The microtubule-associated protein CLASP is translationally-regulated in light-dependent root apical meristem growth

The ability for plant growth to be optimized, either in the light or dark, depends on the intricate balance between cell division and differentiation in specialized regions called meristems. When Arabidopsis thaliana seedlings are grown in the dark, hypocotyl elongation is promoted, whereas root growth is greatly reduced as a result of changes in hormone transport and a reduction in meristematic cell proliferation. Previous work showed that the microtubule-associated protein CLASP sustains root apical meristem (RAM) size by influencing microtubule (MT) organization and by modulating the brassinosteroid (BR) signalling pathway. Here, we investigated whether CLASP is involved in light-dependent root growth promotion, since dark-grown seedlings have reduced RAM activity that is observed in the clasp-1 null mutant. We showed that CLASP protein levels were greatly reduced in the root tips of dark-grown seedlings, which could be reversed by exposing plants to light. We confirmed that removing seedlings from the light led to a discernible shift in MT organization from bundled arrays, which are prominent in dividing cells, to transverse orientations typically observed in cells that have exited the meristem. BR receptors and auxin transporters, both of which are sustained by CLASP, were largely degraded in the dark. Interestingly, we found that despite the lack of protein, CLASP transcript levels were higher in dark-grown root tips. Together, these findings uncover a mechanism that sustains meristem homeostasis through CLASP, and advances our understanding of how roots modulate their growth according to the amount of light and nutrients perceived by the plant. One Sentence Summary The microtubule-associated protein CLASP is regulated at the translational level when root meristem growth is inhibited in dark-grown plants.


INTRODUCTION 77
At the beginning of their lives, plants face a precarious situation. Resources stored in the seed 78 will only sustain life for a limited time, and thus it is critical that plants quickly emerge from the 79 soil to begin the process of photosynthesis and sugar production. Plants must therefore use their 80 initial resources strategically during the first days of development to ensure survival. It is known 81 that when seedlings are germinated and grown in complete darkness (skotomorphogenesis), the 82 hypocotyl grows rapidly, whereas root elongation is inhibited. When grown in light conditions 83 (photomorphogenesis), sucrose derived from aerial organs is sufficient to promote root 84 elongation in Arabidopsis (Kircher and Schopfer, 2012), and to reduce hypocotyl growth. One 85 fundamental question is how growth is promoted in some organs and inhibited in others to meet 86 the needs of a plant during developmental transitions. 87 88 Several signalling pathways are necessary for plants to withstand stress due to lack of nutrients, 89 which is the case in extended periods of darkness. Autophagy is a conserved degradation 90 pathway in eukaryotes that is activated during cellular starvation. When glucose is available, the 91 formation of autophagosomes is inhibited by increased reactive oxygen species (Huang et al., 92 2018). Several proteins are targeted for degradation through the target of rapamycin (TOR) 93 kinase, which is a highly conserved eukaryotic protein that integrates environmental signals with 94 downstream developmental and metabolic pathways, such as translation, protein degradation, 95 and cell division (reviewed in Dobrenel et al., 2016). Previous work has found that sugar, which 96 activates TOR, inhibits autophagy-mediated degradation of the BZR1 transcription factor that 97 normally regulates thousands of genes through the brassinosteroid (BR) signalling pathway. In 98 times of cellular starvation and growth arrest, TOR is inactive and BZR1 is consequently 99 degraded (Zhang et al., 2016). Similarly, a related BR transcription factor BES1 forms a complex 100 with DSK2 and AT8G, which directs cargo to autophagosomes to be degraded via autophagy in 101 times of stress (Nolan et al., 2017). Thus, it is clear that plants respond to stress through crosstalk 102 of BR and autophagy signalling pathways, but these mechanisms likely differ between 103 hypocotyls and roots to control organ-specific growth. 104

