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The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem [1]

['Damiano Martignago', 'Dipartimento Di Bioscienze', 'Università Degli Studi Di Milano', 'Milan', 'Vítor Da Silveira Falavigna', 'Max Planck Institute For Plant Breeding Research', 'Cologne', 'Alessandra Lombardi', 'He Gao', 'Paolo Korwin Kurkowski']

Date: 2023-07

The floral transition occurs at the shoot apical meristem (SAM) in response to favourable external and internal signals. Among these signals, variations in daylength (photoperiod) act as robust seasonal cues to activate flowering. In Arabidopsis, long-day photoperiods stimulate production in the leaf vasculature of a systemic florigenic signal that is translocated to the SAM. According to the current model, FLOWERING LOCUS T (FT), the main Arabidopsis florigen, causes transcriptional reprogramming at the SAM, so that lateral primordia eventually acquire floral identity. FT functions as a transcriptional coregulator with the bZIP transcription factor FD, which binds DNA at specific promoters. FD can also interact with TERMINAL FLOWER 1 (TFL1), a protein related to FT that acts as a floral repressor. Thus, the balance between FT-TFL1 at the SAM influences the expression levels of floral genes targeted by FD. Here, we show that the FD-related bZIP transcription factor AREB3, which was previously studied in the context of phytohormone abscisic acid signalling, is expressed at the SAM in a spatio-temporal pattern that strongly overlaps with FD and contributes to FT signalling. Mutant analyses demonstrate that AREB3 relays FT signals redundantly with FD, and the presence of a conserved carboxy-terminal SAP motif is required for downstream signalling. AREB3 shows unique and common patterns of expression with FD, and AREB3 expression levels are negatively regulated by FD thus forming a compensatory feedback loop. Mutations in another bZIP, FDP, further aggravate the late flowering phenotypes of fd areb3 mutants. Therefore, multiple florigen-interacting bZIP transcription factors have redundant functions in flowering at the SAM.

In this research, we studied how plants regulate the time to flower. This process is highly sensitive to the environment, including seasonal changes in day length. In Arabidopsis thaliana long day conditions, typical of spring and summer, stimulate the production of a specialised protein signal called florigen in leaves to initiate flowering at the shoot (the growing tip of the plant). Florigen proteins move long distance to the shoot where they interact with another set of proteins, the best known of which is FD, belonging to group A bZIPs. Here, we discovered that all group A bZIP proteins can also bind to the florigen. We also found that the bZIP AREB3, present at the shoot like FD, when mutated together with FD caused an aggravated delay in flowering compared with single mutant plants. Mutations in a third bZIP, FDP, resulted in an even later flowering compared with double mutants, showing that the effect of these mutations is cumulative. It appears that the more of this family of regulatory proteins we remove, the later the plant flowers. In conclusion, we discovered that many more bZIPs than previously thought can interact with florigens to regulate flowering time.

Funding: This work was supported by a Research Grant from the HFSP Ref.-No: RGP0011/2019 and from the University of Milan - SEED 2019 - DISENGAGE Ref. no.1236 to LC. DM is supported by a research fellowship co-funded by the European Union - ESF, REACT-EU, PON Ricerca e Innovazione 2014-2020. This work was also funded by the Deutsche Forschungsgemeinschaft through Cluster of Excellence CEPLAS (EXC 2048/1 Project ID: 390686111) to GC, and the laboratory of GC receives core funding from the Max Planck Society. VSF was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 894969 and the von Humboldt Foundation (BRA 1210514 HFST-P). The authors wish to acknowledge the support of the APC central fund of the University of Milan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

