FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) accumulates in homo- and heterodimeric complexes in dynamic and inducible nuclear condensates associated with speckle components

Some nuclear proteins undergo condensation, but the functional importance remains often unclear. The basic helix-loop-helix (bHLH) FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) integrates internal and external signals to control iron acquisition and growth. The previously described C-terminal residues Ser271/272 allow FIT to form active complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into nuclear condensates that we termed FIT nuclear bodies (NBs). FIT NB characteristics were examined using a standardized FIT NB analysis procedure coupled with different types of quantitative and qualitative microscopy-based approaches. FIT condensates were reversible and likely formed by liquid-liquid phase separation. FIT accumulated preferentially in FIT NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. The NB-inducing light conditions were linked with active FIT and elevated FIT target gene expression in roots. Hence, we conclude that inducible, highly dynamic FIT condensates form preferentially when transcription factor complexes are active. Inducible FIT nuclear condensates may affect nuclear mobility and integrate environmental and Fe nutrition signals. Highlights FIT undergoes light-induced, reversible condensation and localizes to nuclear bodies (NBs), likely via liquid-liquid phase separation Functionally relevant Ser271/272 defines an intrinsically disordered region and influences NB formation dynamics NBs are preferential sites for FIT dimerization with FIT and bHLH039, dependent on Ser271/272 FIT NBs colocalize with NB markers related to splicing and light signaling Light conditions inducing NBs are linked with active FIT, in agreement with elevated FIT target gene expression in roots

• Functionally relevant Ser271/272 defines an intrinsically disordered region and 23 influences NB formation dynamics 24 • NBs are preferential sites for FIT dimerization with FIT and bHLH039, dependent 25 on Ser271/272 26 • FIT NBs colocalize with NB markers related to splicing and light signaling 27 • Light conditions inducing NBs are linked with active FIT, in agreement with 28 elevated FIT target gene expression in roots 29 Fluorescence lifetime was decreased for the pair FIT-GFP and bHLH039-mCherry 298 at t=5 within NBs compared to all other measured areas ( Figure 4D). In contrast to that, 299 the fluorescence lifetime decreased for the pair FITmSS271AA-GFP and bHLH039-300 mCherry at t=15 was not different between NBs and NP ( Figure 4E). This indicated that 301 heterodimeric complexes accumulated preferentially in FIT NBs. 302 In summary, heterodimerization of FIT with bHLH039 was spatially concentrated in 303 NBs versus the remaining nuclear space and was less prominent for FITmSS271AA. 304 Hence, the capacity of FIT to form an active TF complex was coupled with its presence in 305 NBs. The occurrence of FIT homo-and heterodimerization preferentially in NBs suggests 306 that FIT protein interaction may drive condensation. We therefore concluded that FIT NBs 307 may be sites with active TF complexes for iron deficiency response regulation. 308

