A gas-and-brake mechanism of bHLH proteins modulates shade avoidance in Arabidopsis thaliana

Plants detect proximity of competitors through reduction in the ratio between red and far red light triggering the shade avoidance syndrome, which includes accelerated shoot elongation and early flowering. Shade avoidance is regulated through PHYTOCHROME INTERACTING FACTORs (PIFs), a group of bHLH transcription factors. Another (b)HLH protein, KIDARI (KDR), which is non-DNA-binding, was identified in de-etiolation studies and proposed to interact with LONG HYPOCOTYL IN FAR-RED 1 (HFR1), a (b)HLH protein that inhibits shade avoidance. Here we establish novel roles of KDR in regulating shade avoidance and investigate how KDR regulates the shade avoidance network. We show that KDR is a positive regulator of shade avoidance and interacts with several negative growth regulators. We identify novel interactors using a combination of yeast two-hybrid screening and dedicated confirmations with bimolecular fluorescence complementation. We demonstrate that KDR is translocated primarily to the nucleus when coexpressed with these newly discovered interactors. A genetic approach confirmed that several of these novel interactions are indeed functional to shade avoidance in Arabidopsis thaliana, whereas we propose that KDR does not interact with HFR1 to regulate shade avoidance. Based on this, we propose that shade avoidance is regulated by a three-layered gas-and-brake mechanism of bHLH protein interactions, adding an additional layer of complexity to what was previously known. One-sentence summary KIDARI is a positive regulator of shade avoidance and part of a three-layer network of bHLH transcription factor interactions.


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Plants harvest light energy during photosynthesis, especially, blue (B) (~400-500 nm 43 waveband) and red (R) (~600-700 nm waveband) light, whilst mostly reflecting far-44 red light (FR) (~700-800 nm waveband). As a consequence, the ratio of R to FR is internode and petiole elongation, early flowering, and upward leaf movement called 50 hyponasty (de Wit et al., 2016;Ballaré et al., 1991;de Wit et al., 2015;Galvāo et al., 51 although it improves individual plant fitness, it may compromise total crop yield 53 (Robson et al., 1996;Boccalandro et al., 2003). Contrary, shade tolerant species, 54 such as those from forest understories, have developed alternative strategies to cope 55 with shade conditions without investing in shade avoidance growth (Gommers et al., 56 2013(Gommers et al., 56 , 2017Molina-Contreras et al., 2019). 57 In an attempt to unravel the strategy of some species suppressing SAS, Gommers et 58 al. (2017) previously described the contrasting shade-tolerant and intolerant 59 responses of two selected Geranium species when exposed to low R:FR. In a 60 transcriptome approach between these species, putative regulators of these two 61 different responses were identified. One of these regulators is a basic helix-loop-helix 62 (bHLH) protein-encoding gene, called KIDARI (KDR)/PACLOBUTRAZOL 63 RESISTANCE 6 (PRE6) (Gommers et al., 2017). The expression of KDR in A. 64 thaliana has been shown to rely on functional PHYTOCHROME-INTERACTING

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KDR promotes shade avoidance response 115 We confirmed the upregulation of KDR in seedlings of A. thaliana exposed to a low  Figure 1B). When the same lines were tested for petiole elongation in rosette plants, 124 a similar suppression of the response was found for kdr-1, but less severely. By  Overexpression of KDR stimulates shade avoidance 130 We created novel lines overexpressing KDR in A. thaliana Col-0 background in order 131 to have improved genetic material over the kdr-D activation tagging line that only 132 mildly overexpresses KDR. We then used four homozygous independent lines to 133 study their response to low R:FR treatment at the seedling stage. We found that most 134 of the novel 35S:KDR transgenic lines showed an even more exaggerated response 135 than found for kdr-D and additionally displayed constitutive hypocotyl elongation in 136 white light ( Figure 2A). Interestingly, the variation in hypocotyl length between the 137 independent transgenic lines correlated with variation in KDR overexpression levels 138 ( Figure 2B and 2C). We selected the two independent lines most strongly 139 overexpressing KDR to continue. When looking more carefully at the phenotype of 140 the selected lines, we observed that KDR overexpression increased elongation of 141 most organs, including hypocotyl, petioles of cotyledons, petioles of true leaves and 142 primary root ( Figure 2D). 143 We also verified if petiole elongation in adult plants was affected in our strong 144 overexpression lines. Interestingly, they did not show an increased petiole elongation 145 response to low R:FR treatment (Supplemental Figure 1A). The KDR overexpression 146 lines in adult stage are relatively small, but they do seem to have relatively elongated 147 petioles with small leaf laminas (Supplemental Figure 1B), reminiscent of a shade 148 avoidance phenotype. Interestingly, another leaf response, upward movement called  Figure   153 2A), which is another established shade avoidance response (Halliday et al., 1994). 154 The KDR overexpressors had very long flowering stems, which at a later life stage 155 started to split open leading to a more bent flowering architecture (Supplemental 156 Figure 2B).