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Research into the function of Cytoplasmic Linker Associated Protein (CLASP) revealed its 106 pivotal function in maintaining root meristem size through control of microtubule (MT) 107 organization. Mutants that lack CLASP are dwarf, with fewer cells in the root division zone, 108 indicating a role for CLASP in cell division (Ambrose et al., 2007). In plants, CLASP is involved 109 in anchoring MTs to the cell cortex and is considered to be a stabilizing factor for MTs 110 (Ambrose and Wasteneys, 2008). Through confocal imaging, CLASP has been found to 111 distribute to the sharp transverse edges of newly-divided cells in the RAM, which normally 112 present a barrier to growing MTs and cause them to undergo catastrophe upon encounter 113 (Ambrose et al., 2011). The presence of CLASP at these edges, however, enables formation of 114 transfacial MT bundles (TFBs) that span the periclinal and transverse faces of the root 115 meristematic cells. TFBs are associated with maintaining the capacity for cell division and are 116 not present in elongating cells. Recent work has concluded that without CLASP, and 117 consequently TFBs, meristem size is reduced because cells prematurely start to elongate instead 118 of continuing to divide. 119 120 Two recent studies demonstrated CLASP's involvement in both auxin and BR hormone 121 signalling. CLASP directly interacts with the retromer component sorting nexin 1 (SNX1) and 122 tethers SNX1-associated endosomes to MTs (Ambrose et al., 2013). Stabilization of SNX1 along 123 MTs fosters recycling of the auxin efflux carrier PIN2 to the plasma membrane (PM), thereby 124 reducing PIN2 transport to vacuoles for degradation (Ambrose et al., 2013). In mutants lacking 125 CLASP expression, PIN2 is depleted at the PM, resulting in auxin accumulation in the root tip, 126 consistent with PIN2's function in directing auxin away from the quiescent centre via the 127 epidermis and cortex tissues (Ambrose et al., 2013 To determine how root growth is regulated in early plant development, we compared the role of 137 CLASP in actively proliferating light-grown meristems to those grown in dark conditions when 138 meristem growth is largely inhibited. Notably, in the absence of BZR1 in the dark, CLASP 139 transcript levels were elevated yet despite this, CLASP protein levels were greatly reduced. In 140 addition, many CLASP-dependent processes such as MT organization and hormone signalling 141 pathways were disrupted. Our work reveals that CLASP is regulated post-transcriptionally based 142 on light signals that control when root growth is desirable for a plant. 143 144 145

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CLASP is required for increased cell proliferation in response to light and/or sucrose. 148 To investigate CLASP's involvement in light-dependent root meristem activity, we compared 149 clasp-1 null-transcript mutants and wild-type root growth in light and dark conditions. Although 150 it is well known that hypocotyl expansion is stimulated and that root growth is inhibited in the 151 dark, we noted that most previous dark-growth investigations with the model system Arabidopsis 152 thaliana included some sucrose in the media. Considering the likelihood that sucrose, a product 153 of photosynthesis, signals successful germination and stimulates root growth, we compared 154 organ growth in culture media either lacking or supplemented with sucrose. 155 156 Root growth responses indicated that CLASP's function is strongly associated with the rapid root 157 growth that is stimulated in the light. Whereas light stimulated a 7-fold length increase in 6 day-158 old wild-type roots from 5.23 ± 0.40 mm to 37.50 ± 3.03 mm, it only caused clasp-1 root length 159 to double from 3.47 ± 0.24 mm to 7.33 ± 0.94 mm (Fig. 1A, C). We also noted that dark-grown 160 wild-type roots were of similar length to clasp-1 mutants, suggesting that dark-grown wild-type 161 meristems are deficient in CLASP. 162 163 We next determined that supplementing the culture medium with a moderate (1% 164 weight/volume) amount of sucrose caused a major increase in the length of dark-grown roots, but 165 that this effect was independent of CLASP. Treatments with 1% sucrose increased dark-grown 166 wild-type root lengths from 5.23 ± 0.40 mm to 20.42 ± 0.88 mm, and increased mean clasp-1 167 root lengths from 3.47 ± 0.24 mm to 15.59 ± 0.83 mm (Fig. 1B, C), which is an approximate 4-168 fold increase for both genotypes. This indicates that the sucrose-dependent growth stimulation is 169 CLASP-independent. By comparison, sucrose had no significant effect on root length for light-170 grown wild-type seedlings and a moderate increase (from 7.33 ± 0.94 mm to 11.56 ± 0.60 mm) 171 for clasp-1. 172 173 Based on the fact that hypocotyl expansion is entirely dependent on cell elongation, we 174 compared the etiolated hypocotyl responses of dark-grown wild-type and clasp-1 mutants to 175 sucrose. Although clasp-1 hypocotyls were significantly shorter than those of wild type under 176 with an approximate 10% length increase (Fig. 1D). These findings demonstrate that although 178 sucrose has relatively little effect on dark-grown hypocotyls, the increase is CLASP independent. 179 180 The observation that dark-grown wild-type and clasp-1 mutants have similar root lengths 181 prompted us to investigate whether the light-induced changes in root elongation can be attributed 182 to CLASP-dependent changes in cell proliferation. Growing seedlings in the light more than 183 doubled the number of cells in wild-type root meristems but only increased the number of cells 184 in clasp-1 mutant root meristems by 10% (Fig. 1E, F). Furthermore, the number of cells in dark-185 grown wild-type seedlings was similar to that found in clasp-1 mutants (Fig. 1E, F). The addition 186 of 1% sucrose to dark-grown seedlings did not affect cell proliferation in either genotype ( The data shown in Figure 1 clearly demonstrate that the inhibition of root growth in the dark is 200 associated with reduced cell proliferation, and that clasp-1 knockout mutants are less affected by 201 these conditions. Taken together, these findings indicate that CLASP mediates the cell 202 proliferation that is stimulated when seedlings are exposed to light. 203

