An Intrinsically Disordered Protein Interacts with the Cytoskeleton for Adaptive Root Growth under Stress.

Intrinsically disordered proteins function as flexible stress modulators in vivo through largely unknown mechanisms. Here, we elucidated the mechanistic role of an intrinsically disordered protein, REPETITIVE PROLINE-RICH PROTEIN (RePRP), in regulating rice (Oryza sativa) root growth under water deficit. With nearly 40% proline, RePRP is induced by water deficit and abscisic acid (ABA) in the root elongation zone. RePRP is sufficient and necessary for repression of root development by water deficit or ABA. We showed that RePRP interacts with the highly ordered cytoskeleton components actin and tubulin, both in vivo and in vitro. Binding of RePRP reduces the abundance of actin filaments, thus diminishing non-cellulosic polysaccharide transport to the cell wall and increasing the enzyme activity of sucrose synthase. RePRP also reorients the microtubule network, which leads to disordered cellulose microfibril organization in the cell wall. The cell wall modification suppresses root cell elongation, thereby generating short roots, whereas increased sucrose synthase activity triggers starch accumulation in "heavy" roots. Intrinsically disordered proteins control cell elongation and carbon reserves via an order-by-disorder mechanism, regulating the highly ordered cytoskeleton for development of "short-but-heavy" roots as an adaptive response to water deficit in rice.

Introduction tubulin is not due to non-specific interactions. 180 We used competition binding assays to verify segmental rOsRePRP2.1 60-179 binding 181 to AG, actin and tubulin ( Figure 1C and 1D). Recombinant OsRePRP2.1 60-179 was first 182 mixed with AG for measuring the binding of rOsRePRP2.1 60-179 to actin and tubulin. 183 In the presence of 250 M AG, rOsRePRP2.1 60-179 retained the binding affinity to  To confirm the binding of rOsRePRP to actin filaments (F-actin) and microtubules, 198 we used high-speed co-sedimentation assays with mixtures of rOsRePRP2.1 60-179 and 199 F-actin and microtubules polymerized to a steady state (Supplemental Figure S3I (Bergersen et al., 2008), so the distance < 60 nm between two sizes of gold 246 particles was assumed to be co-localization. Most of the closest distances between OsRePRP2-OX protoplasts mainly showed cytosolic patterns but few filamentous 288 patterns ( Figure 5B). These results exclude the possibility that reducing F-actin level 289 in OsRePRP2-OX was an artefact caused by the differential staining of phalloidin. Microtubule Orientation is Altered in OsRePRP2-OX 292 We also examined microtubule organization of wild-type, OsRePRP2-OX and 293 OsRePRP-Ri rice root cells under control and ABA treatment by immunofluorescence 294 staining with anti--tubulin antibodies ( Figure 6A to 6F). The microtubule network 295 was less organized in OsRePRP2-OX than wild-type or OsRePRP-Ri root cells 296 ( Figure 6A to 6C). With ABA treatment, the disordered microtubule phenotype was 297 apparent in the wild type but less so in OsRePRP-Ri cells ( Figure 6D and 6F). 298 Further study in the rice root protoplast transient-expression system indicated that 299 GFP-MBD showed a similar filamentous expression pattern in the wild type, 300 OsRePRP2-OX and RePRP-Ri ( Figure 6G to 6L). The microtubule organization in 301 the rice root protoplast transient-expression system did not show the transverse 302 arrangement shown in the whole-mount inmmunostaining of rice roots ( Figure 6A). 303 The results suggested that without the cell wall, the microtubule orientation of 304 OsRePRP2-OX did not differ substantially from the wild type and RePRP-Ri ( Figure   305 6G to 6I).
The cytoskeleton is involved in plant cell shape determination by affecting the 310 patterns by which cell wall materials are deposited (Smith and Oppenheimer, 2005).

311
F-actin plays a role in vesicle trafficking of non-cellulosic polysaccharides (Baluska et 312 al., 2002;Kim et al., 2005). Microtubules maintain the cellulose synthase complexes 313 localized at the plasma membrane to guide cellulose deposition on the cell wall 314 (Paredez et al., 2006;Crowell et al., 2009). Because OsRePRP regulates actin 315 filament distribution and microtubule organization in vivo, we wondered whether 316 OsRePRP affects non-cellulosic polysaccharide secretion on the cell wall and cell-317 wall cellulose microfibril arrangement. By using metabolic click-labeling with fucose 318 alkyne (FucAl) and Alexa Fluor 488-azide (Anderson et al., 2012), we monitored 319 FucAl incorporation into the cell wall by confocal microscopy ( Figure 7A to 7C). OsRePRP2-OX were dispersed and oriented in a multitude of directions ( Figure 7K).