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Overexpression of KDR affects regulation of PIF targets 158 The main function of KDR described in literature is its interaction with the negative 159 growth regulator HFR1, which binds to PIFs and therefore interferes with the  Novel interactors of KDR from Y2H screens 171 We performed a Y2H screen where the coding sequence (CDS) of KDR was cloned  Table 1, including the frequency with which the interactors were found and the strength of their interaction. Among the 176 proteins identified, we focused on PAR1 and PAR2, two known PIF-interacting 177 proteins. We confirmed the interactions by cloning the full-length CDSs of these 178 proteins (rather than the truncated versions from the library) from A. thaliana cDNA 179 into the prey vector and retransformed in yeast to perform a protein-protein 180 interaction assay. In this direct Y2H assay we also tested the previously published 181 interaction of KDR with HFR1 (Hyun and Lee, 2006;Hong et al., 2013), but could not 182 confirm this interaction ( Figure 4). Also, when swapping the bait-prey configuration no 183 interaction was found between HFR1 and KDR (Supplemental Figure 4A). Finally, 184 changing vectors to those used in Hyun and Lee, (2006);Hong et al., (2013) 185 (pGBKT7 for the bait and pGADT7 for the prey) would again not confirm the 186 interaction (Supplemental Figure 4B). As positive controls for Y2H assays, we did 187 confirm interactions of HFR1 with PIF4 and PIF5 [previously published in Hornitschek 188 et al. (2009)], and found that HFR1 can also interact with PIF7 ( Figure 5A). We also 189 found that KDR does not directly interact with PIFs in yeast, while HFR1 and PAR1 190 do ( Figure 5A). Lastly, we confirmed that PIF7 can interact with itself and other PIFs 191 ( Figure 5B), which is consistent with the notion that PIFs form hetero-and 192 homodimers to bind DNA regions and activate the expression of target genes (Bu et 193 al., 2011;Leivar et al., 2008). 194 In order to maximize the number of relevant KDR interactors found, we performed a 195 second Y2H screen using a completely different library consisting of only TFs of A. 196 thaliana cloned in full-length sequence (Pruneda-Paz et al., 2014). Ten putative 197 interactors were discovered and their identity was verified by sequencing (Table 2). 198 We narrowed the selection for further studies to four (b)HLH candidates ATBS1 199 (ACTIVATION-TAGGED BRI1 SUPPRESSOR 1)-INTERACTING FACTOR 2 (AIF2), 200 AIF4, ILI1 BINDING BHLH 1 (IBH1) and IBH1-LIKE 1 (IBL1), since these proteins 201 had previously been linked to growth regulation in association with some regulators 202 of the SAS, but had not been implemented in shade avoidance control before. The 203 strength of interaction was verified by performing a Y2H direct interaction assay. All 204 four candidates were able to grow at least up to the medium lacking histidine (His) 205 and supplemented with 5 mM 3-amino-1, 2, 4-triazole (3-AT), meaning that the indicating that the interaction draws KDR to the nucleus. When coexpressing KDR 221 with HFR1, there was still substantial KDR abundance in the cytoplasm, similar to 222 when KDR was transiently expressed alone, consistent with the lack of interaction 223 found above.