FIT NBs colocalize with speckle components 309
Numerous NB types are known, and they are associated with particular proteins 310 that are indicative of the NB type. To further understand the identity, dynamics, and 311 function of FIT NBs, we co-expressed FIT-GFP with seven different NB markers from The 312 Plant Nuclear Marker collection (NASC) and observed NB formation and protein 313 colocalization before (t=0) and after FIT NB formation (t=5). In cases where we detected 314 a colocalization with FIT-GFP, we analyzed the localization of NB markers also in the 315 single expression at t=0 and at t=5 after the 488 nm excitation, to detect potentially 316 different patterns in single and co-expression. 317 All seven NB markers were expressed together with FIT-GFP, and according to the 318 resulting extent of colocalization we subdivided them into three different types. The first 319 type (type I) did not colocalize with FIT-GFP neither at t=0 nor at t=5. This was the case 320 for the Cajal body markers coilin-mRFP and U2 snRNP-specific protein U2B"-mRFP 321 (Supplemental Figure S3; Lorković et al., 2004;Collier et al., 2006). Coilin-mRFP 322 localized into a NB within and around the nucleolus (Supplemental Figure S3A). The 323 NBs of U2B"-mRFP were also close to the nucleolus (Supplemental Figure S3B). 324 Hence, FIT-GFP was not associated with Cajal bodies. 325 The second type (type II) of NB markers were partially colocalized with  This included the speckle components ARGININE/SERINE-RICH45-mRFP (SR45) and 327 the serine/arginine-rich matrix protein SRm102-mRFP. SR45 is involved in splicing and 328 alternative splicing and is part of the spliceosome in speckles (Ali et al., 2003), and was 329 13 recently found to be involved in splicing of iron homeostasis genes (Fanara et al., 2022). 330 SRm102 is a speckle component (Kim et al., 2016). SR45-mRFP localized barely in the 331 NP but inside few and very large NBs that remained constant at t=0 and t=5. FIT-GFP did 332 not colocalize in those NBs at t=0, however, it colocalized with the large SR45-mRFP NBs 333 at t=5 (Figure 5A). FIT-GFP also localized in typical FIT NBs in the residual NP at t=5 334 ( Figure 5A). SRm102-mRFP showed low expression in the NP and stronger expression 335 in a few NBs that also remained constant at t=0 and t=5. FIT-GFP colocalized with 336 SRm102-mRFP in only few instances at t=5, but not t=0, while most FIT NBs did not 337 colocalize with SRm102-mRFP NBs ( Figure 5B). Both SR45-mRFP and SRm102-mRFP 338 had the same localization pattern at t=0 and t=5, irrespective of FIT-GFP co-expression 339 or 488 nm excitation (Supplemental Figure S4). These type II NB markers seemed to 340 recruit FIT-GFP into NBs after 488 nm excitation that were present (pre-existing) before 341 FIT-GFP NB formation, while FIT-GFP localized additionally in separate FIT NBs. Hence, 342 FIT became associated with splicing components and speckles upon the light trigger. 343 A third type (type III) of three NBs markers, namely UAP56H2-mRFP, P15H1-344 mRFP, and PININ-mRFP, were fully colocalized with FIT-GFP. Until now, these NB 345 marker proteins are not well described in plants. UAP56H2 is a RNA helicase, which is 346 involved in mRNA export (Kammel et al., 2013). P15H1 was found as a putative 347 Arabidopsis orthologue of an exon junction complex component in humans (Pendle et al., 348 2005), while PININ has a redundant role to its paralogue apoptotic chromatin 349 condensation inducer in the nucleus (ACINUS) in alternative splicing (Bi et al., 2021). 350 UAP56H2-mRFP and P15H1-mRFP did not localize in NBs and were not responsive to 351 the 488 nm excitation when expressed alone or together with FIT-GFP at t=0 (Figure 6, 352 A and B and Supplemental Figure S4, C and D). When co-expressed with FIT-GFP and 353 following the 488 nm excitation, at t=5, the two NB markers adopted the FIT NB pattern 354 and colocalized with FIT-GFP in FIT NBs (Figure 6, A and B). PININ-mRFP was also 355 uniformly distributed in the nucleus at t=0 like FIT-GFP and fully colocalized with FIT NBs 356 at t=5 (Figure 6C). But curiously, PININ-mRFP showed a very different localization in the 357 single expression. Predominately, it localized to a very large NB besides several small 358 NBs with no expression in the NP at t=0 and at t=5 (Supplemental Figure S4E). Thus,359 FIT-GFP recruited these type III NB marker and speckle proteins fully into FIT NBs. Since 360 type III NB markers are also potentially involved in splicing and mRNA export from the 361 nucleus, these same functions may be relevant in FIT NBs. 362 14 Taken together, the colocalization studies underlined the dynamic behavior of 363 inducible FIT NB formation. FIT NBs had a speckle function, in which on the one hand FIT 364 was recruited itself into pre-existing splicing-related NBs (SR45-mRFP and SRm102-365   mRFP, type II), while on the other hand it also recruited speckle-localized proteins into  366 FIT NBs (UAP56H2-mRFP, P15H1-mRFP, and PININ-mRFP, type III). 367

PB components influence FIT NB localization and formation 368
PBs are plant-specific condensates which harbor various light signaling 369 components (Kircher et al., 2002;Bauer et al., 2004). Among them are the bHLH TFs of 370 the PIF family. As key regulators of photomorphogenesis, they integrate light signals in 371 various developmental and physiological response pathways (Leivar and Monte, 2014;372 Pham et al., 2018). Indeed, PIF4 may control iron responses in Arabidopsis based on 373 computational analysis of iron deficiency response gene expression networks 374 (Brumbarova and Ivanov, 2019) and both PIF proteins intersect with blue light signaling 375 (Ni et al., 1998;Oh et al., 2004;Pedmale et al., 2016). We tested in the same manner as 376 described above for NB markers, whether FIT NBs coincide with two of the described PB 377 markers, PIF3-mCherry and PIF4-mCherry (Van Buskirk et al., 2014;Qiu et al., 2019Qiu et al., , 378 2021. 379 We detected distinct localization patterns for PIF3-mCherry and PIF4-mCherry. At 380 t=0, PIF3-mCherry was predominantly localized in a single large PB ( Figure 7A). In 381 general, localization of single expressed PIF3-mCherry remained unchanged at t=0 and 382 t=15 (Supplemental Figure S5A). Upon co-expression, FIT-GFP was initially not present 383 in PIF3-mCherry PB at t=0. After 488 nm excitation and at t=5, FIT NBs were still not 384 visible. Instead, FIT-GFP accumulated and finally colocalized with the large PIF3-mCherry 385 PB at t=15, while the typical FIT NBs did not appear ( Figure 7A). 386 PIF4-mCherry localized in two different patterns, and both differed substantially 387 from that of PIF3-mCherry. In the one pattern at t=0, PIF4-mCherry was not localized to 388 any PBs, but instead was uniformly distributed in the NP as was the case for FIT-GFP. 389 Such a pattern was also seen at t=15, and then neither PIF4-mCherry nor FIT-GFP were 390 localized in any PBs/NBs ( Figure 7B). In the other pattern, PIF4-mCherry and FIT-GFP 391 localized in multiple PBs at t=0 and t=15, whereas none of them corresponded 392 morphologically to the typical FIT NBs ( Figure 7C). The same two localization patterns 393 15 were also found for PIF4-mCherry in the single expression, whereby 488 nm excitation 394 did not alter PIF4-mCherry localization (Supplemental Figure S5, B and C). 395 Hence, FIT was able to localize to PBs when co-expressed with PIF3 and PIF4, 396 raising the possibility that FIT is a key regulator to cross-connect iron acquisition regulation 397 and light signaling pathways. 398