343
After ABA treatment, the wild type and OsRePRP2-OX showed dispersed directions, OsRePRP2-OX segments submerged beneath the surface of water, which suggests 353 that OsRePRP2-OX segments were "heavy" ( Figure 7M). After ABA treatment, wild-354 type root-tip segments also submerged, resembling the "heavy" root phenotype of 355 OsRePRP2-OX under the control condition ( Figure 7M). However, after ABA 356 treatment, the "heavy" root phenotype was less apparent in OsRePRP-Ri than the wild 357 type ( Figure 7M). The quantification of dry weight per root length per seedling also 358 confirmed the "heavy" root phenotype ( Figure 7N). 359 We wondered whether storage starch accumulation contributed the biomass 360 accumulation in OsRePRP2-OX, so we used iodine staining to address this question.
Rice roots accumulated starch under PEG stress, as shown by the dark iodine-stained 362 color ( Figure 8A and 8B). OsRePRP2-OX showed more dark-stained roots than the 363 wild type or OsRePRP-Ri did after PEG treatment ( Figure 8B). Sectioning was   Table 3). The co-immunoprecipitation of sucrose synthase in  OsRePRP2-OX and OsRePRP-Ri ( Figure 9B and 9C). The sucrose synthase activity 394 in the cleavage direction was two-fold higher in OsRePRP2-OX than the wild type or  Figure S6A). Thus, we cannot rule out that OsRePRP1 may also 427 play a similar role as OsRePRP2 and conclude that OsRePRPs are sufficient and 428 necessary for ABA/water deficit repression of root development.

429
In the face of adversity and danger, animals can escape by using highly modulated 430 skeletons and muscles. Titin and nebulin rule myosin and actin contraction in muscle 431 cells, controlling muscle contraction (Labeit and Kolmerer, 1995;Wang et al., 1996;432 Gutierrez-Cruz et al., 2001;Ma and Wang, 2002). PEVK motifs of titin are repetitive 433 and intrinsically disordered, with highly charged residues (Labeit and Kolmerer, 1995;434 Gutierrez-Cruz et al., 2001). Tandem repeats of titin and nebulin provide the binding 435 sites for actin and myosin and scaffolding/crosslinking to the filamentous structures 436 (Labeit and Kolmerer, 1995;Wang et al., 1996;Gutierrez-Cruz et al., 2001;Ma and 437 Wang, 2002). OsRePRPs also showed repetitive (PEPK, PQPN and PDPK),   (Schwartz and Ginsberg, 2002), OsRePRP has a distinct role in regulating the 486 very dynamic interactions between the cytoskeleton and cell wall in plants. 487 Although sucrose synthase interacting with actin filaments in vivo is assumed to be a 488 control mechanism (Koch, 2004), we lack direct evidence to support this idea. Herein, 489 we showed increased sucrose synthase enzyme activity in OsRePRP2-OX ( Figure   490 9B), so reducing F-actin may affect the enzyme activity of sucrose synthase in vivo.
Phosphorylation of sucrose synthase in maize has been reported to be associated with 492 its enzyme activity (Huber et al., 1996). However, in our case, the increase in sucrose 493 synthase enzyme activity was not associated with its phosphorylation ( Figure 9D), 494 distinct from the regulation explored in maize sucrose synthase (Huber et al., 1996).

495
In animals, cytoskeleton remodeling releases more free forms of aldolase, thereby OsRePRP2-Ri look quite different in morphology (Fig 8) probably due to the effect of 503 ReOsPRP2 on root elongation. Although the samples were sectioned at the same distance 504 from root tips, i.e., 0.5 cm. they may reflect different root developmental stages caused by the 505 differential expression of OsRePRP2. Nevertheless, the differences of SUS activities can at 506 least partially account for the different level of starch accumulations in these samples. The 507 interaction (direct or indirect) of OsRePRPs with sucrose synthase may not be only 508 related to starch biosynthesis but also to cell wall metabolism by providing NDP-509 glucose as the substrate.