While FD plays a key role in mediating FT signalling at the SAM, fd mutants only partially suppress the early flowering conferred by overexpression of FT [ 19 , 26 ]. This suggests that other genes are also involved in the FT-mediated regulation of flowering. Indeed, some degree of functional redundancy between FD and FD PARALOGUE (FDP) exists, although fd fdp double mutants still retain substantial flowering responsiveness to LDs [ 26 , 27 ]. Other potentially redundant functions to FD may lie within the evolutionarily-related group A bZIP TFs. This group includes several proteins described as mediators of abscisic acid (ABA) signal transduction [ 28 , 29 ], such as ABA INSENSITIVE 5 (ABI5) and related ABRE-binding (AREB) proteins or ABRE-binding factors (ABFs), which were characterised by their common binding to conserved ABA-responsive elements (ABREs, PyACGTGG/TC) [ 30 , 31 ]. Recent studies indicate that ABI5 and TFL1 proteins act in the same protein complex to control seed size and germination [ 32 ], suggesting that other group A bZIP TFs can associate with PEBPs to control different traits. The group A bZIPs ABF3 and ABF4 promote flowering from the leaves in response to drought [ 33 ]. However, it is currently unknown if, besides FD and FDP, other group A bZIPs play any role in relaying FT signalling at the SAM. Here, we studied potential interactions between group A bZIP TFs and the PEBP FT and TFL1. By using CRISPR-Cas9-based mutagenesis and genetics approaches, we demonstrate that AREB3 is a novel interactor of FT, acting redundantly with FD and FDP in flowering-time regulation at the SAM. Confocal microscopy imaging of shoot meristems reveals a striking overlap between AREB3 and FD expression, supporting their redundant role. Notably, AREB3 expression levels are negatively regulated by FD, so higher levels of AREB3 mRNA and its encoded protein are observed in fd mutants. Our results contribute to increasing knowledge of compensatory mechanisms between proteins that play a key role in the photoperiodic regulation of flowering and show that more bZIP TFs than previously known are expressed at the SAM and can interact with FT to finely regulate floral transition.

FT belongs to the phosphatidylethanolamine-binding proteins (PEBPs) superfamily, which includes structurally, but not functionally, related proteins described from bacteria to humans [ 10 – 12 ]. In plants, PEBPs are usually regarded as transcriptional coregulators [ 13 ]. Crystallographic data derived from rice florigen Hd3a describes nuclear-localised hexameric florigen activation complexes (FAC), consisting of pairs of Hd3a proteins, scaffold 14-3-3 proteins, and bZIP (basic leucine zipper) transcription factors (TFs) [ 14 ]. Several independent studies support a general model in which phosphorylated bZIP TFs provide DNA binding selectivity, whereas florigens stimulate transcription at target promoters, possibly stabilizing the formation of the bZIP–DNA complex [ 15 – 18 ]. Phosphorylation of a conserved 14-3-3 binding site at the C terminus of the bZIP TF FD, called SAP motif (RXX(pS/pT)XP), has been described as essential for the formation and function of the FAC [ 14 ]. Although non-phosphorylatable versions of the FD SAP motif showed the impaired formation of the FAC complex, FD still binds to DNA in vivo, even in the absence of FT [ 14 , 15 , 17 ]. FD activates the expression of and directly binds to many flowering-time genes, including SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL), and floral-meristem identity genes such as APETALA1 (AP1) and LEAFY (LFY) [ 8 , 19 – 23 ], and FT is proposed to enhance FD binding to its target genes [ 15 ]. Another PEBP, TERMINAL FLOWER 1 (TFL1), is present at the SAM and antagonises FT function, perhaps by competing for the binding to FD [ 21 , 24 , 25 ]. The TFL1–FD complex formation at FD target chromatin mostly results in transcriptional repression [ 18 , 21 ]. Thus, FD function is key for the assembly of different PEBP complexes at target DNA sequences, causing different transcriptional fates at regulated genes, and ultimately affecting the flowering process.