Blue light has a promoting effect on iron acquisition responses downstream of FIT 399
Previous studies have shown that iron uptake by FIT is diurnally and circadian 400 clock-regulated (Vert et al., 2003;Santi and Schmidt, 2009;Salomé et al., 2013). Knowing 401 that FIT NB formation was light-dependent as well as promoting the interaction of the FIT-402 bHLH039 complex, and that FIT NBs were colocalizing with light signaling and PB 403 components, we reasoned that plants exposed to the FIT NB-forming light cues may show 404 differential FIT-dependent iron uptake responses with respect to control plants exposed 405 to the regular light conditions. To test this, we grew wild-type Arabidopsis plants for 5 d 406 under iron sufficient and iron deficient conditions under white light and exposed the plants 407 additionally for 1 d to blue light. As a control for light quality, we also included exposure to 408 red and far-red light and darkness besides the regular white light. We measured molecular 409 iron uptake responses known to be under the control of FIT-bHLH039 in roots (Gratz et 410 al., 2019). 411 Iron reductase activity is increased in roots when FIT and bHLH039 are activated 412 in response to iron deficiency in our growth system (Gratz et al., 2019). Iron reductase 413 activity was higher under iron deficient conditions compared to iron sufficient conditions 414 under all white light controls, as expected ( Figure 8A-D). Interestingly, when seedlings 415 were exposed to blue light for a day, they induced iron reductase activity in the iron 416 deficient versus iron sufficient condition more than in the white light control ( Figure 8A). 417 On the other hand, exposure to red light did not change the iron reductase activity 418 compared to the white light control (Figure 8B), while exposure to far-red light or darkness 419 abolished the induction (Figure 8, C and D). Hence, only exposure to blue light had an 420 extra promoting effect on iron uptake in the seedlings compared with the other tested light 421 qualities. 422 Increased iron acquisition under iron deficiency and enhanced iron reductase 423 activity require FERRIC REDUCTASE-OXIDASE2 (FRO2) protein (Yi and Guerinot, 1996;424 Robinson et al., 1999), and FRO2 to be induced, along with IRON REGULATED 425 16 TRANSPORTER1 (IRT1; Eide et al., 1996;Korshunova et al., 1999). FRO2 and IRT1 are 426 target genes of FIT-bHLH039 in our growth system (Gratz et al., 2019(Gratz et al., , 2020. As a readout 427 of the activity of the FIT-bHLH039 complex, we examined gene regulation of FRO2 and 428 IRT1 as well as FIT and BHLH039 under the same light conditions as described above. 429 Previously, it was shown that the expression of IRT1 and To sum up, the FIT-bHLH039-dependent target gene expression is similarly light 448 quality-dependent as the FIT-bHLH039-dependent iron reductase activity. Hence, very 449 interestingly, blue light cues which lead to FIT NB formation, promote FIT-bHLH039-450 dependent iron uptake responses, fitting with the above finding that FIT NBs are linked 451 with active TF complexes in condensates. 452