510
Our current study explains the reduced cell length phenotype of OsRePRP2-OX in 511 our previous study (Tseng et al., 2013) and supports that both microtubules and actin 512 filaments are critical for cell expansion (Smith, 2003). Observing disorganization of 513 cell wall cellulose microfibrils and cortical microtubules in OsRePRP2-OX (Figures 6   514 and 7), people may expect to see a phenotype of anisotropic cell expansion rather than 515 just reduced cell elongation. However, previous studies have shown that the degree of growth anisotropy was not correlated with the degree of alignment of either 517 microtubules or microfibrils (Baskin et al., 1999) and cell expansion in longitudinal 518 and radial directions can be regulated independently in roots (Liang et al., 1997; 519 Baskin, 2005). Hence, the phenotype of OsRePRP2-OX may probably support the  This "short-but-heavy root" strategy is similar to the rice flooding-tolerance gene 529 SUBMERGENCE-1, which causes growth quiescence during flooding that is   Ubipro::OsRePRP2-GFP and OsRePRP2.1pro::GUS were generated as previously 595 described (Tseng et al., 2013).  from the 18-nm gold particle to the closest 12-nm gold particle was calculated by 717 using ImageJ, and quantification of the immunogold TEM data was performed as 718 described (Bergersen et al., 2008). For cell-wall cellulose microfibril observation, 8-day-old seedlings (control) and 4-740 day-old seedlings treated with 2 M ABA for 4 days (ABA) were used. Cell-wall 741 preparation was as described (Sugimoto et al., 2000) with slight modifications. The 742 whole roots were cryoprotected in PME buffer 2 (25 mM PIPES, 0.5 mM MgSO 4 , 2.5 743 mM EGTA, pH 7.2) containing 25% and 50% DMSO for 10 min for each step. Root 744 tips were excised, placed on sample carriers and cut by using a glass knife on a Leica 745 Ultracut UCT ultramicrotome equipped with the Leica EM FCS cryo-chamber 746 attachment at -120°C. The remaining root strips were thawed in PME buffer 2 747 containing 50% DMSO, then transferred to PME buffer 2. Samples were treated with acetic acid and nitric acid and distilled water (8:1:2) for 1 hr at 95°C. After a thorough 749 washing in distilled water, samples were dehydrated with an ethanol series (30%, 750 50%, 70%, 95% and 100% three times, 30 min for each step), critical point dried with 751 CO 2 and further mounted on carbon tape-covered stubs with the cut surface facing  For starch staining, 14-day-old seedlings (control) and 14-day-old seedlings treated 761 with 20% PEG6000 for 1 hr (PEG) were used. Rice roots were stained in 1/10 diluted 762 5% Lugol's iodine solution for 10 min, destained with distilled water for 30 min and 763 the root architecture images were captured by using an Epson scanner. For sectioning, 764 1-cm root segments were embedded in 5% agar and cut into 100-to 120-m sections 765 using a DTK-100 microslicer (Dosaka EM). The sections were stained with iodine 766 and observed under a Zeiss Axio Imager Z1 microscope. For starch quantification, 14-767 day-old seedlings (control) and 14-day-old seedlings treated with 20% PEG6000 for 5 768 hr (PEG) were used. Whole roots of 10 to 20 seedlings were ground in liquid nitrogen 769 with a pestle and mortar and dried at 65°C, then dry weight was measured. A starch

774
Whole roots of 10 to 20 14-day-old seedlings were ground into a fine powder in liquid 775 nitrogen by using a pestle and mortar, mixed with 1-mL ice-cold extraction buffer 776 (100 mM HEPES-KOH, pH 7.5, 5 mM MgCl 2 , 5 mM DTT), and centrifuged at 777 11,000 x g for 15 min at 4°C. The supernatant was then collected and used for the 778 measuring sucrose synthase enzyme activity in the cleavage direction according to 779 Sun et al. (1992). The protein extract was quantified by using the Bio-Rad Protein   each OsRePRP-GFP gold particle to the closest actin or tubulin gold particle were 939 measured as described in Methods. The bars represent the distribution of inter-gold-    The distances from each OsRePRP-GFP gold particle to the closest actin or tubulin gold particle were measured as described in Methods. The bars represent the distribution of inter-gold-particle-center distances between OsRePRP-GFP and actin or tubulin ranging from 0 nm to > 100 nm. Ten images were measured, and two biological replicates were performed. Bar = 100 nm in A-B, E-G.  Relative density of root segments in WT, OsRePRP2-OX and OsRePRP-Ri. Two-cm root-tip segments from 12-day-old seedlings (control) and 8-day-old seedlings treated with 2 M ABA for 4 days (ABA) were cut and immerged in the distilled water. Six biological replicates were performed and three independent transgenic lines for each genotype were observed (OsRePRP2-OX #10, #19, #24 and OsRePRP-Ri #5, #6, #7).
(N) Quantitative analysis of total root dry weight (mg) divided by mean root length (cm) per seedling. Whole roots were harvested from WT, OsRePRP2-OX (OX) and OsRePRP-Ri (Ri) 14-day-old seedlings (control) and 8-day-old seedlings treated with 2 M ABA for 6 days (ABA). Significant differences are indicated with asterisks (P < 0.01, two-tailed Mann-Whitney U test). Data are mean±SD of six biological repeats.
Data are mean±SD of six technical repeats. Three biological replicates were performed and two independent transgenic lines for each genotype were measured (OsRePRP2-OX #10, #24 and OsRePRP-Ri #5, #6). Bar = 1 cm in (A-B) and 50 m in (C-Q). treated with 2 M ABA for 2 days. Western blot analysis involved anti-SUS antibodies. The arrow indicates 93-kDa SUS. Two biological replicates were performed. (B-C) Enzyme activity of sucrose synthase in the cleavage (B) and synthetic (C) directions. Total protein extract from 14-day-old whole roots of the wild type (WT), OsRePRP2-OX (OX) and OsRePRP-Ri (Ri) was shown in box plots. The line inside the box indicates the median, and the cross indicates the mean. Box edges are the 25 th to 75 th percentiles; whiskers indicate the range. Significant differences are indicated with asterisks (P < 0.01, two-tailed Mann-Whitney U test). Data are mean/median (Q1-Q3) of nine repeats from three independent biological replicates.
Five biological replicates were performed. (D) PhosTag western blot (WB) and WB analysis with anti-SUS antibodies. Total protein extract from 14-day-old whole roots of the wild type (WT), OsRePRP2-OX (OX) and OsRePRP-Ri (Ri) was assayed. Four biological replicates were performed.