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The BR pathway is dampened in dark-grown roots, leading to an elevation of CLASP 205 transcript levels 206 Previous work (Zhang et al., 2016) showed that dark-induced cellular starvation led to BZR1 207 degradation by autophagy. Using a BZR1-CFP fluorescent reporter, we confirmed that the BZR1 208 but was absent when plants were grown in media lacking sucrose in the dark ( Fig. 2A). By 210 contrast, dark-grown roots with 1% sucrose in the media still showed BZR1-CFP in the nuclei 211 ( Fig. 2B). Since germinating seedlings grown in dark conditions do not yet have a source of 212 sucrose, and the BR-activated transcription factor BZR1 was only degraded under these 213 conditions, our remaining analyses focused on roots grown in the absence of sucrose. 214 215 Given that BZR1 is a negative regulator of CLASP transcription, we hypothesized that CLASP 216 expression would be greater in dark-grown root meristems. Indeed, the CLASP promoter driving 217 the expression of free GFP (CLASP pro :GFP) revealed greater fluorescence in dark-grown roots 218 than in plants grown in the light (Fig. 2C). Interestingly, the level of free GFP in dark-grown 219 roots was especially high in the stele (Fig. 2C). Using qRT-PCR from root-tip RNA, we found an 220 almost 2.5-fold increase in CLASP gene expression in the dark (Fig. 2D), which is consistent 221 with the observed increase in CLASP pro :GFP fluorescence. 222 223 Since BZR1 is a key effector of BR signalling, we predicted that other genes in the BR pathway 224 would be differentially regulated in the dark, minus-sucrose conditions. Genes encoding the BR 225 receptor BRI1 and the BR-biosynthetic enzyme DWF4, had reduced expression levels by about 226 45% and 35% respectively (Fig. 2E), indicating a general loss of the BR signalling pathway in 227 dark-grown roots. To determine if genes encoding other microtubule-associated proteins were 228 affected in a similar manner to CLASP, we evaluated transcript levels of MOR1, which encodes a 229 protein that binds to microtubule plus ends and has domain similarity to CLASP. qRT-PCR 230 analysis showed that plants grown in the dark had a 25% decrease in MOR1 expression (Fig. 2E). 231 Thus, CLASP transcript levels are higher in the absence of BR signalling when roots are exposed 232 to dark conditions, and this pattern is not observed for MOR1, indicating that the increased 233 CLASP expression is a direct consequence of the loss of BR signalling. 234