Discussion 453
In this study, we uncovered a previously unknown phenomenon, the blue light-454 induced and reversible accumulation of FIT condensates in FIT NBs. LLPS was most 455 likely the underlying mechanism for this highly dynamic process. FIT NBs were enriched 456 in active FIT TF complexes that are required for iron deficiency gene regulation. FIT 457 associated with speckles and PBs in a highly dynamic fashion, indicating a function in 458 transcriptional and post-transcriptional control. Blue light exposure resulted in enhanced 459 molecular FIT-bHLH039-dependent iron acquisition responses. Based on these data, we 460 propose that FIT NBs are dynamic microenvironments with active FIT TF complexes that 461 possibly are hubs to enhance and cross-connect transcriptional iron deficiency gene 462 expression with post-transcriptional regulation and light signaling. 463 A standardized procedure for FIT NB induction was crucial to delineate the 464 characteristics of FIT NBs in reliable manner 465 A major aim of this study was to characterize the nature and potential function of 466 light-induced FIT NBs. To be able to apply the quantitative microscopy-based techniques, 467 we needed to control the appearance of NBs in reliable manner. This was clearly a 468 limitation for inspection of root epidermis cells of the root differentiation zone in iron-469 deficient plants in which FIT-controlled iron uptake takes place. Not every root epidermis 470 cell showed NBs and only few FIT NBs were detectable after a delay of 40 min to 120 min. 471 Since condensation depleted FIT protein in the nucleoplasm, the remaining low FIT 472 protein concentration can be the reason why FIT NBs remained few in number in the 473 Arabidopsis root cells. The N. benthamiana protein expression system did not present 474 these limitations and high-quality measurement data were obtained for all experimental 475 series. Furthermore, this expression system is a well-established and widely utilized 476 system in plant biology (Martin et al., 2009;Bleckmann et al., 2010;Leonelli et al., 2016;477 Burkart et al., 2022). The developed standardized assay generated reliable and accurate 478 data for statistical analysis and quantification to conclude about FIT NB characteristics. 479 Condensation likely explains the reduced mobility of FIT-GFP versus 480 FITmSS271AA-GFP seen in a previous study (Gratz et al., 2019). In liquid state, 481 condensates are still more mobile than in the solid one. The condensate formation was 482 reversible, speaking in favor of a liquid state of the condensates. According to FRAP data, 483 FIT NBs maintained a dynamic exchange of FIT protein with the surrounding NP. FIT NBs 484 were also mostly of circular shape. Circular condensates appear as droplets, in contrast 485 to solid-like condensates that are irregularly shaped (Shin et al., 2017). These 486 characteristics speak in favor of liquid-like features, suggesting that LLPS underlies FIT 487 NB formation. A similar situation was described for CRY2 PBs, which were also reversible 488 and of circular shape with mobile protein inside PBs (Wang et al., 2021). bHLH039 was 489 18 found accumulated in cytoplasmic foci at the cell periphery (Trofimov et al., 2019). In such 490 foci, bHLH039 was immobile, and we suspect it was in a non-functional state in the 491 absence of FIT. This underlines the understanding that liquid condensates such as FIT 492 NBs are dynamic microenvironments, whereas immobile condensates point rather 493 towards a solid and pathological state (Shin et al., 2017). 494 In conclusion, the properties of liquid condensation along with the findings that it 495 occurred irrespective of the fluorescence protein tag preferentially with wild-type FIT, but 496 with different dynamics for the mutant FITmSS271AA and not at all for ZAT12, allowed us 497 to coin the term of 'FIT NBs'. 498

IDR Ser271/272 was crucial for interaction and NB formation of FIT 499
FIT NBs were hotspots for FIT interaction, allowing to assume that they are 500 integrated in the iron deficiency response as interaction hubs. FIT formed homodimers 501 and heterodimers with bHLH039 preferentially in NBs compared with the NP. These 502 abilities distinguished wild-type FIT and mutant FITmSS271AA. According to these 503 findings FITmSS271AA was less successful in interacting within NBs, indicating that wild-504 type FIT is a multivalent protein and IDR Ser271/272 is important for that. bHLH proteins 505 interact with other proteins via the helix-loop-helix interface, which may certainly also be 506 the case for FIT. Our study supports previous reports that FIT protein interaction via its C-507 terminus is relevant (Lingam et al., 2011;Le et al., 2016;Gratz et al., 2019). The property 508 of being able to interact via the HLH and via the C-terminal domain allows FIT to be 509 multivalent. It could not be distinguished whether FIT homodimers were a prerequisite for 510 the localization of bHLH039 in NBs or whether FIT-bHLH039 complexes also initiated NBs 511 on their own. 512 The predicted C-terminal FIT IDR Ser271/272 was relevant for NB formation capacity. FIT-GFP accumulated not only in FIT NBs but also in the pre-existing NBs with type II NB 539 markers (SR45 and SRm102) after the FIT NB induction procedure. In this respect, type II 540 markers were similar to PIF3 and PIF4. FIT-GFP was recruited to pre-existing PBs and 541 again only after the light trigger. Interestingly, typical FIT NB formation did not occur in the 542 presence of PB markers, indicating that they must have had a strong effect on recruiting 543 FIT. Overall, the dynamics of FIT colocalization with type II NB and PB markers suggest 544 that these condensates dictated FIT condensation in their own pre-existing NBs/PBs. This 545 recruiting process could be navigated via protein-protein interaction since this is the 546 driving force of condensation (Kaiserli et al., 2015;Emenecker et al., 2020). 547 Speaking in favor of a de novo FIT NB formation is the localization with type III NB 548 markers. The three fully colocalizing type III NB markers (UAP56H2, P15H1 and PININ) 549 accumulated only in FIT NBs upon co-expression with FIT and mostly not on their own. 550 The same was true for bHLH039, that joined FIT in FIT NBs, showing that FIT not only 551 facilitated bHLH039 nuclear localization (Trofimov et al., 2019) but also condensation. 552 Interestingly, FIT was able to change PININ nuclear localization. In single expression, 553 20 PININ was localized to a major large NB, but in colocalization with FIT it joined the typical 554 FIT NBs. This suggests that FIT dictates bHLH039 and type III NB markers and highlights 555 that FIT is also able to set the tone for NB formation. Hence, FIT can recruit other proteins 556 into NBs, and it is possible that FIT forms its own NBs. Protein-protein interaction could 557 underly this recruitment, as evident for bHLH039 (Kaiserli et al., 2015;Emenecker et al., 558 2020). Ultimately, as a high diversification of condensates exists, a combination of newly 559 formed NBs and localization to pre-existing NBs cannot be ruled out. Given the variety of 560 proteins localizing in condensates, effort in isolating FIT NBs and identification of proteins 561 within FIT NBs is necessary to further uncover the driving forces of FIT NB formation. 562