Many plant species detect variations in daylength (photoperiod) and in response to these align their growth and development to the most beneficial environmental conditions. Arabidopsis thaliana responds to long days (LDs), typical of spring/summer at temperate latitudes, to activate flowering and initiate its reproductive cycle [ 1 , 2 ]. Extensive mutagenesis screens led to the definition of a genetic pathway and transcriptional cascade activated by LDs, and major components of this pathway are conserved across species. Photoperiodic flowering involves the transmission of signals from the leaves–the site of photoperiod perception–to the shoot apical meristem (SAM)–where the floral transition and floral development occur. FLOWERING LOCUS T (FT) acts as the main systemic florigenic signal, being produced in the leaf vasculature in response to LDs and moving to the SAM [ 3 – 6 ]. In the SAM, FT triggers extensive transcriptional reprogramming, ultimately causing a change in the identity of lateral organ primordia that switch from forming leaves and axillary branches to forming flowers [ 7 – 9 ].

Results

Widespread interactions between Arabidopsis group A bZIPs and FT or TFL1 To gain insights into the potential interactions between group A bZIPs and FT or TFL1 proteins, a yeast two-hybrid (Y2H) assay was established comprising all group A bZIP sequences fused to the activation domain. This assay confirmed robust interactions between FT/TFL1 and FD/FDP [19,20]. Notably, FT and TFL1 also interacted with nearly all other group A bZIP TFs (Figs 1A and S1). These results suggest that FT can interact with a wider range of group A bZIP TFs than previously proposed and that some of these interactions might contribute to inducing floral transition at the SAM redundantly with FD and FDP. PPT PowerPoint slide

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TIFF original image Download: Fig 1. FT and TFL1 interact with several group A bZIP TFs. (A) Y2H assays were conducted to test protein interactions among group A bZIP TFs and the PEBP proteins FT and TFL1. See S1 Fig for further information. (B) Protein alignments of the C-terminal region of the group A bZIP TFs show strong conservation of the RXX(pS/pT)XP SAP motif. The phosphorylatable T/S residue, which is T282 and S294 for FD and AREB3, respectively, is boxed. The consensus sequence of the SAP motif is depicted in the logo format (made with weblogo.berkeley.edu). (C) Y2H assays of protein interactions among wt FD, wt AREB3, FDT282A and AREB3S294A with FT and TFL1. (D) N. benthamiana co-IP of protein interactions among wt and mutated versions of FD and AREB3 with FT and TFL1. Protein–protein interactions were tested in pairs by co-agroinfiltration of tobacco leaves. FD and AREB protein versions were translationally fused to MyC, whereas both PEBPs were translationally fused to GFP. The input was composed of total proteins recovered before the IP. GFP-fused proteins were pulled down using anti-GFP nanobody (VHH) beads and immunoblotted using α-MyC or α-GFP antibodies. Additional controls are present in S3 Fig. Nt, non-transformed. https://doi.org/10.1371/journal.pgen.1010766.g001 Next, we screened the group A bZIP TFs for the presence of a putative SAP motif in their C terminus. Mode I canonical RXX(pS/pT)XP motifs were described as highly conserved 14-3-3 binding sites, with plants commonly presenting an extended LX(R/K)SX(pS/pT)XP motif [34]. We found that most group A bZIP genes presented at least one splicing form encoding a canonical SAP motif, with a few exceptions at the conserved proline residue (Fig 1B, [28]). In Y2H and EMSA assays, this motif is critical for FD interaction with FT (Fig 1C, [15,19]). Among all group A bZIP TFs, AREB3 (also known as DPBF3, AtbZIP66, At3g56850) has the most similar SAP motif to FD and FDP. Its proposed SAP motif contains a potentially phosphorylatable serine (S294) instead of the threonine of FD (T282) and FDP (T231). By screening publicly available proteomic datasets (S1 Table, [35]), we found that AREB3 is phosphorylated at S294 as well as at other ABA-related sites [36–41].

