abscisic acid». The Plant Cell, Vol. 31, issue 2 (Feb. 2019). DOI 10.1105/tpc.18.00617 This version is avaible at https://ddd.uab.cat/record/203941 under the terms of the license Ortiz-Alcaide et al. 1 Chloroplasts modulate elongation responses to canopy shade by 2 retrograde pathways involving HY5 and ABA 3 4 Miriam ORTIZ-ALCAIDE1, Ernesto LLAMAS1, Aurelio GOMEZ-CADENAS2, Akira 5 NAGATANI3, Jaime F. MARTINEZ-GARCIA1,4,*, Manuel RODRIGUEZ- 6 CONCEPCION1,* 7 8 9 1, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, 08193 10 Barcelona, Spain. 11 2, Universitat Jaume I, 12071 Castelló de la Plana, Spain 12 3, Kyoto University, 606-8502 Kyoto, Japan 13 4, Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain. 14 15 16 (*) Corresponding authors: 17 JMG, jaume.martinez@cragenomica.es 18 MRC, manuel.rodriguez@cragenomica.es 19 20 Short title: Interaction of light and retrograde pathways 21 22 23 24 The authors responsible for distribution of materials integral to the findings 25 presented in this article in accordance with the policy described in the 26 Instructions for Authors (www.plantcell.org) are Jaime F. Martinez-Garcia 27 (jaume.martinez@cragenomica.es) and Manuel Rodriguez-Concepcion 28 (manuel.rodriguez@cragenomica.es). 1 Ortiz-Alcaide et al. 29 ABSTRACT 30 Plants use light as energy for photosynthesis but also as a signal of 31 competing vegetation. By using different concentrations of norflurazon and 32 lincomycin, we found that the response to canopy shade in Arabidopsis thaliana 33 was repressed even when inhibitors only caused a modest reduction in the level 34 of photosynthetic pigments. High inhibitor concentrations resulted in albino 35 seedlings that were unable to elongate when exposed to shade, in part due to 36 attenuated light perception and signaling via phytochrome B and phytochrome- 37 interacting factors. The response to shade was further repressed by a GUN1- 38 independent retrograde network with two separate nodes represented by the 39 transcription factor HY5 and the carotenoid-derived hormone ABA. The unveiled 40 connection between chloroplast status, light (shade) signaling, and 41 developmental responses should contribute to achieve optimal photosynthetic 42 performance under light-changing conditions. 2 Ortiz-Alcaide et al. 43 INTRODUCTION 44 Life on our planet heavily relies on photosynthesis, i.e. the use of solar 45 energy (sunlight) to fix carbon into organic matter linked to the production of 46 oxygen from water. In plants, the quantity and quality of the incoming light 47 strongly influence growth and development. For example, oxidative stress and 48 eventual damage can occur if the amount of light exceeds the photosynthetic 49 capacity of the chloroplast. By contrast, light supply and hence photosynthetic 50 activity can be compromised by the shading of nearby plants. Under a canopy, 51 plants might actually be exposed to moments of both excess light (e.g. sunflecks) 52 and low light (i.e. shading) during the same day. Even in open habitats, plants 53 are usually found in communities where competition for light might result in 54 overgrowing and eventual shadowing by neighbors. 55 Light quality is an important signal that informs plants of potential 56 competitors. Vegetation absorbs light from the visible region (called 57 photosynthetically active radiation or PAR, 400–700 nm). In particular, it absorbs 58 red light (R, 600–700 nm) but transmits and reflects far-red light (FR, 700–800 59 nm), therefore causing a reduction in the R to FR ratio (R/FR). Both PAR (light 60 quantity) and R/FR (light quality) are greatly reduced under a plant canopy, 61 whereas the presence of nearby plants (without direct vegetation shading) 62 involves a more moderate reduction of R/FR without changes in PAR (Casal, 63 2012; Martinez-Garcia et al., 2014; Fiorucci and Fankhauser, 2017). 64 Independently of the PAR level, a drop in R/FR acts as a signal that strongly and 65 differentially affects elongation of shade-avoiding plants such as Arabidopsis 66 thaliana and most crops (Martinez-Garcia et al., 2014). Low R/FR signals also 67 cause a decrease in the levels of photosynthetic pigments (chlorophylls and 68 carotenoids) in seedlings and adult plants (Roig-Villanova et al., 2007; Patel et 69 al., 2013; Bou-Torrent et al., 2015; Llorente et al., 2017). These and other 70 responses triggered by a reduced R/FR are collectively known as the shade 71 avoidance syndrome (SAS) and aim to overgrow neighboring plants, readjust 72 photosynthetic metabolism, and eventually launch reproductive development 73 (Franklin, 2008; Casal, 2012; Gommers et al., 2013; Martinez-Garcia et al., 74 2014). 75 Low R/FR signals indicative of shade are perceived by the phytochrome 76 (phy) family of photoreceptors. Five genes encode the phy family in Arabidopsis: 3 Ortiz-Alcaide et al. 77 phyA to phyE. While phyB is the major phy controlling the responses to shade, 78 other phy members such as phyD and phyE can also redundantly contribute to 79 the control of shade-modulated elongation growth or flowering time (Franklin, 80 2008; Martinez-Garcia et al., 2014). In the case of photolabile phyA, an 81 antagonistic negative role has been reported for the seedling hypocotyl 82 elongation response to shade. Thus, the SAS is induced by phyB deactivation 83 but gradually antagonized by phyA in response to high FR levels characteristic of 84 plant canopy shade (Casal, 2012; Martinez-Garcia et al., 2014). This intrafamily 85 photosensory attenuation mechanism might act to suppress excessive elongation 86 under prolonged direct vegetation shade. It remains unknown whether other SAS 87 responses, including photosynthetic pigment decrease, are also affected by this 88 antagonistic regulation by phyA and phyB. In any case, the balance between 89 positive and negative regulators of the SAS acting downstream phys was found 90 to be instrumental to regulate not only hypocotyl elongation but also carotenoid 91 biosynthesis (Franklin, 2008; Casal, 2013; Bou-Torrent et al., 2015). Positive 92 regulators of the SAS include transcription factors of the basic-helix-loop-helix 93 (bHLH) (e.g. PIFs, BEEs, BIMs) and homeodomain leucine zipper class II 94 (ATHB2, ATHB4, HAT1, HAT2 and HAT3) families, whereas the basic leucine 95 zipper (bZIP) transcription factor HY5 and bHLH family members PIL1, HFR1 96 and PAR1 have negative roles. Among them, PIFs and HY5 have also been 97 found to participate in retrograde signaling during deetiolation, i.e. in the 98 communication between chloroplasts and nucleus when underground seedlings 99 sense the light and change from skotomorphogenic (i.e. heterotrophic) to 100 photomorphogenic (i.e. photosynthetic) development (Ruckle et al., 2007; Martin 101 et al., 2016; Xu et al., 2016). Alterations in the physiological status of the 102 chloroplast in light-grown plants are also signaled to the nucleus by a variety of 103 retrograde pathways that readjust nuclear gene expression accordingly (Baier 104 and Dietz, 2005; Glasser et al., 2014; Chan et al., 2016). Because exposure to 105 shade causes a decrease in the accumulation of chlorophylls and carotenoids 106 that can eventually compromise photosynthesis and photoprotection (Roig- 107 Villanova et al., 2007; Cagnola et al., 2012; Bou-Torrent et al., 2015), we 108 reasoned that the derived effects on chloroplast homeostasis might not be just a 109 consequence but influence the response to shade itself (e.g. in terms of 4 Ortiz-Alcaide et al. 110 elongation) through retrograde signaling. The work reported here aimed to test 111 this possibility. 112 113 RESULTS AND DISCUSSION 114 Functional chloroplasts are required for full response to simulated shade. 115 To initially test whether retrograde signals modulate the elongation 116 response to plant proximity, we used two kinds of inhibitors of chloroplast 117 function associated with retrograde signaling: norflurazon (NF), an inhibitor of 118 carotenoid biosynthesis (Chamovitz et al., 1991) and lincomycin (LIN), an 119 inhibitor of chloroplast protein synthesis (Mulo et al., 2003). Both inhibitors were 120 present in the medium used for seed germination and seedling growth (Figure 1). 121 This medium also contained sucrose to sustain growth even in the absence of 122 photosynthesis. As expected, a concentration-dependent bleaching was 123 observed in wild-type (WT) Arabidopsis plants grown under white light (W) with 124 NF or LIN (Figure 1A). The concentration of inhibitors required to obtain albino 125 seedlings was adjusted to our experimental conditions. For example, an albino 126 phenotype was previously observed in WT seedlings grown without sucrose in 127 the presence of 5 µM NF under 100 μmol m–2 s–1 or with 50 nM NF under 5 μmol 128 m–2 s–1 (Saini et al., 2011). We used intermediate light intensity conditions (20-24 129 μmol m–2 s–1) and, most importantly, added sucrose in the medium, which 130 together made it necessary to adjust NF concentration to 200 nM to obtain 131 completely albino seedlings (Figure 1A). 