FIT NBs might have a transcriptional and post-transcriptional function 563
Since the type II and III markers are splicing components, the colocalization studies 564 suggest that FIT NBs are speckles. On the one hand, the speckle nature coincides well 565 with the dynamic nature of FIT NBs. Like FIT NBs, speckles are highly dynamic. They are 566 forming around transcriptionally active sites in the interchromatin regions recruiting 567 several protein functions like mRNA synthesis, maturation, splicing and export (Reddy et 568 al., 2012;Galganski et al., 2017). The type II speckle component SR45, for instance, was 569 shown to be a highly mobile protein in speckles and required phosphorylation for proper 570 speckle localization (Ali et al., 2003;Reddy et al., 2012). These processes fit well to the 571 described FIT NB attributes. On the other hand, speckle components are also linked with 572 epigenetic mechanisms (Mikulski et al., 2022). The characterization of FIT NBs as 573 speckles is interesting because regulation of splicing and epigenetic regulation is 574 associated with iron deficiency gene expression. Genes were spliced incorrectly in a sr45-575 1 null mutant Arabidopsis line, and gene expression of FIT and FIT target genes was 576 increased in sr45-1 seedlings, showing that an interplay between SR45 and the iron 577 uptake machinery exists (Fanara et al., 2022). Alternative splicing was detected for FIT 578 targets and the BHLH subgroup Ib genes in iron-deficient versus iron-sufficient conditions 579 (Li et al., 2013). Hence, FIT NBs may regulate iron uptake gene expression at 580 posttranscriptional level. Notably, PININ (type III), together with ACINUS, were shown to 581 stabilize SR45 (type II) in plants (Bi et al., 2021). Further, UAP56H2, P15H1, and PININ 582 (type III) are connected to SR45 and SRm102 (type II) in mammalian cells as all being 583 part of the exon junction complex and interacting with each other (Lin et al., 2004;Pendle 584 et al., 2005). This is an interesting parallel, as it suggests that type II and type III marker 585 21 localization is conserved across kingdoms, underlying the ancient nature of condensates. 586 Indeed, SR45 and PININ located to a very large NB in non-induced cells. This opens the 587 possibility that the two proteins might localize to the same speckle, as also might FIT. 588 Taken together, the observations confirm the high diversification and complexity of FIT 589 NBs and speckles (Lorković et al., 2008) and it is tempting to speculate that FIT might 590 regulate splicing and alternative splicing of its target genes by recruiting speckle 591 components. 592 The interesting finding that blue light exposure not only caused the formation of FIT 607 NBs, that we could link with active TF complexes, but also promoted FIT-bHLH039-608 dependent iron mobilization responses can be interpreted as follows: FIT NBs may indeed 609 serve the rapid rearrangement of TFs to enhance target gene expression and subsequent 610 physiological responses. Changes of the light spectrum over the day could serve as a cue 611 for a high photosynthetic period, such a high blue light component, and induce stronger 612 iron uptake. Due to photosynthesis, chloroplasts and photosynthetically active leaves may 613 serve as a major sink for iron. Under far-red light, which is as shade signal, and in 614 darkness, photosynthetic activity is lowered. In turn, lower amounts of iron may be 615 acquired. Regulation of FIT protein abundance occurs rather on the posttranslational level 616 than on transcriptional (Lingam et al., 2011;Meiser et al., 2011;Gratz et al., 2019Gratz et al., , 2020. 617 On the other hand, FIT is itself a target of FIT-bHLH039 (Wang et al. 2007). The fact that 618 22 FIT gene expression is differently regulated from IRT1 and FRO2 depending on light 619 quality is intriguing. Perhaps FIT protein is readily available during the day. The formation 620 and dissociation of NBs could be a fast way to adjust iron uptake and specifically uncouple 621 IRT1 and FRO2 from FIT regulation and therefore adapt even better to environmental 622 changes. Reversibility of FIT condensation might rapidly switch off the process, possibly 623 by phosphorylation triggers leading to inactivation of FIT (Gratz et al., 2020). Reversibility 624 of condensates was shown in the context of light, temperature, or stresses, once the 625 stimulus was removed (Jung et al., 2020;Wang et al., 2021;Zhu et al., 2021;Chen et al., 626 2022;Wang et al., 2022). This supports the hypothesis that plant condensates are hubs 627 for fast adaptation to external cues. 628 As we focused to characterize the phenomenon, the physiological integration and 629 regulation of the induction of FIT NB formation can be subject of future studies. The rapid 630 speed by which FIT NB appeared within 5 min in N. benthamiana leaf cells speaks in favor 631 of protein rearrangement rather than protein synthesis. The long duration of FIT NB 632 formation after blue light induction in Arabidopsis roots suggests that signal transduction 633 was more complex and possibly involved intracellular or even cell-to-cell and long-634 distance leaf-to-root signaling. In how far a long-distance signal or a signaling cascade 635 triggered by light is involved in FIT NB formation in roots remains to be investigated, but 636 CRY1/CRY2 and HY5 are promising candidates for further studies, as their involvement 637 in iron deficiency response regulation has been highlighted (Gao et al., 2021;Guo et al., 638 2021;Mankotia et al., 2023). Since Arabidopsis germinates in the light, it is well 639 conceivable that the FIT NB-forming property is part of a seedling establishment 640 procedure during early phases when roots can be exposed in the light. In order to undergo 641 phase separation, a certain protein concentration must be reached (Bracha et al., 2018). 642 Since FIT protein is subject of proteasomal turnover in roots, FIT NB formation may 643 depend on FIT protein interaction partners in roots that need to be activated (Lingam et 644 al., 2011;Meiser et al., 2011). 645 In summary, FIT localizes to dynamic and reversible NBs. upstream of the CDS was cloned into pGGA000 entry vector using pFIT GG fw and pFIT 660 GG rv primers with respective overhang sequences (Supplemental Table S1). FIT CDS 661 (954 bp, without stop codon) was cloned into pGGC000 entry vector using cFIT GG fw 662 and cFIT GG rv primers with respective overhang sequences (Supplemental Table S1). Root iron reductase activity assay 690 6-d-old seedlings were grown as described in the text. Root iron reductase activity 691 was determined as described in Le et al. (2016). Plants were washed with 0.1 M 692 Ca(NO3)2 · 4H2O and subsequently incubated for 1 h in darkness at room temperature in 693 the iron reductase solution (300 µM Ferrozine, 100 µM FeNaEDTA). The absorbance of 694 the ferrozine-Fe 2+ complex was measured at 562 nm and used to calculate the root iron 695 reductase activity normalized to root fresh weight. 696