Mutations in the SAP motif do not abolish the interaction of bZIP TFs with FT/TFL1 in plant cells We tested the importance of the phosphorylatable S294 residue for FT interaction by constructing the AREB3S294A mutant and found that its interaction with either FT or TFL1 is weakened in a Y2H assay (Fig 1C). Similar results were obtained with AREB3ΔSAP, a truncated version of AREB3 (R291*) lacking the SAP motif cds (S2 Fig). The interaction between AREB3 and FT/TFL1 was further verified through co-immunoprecipitation (co-IP) of transiently expressed, epitope-tagged versions of AREB3 and FT/TFL1 in Nicotiana benthamiana (Fig 1D). As expected, both FD and AREB3 proteins were co-purified with FT and TFL1, supporting theirs in planta interaction. In this assay, the AREB3S294A and FDT282A mutant proteins were also co-immunoprecipitated with FT and TFL1. Similar results were obtained in another tobacco co-IP assay, in which FD and FDΔSAP but not CONSTANS (CO) interacted with FT (S3 Fig). Next, bimolecular fluorescence complementation (BiFC) assays confirmed comparable levels of fluorescence reconstitution in nuclei (n>150) upon co-expression of wild-type (nYFP:AREB3) or truncated (nYFP:AREB3ΔSAP) versions of AREB3 with FT:cYFP in N. benthamiana (S4 Fig). These results show that, in these transient assays, FT and TFL1 interaction with AREB3 and FD can occur in plant cells independently of the SAP motif.