132 The presence of inhibitors had no significant effect on hypocotyl length 133 under W (Figure 1B). However, exposure to FR-enriched W (W+FR) to simulate 134 canopy shade progressively impaired elongation as levels of photosynthetic 135 pigments (chlorophylls and carotenoids) decreased. Importantly, inhibition of 136 shade-triggered elongation growth was observed at concentrations of NF or LIN 137 that only slightly reduced the levels of photosynthetic pigments and had no visual 138 impact on seedling pigmentation (e.g. 25 nM NF or 5 µM LIN), suggesting that 139 even moderate alterations in chloroplast function might influence the response to 140 shade. Hypocotyl elongation in response to shade was completely blocked at 141 concentrations of NF causing more than a 80% loss of chlorophylls, whereas an 142 even lower reduction (50%) was required for a lack of response in LIN-treated 143 seedlings (Figure 1B). In both cases, completely bleached seedlings did not 5 Ortiz-Alcaide et al. 144 elongate at all when exposed to W+FR compared to W controls (Figure 1B), 145 suggesting that functional chloroplasts are required for the elongation response 146 to canopy shade. 147 We next aimed to confirm that the disrupted elongation response to W+FR 148 observed in bleached seedlings was not due to energetic constraints. If non- 149 photosynthetic seedlings lacking chlorophylls maintain an intrinsic capacity to 150 grow, it would be expected that their hypocotyls would elongate when treated 151 with growth-promoting hormones such as brassinosteroids, auxins, or 152 gibberellins. In agreement, seedlings grown in the presence of NF concentrations 153 that completely blocked photosynthetic development (2 µM) were able to 154 elongate very similarly to control green seedlings when treated with any of these 155 hormones (Figure S1). The same hormone treatments caused a similar growth 6 Ortiz-Alcaide et al. 156 response in the case of mutant hdr-3 seedlings, which are unable to produce the 157 precursors for chlorophyll and carotenoid biosynthesis in chloroplasts and hence 158 display an albino phenotype (Pokhilko et al., 2015). We therefore conclude that 159 functional chloroplasts are not required for hormone-mediated hypocotyl 160 elongation (at least in sucrose-supplemented media) but are necessary for 161 growth in response to shade signals. 162 163 Defective chloroplast function impairs phytochrome-mediated shade 164 signaling. 165 Phytochromes are the main photoreceptors involved in shade perception 166 and signal transduction, with phyB having a predominant role in Arabidopsis 167 (Casal, 2012; Martinez-Garcia et al., 2014; Fiorucci and Fankhauser, 2017). To 168 address whether treatment with NF or LIN had an impact on phytochrome 169 signaling, we used transgenic 35S:PHYB-GFP plants expressing a biologically 170 active GFP-tagged version of the phytochrome (Yamaguchi et al., 1999). Under 171 W, the phyB-GFP fusion protein shows a characteristic distribution in nuclear 172 speckles, presumably the site where active photoreceptor proteins interact with 173 other nuclear factors to mediate light signaling (Yamaguchi et al., 1999). We 174 observed that only minutes after exposing 35S:PHYB-GFP seedlings to an end- 175 of-day FR treatment to simulate shade, the green fluorescence associated to the 176 phyB-GFP reporter became more disperse in the nuclei of epidermal hypocotyl 177 cells (Figure 2), likely reflecting phyB inactivation. Strikingly, this shade-mediated 178 inactivation process was clearly delayed in albino seedlings germinated and 179 grown in the presence of NF (5 µM) or LIN (1 mM). While mock (i.e. green) 180 seedlings displayed evenly distributed nuclear phyB-GFP fluorescence in all 181 analyzed cells 90 min after the light treatment, inhibitor-grown (i.e. albino) 182 seedlings still showed nuclear speckles in some cells after 210 min. These 183 results suggest that functional chloroplasts are required for proper phyB 184 inactivation in response to shade signals. Consistent with this conclusion, the 185 shade-triggered and phytochrome-dependent stabilization of photolabile PIF3 186 was attenuated in NF-bleached seedlings (Figure S2). 187 To further confirm whether phytochrome function was altered in albino 188 seedlings, we next analyzed the expression of rapidly shade-induced 189 phytochrome primary target genes in WT plants either treated or not with NF 7 Ortiz-Alcaide et al. 190 (Figure 3). In particular, we chose genes PIL1, ATHB2, HFR1, YUCCA8 and 191 PAR1 (Roig-Villanova et al., 2006). WT plants grown under W for 7 days were 192 exposed to W+FR for 1h and then samples were collected and used for RNA 193 extraction and quantitative RT-PCR (qPCR) analysis. As expected, comparison 194 of W-grown controls and shade-exposed (1h W+FR) samples showed that all 195 genes analyzed were induced by shade in green seedlings, ranging from 2-fold 196 (PAR1) to 80-fold (PIL1). In NF-grown seedlings, however, the induction was 197 much reduced (Figure 3). HFR1, YUCCA8 and PAR1 gene expression hardly 198 changed after W+FR treatment in albino seedlings, whereas PIL1 induction was 199 only 10% compared to that detected in green seedlings and ATHB2 up-regulation 200 was less than half. Together, we conclude that the absence of functional 201 chloroplasts somehow prevents normal light (i.e. shade) perception and signal 202 transduction by phytochromes. 203 Functional phytochrome holoproteins require the covalent attachment of a 204 phytochromobilin (PΦB) chromophore to each phytochrome apoprotein monomer 205 (Rockwell et al., 2006). The synthesis of PΦB occurs in the plastid and the early 206 steps are shared with those required to synthesize heme and chlorophylls (Figure 207 4). To test whether the observed reduction in shade-triggered phytochrome 208 inactivation (and hence hypocotyl elongation) in albino seedlings could result for 209 impaired accumulation of PΦB, we analyzed the elongation response to shade of 8 Ortiz-Alcaide et al. 210 Arabidopsis mutants defective in PΦB synthesis (Parks and Quail, 1991). In 211 particular, we used the hy1-1 allele, which was isolated from a fast-neutron 212 mutagenized population of Landsberg erecta (Ler) and carries a short deletion 213 that disrupts its function (Davis et al., 1999). As shown in Figure 4, elongation in 214 response to W+FR was not repressed but dramatically enhanced in the hy1-1 215 mutant relative to the corresponding WT (Ler). Besides showing a much stronger 216 response to shade under normal growth conditions (i.e. in the absence of 217 inhibitors), hy1-1 seedlings were also able to respond to shade and elongate 218 when treated with NF (Figure 4B). We therefore conclude that treatment with 219 bleaching inhibitors interferes with phytochrome-dependent signaling by 220 mechanisms other than defective chromophore availability. 221 Plastid retrograde signaling has been previously shown to interact with 222 components of light signaling networks to coordinate chloroplast biogenesis with 223 both the light environment and development (Larkin and Ruckle, 2008; Lepisto 224 and Rintamaki, 2012; Ruckle et al., 2012; Martin et al., 2016; Xu et al., 2016). In 225 fact, mutants defective in the PΦB biosynthetic enzymes HY1/GUN2 and 226 HY2/GUN3 (Figure 4A) were isolated in a screen for GENOMES UNCOUPLED 227 (GUN) mutants that retained partial expression of genes encoding 228 photosynthesis-related plastidial proteins after NF treatment (Mochizuki et al., 229 2001). Other GUN proteins such as GUN5 (Mochizuki et al., 2001) participate in 230 a different branch of the tetrapyrrole pathway that leads to the production of 231 chlorophylls (Figure 4A). Unlike other GUN proteins, GUN1 is not an enzyme but 232 a central integrator of retrograde signaling pathways that was proposed to 233 coordinate photomorphogenesis with chloroplast function (Koussevitzky et al., 9 Ortiz-Alcaide et al. 234 2007; Ruckle et al., 2007; Ruckle and Larkin, 2009). Similar to WT plants, 235 mutants gun1-101 (Ruckle et al., 2007) and gun5-1 (Mochizuki et al., 2001) 236 elongated in response to W+FR under normal growth conditions (i.e. when 237 chloroplasts are functional) but not when chloroplast development was blocked 238 with NF (Figure 4). Together, the described results suggest that alteration of 239 chloroplast function impacts a retrograde signaling pathway independent of GUN 240 proteins that modulates the phytochrome-mediated response to shade. 