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To further verify the interactions of KDR identified with the Y2H assays, we examined 226 whether they were also occurring in planta. We performed a BiFC assay where the 227 two parts of the split Venus fluorescent protein (YN or YC) were C-or N-terminally 228 tagged to the proteins of interest and coexpressed in N. benthamiana leaves ( Figure   229 7). We detected the reconstituted YFP signal in the nucleus in all the different 230 samples, apart from the interaction with HFR1, and some differences were noticed 231 when KDR was found to interact with the different candidates. The reconstituted YFP 232 signal was observed in different nuclear compartments, resembling the localization of 233 the targets alone ( Figure 7). The interactions between KDR and PAR1 and PAR2 234 were observed in the nucleoplasm while the interactions with AIF2, AIF4, IBH1 and 235 IBL1 were found in the nucleus with the strongest signal in the nucleolus. Together, 236 the Y2H and the BiFC data indicate that KDR can truly interact with all the identified 237 targets and that their interaction seems to trigger its translocation primarily to sub-238 nuclear complexes, while no interaction with HFR1 could be confirmed.  (Hornitschek et al., 2012;Galstyan et al., 2012Galstyan et al., , 2011Roig-Villanova et al., 2007).

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Also AIF2, AIF4, IBH1 and IBL1 were described as non-DNA-binding (b)HLH proteins 246 Ikeda et al., 2012;Zhiponova et al., 2014) but, while their role is 247 mainly related to elongation growth, nothing is known so far about shade avoidance 248 in mutants for these genes. We first confirmed that in our conditions HFR1, PAR1 249 and PAR2 were also upregulated following a low R:FR treatment in seedlings of Col-250 0 ( Figure 8A). Since AIF2, AIF4, IBH1 and IBL1 were never associated with shade 251 responses, we also verified if their expression level was differentially regulated upon 252 exposure to low R:FR and we found that this was indeed the case ( Figure 8A).

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Next, we studied the response to low R:FR conditions of different mutant and 254 overexpression lines of these bHLH genes relative to Col-0 wild type ( Figures 8B and   255 8C). Figure 8B shows that low R:FR-induced hypocotyl elongation is increased in the 256 hfr1-201, hfr1-5 and par1 mutants. Overexpressing any of these genes was sufficient 257 to severely block the response to low R:FR. These phenotypes are entirely consistent 258 with the roles of these proteins as negative SAS regulators. Instead, for the less 259 known genes AIF2, AIF4, IBH1 and IBL1 we studied the available T-DNA insertion 260 lines, but unfortunately in the SALK lines for IBH1 we could not detect the T-DNA 261 insertion and were therefore discarded. In low R:FR conditions, all the lines except 262 for aif2-2 showed a moderately enhanced hypocotyl elongation response compared 263 to Col-0 wild type ( Figure 8C). In the case of ibl1, a statistically significant difference, 264 although minimal, was already seen in control conditions when compared to the wild 265 type. The relatively mild phenotypes, albeit reproducible and statistically significant, 266 may hint at genetic redundancy between the different KDR targets. Higher order 267 combinations of these mutants, as well as overexpression lines for these genes, 268 would likely help understand the impact of these novel shade avoidance components 269 in more detail. 271 We hypothesized that KDR would act to sequester negative regulators, such as 272 PAR2, by direct interaction. We verified this hypothesis by crossing a PAR2   Finally, we also generated transgenic lines overexpressing KDR in pif7, pif4 pif5 and 287 pif4 pif5 pif7 backgrounds by floral dipping these mutants with a 35S:KDR construct 288 using Agrobacterium-mediated transformation. We then studied their response when 289 exposed to low R:FR using three independent lines for each background, after we 290 verified their expression level ( Figure 10 and Supplemental Figure 6). As expected, 291 pif4 pif5 shows a clear but reduced response to low R:FR, while pif7 and pif4 pif5 pif7 292 lost the hypocotyl response to low R:FR completely. Interestingly, in control white 293 light conditions the overexpression of KDR is able to induce a strong elongation in 294 pif7 and pif4 pif5 backgrounds, and more mildly when overexpressed in the triple 295 knockout pif4 pif5 pif7. When these lines were exposed to low R:FR, pif4 pif5 296 35S:KDR had nearly the same hypocotyl phenotype as the same construct has in 297 wild type background, consistent with the relatively modest role of pif4 pif5 in low 298 R:FR-induced hypocotyl elongation. Contrary, in the pif4 pif5 pif7 mutant, KDR 299 overexpression could not rescue the hypocotyl elongation response to low R:FR, 300 while in the pif7 only mildly. 301 We conclude that KDR interacts with PARs to regulate hypocotyl elongation in 302 response to low R:FR and this subsequently depends on PIFs, probably because 303 PARs directly interact with PIFs to regulate their activity.