Gene expression analysis by RT-qPCR 697
6-d-old seedlings were grown as described in the text. The reverse transcription-698 real time-quantitative polymerase chain reaction (RT-qPCR) procedure and analysis is 699 described in Abdallah and Bauer (2016). Briefly, plant material was harvested, using three 700 biological replicates, and shock frozen. RNA was extracted (peqGOLD Plant RNA Kit, 701 PEQLAB). Subsequently, an amount of 0.5 µg of total RNA was used for cDNA synthesis 702 (Thermo Scientific). Diluted cDNA, SYBR green master mix (Thermo Scientific) and 703 respective primers (Supplemental Table S1) were pipetted in a 96 well plate and qPCR 704 was performed with two technical duplicates with the CFX96 Touch TM Real-Time PCR 705 Detection System (Bio-Rad). qPCR data were analyzed, and quantification was obtained 706 according to the mass standard curve analysis procedure. Normalization was performed 707 using the reference gene ELONGATION FACTOR1Α (EFc). Statistical analysis was 708 performed with absolute normalized gene expression levels. 709

Microscopy of Arabidopsis thaliana seedlings 710
Protein localization studies in roots of 5-d-old seedlings of the Arabidopsis thaliana 711 line FITpro:FIT-GFP and 35Spro:FIT-GFP were performed with the widefield microscope 712 ELYRA PS (Zeiss) equipped with a EMCCD camera. To induce FIT NB formation, whole 713 seedlings were exposed to 488 nm laser light for several minutes. GFP was excited with 714 a 488 nm laser and detected with a BP 495-575 + LP 750 beam splitter. Images were 715 acquired with the C-Apochromat 63x/1.2 W Korr M27 (Zeiss) objective, pixel dwell time of 716 26 1.6 µs and frame size of 512x512. Pictures were processed with the manufacturer's 717 software ZEN lite (Zeiss). 718

Generation of fluorescent constructs 719
All constructs used in this study are listed in Supplemental Table S2. Generation 720 of fluorescent translational C-terminal fusion of PIF3 and PIF4 with mCherry was 721 performed with Gateway Cloning. CDS of PIF3 was amplified with the PIF3 GW fw and 722 PIF3 GW rv primers (Supplemental Table S1), and CDS of PIF4 was amplified with the 723 PIF4 GW fw and PIF4 GW rv primers (Supplemental Table S1