241 242 Retrograde pathways repressing shade-triggered hypocotyl elongation 243 involve HY5 but not GUN1. 244 To identify components of the chloroplast-modulated transduction pathway 245 involved in the response to shade, we next tested the possible role of SAS- 246 related transcription factors known to be involved in both light and retrograde 247 signaling: PIFs (Martin et al., 2016) and HY5 (Ruckle et al., 2007; Xu et al., 248 2016). A role for PIFs as positive regulators of the response to shade (including 249 hypocotyl elongation) is well established (Lorrain et al., 2008; Leivar et al., 2012; 250 Bou-Torrent et al., 2015). However, under our experimental conditions the 251 quadruple pifQ mutant defective in PIF1, PIF3, PIF4 and PIF5 showed a WT 252 phenotype in terms of shade-triggered hypocotyl elongation in both green and 253 albino seedlings (Figure 5). HY5 has been proposed to have a function in the 254 adaptation to prolonged shade and the response to sunflecks, i.e. exposure to 255 sunlight through gaps in the canopy (Sellaro et al., 2011; Ciolfi et al., 2013). The 256 role of this elongation-repressing transcription factor in controlling the shade- 257 promoted growth of seedling hypocotyls, however, remains unclear. Our previous 10 Ortiz-Alcaide et al. 258 work (Bou-Torrent et al., 2015) showed that complete loss of HY5 activity in the 259 null hy5-2 mutant (referred to as hy5 from now on) hardly had an impact in the 260 elongation of Arabidopsis seedlings exposed to a W+FR treatment mimicking 261 vegetation proximity (R/FR = 0.05). As shown in Figure 5, however, hy5 262 seedlings displayed increased hypocotyl elongation compared to the WT when 263 illuminated with light of a lower R/FR (0.02), reminiscent of canopy shade. These 264 results suggest that HY5 is a repressor of hypocotyl elongation in green 265 seedlings exposed to low or very low R/FR conditions. Consistently, shade- 266 triggered hypocotyl growth was inhibited in transgenic seedlings 267 overaccumulating HY5 in a hy5 background (Figure S3). Similar to WT plants, 268 the elongation response to canopy shade of hy5 seedlings was almost 269 completely blocked with LIN (Figure 5). However, the growth response of HY5- 270 deficient seedlings was not abolished but just attenuated in NF-supplemented 271 medium. Similar results were obtained in medium lacking sucrose, but the effects 272 of HY5 gain or loss of function on the elongation response of green or NF-treated 273 seedlings, respectively, were much more obvious in the presence of sucrose 274 (Figure S3). We therefore kept using sucrose-supplemented media for the rest of 275 the work. Double hy5 gun1-101 mutants were also found to display a partial 276 elongation response to shade in NF but not in LIN, similar to that found for the 277 single hy5 mutant (Figure 5). Together, the described results show that HY5 is a 278 repressor of canopy shade-triggered hypocotyl elongation. When this negative 279 regulator is lost, the elongation response to shade can still be blocked by a 280 GUN1-independent retrograde pathway that is active in LIN-treated but not in NF- 281 treated albino seedlings. 11 Ortiz-Alcaide et al. 282 We next analyzed the levels of HY5 transcripts before and after exposure to 283 our shade conditions (Figure 6). In green WT plants (grown without inhibitors) the 284 levels of HY5 transcripts were similar under W and up to 8h of our W+FR 285 treatment (Figure 6A). In contrast, immunoblot analysis of a HY5-GFP reporter in 286 complemented hy5 35S:HY5-GFP plants showed increased protein levels after 287 the simulated shade treatment (Figure 6B). Chromatin immunoprecipitation 288 experiments also detected increased levels of HY5-GFP bound to target 289 promotors in shade-exposed green seedlings (Figure 6C). Although the 290 endogenous HY5 protein might not behave exactly as the overexpressed GFP- 291 tagged version of the protein, our results are in agreement with previous studies 292 using a different reporter (HY5-myc) that concluded that the low R/FR treatment 293 stabilizes HY5 (Pacin et al., 2016). Post-transcriptional HY5 accumulation when 294 R/FR is low or very low in natural environments (such as in deep or canopy 295 shade) might help to prevent seedlings from exhibiting excessive elongation. 296 Both HY5-encoding transcripts (Figure 6D) and HY5-GFP protein (Figure 297 6B) were higher in albino seedlings grown with LIN or NF independent of the light 298 treatment, suggesting that these inhibitors promote HY5 function by increasing 299 gene expression (or/and transcript stability) and decreasing protein turnover. The 300 observation that hypocotyl length is not reduced in W-grown seedlings in the 301 presence of inhibitors (Figure 1B) despite accumulating higher HY5 levels (Figure 302 6B) suggests that hypocotyl elongation is suppressed to a saturating level by 303 multiple pathways under W and hence it would not be further repressed by 304 increasing HY5 function. In response to W+FR, however, enhanced HY5 activity 305 together with reduced light signaling in bleached WT seedlings would result in no 306 hypocotyl elongation. Only when the repressor activity of HY5 is removed (i.e. in 307 HY5-defective mutants), a second pathway that inhibits the elongation response 308 of albino seedlings becomes apparent in the presence of LIN but not in the 309 presence of NF (Figure 5). 310 311 Carotenoid-derived products repress shade-triggered hypocotyl 312 elongation. 313 The distinct mode of action of LIN and NF and particularly their differential 314 effect on carotenoid levels is illustrated by their concentration-dependent impact 315 on photosynthetic pigment accumulation (Figure 1A). HPLC analysis of 12 Ortiz-Alcaide et al. 316 carotenoid contents (Figure S4) confirmed that albino LIN-treated seedlings 317 accumulated low but detectable levels of lutein and violaxanthin as well as traces 318 of β-carotene and neoxanthin. By contrast, NF blocks the desaturation of 319 phytoene, the first committed intermediate of the carotenoid pathway (Figure 7). 320 As expected, NF-treated seedlings accumulated phytoene (which is colorless and 321 hence not detected in the spectrophotometric assay used in Figure 1A) but were 322 virtually devoid of downstream carotenoids (Figure S4). Similar to that observed 323 with LIN, other bleaching inhibitors that prevent chloroplast development and 324 cause albinism without specifically blocking the production of carotenoids, such 325 as the plastid protein synthesis inhibitor chloramphenicol (CAP) or the nitrogen 326 assimilation inhibitor phosphinotricin (PPT), were found to prevent shade- 327 triggered elongation growth in WT and HY5-defective mutants (Figure 7B). By 328 contrast, inhibition of the carotenoid pathway downstream of lycopene by 13 Ortiz-Alcaide et al. 329 blocking the activity of lycopene cyclases with 2-(4-chlorophenylthio)- 330 triethylamine chloride (CPTA) resulted in albino seedlings that were able to 331 respond to shade and elongate when HY5 function was lost (Figure 7). 332 To confirm whether the ability to respond to shade of hy5 seedlings grown 333 in the presence of NF or CPTA was specifically due to the blockage of the 334 carotenoid pathway, we next used Arabidopsis mutants. The enzyme phytoene 335 synthase (PSY) produces phytoene in the first committed step of the carotenoid 336 pathway (Figure 7A). Because PSY is encoded by a single gene in Arabidopsis 337 (Ruiz-Sola and Rodriguez-Concepcion, 2012), the knock-out mutant psy-1 338 (Pokhilko et al., 2015) does not produce phytoene and hence cannot feed the 339 pathway for the biosynthesis of downstream carotenoids (Figure S4). As a 340 consequence, the mutant displays an albino phenotype undistinguishable from 341 that observed in WT seedlings treated with NF or CPTA (Pokhilko et al., 2015). 342 Similar to that described for WT seedlings grown in the presence of carotenoid 343 biosynthesis inhibitors, psy-1 seedlings were unable to elongate when exposed 344 to W+FR (Figure 7B). However, the elongation response was rescued when both 345 HY5 and carotenoids were missing in double hy5 psy-1 mutant seedlings (Figure 346 7B). 347 Pharmacological or genetic blockage of the carotenoid pathway prevents 348 the biosynthesis of carotenoids and derived products, but it might also cause an 349 accumulation of upstream metabolites. Among them, methylerythritol 350 cyclodiphosphate (MEcPP), an intermediate of the pathway that supplies the 14 Ortiz-Alcaide et al. 351 metabolic precursors of carotenoids (Figure 7A), has been shown to act as a 352 retrograde signal in response to stress (Xiao et al., 2012). Blockage of MEcPP 353 production with the inhibitor fosmidomycin (Figure 7A), however, did not prevent 354 the elongation response to shade of NF-treated hy5 seedlings (Figure 7B). We 355 therefore conclude that what allows hy5 seedlings to respond to shade is not the 356 accumulation of a metabolite upstream PSY but the depletion of a carotenoid- 357 derived product synthesized after the step blocked by CPTA, i.e. downstream of 358 lycopene (Figure 7A). 