CLASP protein levels are diminished under dark conditions 236
The high level of CLASP transcript in dark-grown roots is at odds with the shutdown of meristem 237 activity. Since it is impossible to extract sufficient protein from the root apical meristem for 238 immunoblotting, to assess CLASP protein levels we used a GFP-CLASP fluorescent reporter 239 driven by its endogenous promoter (Ambrose et al., 2011). In light-grown root meristems, GFP-240 grown root meristems, by contrast, only a few GFP-CLASP puncta were observed around the 242 cell edges (Fig. 3B), and fluorescence intensity was reduced by about half (Fig. 3C). It is 243 important to note that autofluorescent bodies were also observed in dark-grown root cells using 244 microscope settings equivalent to those used for GFP fluorescence (Supplemental Fig. S1) and 245 should not be confused with GFP-CLASP puncta seen in Fig Recognizing that hypocotyl expansion is stimulated in the dark, we measured CLASP transcript 264 and protein levels in hypocotyls. In rapidly growing dark-grown hypocotyls, GFP-CLASP 265 remained abundant and could be seen distributed along MTs (Supplemental Movie S1). qRT-266 PCR from dark-grown seedlings revealed that CLASP expression was ~ 30% higher in the 267 hypocotyls compared to root tips (Supplemental Fig. S2). This demonstrates that in the dark-268 grown hypocotyls, both GFP-CLASP protein CLASP transcript levels remain high, in contrast to 269 the dark-grown root tips, where transcript levels increased 2.5-fold ( Fig. 2) but protein levels fall 270 (Fig. 3B). To determine if this uncoupling of transcript and protein levels is a general 271 phenomenon for BR-associated components, we measured the expression of DWF4, which 272 encodes a BR biosynthetic enzyme. In contrast to the 30% lower expression of CLASP in the 273 root tip compared to the hypocotyl, we found that that DWF4 expression was reduced by ~ 80% 274 (Supplemental Fig. S2). 275

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The CLASP protein is not degraded via the proteasome or autophagy in the dark 277 To investigate the possibility that CLASP protein was rapidly degraded in the dark, we inhibited 278 two pathways involved in protein degradation and assessed protein abundance using the GFP-279 CLASP fluorescent reporter (Fig. 4). Plants were treated with 50 µM MG132 for 3 hours and 1 280 µM concanamycin A for 6 hours to inhibit the 26S proteasome and autophagy, respectively. No 281 increase in GFP-CLASP was observed with MG132 ( Fig. 4A, B). As a positive control, we 282 observed PIN2-GFP localization after treatment with 50 µM MG132 for 3 hours because this 283 protein is known to be degraded by the 26S proteasome pathway (Laxmi et al. 2008). PIN2 284 fluorescence increased at the plasma membrane and decreased in the vacuole, demonstrating that 285 the MG132 drug treatment was functioning as expected (Fig. 4C). GFP-CLASP levels were also 286 unaffected by Concanamycin A treatments ( Fig. 4D-E). These results demonstrate that CLASP is 287 not actively degraded in the dark, which suggests instead that the protein levels are regulated by 288 suppression of translation. 289 290