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In a comparable way, we found that overexpression of KDR can restore the growth 343 defect of PAR2 overexpression ( Figure 9). This finding places KDR in a new third 344 level of SAS regulation, above PAR1 and PAR2, which suppress PIF activity ( Figure   345 11). Our results also cast severe doubts on the suppressing role of KDR on HFR1, at 346 least in the regulation of SAS.

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In this study, we show that KDR physically interacts in yeast and in planta with a 349 range of negative regulators of cell elongation, i.e. AIF2, AIF4, IBH1 and IBL1. None 350 of them had been previously associated with SAS but they all share some similarities.

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For example, they had been identified already for their interaction with some of the 352 PRE members Ikeda et al., 2013;Zhang et al., 2009)   Col-0, pif4, pif4 pif5 and pif4 pif5 pif7 following the protocol of Zhang et al. (2006).

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Successfully transformed T 1 seeds were selected through the GFP signal in dry 513 seeds. T 2 lines were selected for single insertion of the transgene using the 514 selectable marker bar, which confers resistance to the herbicide Basta (25 µg/ml) 515 (DL-Phosphinothricin, Duchefa Biochemie). Finally, T 3 seeds were screened for 516 homozygosity using the GFP signal and the insertion of the transgene was confirmed 517 by PCR reaction performed on genomic DNA (gDNA) extracted from homozygous 518 plants using the primers listed in Supplemental Table 4. Experiments were performed 519 using T 3 or T 4 seeds.

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Leaf material was used to extract gDNA from transgenic lines 35S:KDR #1, 3, 8 and 522 9, created using the pFAST-G02 vector, as described above. The gDNA was used to 523 perform a TAIL-PCR reaction as described in Liu et al. (1995) with minor 524 modifications, using arbitrary degenerate (AD), T-DNA left border (LB) end primers 525 (Supplemental Table 5) and DreamTaq DNA polymerase (Thermo Fisher Scientific).

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The cycle settings used for the TAIL-PCR reactions were adjusted based on the 527 characteristics of the polymerase and primers used and listed in Supplemental Table   528 6. Purified fragments obtained in the second or third TAIL-PCR reactions were The procedure for cloning KDR CDS was as described before but with the use of the 534 primers listed in Supplemental Table 7   The plasmid DNA was then extracted using the QIAprep® Spin Miniprep kit (Qiagen).

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Yeast prey plasmid cDNA library of A. thaliana 553 The yeast prey plasmid cDNA library of A. thaliana was kindly provided by Prof. Dr.

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Guido van den Ackerveken (Utrecht University, The Netherlands) and created using  constructs cloned into the pDEST22 or pGADT7 were transformed into the strain 575 Y8800 using the LiAc (Sigma-Aldrich) method (Schiestl and Gietz, 1989). Yeast 576 transformed with the expression vectors pDEST32 and pGADT7 were plated on SC 577 medium lacking the selective AA leucine (Leu). The same was done for the colonies 578 transformed with the pDEST22 and pGBKT7, but in this case SC was used without 579 the selective AA Trp. 4-day-old single colonies growing at 30°C were isolated and the 580 insertion of the plasmids was confirmed with colony PCR using the primers listed in 581 Supplemental Table 8. Positive transformed colonies were resuspended in YEPD 582 containing 24% glycerol and stored at -80°C. To test for auto-activation of the bait 583 constructs, yeast strain Y8930 carrying the expression vectors pDEST32 or pGBKT7 584 were grown on a SC medium lacking His and adenine (Ade) in the following 585 combinations: -Leu -His; -Leu -His + 2 mM 3-AT; -Leu -His + 5 mM 3-AT and -Leu -586 Ade for pDEST32 constructs, in the case of colonies carrying the pGBKT7 the 587 medium was lacking Trp instead of Leu. Colonies expressing the proteins PIF4 or 588 PIF5 were able to activate the HIS3 and ADE2 reporter genes and for this reason 589 they were not used in the experiments in the bait conformation.