359 As represented in Figure 7A, lycopene cyclization leads to the production of 360 carotenoids with two β rings (β,β carotenoids such as β-carotene and derived 361 xanthophylls) of with one β and one ε ring (β,ε carotenoids such as lutein). The 362 production of β,ε carotenoids in Arabidopsis is completely blocked in the green 363 lut2 mutant (Figure S4) (Emiliani et al., 2018), which is defective in the only gene 364 encoding lycopene ε-cyclase (LCYE/LUT2) in this plant species (Figure 7A) 365 (Ruiz-Sola and Rodriguez-Concepcion, 2012). Loss of β,ε carotenoids did not 366 change the elongation response to shade of single lut2 (vs. WT) or double hy5 367 lut2 (vs. hy5) seedlings (Figure S5). We therefore concluded that the effect 368 observed with CPTA (Figure 7B) is not due to the absence of β,ε carotenoids but 369 most likely to defects in the β-carotene branch of the carotenoid pathway (Figure 370 7A). Considering all these data together, we speculated that unidentified products 371 derived from β,β carotenoids can repress shade-induced elongation growth in 372 seedlings bleached with LIN and other inhibitors that do not target the carotenoid 373 biosynthesis pathway. The absence of these products in seedlings treated with 374 NF or CPTA, or in the psy-1 mutant, allows hypocotyl elongation in response to 375 shade but only when the growth-inhibitory effect of HY5 is released. 376 377 ABA represses the elongation response to shade. 378 Among the biologically active metabolites derived from β,β carotenoids 379 (Hou et al., 2016), we decided to evaluate the role of ABA as this plant hormone 380 was found to participate in the transduction of chloroplast-derived ROS/redox 381 signals (Baier and Dietz, 2005; Glasser et al., 2014; Chan et al., 2016), to 382 modulate hypocotyl growth (Lau and Deng, 2010; Humplik et al., 2017) and to act 383 together with HY5 in the regulation of several plant cell responses (Chen et al., 384 2008; Xu et al., 2014). Furthermore, treatment with low R/FR was reported to 15 Ortiz-Alcaide et al. 385 induce ABA production and signaling in tomato and Arabidopsis (Cagnola et al., 386 2012; Gonzalez-Grandio et al., 2013; Holalu and Finlayson, 2017). Indeed, ABA 387 contents were also found to slightly increase in green seedlings soon (1h) after 388 exposure to our simulated canopy shade conditions, even though the change 389 was not statistically significant (Figure S6). As expected, ABA was absent in NF- 390 treated seedlings but it could be detected in LIN-grown seedlings (Figure S6). If 391 the presence of ABA in LIN-treated hy5 seedlings contributed to inhibit their 392 response to shade, it would be expected that preventing the formation of this 393 hormone would be sufficient to rescue their response to shade. In agreement, a 394 genetic blockage of the last step of ABA biosynthesis, catalyzed by the ABA2 395 protein (Figure 7A), allowed LIN-treated double hy5 aba2 seedlings to elongate in 396 response to shade (Figure 8). Single hy5 and double hy5 aba2 seedlings had a 397 very similar response to shade in NF-supplemented medium. By contrast, in 398 green seedlings grown in the absence of inhibitors (i.e. with functional 399 chloroplasts) the double mutant elongated more than single hy5 seedlings when 400 exposed to shade (Figure 8A). We therefore concluded that HY5 and ABA likely 401 repress shade-induced hypocotyl elongation by independent pathways. This 402 conclusion was confirmed by treating NF-grown WT and hy5 seedlings with 403 increasing concentrations of ABA (Figure 8B). While no effect was observed in 16 Ortiz-Alcaide et al. 404 the WT, the ability of hy5 seedlings to elongate in response to W+FR exposure 405 was progressively repressed as ABA concentration increased. At concentrations 406 of the hormone of 200 nM or higher, which are within the physiological range 407 (Waadt et al., 2014), NF-treated hy5 seedlings did not respond to shade (Figure 408 8B), similar to that observed with the LIN treatment. 409 Exogenous ABA treatment was also able to repress shade-promoted 410 hypocotyl elongation in green WT seedlings grown without inhibitors (Figure 9). 411 We next used this phenotype to identify ABA-related transcription factors 412 involved in this response. Mutants defective in ABI3 and ABI4 elongated slightly 413 more than WT seedlings when illuminated with W+FR and this response was not 414 repressed by ABA. By contrast, ABI5-defective seedlings showed a WT 415 phenotype in terms of sensitivity of shade-triggered elongation to ABA treatment 416 (Figure 9A). These results suggest that ABI3 and ABI4 but not ABI5 are required 417 for ABA to inhibit hypocotyl elongation. If these transcription factors also 418 transduce the ABA signal in the molecular pathway that blocks elongation in 419 shade-exposed albino seedlings, it would be expected that double mutants 420 lacking both HY5 and ABI3 or ABI4 and grown in the presence of LIN would be 421 able to elongate when exposed to W+FR. Indeed, these double mutants 422 elongated more than their parental lines when both mock (green) and LIN-treated 423 (albino) seedlings were grown under simulated shade (Figure 9B). Shade- 424 triggered elongation of LIN-treated hy5 abi3 and hy5 abi4 seedlings, however, 425 was reduced compared to that of ABA-defective hy5 aba2 seedlings (Figure 9B). 426 These results suggest that ABI3 and ABI4 might not participate in the same ABA 427 signaling pathway eventually repressing hypocotyl elongation but have partially 428 redundant roles in this process (Figure 10). 429 430 A mechanistic model for the modulation of shade elongation responses by 431 plastid-dependent signals. 432 A model generated based on the described results is shown in Figure 10. In 433 high plant density environments, like those found in forests, prairies or orchard 434 communities, a set of R/FR-dependent adaptive responses are unleashed in 435 shade-avoiding plants. Compared to plant proximity (without direct vegetative 436 shading), canopy shade in nature involves lower R/FR values associated with a 437 reduction in the amount of PAR. Although phyB is the major phytochrome 17 Ortiz-Alcaide et al. 438 controlling these responses, the photolabile phyA has an antagonistic negative 439 role in the shade-mediated regulation of hypocotyl elongation (Ciolfi et al., 2013; 440 Martinez-Garcia et al., 2014; Wang et al., 2018; Zhang et al., 2018). 441 Independently of the PAR level, phyB is deactivated by shade of intermediate, 442 low and very low R/FR, whereas phyA signaling is activated by shade of low and 443 very low R/FR. As a result, hypocotyl elongation is derepressed under conditions 444 mimicking vegetation proximity (a response aimed at overgrowing neighbors for 445 optimal light exposure). Under R/FR values typical of canopy shade, however, 446 phyA activation prevents seedlings from exhibiting excessive elongation (Figure 447 10). Our results reported here and elsewhere (Bou-Torrent et al., 2015) suggest 448 that HY5 represses the hypocotyl elongation response more strongly under 449 canopy shade. As previously proposed, HY5 might be principally involved in the 450 phyA-dependent pathway (Ciolfi et al., 2013; Wang et al., 2018; Zhang et al., 451 2018) whereas other transcription factors, including growth-promoting PIFs, 452 would be mostly associated to the phyB-dependent pathway (Figure 10). These 453 antagonistic phyB/PIFs and phyA/HY5 pathways likely provide young seedlings 454 with the capacity to rapidly elongate when impending competition is nearby but 455 also to attenuate excessive growth when growing under a canopy. 456 During seedling deetiolation, the phyB/PIFs pathway converges with a 457 GUN1-dependent retrograde pathway to antagonistically regulate the 458 transcriptional photomorphogenic network (Martin et al., 2016). The GUN1- 459 mediated retrograde signal involved in this particular process was proposed to 460 attenuate photomorphogenesis when chloroplast function is challenged and to be 18 Ortiz-Alcaide et al. 461 independent of ABI4 and HY5 (Martin et al., 2016). Our results reported here 462 suggest that in shade-exposed seedlings, a completely different retrograde 463 network that is independent of GUN1 but does depend on HY5, ABI3 and ABI4 464 modulates the antagonistic action of phyA/HY5 and phyB/PIFs signaling 465 pathways (Figure 10). 