Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness.

Seedling emergence in monocots depends mainly on mesocotyl elongation, requiring coordination between developmental signals and environmental stimuli. Strigolactones (SLs) and karrikins are butenolide compounds that regulate various developmental processes; both are able to negatively regulate rice (Oryza sativa) mesocotyl elongation in the dark. Here, we report that a karrikin signaling complex, DWARF14-LIKE (D14L)-DWARF3 (D3)-O. sativa SUPPRESSOR OF MAX2 1 (OsSMAX1) mediates the regulation of rice mesocotyl elongation in the dark. We demonstrate that D14L recognizes the karrikin signal and recruits the SCFD3 ubiquitin ligase for the ubiquitination and degradation of OsSMAX1, mirroring the SL-induced and D14- and D3-dependent ubiquitination and degradation of D53. Overexpression of OsSMAX1 promoted mesocotyl elongation in the dark, whereas knockout of OsSMAX1 suppressed the elongated-mesocotyl phenotypes of d14l and d3. OsSMAX1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs, regulating downstream gene expression. Moreover, we showed that the GR24 enantiomers GR245DS and GR24ent-5DS specifically inhibit mesocotyl elongation and regulate downstream gene expression in a D14- and D14L-dependent manner, respectively. Our work revealed that karrikin and SL signaling play parallel and additive roles in modulating downstream gene expression and negatively regulating mesocotyl elongation in the dark.

, cytokinins (Hu et al., 2014), Here, we report that mesocotyl elongation in the dark is regulated by the 145 D14L-D3-Oryza sativa SUPPRESSOR OF MAX2 1 (OsSMAX1) module in rice. deficiency in SL biosynthesis or signaling leads to the accumulation of D53 and 165 results in tiller bud outgrowth (Jiang et al., 2013;Zhou et al., 2013). As a 166 gain-of-function SL-insensitive mutant, the shoot-branching phenotype of d53 is 167 similar to that of the loss-of-function SL biosynthesis mutants d27, d17 and d10 and 168 the loss-of-function SL signaling mutants d14 and d3 (Jiang et al., 2013;Zhou et al., 169 2013). Compared with the WT, both the SL-deficient mutants (d27, d17 and d10) and 170 the SL-insensitive mutants (d14 and d3) have longer mesocotyls (Hu et al., 2010). To 171 investigate the role of D53 in the inhibition of mesocotyl elongation in the dark, we 172 measured the mesocotyl length of d53, which was as long as that of d14 ( Figure 1A 173 and 1B). ACT:D53m-GFP transgenic seedlings, which constitutively express d53 and 174 GFP fusion proteins, exhibited a shoot-branching phenotype similar to that of d53 175 (Supplemental Figure 1A and 1B) (Jiang et al., 2013). The mesocotyl length of the 176 ACT:D53m-GFP transgenic seedlings was also similar to that of d14 (Supplemental 177 Figure 1C and 1D). These results suggested that D53 accumulation in these seedlings 178 promotes mesocotyl elongation in the dark and confirmed that D14-and 179 D3-dependent D53 degradation is involved in the inhibition of rice mesocotyl 180 elongation in the dark. However, the d3 mesocotyl length was longer than that of 181 other SL mutants, including d14 and d53, strongly suggesting that dark-induced 182 mesocotyl elongation in rice is also regulated by an SL-independent pathway. D14L, conditions, mesocotyl elongation was not observed in d14, d14l,d3,d14 d14l,or WT 199 seedlings (Supplemental Figure 4). In the dark, the mesocotyl length of d14l was 200 similar to that of d14, which was longer than that of the WT but shorter than that of 201 d3 (Figure 1A and 1B). The d14l mesocotyl phenotype in this study is consistent with 202 the phenotypes reported previously (Gutjahr et al., 2015b;Kameoka and Kyozuka, 203 2015). However, unlike the D14L RNAi d14 seedlings in previous study (Kameoka 204 and Kyozuka, 2015) the mesocotyl length of the d14 d14l double mutant was as long 205 as that of d3 (Figure 1A and 1B). In addition, the mesocotyl of the d53 d14l double 206 mutant was longer than that of d14l and similar to that of d3 (Supplemental Figure   207 1E and 1F). These results suggested that the D14L-mediated pathway acts additively negatively regulates mesocotyl elongation (Xiong et al., 2017). Deficiency in either 213 D14 or D14L resulted in significant downregulation of GY1 expression ( Figure 1D). 214 The downregulation of OsTCP5 and GY1 expression in d14 d14l and d3 was similar 215 to that in d14 and d14l ( Figure 1C and 1D), indicating that the D14-and 216 D14L-mediated pathways potentially regulate common target genes to modulate 217 mesocotyl elongation in the dark. 218 Next, we compared the gene expression profiles of WT,d14,d14l,d3, and d14 219 d14l seedlings grown in the dark. More than two-thirds of the differentially expressed 220 genes (DEGs) between the WT and d3 overlapped with the DEGs between the WT 221 and d14 d14l. Interestingly, more than one-third of the DEGs between the WT and 222 d14 overlapped with the DEGs between the WT and d14l (Figure 1E and 1F). Gene 223 Ontology (GO) analysis of these DEGs revealed a significant enrichment of genes 224 associated with the responses to abiotic stress and light stimuli, which are likely 225 subject to regulation by both the D14-and D14L-mediated pathways ( Figure 1G). 226 Loss of function of either D14 or D14L resulted in a decrease in the expression of 227 D14L ( Figure 1H). The expression of D14L2 has been suggested to be dependent on 228 D14L (Gutjahr et al., 2015b). Indeed, disrupting either D14 or D14L function led to a 229 dramatic decrease in D14L2 expression ( Figure 1I). Moreover, the expression of 230 D14L3 also depended on the function of both D14 and D14L ( Figure 1J). Taken 231 together, these results suggested that, in terms of the regulation of rice mesocotyl 232 elongation in the dark, the D14L-and D14-mediated pathways act additively and in 233 parallel, and both depend on the function of D3.