Y2H cDNA library screening and individual interactions 591
The Y2H library screening was performed using a mating-based approach described  Table 8 were used to perform the colony PCR.

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For in planta localization and colocalization experiments, cDNA deriving from A. 663 thaliana was used to amplify the CDSs of KDR, HFR1, PAR1 and PAR2, while AIF2, 664 AIF4, IBH1 and IBL1 were amplified from the respective cloned in pDEST22 665 (previously described) using the primers listed in the Supplemental Table 9, which 666 were designed in a way that the CDSs were in frame with a C-terminal tag and Growth data were analyzed by 2-way ANOVA followed by post-hoc Tukey test, while 718 qRT-PCR data were analyzed by Student's t-test or 1-way ANOVA followed by post-719 hoc Tukey test. All the analyses were done using GraphPad Prism.   MRS2-1 1 1 1 The strength of interaction is defined as weak when the yeast grew only on SC -Leu -Trp -His medium, as mild when growing up to SC -Leu -Trp -His + 2 mM 3-AT, as medium when yeast grew with the 3-AT increased to 5 mM and as strong when able to grow on SC -Leu -Trp -Ade.        The interaction of KDR with PAR1, PAR2, AIF2, AIF4, IBH1 and IBL1 was visualized as the reconstituted YFP signal in different nucleus compartments based on the type of interaction. No interaction was found between HFR1 and KDR. The autofluorescence of the chloroplasts is shown in red and the BiFC signal of Venus (YFP) in green. Images were taken 2 days after agroinfiltration. The scale bar represents 10 μm.  (hfr1-201, hfr1-5, par1, par2-1, aif2-1, aif2-2, aif4 and ibl1) grown in control light condition (R:FR = 2) or low R:FR (R:FR = 0.2) after 5 days of light treatment. Data represent mean ± SE, n = 38. Different letters indicate statistically significant differences (2-way ANOVA with post-hoc Tukey test, p < 0.05).   Relative expression of known PIF targets determined by qRT-PCR in shoots of wild type Col-0 and two independent homozygous transgenic lines overexpressing KDR, grown in control white light condition (R:FR = 2) for 4 days. Data represent mean ± SE, n = 4. Different letters indicate statistically significant differences (1-way ANOVA with post-hoc Tukey test, p < 0.05).

Supplemental Figure 4: No interaction found for KDR and HFR1 in Y2H protein-protein interaction assays.
(A) In the GAL4 Y2H assay, the GAL4 DNA-binding domain (BD) fused to HFR1 was coexpressed with the GAL4-activation domain (AD) fused to KDR. The mating of the yeast was confirmed through growth on non-selective medium (-LT). No interaction was found, as shown for lack of growth in all the different selective media (-LTH + 2 or 5 mM 3-AT and -LTA). For this experiment, the pDEST32 (bait) and pDEST22 (prey) were used. (B) The interaction between KDR and HFR1 was studied using KDR as bait and HFR1 as prey in the left picture. The same interaction was studied in the other conformation, as shown in the right picture. For this experiment, the vectors pGBKT7 (bait) and pGADT7 (prey) were used. No interaction was found also in this experiment. L: leucine; T: tryptophan; H: histidine; A: adenine; 3-AT: 3-amino-1, 2, 4-triazole. The insertion sites drawn with a triangle show the orientation of the T-DNA in the genome and with the numbers below is indicated the distance (bp) either from the start codon or from the stop codon relatively to the two closest genes. Genes are in scale.