466 Prolonged exposure to shade causes a decrease in the accumulation of 467 chlorophylls and carotenoids that can eventually compromise photosynthesis and 468 photoprotection (Roig-Villanova et al., 2007; Cagnola et al., 2012; Bou-Torrent et 469 al., 2015). Our results suggest that such a challenge to the chloroplast functional 470 status might in turn feedback-regulate the response to shade (Figure 10). 471 Treatment with low concentrations of NF or LIN (i.e. those causing weak to 472 moderate reduction in the level of photosynthetic pigments) was sufficient to 473 repress the hypocotyl elongation response to low R/FR (Figure 1), likely due to 474 delayed phyB deactivation after a reduction in R/FR (Figure 2). Decreased phyB 475 deactivation correlated with impaired PIF accumulation (Figure S2) and 476 attenuated gene expression changes (Figure 3). NF or LIN treatments also 477 caused an enhanced accumulation of HY5 transcripts and increased the stability 478 of the HY5-GFP reporter protein (Figure 6). Together, our findings suggest that 479 retrograde signals inhibit the SAS by repressing the (positive) phyB/PIFs pathway 480 and by promoting the (negative) phyA/HY5 pathway (Figure 10). 481 Our work further unveiled ABA as another component of the feedback 482 mechanism. This carotenoid-derived hormone was found to repress shade- 483 triggered hypocotyl elongation (Figure 8), likely through the action of the 484 transcription factors ABI3 and ABI4 (Figure 9). ABI4 has been proposed to 19 Ortiz-Alcaide et al. 485 participate in GUN1-dependent retrograde signaling (Koussevitzky et al., 2007; 486 Sun et al., 2011; Guo et al., 2016; Xu et al., 2016). However, the results 487 supporting this claim have been repeatedly challenged (Kacprzak et al., 2018). 488 Our data suggest that ABI4 (and ABI3) may act redundantly to transduce the 489 ABA-dependent signal that represses shade-triggered hypocotyl elongation in 490 response to chloroplast dysfunction (Figure 9). While HY5 was previously shown 491 to directly bind and activate the promoter of ABI5 to promote light-induced 492 hypocotyl inhibition during deetiolation (Chen et al., 2008; Xu et al., 2014), our 493 results suggest that this mechanism does not participate in the control of shade- 494 dependent hypocotyl growth. First, HY5 and ABA appear to repress hypocotyl 495 growth by independent pathways (Figure 8). And second, ABI5 is not required to 496 transduce the ABA signal eventually repressing the response to shade (Figure 9). 497 Arabidopsis mutants defective in phyB were found to accumulate greater 498 amounts of ABA under well-watered conditions and to be less sensitive to 499 exogenous ABA treatments (Gonzalez et al., 2012). Further supporting a 500 negative role of light for ABA synthesis, dark treatment of previously light-grown 501 plants resulted in increased ABA contents (Weatherwax et al., 1996). A shade- 502 triggered increase in ABA production was reported here (Figure S6) and 503 elsewhere (Cagnola et al., 2012; Gonzalez-Grandio et al., 2013; Holalu and 504 Finlayson, 2017). It is possible that W+FR treatment might promote ABA 505 production to repress the elongation response to shade as part of the mechanism 506 that prevents a too intense commitment (Figure 10). These results together 507 support ABA as a central signal connecting the functional status of the 508 chloroplast with light responses. Interestingly, the plastid-synthesized metabolite 509 3’-phosphoadenosine 5’-phosphate (PAP), which functions as a retrograde signal 510 during oxidative stress caused by high light exposure and drought, was recently 511 shown to act in concert with ABA signaling in guard cells to mediate stomatal 512 closure and in seeds to mediate dormancy and germination (Pornsiriwong et al., 513 2017). PAP accumulates when the SAL1 phosphatase that normally degrades 514 this metabolite is inactivated during oxidative stress (Estavillo et al., 2011). SAL1- 515 defective mutants show a short hypocotyl phenotype in the light, indicating that 516 accumulation of PAP can repress hypocotyl elongation (Kim and von Arnim, 517 2009; Chen and Xiong, 2011). This phenotype is rescued (at least partially) in 518 double sal1 phyB and sal1 hy5 mutants (Kim and von Arnim, 2009; Chen and 20 Ortiz-Alcaide et al. 519 Xiong, 2011), suggesting that functional phyB and HY5 are required for the PAP- 520 promoted and light-dependent repression of hypocotyl growth. Futher 521 experiments should explore whether PAP is the retrograde signal deduced from 522 our data to attenuate the response to shade in terms of hypocotyl elongation by 523 independently inhibiting phyB deactivation, increasing HY5 accumulation, and 524 promoting ABA signaling (Figure 10). 525 Besides ABA, it is possible that other carotenoid-derived products might 526 also contribute to the repression of shade-triggered hypocotyl elongation 527 detected in hy5 seedlings bleached with LIN, CAP or PPT but not with NF or 528 CPTA (Figure 7). In particular, strigolactones are hormones derived from β- 529 carotene (Figure 7A) that inhibit hypocotyl elongation in the light by a mechanism 530 requiring phytochromes and involving upregulation of HY5 expression and 531 protein (Tsuchiya et al., 2010; Jia et al., 2014). Other metabolites produced after 532 cleavage of carotenoids include β-cyclocitral, and unknown compounds that 533 modulate developmental and stress responses (Hou et al., 2016). While β- 534 cyclocitral is a relatively well-established retrograde signal associated to oxidative 535 stress (Ramel et al., 2012), its contribution to hypocotyl elongation is unknown. 536 Similarly, no hypocotyl growth alterations have been reported in mutants lacking 537 carotenoid-derived signals that do have an impact on leaf development (van 538 Norman et al., 2007; Avendaño-Vazquez et al., 2014). Whether any of these 539 carotenoid-related metabolites participate in the elongation response to shade 540 remains to be investigated. 541 Collectively, our data support the notion that chloroplasts are plant cell 542 compartments with fundamental roles not only for photosynthesis and 543 metabolism but also for environmental (light) sensing and signaling. Here we 544 show that HY5 and ABA (via ABI3 and ABI4) are nodes of a plastid-modulated 545 network that attenuates the response to shade in terms of hypocotyl elongation. 546 In green plants with functional chloroplasts, light signals associated with canopy 547 shade rapidly promote hypocotyl elongation via the phyB/PIFs pathway. 548 Exposure to low R/FR also triggers negative (growth-repressing) circuits involving 549 the phyA/HY5 pathway and the carotenoid-derived hormone ABA, likely to 550 prevent an excessive response and facilitate the return to non-shade conditions if 551 the low R:FR signal disappears (e.g. if a commitment to the shade-avoidance 552 lifestyle is unnecessary). When maintained, shade further causes a decrease of 21 Ortiz-Alcaide et al. 553 chlorophyll and carotenoid contents which might eventually disrupt chloroplast 554 homeostasis. Such situation would be then signaled to feedback-regulate the 555 response to the light signal by independently inhibiting phyB deactivation, 556 increasing HY5 accumulation, and promoting ABA signaling. This mechanism 557 connecting the metabolic status of the chloroplast with light (shade) signaling and 558 developmental responses likely contributes to achieve optimal photosynthetic 559 performance. 560 561 MATERIALS AND METHODS 562 Plant material 563 All mutants used in this work are listed in Table S1. Arabidopsis thaliana lines 564 used here were in the Columbia (Col) background with the only exception of hy1- 565 1, a Landsberg erecta (Ler) mutant (Rodriguez-Concepcion et al., 2004). Some 566 of those lines were already available in our lab and previously used in published 567 works, including hdr-3 (Pokhilko et al., 2015), gun1-101 (Llamas et al., 2017), 568 gun5-1 (Llamas et al., 2017), pifQ (Toledo-Ortiz et al., 2010), hy5-2 (Bou-Torrent 569 et al., 2015), psy-1 (Pokhilko et al., 2015), lut2 (Emiliani et al., 2018), aba2 (Ruiz- 570 Sola et al., 2014), and hy5 35S:HA-HY5 (Toledo-Ortiz et al., 2014). Lines abi3-8 571 (Nambara et al., 2002), abi4-1 (Finkelstein et al., 1998), abi5-7 (Tamura et al., 572 2006), and 35S:GUS-PIF3 (Monte et al., 2004) were requested. For generation of 573 double mutants, single homozygous plants were crossed and the F2 progeny 574 was first screened for the characteristic long hypocotyl phenotype associated to 575 the hy5 mutation in homozygosis. Long individuals were then PCR-genotyped to 576 identify homozygous mutants for the second gene and confirm that they were 577 also homozygous for hy5. For the generation of the 35S:HY5-GFP construct, the 578 full coding region of the Arabidopsis HY5 cDNA was PCR-amplified using primers 579 HY5‐attB1-F and HY5‐attB2-R (Table S2) and cloned into Gateway pDONR-207. 