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The D53-like/SMAXL protein family has nine members in rice (Jiang et al., 2013). In 236 the presence of SL, D14 recruits D3 to degrade D53 (Jiang et al., 2013;Zhou et al., 237 2013). We hypothesized that D14L may be similar to D14 with respect to the  Figure 7). We therefore refer to 248 LOC_Os08g15230 as OsSMAX1 hereafter. Pull-down assays confirmed that 249 OsSMAX1 interacts with D14L but not D14 ( Figure 2B). Moreover, HA-OsSMAX1 250 was coprecipitated by GFP-D14L but not by GFP-D14 when tagged proteins were 251 transiently expressed in rice protoplasts, further confirming the interaction between 252 OsSMAX1 and D14L ( Figure 2C). 253 We found that D14L interacts with OsSMAX1 but not with other 254 D53-like/SMAXL proteins in the yeast two-hybrid assay (Supplemental Figure 6). 255 To identify the OsSMAX1 domain responsible for its interaction with D14L, various 256 OsSMAX1 fragments were tested via yeast two-hybrid assays and pull-down assays.

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To detect the interaction between D14L and D3, we performed a pull-down 268 assay. Nus-D14L was pulled down by glutathione S-transferase (GST)-D3 but not by 269 GST, suggesting that D14L can interact with D3 ( Figure 2E). Both HA-D3 and and D3 in vivo, we used OsSMAX1 antibodies to measure OsSMAX1 protein levels 280 in the WT, d14, d14l, d3, and d14 d14l seedlings (Supplemental Figure 10).