Microtubules do not form transfacial bundles in dark-grown meristems 291
Since CLASP binds to MTs and influences their organization, we hypothesized that CLASP-292 dependent MT arrays would be disrupted in dark-grown roots. In roots grown in the light, 293 CLASP mediates the formation of transfacial MT bundles that are specific to cells in the division 294 zone (Fig. 5A, B, C). Transfacial MT bundles were completely absent in dark-grown root tips 295 and instead, MTs displayed a predominately transverse array characteristic of cells in the clasp-1 296 knockout mutant (Fig. 5D, E, F). In the elongation zone of light-grown roots, MTs are organized 297 transverse to the axis of growth. In contrast, the MTs in dark-grown elongating root cells were 298 oriented along the long axis of the cell (Fig. 5G, Supplemental Movie S2). When increasing 299 amounts of sucrose were added to the growth medium of dark-grown plants, the angle of MT 300 alignment in division-zone cells had a tendency towards more longitudinal than transverse, but 301 was not fully restored to light-grown conditions (Fig. 5H). The addition of sucrose did not have a 302 significant effect on MT alignment in light-grown seedlings (Fig. 5H). These results suggest that 303 306 In a previous study, gibberellins (GAs) were identified as regulators of MT organization through 307 the interaction of DELLA proteins and the prefoldin complex, which promotes proper tubulin 308 folding (Locascio et al., 2013). This work showed that when GA is absent, the prefoldin complex 309 is sequestered in the nucleus and decreases the amount of free tubulin heterodimers available for 310 incorporation into the MT. Treatment with GA results in degradation of the DELLA proteins and 311 the movement of the prefoldin complex to the cytoplasm. Based on the sparse MT population 312 observed in dark-grown roots, we hypothesized that GA treatment would induce the prefoldin 313 complex to become functional in the cytoplasm and increase the assembly of MTs, which in turn 314 would result in greater accumulation of CLASP at cell edges. In all cases examined, application 315 of gibberellic acid did not affect MT density or organization (Supplemental Fig. S3). This 316 suggests that the lack of transfacial MT bundles and CLASP protein cannot be rescued by a more 317 active prefoldin complex in dark-grown roots. 318 319

Auxin transporters and BR receptors accumulate in large central vacuoles in dark-grown 320 roots 321
Previous work has shown that CLASP promotes recycling of the BRI1 receptor (Ruan et al., 322 2018) and PIN2 transporter (Ambrose et al., 2013) to the plasma membrane. Since CLASP 323 protein levels were depleted in dark-grown seedlings, we hypothesized that the distribution and 324 abundance of BRI1 and PIN2 would be affected in light-versus dark-grown seedlings. We first 325 noticed the formation of large central vacuoles in meristematic cells of dark-grown seedlings, 326 which were absent from cells of the same developmental stage exposed to light (Fig. 6 A-D, 327 brightfield images). GFP-tagged BRI1 and PIN2 were then observed in light and dark conditions 328 ( Fig. 6 A-D). In the light treatments, fluorescence intensity was highest at the plasma membrane, 329 both with and without sucrose (Fig. 6A, B, Supplemental Fig. S4). For seedlings grown in the 330 dark with 1% sucrose, PIN2-GFP and BRI1-GFP were still detected at the plasma membrane, 331 although the fluorescence intensity was considerably reduced (Supplemental Fig. S4). In dark 332 conditions in the absence of sucrose, both BRI1 and PIN2 were localized to the vacuoles, 333 indicating that these proteins were undergoing degradation (Fig. 6C, D). Additionally, DR5:GFP 334 labelling in light-and dark-grown roots showed an accumulation of auxin in the meristem of 335 dark-grown roots compared to light-grown roots (Fig. 6E). The transport of BRI1 and PIN2 to 336 339

DISCUSSION 340
Root meristem growth is essential for plant development, and can be readily adjusted based on 341 light availability, nutrients, or abiotic stress. Here, we show how modulation of CLASP protein 342 levels are coincident with either rapid root growth in light conditions, or a cessation of growth in 343 dark conditions (Fig. 7). We showed through fluorescence imaging that BZR1, a negative 344 regulator of CLASP, is degraded in dark-grown root meristems. As expected, due to the loss of 345 negative regulation by BZR1, CLASP transcript levels were increased in dark-grown root tips. 346 Despite the two-and-one-half fold-elevated transcript level, we detected greatly reduced CLASP 347 protein abundance both when CLASP was expressed under its endogenous promoter or 348 overexpressed with the 35S promoter. We also determined that CLASP is not actively degraded 349 in dark-grown roots, leading us to conclude that there is a 'translational checkpoint' that 350 maintains low levels of CLASP when root cell division and elongation are not necessary, such as 351 in the dark when plants must prioritize hypocotyl expansion. It is important to note that in early 352 seedling development and prior to becoming photosynthetically active, plants must ration sugar 353 reserves. Our work also clearly demonstrates that supplementing growth media with sucrose 354 while growing plants in the dark obscures the dramatic phenotypes associated with cellular 355 starvation, which plants would likely experience when photosynthetic output is limited. 356