AREB3 distribution at the SAM overlaps with FD The stronger late-flowering phenotype observed in fd-3 areb3 mutants compared to fd-3 may be due to AREB3 partially compensating for the loss of FD at the SAM. To test whether AREB3 is expressed in a similar temporal and spatial pattern to FD, stable transgenic lines expressing a fusion of AREB3 to the VENUS fluorescent protein (pAREB3:VENUS:AREB3) were constructed in the areb3-1 background. Four independent homozygous T3 lines were obtained, and RT-PCR experiments confirmed the expression of the mRNA of the chimeric version of AREB3 at similar levels to the endogenous gene (S10 Fig). Confocal laser microscopy imaging of shoot apices of pAREB3:VENUS:AREB3 areb3-1 (#11.4) plants revealed that AREB3 was detectable in most cells of the vegetative SAM, including the L1, L2 and L3 meristematic cell layers, and young leaf primordia (Fig 3A). After the floral transition, AREB3 was also present throughout the inflorescence meristem, in young flower primordia and stems. One representative pAREB3:VENUS:AREB3 line (#11.4) was crossed with areb3-1 fd-3 to obtain pAREB3:VENUS:AREB3 areb3-1 fd-3. The confocal analysis did not reveal obvious changes in the AREB3 spatial distribution at the SAM in the areb3-1 fd-3 background in comparison to areb3-1, despite their different developmental stages (Fig 3A). Still, the insertion of AREB3:VENUS:AREB3 into areb3-1 fd-3 double mutants complemented the later-flowering phenotype caused by areb3-1, indicating that the VENUS:AREB3 fusion protein was functional (S10 Fig). In contrast, Basta-resistant areb3-1 fd-3 T2 transgenic lines carrying a mutant construct lacking the SAP motif of AREB3 (pAREB3:VENUS:AREB3ΔSAP) flowered as late as areb3-1 fd-3 (S10 Fig), supporting the importance of the AREB3 SAP motif for floral promotion. To understand if the lack of the SAP motif could influence AREB3 protein accumulation or subcellular localisation at the SAM, we analysed homozygous T3 pAREB3:VENUS:AREB3ΔSAP (#1.7) plants at different developmental stages. The VENUS:AREB3ΔSAP protein was nuclear localised and expressed in a similar spatio-temporal pattern to that of the wild-type VENUS:AREB3 protein during floral transition (Fig 3B). Thus, while an FT–AREB3ΔSAP complex could potentially form at the SAM, no FT signalling is elicited in the absence of the SAP motif. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Gene expression and protein localisation of AREB3 and FD. (A) Confocal analysis of dissected apices during the floral transition of pAREB3:VENUS:AREB3 in areb3-1 and areb3-1 fd-3 backgrounds. (B) Dissected apices of pAREB3:VENUS:AREB3ΔSAP in areb3-1 fd-3 were analysed by confocal microscopy. (C) Confocal analysis of dissected apices of pFD:VENUS:FD in fd-3 and fd-3 areb3-1 backgrounds. In these assays, at least three apices of each line were analysed and a representative image of the spatial distribution of the proteins was selected. The star indicates the establishment of the inflorescence meristem. Scale bars, 100 μm. (D) mRNA levels of AREB3 in wt and the fd-3 mutant in SAM-enriched tissue. (E) AREB3:VENUS protein abundance in SAMs of areb3-1 (lane 1) and areb3-1 fd-3 (lane 2) isogenic lines #11.4. Upper panel: immunoblot analysis of AREB3:VENUS detected with αGFP antibodies; Rel. Q. indicates protein quantity relative to the sample loaded in lane1. Lower panel: immunoblot analysis of UGPase, used as a loading control. For each panel, numbers indicate molecular weights. (F) mRNA levels of FD in wt and areb3-1 mutant in SAM-enriched tissue. In both experiments, Arabidopsis plants were grown for two weeks in short-day (SD) conditions and then shifted to long days (LD). Samples were harvested at ZT8 before the shift (D0) and 1, 3, and 7 days (D1, D3, and D7, respectively) after the shift to flowering-inducing photoperiod. Each point represents an independent pool of around five meristems. The experiment was performed twice with similar results. https://doi.org/10.1371/journal.pgen.1010766.g003 We next asked whether changes in FD function might influence AREB3 accumulation or vice versa. Wild-type, areb3-1 or fd-3 plants were shifted from SDs to LDs to activate flowering, and the transcript levels of AREB3 or FD were assayed by RT-qPCR from manually dissected shoot apices. Notably, AREB3 mRNA levels were higher in fd-3 mutants than in wild-type plants (Fig 3D). In agreement, immunoblot analysis of shoot apices from pAREB3:VENUS:AREB3 in areb3-1 or areb3-1 fd-3 backgrounds collected 3 days after the shift to LDs revealed an increase (1.6X) in VENUS:AREB3 protein accumulation in fd-3 compared to the isogenic FD background (Fig 3E). Thus, the increase in AREB3 transcript levels detected in fd mutants translates into more protein accumulation in this genetic background. Comparable transcript levels of FD were identified between areb3-1 mutants and wild-type plants at all time points analysed (Fig 3F). Similar to AREB3 (Fig 3A), no clear changes in the spatial distribution of FD at the SAM were observed when comparing fd-3 to fd-3 areb3-1 (Fig 3C). These results suggest that the AREB3 upregulation in fd mutants may partially compensate for the effect of loss of FD activity on flowering time. For this compensation to occur, FD and AREB3 proteins should share similar spatial and temporal localisation during floral transition. To test this possibility, transgenic lines expressing a fusion of FD to the mCHERRY fluorescent protein (pFD:mCHERRY:FD) were constructed in the fd-3 background, and three homozygous single-copy lines were obtained. These lines complemented the late-flowering phenotype of fd-3 and showed similar spatial and temporal localisation to VENUS:FD [26]. Next, double hemizygous pFD:mCHERRY:FD pAREB3:VENUS:AREB3 fd-3 areb3-1 lines were then examined and showed a strong overlap in the accumulation of mCHERRY:FD and VENUS:AREB3 at the SAM (Fig 4). Yet, AREB3 was consistently identified in the L1 meristematic layer, young flower primordia and developing stems, regions in which FD is absent [7,26]. These results suggest that the partially redundant genetic relationship between AREB3 and FD may be due to their encoded proteins having overlapping spatial patterns of expression and related biochemical functions in the SAM. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Colocalisation analysis of FD and AREB3 in the SAM. pAREB3:VENUS:AREB3 areb3-1 and pFD:mCHERRY:FD fd-3 plants were crossed and the F1 progeny was analysed by confocal microscopy. Dissected apices were sampled at 11, 13, 15 and 18 long days (LD) and simultaneously imaged for VENUS, mCHERRY and Renaissance 2200 fluorescence. FM, floral meristem. Scale bars, 100 μm. https://doi.org/10.1371/journal.pgen.1010766.g004

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