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OsSMAX1 protein accumulated in d14l, d3, and d14 d14l but not in the WT or d14 282 ( Figure 3A). These results indicated that the regulation of OsSMAX1 protein stability 283 depends on the function of D14L and D3 but not on that of D14. By contrast, we 284 found that the accumulation of D53 in d14l was similar to that in the WT ( Figure   285 3B), suggesting that D14L has little effect on the abundance of D53.

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In d53 plant, the lack of the GKT motif prevents d53 from being subjected to 287 SL-induced degradation. When the GKT motif-mutated D53 (D53m) was 288 overexpressed in the WT, the shoot-branching phenotypes were similar to those of 289 d53 (Supplemental Figure 1A and 1B) (Jiang et al., 2013). Since the GKT motif 290 region of OsSMAX1 is highly similar to that of D53, we generated a mutated 291 OsSMAX1 (OsSMAX1m) by an in-frame deletion of the GKT motif at 744-747 292 (Gly-Lys-Thr-Ala) (Figure 3C), which is similar to that in D53 at 813-817 293 (Gly-Lys-Thr-Gly-Ile). Since d53 interacts with D14 as well as D53, yeast two-hybrid 294 assays ( Figure 3D) and pull-down assays ( Figure 3E) were conducted to verify the 295 interaction between OsSMAX1 and D14L. The results showed that OsSMAX1m 296 interacts with D14L as well as OsSMAX1.

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To further evaluate the stability of D53/SMAXL family proteins, we performed a  Figure 11A). To verify the viability of this assay, we first 300 tested the effects of rac-GR24 on the stability of D53 and D53m reporters that were 301 transiently expressed in WT, d3, d14, d14l, and d14 d14l protoplasts (Supplemental 302 Figure 11B). The FF/REN ratio of the D53 reporter in d14, d3, and d14 d14l treated 303 with 1 M rac-GR24 was higher than that in the WT and d14l. By contrast, there 304 were no obvious differences in the FF/REN ratio of the D53m reporter in response to 305 rac-GR24 treatment among the WT, d3, d14, d14l, and d14 d14l (Supplemental 306 Figure 11B). To evaluate the stability of OsSMAX1 and OsSMAX1m,

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StrigoQuant-like reporters of OsSMAX1 and OsSMAX1m were transiently expressed 308 in WT, d14, d14l, d3 and d14 d14l protoplasts. The stability of OsSMAX1, indicated 309 by the FF/REN ratio, was higher in d14l, d3, and d14 d14l than in the WT, while the 310 stability of OsSMAX1 in d14 was similar to that in the WT ( Figure 3F). However, 311 the stability of OsSMAX1m did not obviously differ in d14, d14l, d3, or d14 d14l 312 compared to that in the WT ( Figure 3F). These results suggested that the stability of 313 OsSMAX1 is regulated by D14L and D3 but not by D14 and that the mutation of the 314 GKT motif of OsSMAX1 enables OsSMAX1 to resist D14L-and D3-dependent 315 degradation.

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To test whether the elongation of the mesocotyls of d14l, d3, and d14 d14l 317 resulted from the accumulation of OsSMAX1 in these mutants, we constructed

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To determine the role of karrikins in the inhibition of rice mesocotyl elongation in the 342 dark, we added either KAR 1 or KAR 2 to the medium used for seedling growth in the  M GR24 ent-5DS but not with 10 M GR24 5DS ( Figure 4D). However, the abundance 361 of OsSMAX1m was not affected by treatment with either GR24 ent-5DS or GR24 5DS

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( Figure 4D). Moreover, the results showed that GR24 ent-5DS treatment could induce 363 the ubiquitination of OsSMAX1 but not of OsSMAX1m ( Figure 4E). Taken together, 364 these results indicated that different GR24 stereoisomers have distinct effects on the 365 induction of OsSMAX1 degradation and that the mutation of the GKT motif of 366 OsSMAX1 is resistant to GR24 ent-5DS induced ubiquitination and degradation.