580 Cloning into Gateway pGWB405 eventually generated the construct for the 35S 581 promoter-driven expression of a C-terminal fusion of the sGFP reporter protein to 582 HY5. This construct was used to transform the hy5-2 mutant by floral dipping. 583 The hy5 35S:HY5-GFP line used for the experiments reported here was selected 584 based on complete complementation of the long hypocotyl phenotype associated 585 with the hy5 mutation and high levels of nuclear GFP fluorescence. Line 22 Ortiz-Alcaide et al. 586 35S:PHYB-GFP was generated by transforming Col-0 plants with the same 587 construct previously found to work in an Arabidopsis phyB mutant in the Ler 588 background (Yamaguchi et al., 1999). From the resulting transformants, we 589 selected for further experiments one of the lines showing a clearer accumulation 590 of the phyB-GFP protein in nuclear bodies under W. 591 592 Growth conditions and treatments 593 Seeds were surface-sterilized and germinated on solid Murashige and Skoog 594 (MS) medium supplemented with 10 mg/ml of sucrose to provide carbon and 595 energy for albino seedlings to grow. When indicated, the medium was further 596 supplemented with different concentrations or norflurazon (NF, Zorial), lincomycin 597 (LIN, Sigma) or abscisic acid (ABA, Sigma). Other chemicals added to the 598 medium included epibrassinolide (1 μM), gibberellic acid (10 μM), picloram (5 599 μM), 2-(4-chlorophenylthio)-triethylamine chloride (25μM), fosmidomycin (500 600 μM), chloramphenicol (50 μM), or phosphinotricin (100 μM). When comparing 601 different lines (e.g. WT vs. mutant), they were grown together on the same plate 602 instead of growing each line on a different plate. After stratification for at least 3 603 days at 4°C in the dark, plates were incubated in growth chambers at 22°C under 604 W of 20-24 μmol m–2 s–1 PAR (R/FR = 1.6). When indicated, W was 605 supplemented with FR provided by GreenPower LED module HF far-red (Philips) 606 QB1310CS-670-735 light-emitting diode hybrid lamps (Quantum Devices) to 607 simulate canopy shade (20-24 μmol m–2 s–1 PAR, R/FR = 0.02). Fluence rates 608 were measured using a Spectrosense 2 meter associated with a 4-channel 609 sensor (Skye Instruments Ltd.) as described (Martinez-Garcia et al., 2014). 610 Grown seedlings were laid out flat on the growth media and digital images were 611 taken to quantify hypocotyl length using the NIH ImageJ software. 612 613 Microscopy 614 Whole 35S:PHYB-GFP seedlings germinated and grown under W for 7 days on 615 media with or without 5 μM NF or 1 mM LIN were exposed to a 5 min pulse of FR 616 (735 nm, 60 μmol/m2/s) and then kept in the dark. At different timepoints, treated 617 seedlings were placed on glass slides under a safety green light and kept in the 618 dark until observation with an Olympus BX60 FLUOVIEW FV300 microscope. 619 Confocal laser scan images of the hypocotyl area closer to the cotyledons were 23 Ortiz-Alcaide et al. 620 obtained at different timepoints in the dark with a combination of 488 nm laser 621 excitation and 515 nm longpass filter (LP515; Carl Zeiss Jena). For each 622 timepoint, three sequential images from different focus planes were recorded 623 automatically. 624 625 Chromatin immunoprecipitation 626 About 800 μl of seeds from hy5 35S:HY5-GFP plants were plated on 8 square 627 (10 cm x 10 cm) plates of sucrose-supplemented medium. After growth for 2 628 days under W, 4 plates were left under W and 4 were transferred to W+FR for 5 629 additional days. For chromatin immunoprecipitation (Moon et al., 2008), each 630 sample was divided in 3 aliquots after crosslinking and sonication: one input, one 631 to be incubated with a 1:1000 dilution of anti-GFP antibody (Life Technologies), 632 and the last one to be processed similarly but without antibody. After DNA 633 isolation, the three samples were used for qPCR analysis of promoter sequence 634 abundance with the primers shown in Table S2. After normalization with the 635 input, enrichment was calculated as the ratio of the signal with vs. without 636 antibody. 637 638 Gene expression and immunoblot analyses 639 Total RNA was extracted from whole seedlings and used for qPCR analysis as 640 described (Llamas et al., 2017) with the gene-specific primers listed in Table S2. 641 Protein extraction, immunoblot analysis, and quantification of protein abundance 642 were performed as described (Llamas et al., 2017) using a 1:1000 dilution of anti- 643 GFP serum (Life Technologies). 644 645 Quantification of metabolite levels 646 Whole seedlings were frozen in liquid nitrogen, lyophilized, and ground in a 647 mortar for extraction and quantification of photosynthetic pigments and ABA. 648 Chlorophyll and carotenoid levels were measured either by spectrometric 649 methods or by HPLC (Bou-Torrent et al., 2015). ABA content was quantified by 650 LC/ESI-MS/MS as described (Ruiz-Sola et al., 2014). 24 Ortiz-Alcaide et al. 651 ACKNOWLEDGEMENTS 652 We thank Elena Monte (CRAG) for comments on the manuscript. Technical 653 support from M. Rosa Rodríguez-Goberna and members of the CRAG core 654 facilities is greatly appreciated. This work was funded by grants BIO2014‐59895‐ 655 P, BIO2014-59092-P, BIO2015-71703-REDT, BIO2017-90877-REDT, BIO2017- 656 85316-R and BIO2017-84041-P from the Spanish Ministry of Science, 657 Innovation, and Universities (MICINN) and grants 2017SGR-1211 and 2017SGR- 658 710 from Generalitat de Catalunya to JFMG and MRC. Funding from the Japan 659 Society for the Promotion of Science (JSPS) KAKENHI grant JP-15H04389 to AN 660 is also acknowledged. We thank the financial support of the MINECO Severo 661 Ochoa Programme for Centres of Excellence in R&D 2016-2019 (SEV‐2015‐ 662 0533) and the Generalitat de Catalunya CERCA Programme to CRAG. MOA and 663 EL were supported by PhD fellowships from Spanish MINECO (BES-2012- 664 052597) and Mexican CONACYT (421688 and SEP “beca complemento”), 665 respectively. 666 667 AUTHOR CONTRIBUTIONS 668 MOA, JFMG and MRC designed the research; MOA, EL, and AGC performed 669 research; AN contributed analytic tools; MOA, EL, AN, JFMG and MRC analyzed 670 data; JFMG and MRC wrote the paper. 25 Ortiz-Alcaide et al. 671 26 Parsed Citations Avendaño-Vázquez, A.O., Cordoba, E., Llamas, E., San Román, C., Nisar, N., De la Torre, S., Ramos-Vega, M., Gutiérrez-Nava, M.D., Cazzonelli, C.I., Pogson, B.J., and León P. (2014). An Uncharacterized Apocarotenoid-Derived Signal Generated in ζ-Carotene Desaturase Mutants Regulates Leaf Development and the Expression of Chloroplast and Nuclear Genes in Arabidopsis. The Plant cell 26, 2524-2537. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Baier, M., and Dietz, K.J. (2005). Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology. Journal of experimental botany 56, 1449-1462. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Bou-Torrent, J., Toledo-Ortiz, G., Ortiz-Alcaide, M., Cifuentes-Esquivel, N., Halliday, K.J., Martinez-Garcia, J.F., and Rodriguez- Concepcion, M. (2015). Regulation of Carotenoid Biosynthesis by Shade Relies on Specific Subsets of Antagonistic Transcription Factors and Cofactors. Plant physiology 169, 1584-1594. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Cagnola, J.I., Ploschuk, E., Benech-Arnold, T., Finlayson, S.A., and Casal, J.J. (2012). Stem transcriptome reveals mechanisms to reduce the energetic cost of shade-avoidance responses in tomato. Plant physiology 160, 1110-1119. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Casal, J.J. (2012). Shade avoidance. Arabidopsis Book 10, e0157. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Casal, J.J. (2013). Photoreceptor signaling networks in plant responses to shade. Annual review of plant biology 64, 403-427. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ciolfi, A., Sessa, G., Sassi, M., Possenti, M., Salvucci, S., Carabelli, M., Morelli, G., and Ruberti, I. (2013). Dynamics of the Shade Avoidance Response in Arabidopsis thaliana. Plant physiology. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Chamovitz, D., Pecker, I., and Hirschberg, J. (1991). The molecular basis of resistance to the herbicide norflurazon. Plant molecular biology 16, 967-974. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Chan, K.X., Phua, S.Y., Crisp, P., McQuinn, R., and Pogson, B.J. (2016). Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond. Annual review of plant biology 67, 25-53. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Chen, H., Zhang, J., Neff, M.M., Hong, S.W., Zhang, H., Deng, X.W., and Xiong, L. (2008). Integration of light and abscisic acid signaling during seed germination and early seedling development. Proceedings of the National Academy of Sciences of the United States of America 105, 4495-4500. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Chen, H. and Xiong, L. (2011). Genetic interaction of two abscisic acid signaling regulators, HY5 and FIERY1, in mediating lateral root formation. Plant signaling and behavior 6, 123-125. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Davis, S.J., Kurepa, J., and Vierstra, R.D. (1999). The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases. Proceedings of the National Academy of Sciences of the United States of America 96, 6541-6546. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Emiliani, J., D'Andrea, L., Lorena Falcone Ferreyra, M., Maulion, E., Jose Rodriguez, E., Rodriguez-Concepcion, M., and Casati, P. (2018). A role for beta,beta-xanthophylls in Arabidopsis UV-B photoprotection. Journal of experimental botany 69, 4921-4933. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Estavillo, G.M., Crisp, P.A., Pornsiriwong, W., Wirtz, M., Collinge, D., Carrie, C., Giraud, E., Whelan, J., David, P., Javot, H., Brearley, C., Hell, R., Marin, E., and Pogson, B.J. (2011). Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in arabidopsis. The Plant cell 23, 3992–4012. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Finkelstein, R.R., Wang, M.L., Lynch, T.J., Rao, S., and Goodman, H.M. (1998). The Arabidopsis abscisic acid response locus ABI4 encodes an APETALA 2 domain protein. The Plant cell 10, 1043-1054. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Fiorucci, A.S., and Fankhauser, C. (2017). Plant Strategies for Enhancing Access to Sunlight. Curr Biol 27, R931-R940. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Franklin, K.A. (2008). Shade avoidance. The New phytologist 179, 930-944. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Glasser, C., Haberer, G., Finkemeier, I., Pfannschmidt, T., Kleine, T., Leister, D., Dietz, K.J., Hausler, R.E., Grimm, B., and Mayer, K.F. (2014). Meta-analysis of retrograde signaling in Arabidopsis thaliana reveals a core module of genes embedded in complex cellular signaling networks. Molecular plant 7, 1167-1190. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Gommers, C.M., Visser, E.J., St Onge, K.R., Voesenek, L.A., and Pierik, R. (2013). Shade tolerance: when growing tall is not an option. Trends in plant science 18, 65-71. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Gonzalez-Grandio, E., Poza-Carrion, C., Sorzano, C.O., and Cubas, P. (2013). BRANCHED1 promotes axillary bud dormancy in response to shade in Arabidopsis. The Plant cell 25, 834-850. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Gonzalez, C.V., Ibarra, S.E., Piccoli, P.N., Botto, J.F., and Boccalandro, H.E. (2012). Phytochrome B increases drought tolerance by enhancing ABA sensitivity in Arabidopsis thaliana. Plant, cell & environment 35, 1958-1968. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Guo, H., Feng, P., Chi, W., Sun, X., Xu, X., Li, Y., Ren, D., Lu, C., David Rochaix, J., Leister, D., and Zhang, L. (2016). Plastid-nucleus communication involves calcium-modulated MAPK signalling. Nature communications 7, 12173. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Holalu, S.V., and Finlayson, S.A. (2017). The ratio of red light to far red light alters Arabidopsis axillary bud growth and abscisic acid signalling before stem auxin changes. Journal of experimental botany 68, 943-952. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Hou, X., Rivers, J., Leon, P., McQuinn, R.P., and Pogson, B.J. (2016). Synthesis and Function of Apocarotenoid Signals in Plants. Trends in plant science 21, 792-803. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Humplik, J.F., Bergougnoux, V., and Van Volkenburgh, E. (2017). To Stimulate or Inhibit? That Is the Question for the Function of Abscisic Acid. Trends in plant science 22, 830-841. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Jia, K.P., Luo, Q., He, S.B., Lu, X.D., and Yang, H.Q. (2014). Strigolactone-regulated hypocotyl elongation is dependent on cryptochrome and phytochrome signaling pathways in Arabidopsis. Molecular plant 7, 528-540. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Kacprzak, S.M., Mochizuki, N., Naranjo, B., Xu, D., Leister, D., Kleine, T., Okamoto, H., and Terry, M.J. (2018). Plastid-to-nucleus retrograde signalling during chloroplast biogenesis does not require ABI4. Plant physiology. pii: pp.01047.2018. doi: 10.1104/pp.18.01047 Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Kim, B.H. and von Arnim, A.G. (2009). FIERY1 regulates light-mediated repression of cell elongation and flowering time via its 3'(2'),5'- bisphosphate nucleotidase activity. The Plant journal : for cell and molecular biology 58, 208-219. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Koussevitzky, S., Nott, A., Mockler, T.C., Hong, F., Sachetto-Martins, G., Surpin, M., Lim, J., Mittler, R., and Chory, J. (2007). Signals from chloroplasts converge to regulate nuclear gene expression. Science 316, 715-719. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Larkin, R.M., and Ruckle, M.E. (2008). Integration of light and plastid signals. Current opinion in plant biology 11, 593-599. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Lau, O.S., and Deng, X.W. (2010). Plant hormone signaling lightens up: integrators of light and hormones. Current opinion in plant biology 13, 571-577. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Leivar, P., Tepperman, J.M., Cohn, M.M., Monte, E., Al-Sady, B., Erickson, E., and Quail, P.H. (2012). Dynamic antagonism between phytochromes and PIF family basic helix-loop-helix factors induces selective reciprocal responses to light and shade in a rapidly responsive transcriptional network in Arabidopsis. The Plant cell 24, 1398-1419. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Lepisto, A., and Rintamaki, E. (2012). Coordination of plastid and light signaling pathways upon development of Arabidopsis leaves under various photoperiods. Molecular plant 5, 799-816. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Lorrain, S., Allen, T., Duek, P.D., Whitelam, G.C., and Fankhauser, C. (2008). Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. The Plant journal : for cell and molecular biology 53, 312-323. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Llamas, E., Pulido, P., and Rodriguez-Concepcion, M. (2017). Interference with plastome gene expression and Clp protease activity in Arabidopsis triggers a chloroplast unfolded protein response to restore protein homeostasis. PLoS genetics 13, e1007022. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Llorente, B., Martinez-Garcia, J.F., Stange, C., and Rodriguez-Concepcion, M. (2017). Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light. Current opinion in plant biology 37, 49-55. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Martin, G., Leivar, P., Ludevid, D., Tepperman, J.M., Quail, P.H., and Monte, E. (2016). Phytochrome and retrograde signalling pathways converge to antagonistically regulate a light-induced transcriptional network. Nature communications 7, 11431. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Martinez-Garcia, J.F., Gallemi, M., Molina-Contreras, M.J., Llorente, B., Bevilaqua, M.R., and Quail, P.H. (2014). The shade avoidance syndrome in Arabidopsis: the antagonistic role of phytochrome a and B differentiates vegetation proximity and canopy shade. PloS one 9, e109275. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Mochizuki, N., Brusslan, J.A., Larkin, R., Nagatani, A., and Chory, J. (2001). Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proceedings of the National Academy of Sciences of the United States of America 98, 2053-2058. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Monte, E., Tepperman, J.M., Al-Sady, B., Kaczorowski, K.A., Alonso, J.M., Ecker, J.R., Li, X., Zhang, Y., and Quail, P.H. (2004). The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development. Proceedings of the National Academy of Sciences of the United States of America 101, 16091-16098. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Moon, J., Zhu, L., Shen, H., and Huq, E. (2008). PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 105, 9433-9438. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Mulo, P., Pursiheimo, S., Hou, C.X., Tyystjarvi, T., and Aro, E.M. (2003). Multiple effects of antibiotics on chloroplast and nuclear gene expression. Funct Plant Biol 30, 1097-1103. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Nambara, E., Suzuki, M., Abrams, S., McCarty, D.R., Kamiya, Y., and McCourt, P. (2002). A screen for genes that function in abscisic acid signaling in Arabidopsis thaliana. Genetics 161, 1247-1255. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Pacin, M., Semmoloni, M., Legris, M., Finlayson, S.A., and Casal, J.J. (2016). Convergence of CONSTITUTIVE PHOTOMORPHOGENESIS 1 and PHYTOCHROME INTERACTING FACTOR signalling during shade avoidance. The New phytologist 211, 967-979. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Parks, B.M., and Quail, P.H. (1991). Phytochrome-Deficient hy1 and hy2 Long Hypocotyl Mutants of Arabidopsis Are Defective in Phytochrome Chromophore Biosynthesis. The Plant cell 3, 1177-1186. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Patel, D., Basu, M., Hayes, S., Majlath, I., Hetherington, F.M., Tschaplinski, T.J., and Franklin, K.A. (2013). Temperature-dependent shade avoidance involves the receptor-like kinase ERECTA. The Plant journal : for cell and molecular biology 73, 980-992. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Pornsiriwong, W., Estavillo, G.M., Chan, K.X., Tee, E.E., Ganguly, D., Crisp, P.A., Phua, S.Y., Zhao, C., Qiu, J., Park, J., Yong, M.T., Nisar, N., Yadav, A.K., Schwessinger, B., Rathjen, J., Cazzonelli, C.I., Wilson, P.B., Gilliham, M., Chen, Z.H., and Pogson, B.J. (2017). A chloroplast retrograde signal, 3'-phosphoadenosine 5'-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination. Elife 6, e23361. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Pokhilko, A., Bou-Torrent, J., Pulido, P., Rodriguez-Concepcion, M., and Ebenhoh, O. (2015). Mathematical modelling of the diurnal regulation of the MEP pathway in Arabidopsis. The New phytologist 206, 1075-1085. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ramel F, Birtic S, Ginies C, Soubigou-Taconnat L, Triantaphylidès C, and Havaux M. (2012). Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants. Proceedings of the National Academy of Sciences of the United States of America 109, 5535-5540. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Rockwell, N.C., Su, Y.S., and Lagarias, J.C. (2006) Phytochrome structure and signaling mechanisms. Annual Review of Plant Biology 57, 837-858. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Rodriguez-Concepcion, M., Fores, O., Martinez-Garcia, J.F., Gonzalez, V., Phillips, M.A., Ferrer, A., and Boronat, A. (2004). Distinct light- mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. The Plant cell 16, 144-156. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Roig-Villanova, I., Bou, J., Sorin, C., Devlin, P.F., and Martinez-Garcia, J.F. (2006). Identification of primary target genes of phytochrome signaling. Early transcriptional control during shade avoidance responses in Arabidopsis. Plant physiology 141, 85-96. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Roig-Villanova, I., Bou-Torrent, J., Galstyan, A., Carretero-Paulet, L., Portoles, S., Rodriguez-Concepcion, M., and Martinez-Garcia, J.F. (2007). Interaction of shade avoidance and auxin responses: a role for two novel atypical bHLH proteins. EMBO J 26, 4756-4767. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ruckle, M.E., and Larkin, R.M. (2009). Plastid signals that affect photomorphogenesis in Arabidopsis thaliana are dependent on GENOMES UNCOUPLED 1 and cryptochrome 1. The New phytologist 182, 367-379. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ruckle, M.E., DeMarco, S.M., and Larkin, R.M. (2007). Plastid signals remodel light signaling networks and are essential for efficient chloroplast biogenesis in Arabidopsis. The Plant cell 19, 3944-3960. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ruckle, M.E., Burgoon, L.D., Lawrence, L.A., Sinkler, C.A., and Larkin, R.M. (2012). Plastids are major regulators of light signaling in Arabidopsis. Plant physiology 159, 366-390. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ruiz-Sola, M.A., and Rodriguez-Concepcion, M. (2012). Carotenoid biosynthesis in Arabidopsis: a colorful pathway. Arabidopsis Book 10, e0158. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ruiz-Sola, M.A., Arbona, V., Gomez-Cadenas, A., Rodriguez-Concepcion, M., and Rodriguez-Villalon, A. (2014). A root specific induction of carotenoid biosynthesis contributes to ABA production upon salt stress in arabidopsis. PloS one 9, e90765. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Saini, G., Meskauskiene, R., Pijacka, W., Roszak, P., Sjögren, L.L., Clarke, A.K., Straus, M., and Apel, K. (2011). 'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings. The Plant journal : for cell and molecular biology 65, 690-702. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Sellaro, R., Yanovsky, M.J., and Casal, J.J. (2011). Repression of shade-avoidance reactions by sunfleck induction of HY5 expression in Arabidopsis. The Plant journal : for cell and molecular biology 68, 919-928. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Sun, X., Feng, P., Xu, X., Guo, H., Ma, J., Chi, W., Lin, R., Lu, C., and Zhang, L. (2011). A chloroplast envelope-bound PHD transcription factor mediates chloroplast signals to the nucleus. Nature communications 2, 477. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Tamura, N., Yoshida, T., Tanaka, A., Sasaki, R., Bando, A., Toh, S., Lepiniec, L., Kawakami, N. (2006). Isolation and characterization of high temperature-resistant germination mutants of Arabidopsis thaliana. Plant cell and physiology 47, 1081-1094. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Toledo-Ortiz, G., Huq, E., and Rodriguez-Concepcion, M. (2010). Direct regulation of phytoene synthase gene expression and carotenoid biosynthesis by phytochrome-interacting factors. Proceedings of the National Academy of Sciences of the United States of America 107, 11626-11631. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Toledo-Ortiz, G., Johansson, H., Lee, K.P., Bou-Torrent, J., Stewart, K., Steel, G., Rodriguez-Concepcion, M., and Halliday, K.J. (2014). The HY5-PIF Regulatory Module Coordinates Light and Temperature Control of Photosynthetic Gene Transcription. PLoS genetics 10, e1004416. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Tsuchiya Y, Vidaurre D, Toh S, Hanada A, Nambara E, Kamiya Y, Yamaguchi S, and McCourt P. (2010). A small-molecule screen identifies new functions for the plant hormone strigolactone. Nature Chemical Biology 6, 741-749. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Van Norman JM, and Sieburth LE. (2007). Dissecting the biosynthetic pathway for the bypass1 root-derived signal. The Plant journal 49, 619-628. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Waadt, R., Hitomi, K., Nishimura, N., Hitomi, C., Adams, S.R., Getzoff, E.D., and Schroeder, J.I. (2014). FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis. Elife 3, e01739. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Wang, F., Wu, N., Zhang, L., Ahammed, G.J., Chen, X., Xiang, X., Zhou, J., Xia, X., Shi, K., Yu, J., Foyer, C.H., and Zhou, Y. (2018). Light Signaling-Dependent Regulation of Photoinhibition and Photoprotection in Tomato. Plant physiology 176, 1311-1326. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Weatherwax, S.C., Ong, M.S., Degenhardt, J., Bray, E.A., and Tobin, E.M. (1996). The interaction of light and abscisic acid in the regulation of plant gene expression. Plant physiology 111, 363-370. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Xiao, Y., Savchenko, T., Baidoo, E.E., Chehab, W.E., Hayden, D.M., Tolstikov, V., Corwin, J.A., Kliebenstein, D.J., Keasling, J.D., and Dehesh, K. (2012). Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes. Cell 149, 1525-1535. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Xu, D., Li, J., Gangappa, S.N., Hettiarachchi, C., Lin, F., Andersson, M.X., Jiang, Y., Deng, X.W., and Holm, M. (2014). Convergence of Light and ABA signaling on the ABI5 promoter. PLoS genetics 10, e1004197. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Google Scholar: Author Only Title Only Author and Title Xu, X., Chi, W., Sun, X., Feng, P., Guo, H., Li, J., Lin, R., Lu, C., Wang, H., Leister, D., and Zhang, L. (2016). Convergence of light and chloroplast signals for de-etiolation through ABI4-HY5 and COP1. Nature plants 2, 16066. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Yamaguchi, R., Nakamura, M., Mochizuki, N., Kay, S.A., and Nagatani, A. (1999). Light-dependent translocation of a phytochrome B-GFP fusion protein to the nucleus in transgenic Arabidopsis. The Journal of cell biology 145, 437-445. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Zhang, S., Li, C., Zhou, Y., Wang, X., Li, H., Feng, Z., Chen, H., Qin, G., Jin, D., Terzaghi, W., Gu, H., Qu, L.J., Kang, D., Deng, X.W., and Li, J. (2018). TANDEM ZINC-FINGER/PLUS3 Is a Key Component of Phytochrome A Signaling. The Plant cell 30, 835-852. Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title