Confocal microscopy 745
For localization studies a confocal laser scanning microscope LSM780 (Zeiss) was 746 used. Imaging was controlled by the ZEN 2.3 SP1 FP3 (Black) (Zeiss) software. GFP was 747 excited with a 488 nm laser and detected in the range of 491-553 nm. mCherry and mRFP 748 were excited with a 561 nm laser and detected in the range of 562-626 nm. Fluorophore 749 crosstalk was minimized by splitting of the excitation tracks and reduction of emission 750 spectrum overlap. Images were acquired with the C-Apochromat 40x/1.20 W Korr M27 751 (Zeiss) objective, zoom factor of 8, pinhole set to 1,00 AU, pixel dwell time of 1.27 µs and 752 frame size of 1.024x1.024. Z-stacks for quantification were taken with the same settings, 753 except with pixel dwell time of 0.79 µs and frame size of 512x512. Pictures were 754 processed with the manufacturer's software ZEN lite (Zeiss). 755

Standardized FIT NB analysis procedure 756
Following Nicotiana benthamiana leaf infiltration with Rhizobium radiobacter, FIT-757 GFP protein expression was induced after 2-3 d by β-estradiol, as described above. 16 h 758 later, a leaf disc was excised and FIT-GFP fluorescence signals were recorded (t=0). The 759 leaf disc was excited with 488 nm laser light for 1 min. 5 min later, FIT-GFP accumulation 760 in FIT NBs was observed (t= 5 min). See Supplemental Figure S1. This procedure was 761 modified by using different time points for NB analysis and different constructs 762 (Supplemental Table S2) and co-expression as indicated in the text. Imaging was 763 performed at the respective wavelengths for detection of GFP and mRFP/mCherry. 764

Anisotropy (homo-FRET) measurements 782
Anisotropy measurements (Stahl et al., 2013;Weidtkamp-Peters et al., 2022) were 783 performed at the confocal laser scanning microscope LSM780 (Zeiss) equipped with a 784 polarization beam splitter, bandpass filter (520/35), and a single-photon counting device 785 HydraHarp (PicoQuant) with avalanche photo diodes (τ-SPADs). Emission was detected 786 in parallel and perpendicular orientation. Rhodamine 110 was used to determine the G 787 factor to correct for the differential parallel and perpendicular detector sensitivity. 788 Calibration of the system was performed for every experiment and measurements were 789 conducted in darkness. Free GFP and GFP-GFP were used as references for mono-and 790 dimerization, respectively. GFP was excited with a linearly polarized pulsed (32 MHz Counter' -'3D object counter' was selected. Threshold for the intensity limit (areas below 835 that limit were not considered for quantification) was set manually for every z-stack. 836 Calculated values were further processed in Excel (Microsoft Corporation). Only size 837 between 0,01-15 µm³ was considered. 838

Protein domain prediction 839
IDRs in FIT/FITmSS271AA were predicted with the tool PONDR-VLXT 840 (www.pondr.com, Molecular Kinetics, Inc.). According to the sequence of the protein, a 841 PONDR score was determined for each amino acid. A score above 0.5 indicates intrinsic 842 30 disorder. The bHLH domain of FIT was predicted with InterPro (www.ebi.ac.uk/interpro, 843 EMBL-EBI). 844

Statistical analysis 845
Line and bar diagrams represent the mean and standard deviation. Box plots show 846 25-75 percentile with min-max whiskers, mean as small square and median as line. 847 Graphs and statistical analysis were created and performed with OriginPro (OriginLab 848 Corporation). Data was tested for normal distribution with the Shapiro-Wilk test. Statistical 849 significance of data with normal distribution was tested by one-way Anova with Tukey 850 post-hoc test. Statistical significance of data with non-normal distribution was tested by 851 Mann-Whitney test. Different letters indicate statistically significant differences (P < 0.05). 852 Illustrations were created with BioRender.com. 853

Accession numbers 854
Sequence data from this article can be found in the EMBL/GenBank data    Table S1. List of primers used in this study. 892 Supplemental Table S2. List of vectors used in this study. 893