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To further determine whether karrikins could enhance the interaction between 368 D14L and OsSMAX1 in a manner similar to that of SLs with respect to the interaction 369 between D14 and D53, we performed a yeast two-hybrid assay with different small 370 molecules. The results showed that the addition of 20 M rac-GR24 or GR24 5DS 371 could enhance the interaction between D14 and D53, but neither rac-GR24 and 372 GR24 5DS nor KAR 1 and GR24 ent-5DS could enhance the interaction between D14L and 373 OsSMAX1 (Supplemental Figure 13). The pull-down assay also revealed that 374 addition of 20 M rac-GR24, KAR 1, GR24 5DS , or GR24 ent-5DS has no obvious effect 375 on the interaction between D14L and OsSMAX1. Even when the concentration of 376 these chemicals was increased to 50 M, KAR 1 and GR24 ent-5DS still had no obvious 377 effect on the interaction between D14L and OsSMAX1 (Supplemental Figure 14). 378 These results indicated that there may be different recognition mechanisms between

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KLs and D14L and between SLs and D14.

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We observed that the plant height was reduced both in the OsSMAX1 and

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OsSMAX1m overexpression plants (Supplemental Figure 17G) and in the Ossmax1 463 plants ( Figure 6C). Similarly, the plant height of Ossmax1 d3, Ossmax1 d14, and 464 Ossmax1 d10 was also reduced compared to that of d3, d14, and d10, respectively OsSMAX1-downregulated gene LP2 was induced in response to both KAR 1 and 527 rac-GR24 in WT and d17 and induced in response to KAR 1 in d14 ( Figure 8F).

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Expression of the OsSMAX1-upregulated genes LOC_Os04g15840 and 529 LOC_Os06g32355 were not induced in response to KAR 1 or rac-GR24 in the WT 530 ( Figure 8G and Figure 8H). Deficiency of the SL or karrikin pathway led to 531 increased expression of these two genes, such that their expression was inhibited in 532 response to both KAR 1 and rac-GR24 in the d17 mutants ( Figure 8G and  Figure 9C and 9D). Taken together, these results showed that D14L responds 558 specifically to GR24 ent-5DS in the regulation of mesocotyl development.

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All the aerial parts of dark-grown 7-day-old seedlings were sampled for total RNA The RNA-Seq information has been deposited in the BioProject ID PRJNA553596.

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Sequence data from this article can be found in the GenBank/EMBL libraries under 1040 the following accession numbers:  Table 1 Primers used for vector construction.

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Supplemental Table 2 Primers used for RT-qPCR.     Step/Event Figure 10. The KL signaling pathway mirrors the SL signaling pathway. A working model of karrikin signaling mediated by the D14L-D3-OsSMAX1 complex. Karrikin signaling mirrors the SL signaling complex in rice. In the absence of ligands, both OsSMAX1 and D53 are able to interact with TPR transcriptional corepressors and repress the expression of downstream genes. In the presence of ligands, D14L and D14 perceive specific ligands (such as GR24 ent-5DS and GR24 5DS ) and recruit the SCF D3 complex to ubiquitinate OsSMAX1 and D53 for degradation by the 26S proteasome. In turn, this releases OsSMAX1-and D53-mediated repression of the activity of their interacting transcription factors, thus regulating the expression of downstream target genes. SL signals specifically regulate shoot branching, and KL signals might specifically regulate root colonization by AM fungi. The specificity of the output of SL signals and KL signals is likely determined by transcription factors that interact specifically with D53 or OsSMAX1. It is possible that some common transcription factors both interact with D53 and are responsible for the expression of a subset of common genes, which could respond to both KL signaling and SL signaling. During skotomorphogenesis, KL and SL signals act through the D14L-D3-OsSMAX1 complex and D14-D3-D53, respectively, and act in parallel and/or additively to trigger the expression of their specific or commonly regulated downstream genes, which leads to the inhibition of rice mesocotyl elongation.

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