357
Our study has identified a very specific and specialized regulatory system that controls CLASP 358 protein levels in response to light/dark conditions, and further underlines the importance of 359 CLASP as a regulator of meristem size in the Arabidopsis root tip (Ambrose et al., 2007). redistribution is consistent with a lack of CLASP, which normally promotes recycling of PIN2 391 and BRI1 to the plasma membrane through tethering of SNX1 vesicles (Ambrose et al., 2013;392 Ruan et al., 2018). Cells in the division zone of roots normally have several small vacuoles, 393 whereas an enlarged central vacuole is characteristic of elongating and differentiated cells. It 394 may be that cell wall properties play a role in vacuolar expansion in these cells, as mutants of 395 extracellular leucine-rich repeat extensins and the receptor-like kinase FERONIA displayed 396 enlarged vacuoles in root meristematic cells (Dünser et al., 2019), similar to what we observed in 397 our study. 398 399 What mechanism controls translation in Arabidopsis root tips during carbon-limited conditions? 400 Plants maintain elaborate nutrient-sensing signalling pathways that impinge on transcriptional 401 and translational network remodelling. Carbon derived from the shoot activates TOR signalling 402 to induce meristem growth in the root. In addition to activating genes related to amino acid 403 synthesis, the cell cycle, and RNA synthesis and processing, TOR phosphorylates the E2Fa 404 transcription factor that activates S-phase related genes (Xiong et al., 2013). When these 405 processes are inhibited under nutrient starvation, mature ribosomes are degraded by autophagy (a 406 process termed ribophagy) that has so far been described in yeast (Kraft et al., 2008) and 407 mammals (An and Harper, 2018;Wyant et al., 2018). Work on ribophagy has been limited in 408 plants, although Floyd et al. (2015)  To quantify meristem cell number, root tips were stained in a 10 μg/mL solution of propidium 451 iodide dissolved in distilled water for 1 minute. This was followed by three 1 minute rinses in 452 dH 2 O before mounting on a glass slide for microscopy. 453 454 Confocal Microscopy 455 6-day-old seedlings were mounted in either ½ MS (no sucrose) liquid or drug solutions on glass 456 slides for imaging. Fluorescent protein reporters were imaged using a spinning-disk confocal 457 microscope (Leica DMi8 inverted microscope, Perkin-Elmer UltraView spinning-disk system, 458 and a Hamamatsu 9100-02 Camera) with either a 40x/NA 1.25 oil or 63x/NA 1.3 glycerol lens. 459 CFP was imaged with a 440 nm laser and 480/40 emission filter, GFP was detected using a 488 460 nm laser and 525/36 emission filter, and YFP was imaged using a 514 nm laser and 540/30 461 emission filter. For propidium iodide and FM4-64, excitation was with a 561 nm laser and 462 595/50 emission filter. The slice thickness for z-projections was 0.3 μm. Images were captured 463 using the Volocity 6.3 software package (Perkin Elmer). 464 465

Image Analysis 466
Confocal images were processed using ImageJ (https://imagej.nih.gov/ij/). Root length was 467 quantified using the NeuronJ plugin (Meijering et al., 2004). Fluorescence intensity of GFP-468 CLASP in root meristems was measured by drawing regions that encompassed epidermal cells 469 from the quiescent centre to the beginning of the elongation zone from compressed z-stacks. The 470 "corrected fluorescence" was calculated by subtracting the mean background fluorescence and 471 then dividing by area of the measured region and number of z slices to facilitate comparisons 472 between light and dark samples. Microtubule orientation within meristematic cells was 473 calculated using the ImageJ plugin FibrilTool (Boudaoud et al., 2014). BRI1 and PIN2 474 fluorescence was measured in two cell files from six independent roots in cells extending from 475 the quiescent centre to the elongation zone. Briefly, the sum slices tool was used to generate z-476 projections that extended from the top-most part of the cell to 8-9 μm deep. The plasma 477