Supplemental Movie S1. Light induction triggers the formation of NBs with FIT and 894
FITmSS271AA with different dynamics. (Supports Figure 1 and 2   A, Confocal images of nuclear localization of FITmSS271AA-GFP at t=0 and t=15 min.
FITmSS271AA-GFP accumulated in NBs, but NB formation required a longer time compared to FIT-GFP.
See also Supplemental Movie S1    Confocal images showing localization of FIT-GFP and NB markers (type II) upon co-expression in the nucleus at t=0 and t=5 min. Co-expression of FIT-GFP with A, SR45-mRFP, and B, SRm102-mRFP.
Type II NB markers localized inside NBs at t=0 and t=5 min. Similar localization patterns were observed upon single expression, showing that SR45 and SRm102 are present in distinct NB types (compare with Supplemental Figure S4, A and B). FIT-GFP colocalized with type II markers in their distinct NBs at t=5 min, but not t=0. FIT-GFP additionally localized in FIT NBs at t=5 min. Type II markers were not present in FIT NBs, while FIT-GFP became recruited into the distinct type II NBs upon the light trigger. Hence, FIT NBs could be associated with speckle components.
Scale bar: 2 µm. Filled arrowheads indicate colocalization in NBs, empty arrowheads indicate no colocalization in NBs. G = GFP; R = mRFP. Fluorescence protein analysis was conducted in transiently transformed N. benthamiana leaf epidermis cells, following the standardized FIT NB analysis procedure.
Representative images from two to five independent experiments are shown. For data with type I markers (no colocalization) and type III markers (full colocalization) see Supplemental Figure S3 and Figure 6. Confocal images showing localization of FIT-GFP and NB markers (type III) upon co-expression in the nucleus at t=0 and t=5 min. Co-expression of FIT-GFP with A, UAP56H2-mRFP, B, P15H1-mRFP, and C, PININ-mRFP. All three type III NB markers were homogeneously distributed and colocalized with FIT-GFP in the nucleus at t=0, while they colocalized with FIT-GFP in FIT NBs at t=5 min. UAP56H2-mRFP and P15H1-mRFP showed homogeneous localization in the single expression at both t=0 and t=5 min (compare with Supplemental Figure S4, C and D), while PININ-mRFP localized mainly in one large and several small NBs upon single expression at t=0 and t=5 min (compare with Supplemental Figure S4E). Hence, these three markers adopted the localization of FIT-GFP upon co-expression and suggest that FIT NBs have a speckle function.  Confocal images showing localization of FIT-GFP and PB markers upon co-expression in the nucleus at t=0 and t=15 min. Co-expression of FIT-GFP with A, PIF3-mCherry, and B and C, PIF4-mCherry, in B, showing a typical pattern with absence of NBs (ca. 50% of nuclei), in C, showing a typical pattern with presence of NBs (ca. 50% of cells). When FIT-GFP was co-expressed with PB markers, FIT NBs did not appear at t=5 min, but instead, FIT-GFP colocalized with PB markers in PBs at t=15 min. A, PIF3-mCherry localized predominantly to a single large PB at t=0 and t=15 min. FIT-GFP colocalized with PIF3-mCherry in this single large PB at t=15 min. B, PIF4-mCherry and FIT-GFP were both homogeneously distributed in the nucleoplasm at t=0 and t=15 min. In C, FIT-GFP colocalized with PIF4-mCherry in PBs at t=0 and t=15 min.
The same localization patterns were found for PIF3-mCherry and PIF4-mCherry upon single expression (compare with Supplemental Figure S5). Hence, FIT-GFP was recruited to the two distinct types of PIF3 and PIF4 PBs, whereas PIF3 and PIF4 were not recruited to FIT NBs. This suggests that FIT NBs are affected by the presence of PIF3-and PIF4-containing PBs and a connection to light signaling exists.   Root iron reductase activity assay on 6-d-old Arabidopsis seedlings grown under white light for 5 d under iron deficient and iron sufficient conditions and then exposed for 1 d to blue, red, far-red light or darkness, or in parallel as control to white light. A, Induction of iron reductase activity upon iron deficiency versus sufficiency was higher under blue light than compared to the white light control. B, Exposure to red light did not change the iron reductase activity compared to the white light control. C-D, Plants exposed to far-red light and darkness did not show induction of iron reductase activity under iron deficient conditions compared to the iron deficient conditions.
Two experiments were conducted, one representative result is shown. Bar diagrams represent the mean and standard deviation of four replicates with four seedlings each (n=4). Statistical analysis was performed with one-way ANOVA and Tukey post-hoc test. Different letters indicate statistically significant differences (P < 0.05).  Gene expression analysis of iron deficiency genes FIT, BHLH039, FRO2, and IRT1 on 6-d-old Arabidopsis seedlings grown under white light for 5 d under iron deficient and iron sufficient conditions and then exposed for 1 d to blue, red, far-red light or darkness, or in parallel as control to white light. A-B, FIT and bHLH039 gene induction in response to low iron supply did not change after blue light exposure compared to the white light control. C-D, FRO2 and IRT1 gene expression increased after blue light exposure in iron deficient conditions compared to respective iron sufficient condition and white light control.
E, FIT gene expression did not change after red light exposure compared to the white light control. F-G, Two experiments were conducted, one representative result is shown. Bar diagrams represent the mean and standard deviation of three replicates with twelve seedlings and two technical replicates each (n=4). Statistical analysis was performed with one-way ANOVA and Tukey post-hoc test. Different letters indicate statistically significant differences (P < 0.05).